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APPENDIX
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EBMT Officers
EBMT Coordination Offices
President Dietger Niederwieser
EBMT Secretariat & JACIE Accreditation Office
Division of Haematology & Oncology University Hospital Leipzig Johannisallee 32 A 04103 Leipzig GERMANY Tel: +49 341 971 3050 Fax: +49 341 971 3059
[email protected]
Hospital Clínic Villarroel 170, 08036 Barcelona SPAIN Tel: +34 93 454 9543 Fax: +34 93 453 1263
[email protected] [email protected]
Secretary Per Ljungman
12th Floor Tower Wing Guy's Hospital Great Maze Pond London SE1 9RT UNITED KINGDOM
Department of Haematology Karolinska University Hosp/Huddinge 141 86 Stockholm SWEDEN Tel.: +46 8 585 80000 Fax: +46 8 774 8725
[email protected]
Treasurer (2002 – 2008) Harry Schouten University Hospital Maastricht Dept. Hematology P.O. Box 58000 6202 AZ Maastricht The Netherlands Tel.: +31 43 3 877025 Fax: +31 43 3 875006
[email protected]
EBMT Clinical Trials & Registry Office
Registry Tel: +44 207 188 8408 Fax: +44 207 188 8411
[email protected]
Clinical Trials Tel: +44 207 188 8402 Fax: +44 207 188 8406
[email protected]
EBMT Data & Study Office Faculté de Médecine Saint-Antoine 27, rue Chaligny, 75571 Paris Cedex 12 FRANCE Tel: +33 1 40 46 95 07 Fax: +33 1 40 46 96 07
[email protected]
EBMT Clinical Trials & Study Office Department of Medical Statistics & Bioinformatics, Postzone S-05-P LUMC, PO Box 9600 2300 RC Leiden THE NETHERLANDS
[email protected] HAEMATOPOIETIC STEM CELL TRANSPLANTATION
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EBMT Committees Cord Blood Committee Chair: Eliane Gluckman
Outreach Committee Co-Chairs: Vladimir Koza
[email protected]
[email protected]
Eliane Gluckman Developmental Committee Co-Chairs: Katarina Leblanc Willem Fibbe
Prospective Clinical Trials Chair: Gérard Socié
[email protected]
[email protected]
Education Committee Chair: Tamás Masszi
Quality Assessment of Autografts Chair: Francesco Lanza
[email protected]
[email protected]
JACIE Executive Committee President: Ineke Slaper-Cortenbach
Registry Committee Chair: Carmen Ruiz de Elvira
[email protected]
[email protected]
[email protected]
Vice-President: Jane Apperley
[email protected] Nuclear Accident Committee Chair: Ray Powles
[email protected]
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Statistical Committee Chair: Myriam Labopin
[email protected]
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EBMT Working Parties
Please refer to the following website address:
http://www.ebmt.org/5WorkingParties/wparties1.html
EBMT Nurses Group Board President Erik Aerts
Secretary Michelle Davies
University Hospital Zürich Dept. of Medicine 8091 Zürich SWITZERLAND Tel.: +41 44 255 2633 Fax: +41 44 255 9781
[email protected]
Adult Leukaemia Unit Christie Hospital Wilmslow Road Manchester M20 4BX UNITED KINGDOM
[email protected]
President Elect Arno Mank Academic Medical Centre Dept. Hematology Meibergreef 9, NL-1105 AZ Amsterdam NETHERLANDS Tel: +31 20 566 7905 Fax: +31 20 566 9030
[email protected]
Treasurer Joachim Blankart AK St. Georg Dept. of Hematology G3, STE Lohmuehlenstrasse, 5 20099 Hamburg GERMANY
[email protected]
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Affiliated Organisations/Websites International Organisations: ASBMT ASCO ASH BMDW EBMT EHA EORTC ESH EUROCORD IBMTR ISCT Europe ISEH NETCORD WMDA
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www.asbmt.org www.asco.org www.hematology.org www.bmdw.org www.ebmt.org www.ehaweb.org www.eortc.be www.esh.org www.eurocord.org www.ibmtr.org www.celltherapysociety.org/Related_Organizations/ ISCT_Europe/ www.iseh.org www.netcord.org www.worldmarrow.org
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Published by
Via Martin Piaggio, 17/6 16122 Genoa, Italy tel +39 10 83794229 - fax +39 10 83794260
[email protected] - www.accmed.org
© 2008 EBMT and ESH Printed by Litoprint (Genoa, Italy)
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CHAPTER 1
Stem cell transplant organisations
J. Apperley, A. Keating
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CHAPTER 1 • Transplant societies
Haematopoietic stem cell transplantation (HSCT) is a professional medical activity and like other medical specialities, lends itself to the development of groups of individuals with similar and/or overlapping interests. It is therefore not surprising that there are a number of national and international HSCT societies that carry out activities with common aims, i.e. the continuous development of their speciality and improvement in outcome for patients. Such activities usually revolve around one or more annual meetings at which new information can be presented, discussed and disseminated. The organisations may have accompanying infrastructures for education, professional standards, production of guidelines and lobbying. However there is one additional characteristic of the HSCT societies that sets them apart from many other organisations and that is the collection, analysis and reporting of outcome of all transplants performed by their member centres. Worldwide there are three international HSCT societies - European Group for Blood and Marrow Transplantation (EBMT) - Center for International Blood and Marrow Transplantation Research (CIBMTR) - Asia Pacific Blood and Marrow Transplantation (APBMT). There are several national HSCT societies in Europe (Table 1) and there are welldeveloped national groups elsewhere including those in Australia, Canada, Japan, New Zealand and North America. The largest of these is the ASBMT in the USA and their official journal is Biology of Blood and Marrow Transplantation.
Table 1: National HSCT Societies and Registries in Europe Country
E-mail address
Austrian SCT Registry
[email protected]
Czech SCT Registry
[email protected]
Societé Francaise de Greffe de Moelle et de Thérapie Cellulaire
[email protected]
Deutsches Register für Stammzelltransplantationen (DRST)
[email protected]
Italian National BMT Registry (GITMO)
[email protected]
Dutch National Registry (TYPHON)
[email protected]
Spanish Haematopoietic Transplantation Group & Cellular Therapy (GETH)
[email protected]
Swiss National Stem Cell Transplant Registry (STABMT)
[email protected]
Turkish Transplant Registry
[email protected]
British Society of Blood and Marrow Transplant
[email protected]
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Laboratory processing of haematopoietic stem cells typically involves a group of professionals with skills that are complementary to those of the average transplant physician and they are represented by the International Society of Cellular Therapy. There is clearly a considerable overlap with the interests of the physician orientated HSCT societies and indeed these professional groups work closely together, most notably in the development of an accreditation system that has established and inspects against, a series of standards for the clinical, collection and laboratory facilities, i.e. FACT-JACIE. The principles of this accreditation system will be discussed in Chapter 4 and will not be discussed in detail in this Chapter. All these societies (EBMT, CIBMTR, APBMT, ISCT) have a similar structure in which a Board of Directors (usually elected by the professional membership) provide and manage the infrastructure, which in turn permits the work of the society. Although the terminology may differ a number of committees or working groups oversee the scientific work and parallel groups of individuals focus on policy issues such as education, meeting organisation, legal and regulatory matters etc. In addition the provision of donors (bone marrow, peripheral blood derived stem cells, cord blood, cellular sub-populations) has also necessitated the development of local and national donor organisations, whose focus is rather different from the professional societies. They have established mechanisms for the recruitment, assessment, provision and follow-up of donors, and are all members of an umbrella organisation, the World Marrow Donor Association (WMDA), that oversees the development of standards and guidelines for the management of these volunteer individuals. NETCORD was developed to share best practice in cord blood procurement. In order to facilitate rapid identification of suitable HLA-matched unrelated donors for individual patients, all the donor registries submit their typing data to an organisation known as Bone Marrow Donors Worldwide (BMDW).
1. European Group for Blood and Marrow Transplantation (EBMT) 1.1. History Over the past three decades the EBMT has grown from a small group of enthusiasts to a society representing more than 500 teams from 57 countries. In 1975 teams from Paris, Leiden, London and Basel met for the first time to discuss general problems and individual patients, and to draw up protocols with a view to improving results. They continued to meet regularly in the Swiss or French Alps and the group, then called the European Cooperative Group for Bone Marrow Transplantation, attracted an ever-increasing number of participants. In 1979, the European Foundation for Bone Marrow Transplantation (EBMT) was formally established with legal status in 20
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the Netherlands. Four Working Parties were created, each with its own registry, i.e. Aplastic Anaemia, Acute Leukaemia, Inborn Errors and Transplantation Immunology. In 1989, a new constitution created the “European Group for Bone Marrow Transplantation”, the abbreviation EBMT being retained. Subsequently, the working parties were restructured and new ones added, i.e. Acute Leukaemia, Aplastic Anaemia, Autoimmune Disease, Chronic Leukaemia, Immunobiology, Inborn Errors, Infectious Diseases, Late Effects, Lymphoma, Nursing, Paediatrics and Solid Tumours. In 1995 the name of the group was changed once more to “European Group for Blood and Marrow Transplantation”, though the acronym “EBMT” again remained. 1.2. Aims The overall objective of the EBMT is to improve the outcome of HSCT by a number of connected activities. The most long-standing of these activities is the collection, validation and analysis of outcome data of all transplants performed by member teams. However the work of the group has expanded to include accreditation, education and outreach, prospective clinical trials and regulatory affairs. 1.3. Organisation EBMT members may be full members, associate members, individual members or corporate patrons. Full and associate memberships are granted to teams rather than individuals. EBMT members have the right to propose and elect office holders, stand for election, attend annual meetings and participate in the Working Parties and EBMT studies. Full membership implies a duty to report individual transplant data to the EBMT registries. The EBMT Board is composed of elected individuals including the President, Secretary, Treasurer and the Working Party Chairpersons. The Board co-ordinates EBMT activities and is responsible for the budget. The organisational structure is shown in Figure 1. The Working Parties are the heart of EBMT, performing retrospective and prospective studies relating to their particular disease or other interest. However the Board of the EBMT have long recognised the need for activities that cross Working Party activities and this has led to the development of EBMT committees. Unlike the Working Party Chairs, who are elected by the member centres and who have permanent positions on the EBMT Board, the Chairs of the committees are appointed by the Board. Committees are designed to be responsive to the needs of the organisation and can be dissolved which there is no longer any requirement for the particular activity. The journal Bone Marrow Transplantation is the official journal of the EBMT.
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Figure 1: Organisational structure of EBMT EBMT Board Working Parties
Committees
Registries
Administration
Acute Leukaemia Aplastic Anaemia Autoimmune Chronic Leukaemias Immunobiology Infectious Diseases Inborn Errors Late Effects Lymphoma Nurses group Paediatrics Solid tumours
Accreditation Clinical Trials Education Developmental Graft Engineering Nuclear Accidents Outreach Statistical
Data Managers Study Coordinators Statisticians
EBMT Secretary EBMT Treasurer Executive Secretary JACIE Office
1.4. Committees The current committees are shown in Table 2. The work of some of these committees will be discussed in more detail below. 1.4.1. Education and Outreach The Education committee was established in 1995 with the intention to provide a more structural approach to training in HSCT. Together with the European School for Haematology (ESH) they have successfully organised the annual residential training course in which senior transplant physicians and scientists interact in formal and informal settings with less experienced colleagues. This committee have also issued this ESH-EBMT transplant manual, now in its fifth edition. Most recently this committee has developed an Outreach project to assist transplant units in economically disadvantaged countries. The EBMT are not alone in this concept and are actively integrating their ideas with the ESH, and are aware of similar programmes within the American Society of Hematology (ASH), CIBMTR, the European Hematology Association (EHA) and the World Health Organisation (WHO). This project supports educational initiatives including training courses in clinical and laboratory techniques, exchange programmes, fellowships, clinical trial participation and twinning programmes.
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Table 2: EBMT Committees Sub-committee
Responsibilities
Accreditation
JACIE, audit and activity survey
Developmental
Emerging stem cell therapies for nonhaematological disorders
Education
Training courses, handbook (jointly with ESH), outreach initiatives
Nuclear accident
Establishment of network of units with haematological expertise to respond to the need for care of neutropenic patients in the event of nuclear accidents
Outreach
Dissemination of excellence in HSCT
Prospective clinical trials
Infrastructure for prospective clinical trials
Quality assessment of autografts
Standardisation of assessment of haematopoietic cell product quality
Statistics
Ensure statistical accuracy within EBMT studies. Develop new statistical tools, production of guidelines
1.4.2. Prospective Clinical Trials This committee was established in 2003 to assist the EBMT in their progress from a retrospective study organisation to a prospective clinical trials group. The introduction into European Legislation of the EU Directive on Clinical Trials (2001/20/EC) has highlighted the necessity of creating an appropriate infrastructure for the conduct of these trials. Most recently the EBMT together with the CIBMTR and the University of Central Lancashire (UCLAN) have been successful in obtaining funding from the European Commission to develop international prospective clinical trials within the transplant community, a project that has the acronym CLINT. 1.4.3. Accreditation The Accreditation committee has a number of diverse functions, which have developed over many years. The committee was originally responsible for a simple form of accreditation for allogeneic sibling transplant in which centres requested accreditation on the basis of performance of a minimum number of transplants annually. Each year, one-third of centres is chosen at random and alerted to the possibility of an audit of data and centre validation. Later, a proportion of these “at risk” centres are visited by senior transplant physicians and data are checked at source.
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One of the most valuable activities of this committee and indeed of EBMT is also one of the simplest. In 1990, Professor Alois Gratwohl began to collect information relating to the annual transplant activity of all transplant centres in Europe (including those centres that are not EBMT members). As a result the EBMT now hold important data relating to activity, geographical variation, changing trends and the development of new technologies. Activity is published annually and has proved invaluable to the funding agencies (public and private) in identifying transplant needs. More recently the bulk of the work if the accreditation committee has been the development and introduction of the FACT-JACIE accreditation programme for all transplant units. This is discussed in more detail in Chapter 4. 1.5. Data collection and data-flow Since its inception the essence of EBMT has been to evaluate and optimise the outcome of patients treated by HSCT. In order to do this it was always deemed necessary to collect patient data, and so the EBMT registry was formed. As of today the registry holds information on almost 300,000 transplants performed by the member teams, and this has proved an invaluable resource in the analysis of factors affecting transplant outcome, in identifying complications and their optimal treatment, in determining trends in management and in evaluating new technologies. Member teams are required to report all their transplant activity. “Minimal Essential Data” (MED) are collected on a number of different forms. MED-A data form the basis of the registry. Relatively simple and limited data collected in this way allow the analyses of overall survival, disease free survival, transplant related mortality, and relapse risk. These data are identical to those collected by the CIBMTR on their Transplant Essential Data (TED) form, and the facility exists to allow cross reporting to both registries. More detailed information is collected on MED-B forms which relate to the specific disease and to the procedure (allograft or autograft). The submission of these data by members is voluntary as opposed to MED A data which are obligatory. Information relating to specific studies is collected via MED-C forms. Until recently the term “form” literally referred to hard copy in virtually all cases. Paper forms were sent to the EBMT and transcribed onto a central electronic database, but the complexity of the programme and the relative lack of familiarity with computers at the time precluded widespread use of this approach. The EBMT has now developed an on-line Internet based reporting system, Project Manager Internet Server or ProMISe. Data reported in this way are received simultaneously by the EBMT registries in Genoa, London, Leiden and Paris but can also be downloaded for local use. This system has revolutionised data reporting with increasing numbers of centres adopting ProMISe. In 2006, 458 users from 364 centres accessed the database to enter data for more than 25,000 transplants. 24
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CHAPTER 1 • Transplant societies
The EBMT has welcomed the development of national transplant societies and has understood the desire of some of these groups to have their own national databases. In the past, for member centres in countries with national registries, data flowed from the centre to the national registry and then to EBMT. Centres in countries without a national registry reported directly to EBMT. Now if the centre is utilising ProMISe, the data are deposited simultaneously in the national and EBMT registries.
2. Center for International Blood and Marrow Transplantation Research (CIBMTR) 2.1. History The CIBMTR is a relatively new organisation having developed in 2004 from an affiliation of the International Blood and Marrow Transplant Registry (IBMTR), a sister organisation of EBMT based at the Medical College of Wisconsin, and the research interests of the US National Marrow Donor Program (NMDP). The IBMTR was established in 1972 to link transplant centres internationally and to collect data derived from allogeneic transplants for the purposes of analysis and reporting. As autografting became more extensively used these transplants were added to the IBMTR databases but collection was confined to procedures performed in North and South America. The NMDP was established in 1987 to facilitate the search, identification, and procurement of volunteer unrelated donor haematopoietic stem cells. At that time the NMDP also had the remit to collect and analyse data emanating from these alternative donor transplants, a task which has now been subsumed into the activities of CIBMTR. The NMDP, like several of the unrelated donor registries, also maintains a tissue biobank of donor and recipient samples. The CIBMTR now has 459 members in 52 countries. Based on data collected in the Centers for Disease Control Hospital Surveys and the US Government Accounting Office and worldwide surveys of transplant activity, approximately 40% of allogeneic transplants worldwide and about 50% of autografts performed in North and South America are registered with the CIBMTR. 2.2. Aims The CIBMTR is a clinical research programme whose major mission is to provide a resource of data and statistical expertise to the HSCT community. They aim to define key areas for future research, secure funding for research, design and implement clinical studies and make available their resources including the clinical database and the tissue bank.
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2.3. Organisation The organisational structure of the CIBMTR is shown in Figure 2. The Affiliation Board and Executive Director have managerial oversight. The Chief Scientific Director has primary responsibility for administrative and scientific operations. CIBMTR Scientific Working, Executive and Advisory Committees provide policy and scientific oversight for this work. The activities of the CIBMTR are funded primarily by grants and contracts from the US government. The CIBMTR Assembly is the voting membership, comprised of a single representative from each CIBMTR Research Center. The CIBMTR Advisory Committee meets biannually to review scientific and other activities of the CIBMTR. The CIBMTR Executive Committee is a subcommittee of the Advisory Committee that provides ongoing advice and counsel to the CIBMTR Statistical Center between meetings of the Advisory Committee.
Figure 2: Organisational structure of CIBMTR
NMDP - Research
Affiliation Committee
Assembly
Executive Director
Advisory Committee
Executive Committee
MCW - IBMTR
Senior Research Advisor
Chief Scientific Director Working/Steering Committees Statistical Methodology
Clinical Trials Support
BMT CTN*
Immunobiology
RCI BMT
Observational Research
Clinical Outcomes
Health Policy
* The BMT CTN Data and Coordinating Center is a Collaboration of CIBMTR, NMDP and the EMMES Corporation; NMDP: National Marrow Donor Program; IBMTR: International Bone Marrow Transplant Registry; MCW: Medical College of Wisconsin; BMT CTN: Blood and Marrow Transplant Clinical Trials Network; RCI BMT: Resource of Clinical Investigations in Blood and Marrow Transplantation 26
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2.4. Committees The Scientific Working Committees are analogous to the EBMT Working Parties and set priorities for observational studies. There are 17 Working Committees: Acute Leukaemia; Chronic Leukaemia; Lymphoma; Plasma Cell Disorders; Solid Tumours; Paediatric Cancer; Non-malignant Marrow Disorders; Immune Deficiencies/Inborn Errors of Metabolism; Autoimmune Diseases; Graft Sources and Manipulation; Graft versus Host Disease; Late Effects and Quality of Life; Immunobiology; Infection and Immune Reconstitution; Regimen-related Toxicity and Supportive Care; Health Services and Psychosocial Issues; Donor Health and Safety. Membership on CIBMTR Working Committees is open to anyone willing to take an active role in studies using CIBMTR data and/or resources. The CIBMTR also functions as the Data and Coordinating Centre of the US Blood and Marrow Transplant Clinical Trials Network (BMT CTN). The BMT CTN was established by the National Heart, Lung, and Blood Institute (NHLBI) and the National Cancer Institute (NCI) in order to design, develop, and execute prospective clinical trials to improve the safety, applicability, and efficacy of HSCT. Sixteen core centres were awarded cooperative agreements with the NHLBI/NCI and a broader network of over 60 transplant centres have participated in eight prospective trials enrolling over 1,500 patients in the first four years of activity. 2.5. Data collection The CIBMTR collects data at two levels. Registration data (Transplant Essential Data–TED) are identical to the Med-A data of the EBMT and the facility exists to allow cross reporting to both registries. All CIBMTR teams contribute registration data. Research data are collected on subsets of registered patients and includes comprehensive pre- and post-transplant clinical information.
3. The Asia Pacific Blood and Marrow Transplantation (APBMT) The Asia Pacific Blood and Marrow Transplantation (APBMT) group is an organisation, akin to EBMT and CIBMTR, which allows HSCT physicians in Asian countries to share their experience and develop co-operative studies. The APBMT group was established in 1990 and initial meetings were held in China and Japan. Since 1994 annual meetings have been an integral part of the organisation. The APBMT aims to promote all aspects associated with HSCT including basic and clinical research. A registry to collect the results of HSCT performed in member centres began in 2006.
4. Worldwide Network for Blood and Marrow Transplantation (WBMT) In 2007 the concept developed of an organisation that would unite the three
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international HSCT societies (EBMT, CIBMTR and APBMT) together with the WMDA with the aim of unifying data collection and reporting systems, developing harmonised guidelines, providing an effective professional lobby to the various national and international regulatory agencies and strengthening the research database. The name Worldwide Network for Blood and Marrow Transplantation was adopted and the constitution is currently in development.
5. International Society for Cellular Therapy (ISCT) 5.1. History ISCT was established in 2002 as a professional organisation for those working or interested in cell-based research, processing, manipulation, and clinical translation. It evolved from the US and European branches of the International Society for Hematotherapy and Graft Engineering (ISHAGE) when the change in name and focus was considered essential to encompass the use of cellular therapy for a wider range of diseases and applications. ISHAGE had itself been established in 1991 when it had become increasingly clear that an organisation was required to represent the interests and needs of those involved in graft manipulation. 5.2. Aims The mission of ISCT is to provide a global forum for the development and validation of standardised technology and for representation of the membership to other professional organisations and regulatory and governmental bodies. They also have a strong commitment to education and training. 5.3. Organisation In contrast to the other HSCT organisations, the membership of the ISCT is largely composed of individuals although there are arrangements for laboratory and corporate membership. In 2007, the Society had over 1200 members from 6 continents. Leadership of the ISCT is provided by an Executive Committee which manages the affairs of the Society and oversees the work of the committees and working groups. An advisory committee, composed largely of previous elected officers, provides input from the membership to the Executive Committee and also advise on the long-term strategy of the Society. ISCT is composed of several standing committees, representing all facets of ongoing activities of the Society, and short-term working groups, designed to address immediate concerns within cell therapy, drafting position statements and guidance for broad dissemination or submission to the FDA. The current committees and groups are shown in Table 3. 28
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Table 3: Working Parties and Standing Committees of ISCT Working Parties
Standing Committees
- Microbial testing - Storage & transportation - Devices - Facility sanitisation - Homologous use - Release testing
- Cell therapy commercialisation - Cell and tissue evaluation - Education and publication - Gene therapy - Haematopoietic stem cells - Immunotherapy & dendritic cells - Laboratory practices - Regulatory affairs - Europe - Regulatory affairs - America - Regulatory affairs - Asia - Mesenchymal and tissue stem cells
ISCT organises an annual meeting of diverse groups of investigators, practitioners, and technologists with a focus on research and education. Cytotherapy is the official journal of ISCT.
6. World Marrow Donor Association (WMDA) 6.1. History The WMDA was created in 1994 to address obstacles faced when transplantation involved donors and recipients in different countries. It is a voluntary organisation of representatives of HSCT donor registries, cord blood banks, and other institutions and individuals involved in the use of unrelated donors. Currently the WMDA represents 11.8 million donors in 67 stem cell registries and 292,000 units in 47 cord blood banks from 48 countries. The individual donor registries are too numerous to mention here but include the National Marrow Donor Program (NMDP) in the United States, the Anthony Nolan Trust in the UK and the German. 6.2. Aims To improve and simplify stem cell donation for donors and patients. 6.3. Organisation (Figure 3) The membership of the WMDA is comprised of both donor (blood, marrow and cord blood) organisations and individuals. Much of the work of the WMDA is done through its working groups (Figure 3), which are focussed on developing best practice in the medical, ethical, technical, quality and financial activities of international HCT as they relate to volunteer donors. Importantly, the WMDA established an
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Figure 3: Organisational structure of WMDA President Vice Presidents Past President Chief President Elect President Emeritus North & South America Operating Asia, Australia, Officer Secretary General Pacific Islands, Europe Treasurer
Working Groups Clinical Registries Information Technology Ethics Quality Assurance
Accreditation Committee
Secretaries North & South America Asia, Australia, Pacific Islands, Europe
WMDA Administrative Office Leiden, NL
accreditation program for donor registries in 2003, to certify adherence to WMDA standards for registry operation. These standards cover the general organisation of the registry, donor recruitment and consent procedures, donor characterisation and evaluation, information technology, facilitation of search requests, second and subsequent donations, graft procurement and transport, assessment of donor and transplant outcomes and financial and legal liabilities. The standards are exacting and to date 12 registries have received accreditation. The aim is clearly to extend the process to all their registry members.
7. Alliance for harmonisation of cellular therapy accreditation (AHCTA) The process of HSCT has recently come under extensive scrutiny by national and international regulatory agencies and this has resulted in increasing stringency in the legislation regarding the procurement and use of stem cells. In 2006 it became clear that there was a real risk that regulations could be introduced nationally that would conflict with processes elsewhere in the world. This is particularly relevant to the requirements for import and export of cells. As it is common practice in alternative donor transplantation to use stem cells collected in one country for a patient treated in another country there was some urgency 30
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to present our concerns in a unified manner. Furthermore there was clearly a need for the relevant parties to agree common policies, procedures and protocols. AHCTA was developed to harmonise the standards of all the involved organisations (Figure 4) with the objective of creating a single set of quality, safety and professional requirements for cellular therapy. These standards will comprehensively cover all aspects of the process from assessment of donor eligibility to transplantation and clinical outcome. AHCTA regards regulatory authorities as partners in the application of these global standards, essential to their successful adoption and will endeavour to inform and support these authorities in the area of cellular therapy regulation.
Figure 4: Membership of AHCTA European Federation for Immunogenetics
European Group for Blood & Marrow Transplantation
Foundation for the Accreditation of Cellular Therapy
Joint Accreditation Committee ISCT-EBMT
International NETCORD Foundation
World Marrow Donor Association
International Society for Cellular Therapy (Europe)
8. Bone Marrow Donors Worldwide (BMDW) 8.1. History BMDW began as an initiative of the Immunobiology Working Party of the EBMT in 1988. It represents a voluntary collaborative effort of the stem cell donor registries and cord blood banks to provide centralised information on the HLA phenotypes and other relevant data of unrelated stem cell donors and cord blood units and to make this information easily accessible to the physicians of patients in need of HSCT. The first edition of a publication containing the donor files of eight registries with a total of 155,000 volunteer stem cell donors was
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issued in 1988. The current number of donors and cord blood units in the BMDW database is almost 12 million! 8.2. Aims The original goal was to collect the HLA phenotypes of volunteer stem cell donors and cord blood units, and to maximise the chance of finding a stem cell donor or cord blood unit by providing access to all stem cell donors and cord blood units available in the world. 8.3. Organisation The BMDW Editorial Board consists of one representative of each stem cell donor registry or cord blood bank participating in BMDW, and meets twice a year to discuss achievements and necessary improvements. The office of BMDW remains in Leiden in the Netherlands and the service is provided and managed by Europdonor Foundation. The HLA types and matching programmes are available online. More recently programmes for mismatched donors and cord blood donors have been added together with a haplotype frequency analysis on phenotype data submitted by the participating registries. Only HSCT professionals who have received authorisation and participating registries can access these services. A username and password are required, and a secure protocol has been implemented to encrypt all transmitted data.
9. NETCORD 9.1. History NETCORD is a global non-profit organisation, which was established in 1998 to connect the existing cord blood banks in order to share best practice in this rapidly evolving field. 9.2. Aims The goal of the organisation is to maintain compliance with quality standards so as to ensure uniformity and quality assurance of the cord blood units supplied by their members. In addition they provide access for registered transplant units to a centralised web-based search process, support research to constantly improve the processes of procurement and processing and develop educational programmes to increase the donor pool and the subsequent use of the products. 9.3. Organisation Member cord blood banks are expected to have inventories of at least 1000 cord 32
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CHAPTER 1 • Transplant societies
blood units of documented high quality. Thus far, NETCORD has 23 members and as of late 2007 has an inventory of 153,830 units, an increase of nearly 30,000 units or 23% in the past 12 months. NETCORD has facilitated the use of over 3,000 cord blood units worldwide both for children and adults. In order to ensure high and uniform quality of all cord blood (CB) units, NETCORD established quality standards in collaboration with FACT for the collection, cryopreservation, storage and release of units. All NETCORD banks are expected to obtain accreditation by NETCORD-FACT and to comply with their quality standards. To facilitate direct searches for CB units by transplant centres and to avoid allocation conflicts for patients, NETCORD developed the Virtual Office (VO), which contains a real-time search programme for compatible and available units. Details include high resolution HLA typing, volume, cell number and infectious disease markers. By submitting a search request to the VO, transplant centres no longer have to search multiple databases of the different cord blood banks, but receive a single unified search report on all acceptable units that are contained in the inventory. The collection of clinical outcome data is coordinated by CIBMTR or Eurocord. The Eurocord registry has operated on behalf of EBMT since 1995. Thanks to the NETCORD-Eurocord collaboration, 3286 cord blood transplantations have been reported to the Eurocord registry from 177 EBMT (64% of cases) and 187 non-EBMT (36% of cases) centers from 43 European and nonEuropean countries. In summary, the intense activity within all these societies reflects the on-going enthusiasm for HSCT and cellular therapy. Further information can be obtained from the specific websites (Table 4).
Table 4: Web addresses of transplant related organisations EBMT
www.ebmt.org
ESH
www.esh.org
CIBMTR
www.cibmtr.org
APBMT
www.apbmt.org
ISCT
www.celltherapysociety.org
WMDA
www.worldmarrow.org
AHCTA
www.ahcta.org
BMDW
www.bmdw.org
NETCORD
www.netcord.org
Eurocord
www.eurocord.org
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*
CHAPTER 2
Biological properties of haematopoietic stem cells
A. Wodnar-Filipowicz
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1. Introduction Until a few years ago, an interest in stem cells, mostly of haematopoietic origin, was limited to a relatively small representation of scientists and clinicians seeking to understand the role of these rare cells in tissue homeostasis and to utilise their remarkable potential to regenerate an adult tissue following transplantation. Over the last few years, stem cells have captured the imagination of scientists, clinicians and the lay public alike with the promise of representing a future remedy for the major degenerative diseases of our civilisation and even dysfunctions associated with normal ageing. The progress in technologies to detect and enumerate stem cells in vivo led to discovery of stem cells residing in most mammalian tissues, contributing to their generation, homeostasis and probably repair. A major breakthrough has been achieved in the development of methods to propagate human embryonic stem (ES) cells in culture and to drive their in vitro differentiation into specialised human tissues. The concept of cancer stem cells has emerged, as cells responsible for generation and persistence of tumours. Here we discuss the stem cell field by summarising the current knowledge of the phenotype of these cells, their interactions with the microenvironment in which they reside, and the mechanisms regulating their functions. The central place will be taken by haematopoietic stem cells (HSCs), representing the first-discovered, the best-understood and, at present, the only clinically-applicable population of stem cells. We also summarise the current state of knowledge on the functional plasticity of somatic tissue stem cells and on human ES cells. The therapeutic relevance gained from the basic research findings will be emphasised.
2. Stem cell definition Stem cells are defined as a population of undifferentiated cells with the capacity to divide for indefinite periods, to self-renew and to generate a functional progeny of highly specialised cells. This common definition includes cells present in different physical locations and having fundamentally different proliferative properties and functions (Table 1). A fertilised egg (zygote) represents a totipotent stem cell, a cell with unrestricted differentiation potential and the only cell with the capacity to give rise to all cells necessary for the development of foetal and adult organs. ES cells forming a cluster of cells inside the blastocyst are pluripotent stem cells, capable of generating a variety of specialised cell types, but limited in their differentiation potential by the inability to support the development of a foetus. Further specialisation results in generation of multipotent stem cells residing in adult somatic tissues. Their physiological functions are to replenish mature cell populations of the given tissue
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Table 1: Definition of stem cell types Stem cell
Developmental properties
Fertilised egg
- Totipotent - Unrestricted differentiation potential
ES cells
- Pluripotent - Give rise to a variety of specialised cell types
Adult somatic stem cells
- Multipotent - Limited to specific tissues
or organ, and to respond to stress by repairing the damage. HSCs represent the prototype of multipotent adult tissue stem cells (1). In humans, HSCs can be found in cord blood as a result of stem cell migratory properties during foetal development, whereas post-natally, the only organ harbouring HSCs and pursuing active multilineage haematopoiesis is the bone marrow.
3. Characteristics of stem cells in the bone marrow More than 40 years of research on bone marrow-derived stem cells, initiated in the 1960s by Till and McCulloch, marked an ongoing improvement in methods to quantitate and isolate these cells. Assays for clonogenic precursors of the myeloerythroid lineages in vitro, defined as long term culture-initiating cells (LTCICs) and committed colony forming units (CFUs) were followed by the development of a model of immunocompromised non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, which allows the study of the repopulating ability of human haematopoietic cells in vivo (2). These functional assays are paralleled by progress in the phenotypic characterisation of haematopoietic cells by flowcytometry, owing to monoclonal antibodies specifically recognizing cell surface molecules (Figure 1). The phenotypic properties of murine HSCs have been precisely defined as cells devoid of lineage markers and expressing the stem cell antigen (sca1) and the receptor c-kit. The Lin-sca1+c-kit+ (LSK) cell population has self-renewing and long-term repopulating activity in vivo. Other cell surface markers defining the HSC compartment in mice include the tie2 and flt3 receptors and CD150. Characteristically, c-kit, flt3 and tie2 function as receptors for early-acting haematopoietic growth factors: stem cell factor, flt3 ligand and angiopoietin, which act as key positive regulators of haematopoiesis (3). The most primitive human HSCs express CD34 and lack CD38 cell surface antigens and have the capacity to reconstitute a sublethally irradiated NOD/SCID host. The CD34+CD38- HSC compartment, which constitutes ±0.1% of bone marrow cells, is 36
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Figure 1: The immunophenotypic characteristics of human and mouse long-term repopulating haematopoietic stem cells (LT-HSC)
Human HSC
Mouse HSC
LinCD34+ CD38c-kit+ CD133+ SP
Linc-kit+ sca1+ Tie2+ CD150+ CD244CD48SP
LT-HSC
Lin: lineage; SP: side population cells
heterogeneous and contains also c-kit-, flt3-, and CD133-expressing cells. Both mouse and human HSCs are present among the side population (SP) cells which express the drug transporter protein Abcg2 and therefore have the ability to actively efflux the DNA-intercalating dye Hoechst. Human HSCs remain at present less well defined than murine HSCs. Despite the availability of methods which greatly facilitate and enhance the precision of studies with well defined cell populations, it is most likely that pure human HSCs have not yet reached the hands of the scientists. Even less defined remain the properties of stem cells from tissues other than bone marrow, primarily because the isolation of stem cells of skin, muscle, brain or liver remains difficult. Nevertheless, the flow cytometry-based characterisation of somatic tissue stem cells indicates that several cell surface markers are shared, including CD34, c-kit, sca1 and CD133, underlying common features of these rare and not easilyaccessible stem cell populations. Haematopoiesis occurs in close physical contact with stroma lining the bone marrow niches. Recently, much attention has focused on the differentiation properties of stroma cells themselves. A marrow stromal cell population, termed mesenchymal stem cells, has been shown to give rise to numerous tissue types, including cartilage, bone, fat, and muscle (4). In addition, a minor population of adherent multipotent adult progenitor cells (MAPCs) was found to be capable of differentiation into functional hepatocytes, endothelial cells, skeletal myeloblasts, osteoblasts, chondrocytes and, importantly, haematopoietic cells (5). This suggests the capacity of the bone marrow for multilineage tissue regeneration is present in
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both haematopoietic and non-haematopoietic stem cell populations. The therapeutic implications of non-haematopoietic bone marrow cells have already been clinically tested in the treatment of osteogenesis imperfecta in transplanted children (6) (see also Chapter 34). Improvement of clinical parameters suggests that bone marrow derived mesenchymal progenitor cells carry a potential for repair of bone and cartilage tissue.
4. Stem cell niches The concept of the stem cell niche defines a microenvironment, where important interactions between adhesion molecules and their ligands, and between cytokines, chemokines and their corresponding receptors control the fate of stem cells and their progeny (7). In the adult bone marrow, HSCs are located in the trabecular endosteum, where osteoblastic cells are critical components sustaining the quiescence or selfrenewal of HSCs, the properties essential for long-term haematopoiesis (8). Both intrinsic and extrinsic mechanisms define the state of either quiescence or cycling and differentiation of HSCs (Figure 2). The intrinsic mechanisms include transcription Figure 2: The mechanisms regulating the HSC niche
Extrinsic mechanisms PTH
nerves Osteoblast
Cadherins VCAM - VLA ICAM - LFA SDF-1 - CXCR4
cell-cell adhesion migration
quiescence self-renewal expansion
SCF - c-kit Flt3-L - Flt3 Ang-1 - tie2 Jagged/Delta - Notch Wnt - Frizzled
Signal transduction Gene expression HSC
Intrinsic mechanisms
A graphical representation of extrinsic and intrinsic mechanisms in the niche based on the functional interaction of the parathyroid hormone (PTH) and nervous system with osteoblasts, the homing of HSCs in response to chemokines (SDF-1), the physical interactions involving the cadherins and integrins (VLA), and regulation of HSC quiescence, self-renewal and expansion by cytokines: stem cell factor (SCF), Flt3 ligand (Flt3-L), angiopoietin-1 (ang-1), notch ligands (jagged and delta) and wnt ligands, followed by signaling downstream from the cognate receptors and initiating the gene expression 38
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factors and epigenetic regulators acting through chromatin remodelling. The extrinsic mechanisms are dictated by the environment of stromal and osteoblastic cells. Chemokines are responsible for HSC homing into the niche. The direct physical interaction between HSCs and the niche cells are mediated by adhesion molecules, such as integrins and cadherins. The membrane-bound and locally secreted cytokines define the HSC fate by initiating specific signalling pathways within the cell. The most prominent examples are stem cell factor, flt3 ligand, angiopoietin, Notch ligands and wnt ligands, which act synergistically. Also ex vivo, these cytokines allow an expansion of HSC numbers. According to the most recent findings, the extrinsic environmental cues in the HSC niche include hormonal regulators, such as parathyroid hormone, and the influence of a sympathetic nervous system, both signalling through the osteoblastic niche component, as well as the regulation by oxidative conditions in the niche. The information on the structure and cellular composition of the niche is only beginning to be revealed, and particularly in the human system the knowledge is only just emerging.
5. Leukaemic stem cells The available data suggest that leukaemia is a stem cell disease, in which the stem cell self-renewal mechanisms are preserved but the tight growth control is lost due to malignant transformation. As an additional mechanism, the oncogenic events might be imposing the self-renewal capacity at the level of committed progenitor cells (9). Consequently, leukaemic stem cells (LSCs) share many molecular mechanisms that regulate the function of normal HSCs. At present, phenotypic features specific for LSCs are not defined. LSCs are thought to reside within the CD34+CD38- cell population, which contains transplantable cells giving rise to human leukaemia in NOD/SCID mice. HSC-characteristic adhesion molecules, such as integrins, are involved in LSC interaction with stroma. Hence, dissimilarities between normal and leukaemic cells most likely have their origin in differences in intracellular signal transduction pathways (10). As an example, oncogenic lesions in the cytokine receptors, c-kit and flt3, are responsible for constitutive activation of downstream signalling in cells at the earliest stages of haematopoietic differentiation, resulting in HSC to LSC transition. A better understanding of the properties of LSCs is of major therapeutic relevance for the design of LSC-targeted therapies (Figure 3). Conventional chemotherapy-based treatment of leukaemia, and cancer in general, is primarily directed against the bulk of malignant cells, and thus does not eliminate the abnormal stem cells. These cells are the origin of cancer recurrence and are responsible for relapse. Current efforts are focussed on the development of methodology to isolate these cells to better
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Figure 3: The therapeutic relevance of LSCs
Conventional cancer therapy
Specific cancer stem cell therapy
Leukaemia
Leukaemia relapse
Leukaemia regression
Leukaemic blasts are depicted in grey, and LSCs in blue
homogeneity, and to dissect the differences in molecular mechanisms used by normal HSC and LSC for their self-renewal and interaction with the microenvironment in the bone marrow (11).
6. Embryonic stem cells Human ES cells can be isolated from the blastocyst 4–5 days after fertilisation, and cultured in vitro to give rise to immortalised cell lines. Depending on the culture conditions, differentiation into cells bearing characteristics of various somatic tissue types including haematopoietic, neural, muscle and other tissues, can be achieved. This work, initiated in 1998 by Thompson et al. (12) who defined methods to isolate and propagate human ES cells from the fertilised oocyte at the 30 cell stage, is of significant value for studies on human developmental biology. Importantly, in vitro cultured human ES cells represent valuable tools for drug screening. The most publicised and controversial aspect of ES-related research is associated with the origin of these cells, being a donated surplus human blastocyst from in vitro fertilisation procedures. Destruction of human embryos in order to obtain the ES cells has been of serious ethical concern. Novel findings describe derivation of human ES cell lines from single blastomeres at the 8-cell stage, without embryo destruction. ES cells are the subject of intense research which aims at understanding the molecular basis of “stemness”. In parallel, cellular biology techniques have been
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developed which allow manipulations such as nuclear transfer from a somatic cell to the enucleated oocyte, and the further generation of ES cell lines bearing genetic information defined by the donated nucleus. The multilineage developmental potential of human ES cells opens new therapeutic avenues for the restoration of damaged or diseased tissue. The possibility to provide these cells with genetic information from the patient by nuclear transfer, is an approach which – in the future – might yield transplantable tissue of a full immunological compatibility. Ultimately, the choice of an approach will require the consideration of advantages and disadvantages associated with the cell source for transplantation (Table 2).
Table 2: Potential therapeutic value of ES cells and adult somatic stem cells Stem cell source
Advantages
Disadvantages
ES cells
- perfect plasticity - easy to programme - stable in culture - no risk of infection transmission
- ethical concern - limited source - rejection danger - carcinogenicity?
Adult somatic stem cells
- accessible source (HSCs) - no rejection (autologous) - no ethical concern - no carcinogenicity
- less/no plasticity?
7. Stem cell plasticity The term “adult stem cell plasticity” defines the ability of tissue-specific stem cells to acquire, under certain microenviromental conditions, the fate of cell types different from the tissue of origin and belonging to all three germ layers, i.e. similar to the differentiation ability of ES cells. Traditionally, the development of adult stem cells has been depicted along a well-defined path of a linear and irreversible progression concluding in terminally differentiated cell types. Furthermore, the differentiation and regenerative potential of adult stem cells has been regarded as restricted to tissues in which they reside. These traditional concepts have been challenged in the recent years by numerous studies performed by transplantation of stem cells derived from bone marrow and other organs, and which demonstrated the presence of cells of interest in tissues other than those in which they normally reside. The findings from murine studies, which suggested that stem cells may be recruited out of a circulation and engaged in regeneration of diverse tissues at distal sites, have initiated a search for “unusual” locations of donor-derived stem cells
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in patients receiving organ transplants. In some cases transitions have been documented, and are likely to reflect a healing response by cells summoned to the site of injury and instructed by the local environment of the damaged tissue. However, donor-host cell fusions rather than functional stem cell plasticity may represent the underlying mechanism (13). This concept of plasticity of somatic tissue stem cells has a potential clinical impact and may revolutionise tissue transplantation therapies and regenerative medicine. According to this novel view, at least a subset of stem cells may alter their fate in a manner that is more plastic and dynamic then previously thought, causing a fascination with these cells that has spread to nearly all clinical disciplines. There are more questions raised then answers. Following a phase of excitement and rapid accumulation of results in favour of stem cell plasticity, research in this field is now going through a less spectacular phase of verification of the existing data with refined techniques. It is too soon to discard the basic paradigm of developmental biology of the mesodermal, endodermal and ectodermal germ layer origin of mammalian organs, but a need for possible revisions to the unidirectional view of cell fate in post-embryonic development may arise.
8. Conclusions and future perspectives The ultimate goal for regenerative medicine is to channel the multipotent and/or pluripotent stem cells with high proliferative capacity into specified differentiation programs within the body for a multitude of therapeutic uses. These envisaged uses may include the generation of neurons for treatment of Alzheimer’s disease, Parkinson’s disease or spinal cord injuries, the generation of insulin-secreting pancreatic cells for the treatment of diabetes, or the generation of heart muscle cells for treatment of congenital disorders or heart attacks. Recent major technical advancements in the isolation, expansion and controlled differentiation of human ES cells and adult stem cells from at least some tissues, and additionally, the establishment of nuclear transfer techniques from the somatic cell to an enucleated donor oocyte opened a number of potential new therapeutic approaches for the restoration of damaged or diseased tissue. The new challenge in stem cell biology is related to understanding the molecular and the functional programmes of leukaemic versus normal stem cells. The principle of self-renewal and lineage-making decisions of stem cells of different tissue-origin requires understanding in molecular terms. Gene expression profiles bring evidence of the overlapping genetic programs of ES cells, haematopoietic and other adult tissue stem cells, both normal and transformed (14). Determining how epigenetic features relate to the transcriptional signatures of ES and various types 42
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of adult stem cells, is a new key challenge for the future (15). Understanding of the stem cell niche is essential for advancing the approaches to control developmental pathways by both cell-autonomous and microenvironmental cues. All this information will be used as guidance for specifically targeting the fate of normal and malignant stem cells in clinical settings.
Acknowledgments This work has been supported by the Swiss National Science Foundation grant 3100-110511.
References 1. Bryder D, Rossi DJ, Weissman IL. Hematopoietic stem cells: The paradigmatic tissue-specific stem cell. Am J Pathol 2006; 169: 338-346. 2. Bhatia M, et al. A newly discovered class of human hematopoietic cells with SCIDrepopulating activity. Nat Med 1998; 4: 1038-1045. 3. Arai F, SudaT. Maintenance of quiescent hematopoietic stem cells in the osteoblastic niche. Ann NY Acad Sci 2007; 1106: 41-53. 4. Pittenger MF, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 284: 143-147. 5. Jiang Y, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002; 418: 41-49. 6. Horwitz EM, et al. Clinical responses to bone marrow transplantation in children with severe osteogenesis imperfecta. Blood 2001; 97: 1227-1231. 7. Scadden DT. The stem-cell niche as an entity of action. Nature 2006; 441: 1075-1079. 8. Wilson A, Trumpp A. Bone-marrow haematopoietic-stem-cell niches. Nat Rev Imunol 2006; 6: 93-106. 9. Huntly BJ, Gilliland DG. Leukaemia stem cells and the evolution of cancer-stem-cell research. Nat Rev Cancer 2005; 5: 311-321. 10.Rizo A, et al. Signaling pathways in self-renewing hematopoietic and leukemic stem cells: Do all stem cells need a niche? Hum Mol Genet 2006; 15 Spec No 2: R210-9. 11.Clarke MF, et al. Cancer Stem Cells – Perspectives on Current Status and Future Directions: AACR Workshop on Cancer Stem Cells. Cancer Res 2006; 66: 9339-9344. 12.Thomson JA, et al. Embryonic stem cell lines derived from human blastocysts. Science 1998; 282: 1145-1147. 13.Serafini M, Verfaillie CM. Pluripotency in adult stem cells: State of the art. Semin Reprod Med 2006; 24: 379-388. 14.Forsberg EC, Bhattacharya D, Weissman IL. Hematopoietic stem cells: Expression profiling and beyond. Stem Cell Rev 2006; 2: 23-30. 15.Spivakov M, Fisher AG. Epigenetic signatures of stem-cell identity. Nat Rev Genet 2007; 8: 263-271.
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Multiple Choice Questionnaire To find the correct answer, go to http://www.esh.org/ebmt-handbook2008answers.htm 1. Haematopoiesis takes place in the following adult human organs: a) Bone marrow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Peripheral blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Spleen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. The stem cell compartment in the bone marrow consists of: a) Clonogenic CFU and LTC-IC progenitors only . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Haematopoietic, mesenchymal and endothelial cell progenitors . . . . . . . . . . c) NOD/SCID repopulating cells only . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Haematopoietic, liver and neural stem cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Cell surface antigen CD34 is expressed on: a) Long-term repopulating haematopoietic stem cells . . . . . . . . . . . . . . . . . . . . . . . . b) Short-term repopulating haematopoietic stem cells . . . . . . . . . . . . . . . . . . . . . . . . c) Haematopoietic and non-haematopoietic stem and progenitor cells . . . . . . d) Lineage-committed progenitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Embryonic stem cells are characterised by: a) Lineage-restricted differentiation potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Potential to become a variety of specialised cell types . . . . . . . . . . . . . . . . . . . . c) Ability to generate placenta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Ability to form a blastocyt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Osteoblast components of the stem cell niche are involved in: a) Supporting stem cell self-renewal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Supporting the stem cell differentiation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Inhibiting the bone marrow stroma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Promoting the stem cell exit to the peripheral blood . . . . . . . . . . . . . . . . . . . . . .
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*
CHAPTER 3
Immunogenetics of allogeneic HSCT
*
3.1
The role of HLA in HSCT J.M. Tiercy
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CHAPTER 3.1 • HLA and donor matching
1. Introduction Tissue compatibility is determined by genes of the major histocompatibility complex (MHC), known as the HLA system in man, that are clustered on the short arm of chromosome 6. The HLA region is a multigenic system that encodes structurally homologous cell surface glycoproteins characterised by a high degree of allelic polymorphism in human populations. Immune responses against incompatible HLA antigens represent a major barrier to haematopoietic stem cell transplantation (HSCT). The accuracy of histocompatibility testing and matching criteria will therefore have important consequences on transplant outcome. This is particularly true in the case of transplantation with HSC from unrelated donors, where serologically hidden incompatibilities may account for the increased rate of post-transplant complications.
2. HLA antigens The homologous HLA Class I (HLA-A, -B, -C) and Class II (HLA-DR, -DQ, -DP) antigens are codominantly expressed and differ in their structure (Figure 1), tissue distribution and characteristics in peptide presentation to T-cells (1). The biological function of HLA molecules is to present peptide antigens to T-cells, thereby playing a central role in T-cell-mediated adaptive immunity. HLA Class I molecules, which are expressed on most nucleated cells, are composed of an a-chain (encoded in the MHC) non covalently associated with b2-microglobulin (encoded on chromosome 15) (Figure 1). The two outermost a1 and a2 domains of the heavy chain form the peptide binding site. Peptides (usually of 8–10 amino acids) presented by Class I
Figure 1: Schematic representation of HLA Class I and Class II molecules
The two polymorphic domains a1/a2 of HLA Class I are encoded by exons 2 and 3, respectively, and exon 3 codes for the most proximal a3-domain that interacts with b2-microglobulin (b2m) and CD8. The HLA Class I b-chain is non covalently associated with b2m. The Class II molecules are heterodimers composed of an a and a b-chain. The most distal domains of each of the two chains form the peptidebinding site
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molecules are derived from proteolytic degradation of cytoplasmic proteins by the proteasome. These are transported across the endoplasmic reticulum where they bind to Class I antigens. Pathogen-derived peptides presented to Class I antigens are usually recognised by CD8+ cytotoxic T-lymphocytes (CTL) (1). HLA Class II antigens are expressed on a subset of cells of the immune system comprising dendritic cells, B cells, activated T-cells, macrophages, collectively referred to as antigen presenting cells (APC). They are heterodimers composed of the two membrane-bound a- and b-chains that are encoded by two genes that colocalise in the MHC. The peptide-binding pocket is formed by the most distal domains of the two chains. Extracellular antigens internalised by endocytosis/phagocytosis are degraded in an endocytic compartment into peptides of 10–30 amino acids that bind Class II molecules. HLA Class II-peptide complexes expressed on the membrane are usually recognised by CD4+ T-helper cells (1). Peptide-HLA complexes are the ligands of clonally distributed T-cell receptors (TCRs). TCRs are also able to recognise allogeneic HLA molecules at a high frequency, so that 1–10% of the peripheral blood lymphocytes of a donor can respond to a given allo-MHC antigen (2). Immune responses against incompatible HLA antigens may be extreme, such as in the case of graft-versus-host disease (GvHD) mediated by alloreactive cytotoxic T-lymphocytes (CTL), and thus represent a major barrier to HSCT.
3. Genomic organisation of the HLA system The MHC comprises 12 classical HLA genes located on a 3.6 Mb segment of the short arm of chromosome 6. Three HLA Class I genes (A, B, C) (Figure 2) encode for the heavy chains of HLA-A, B and -C antigens. Polymorphic residues are essentially located in the a1- and a2-domains encoded by exons 2 and 3, respectively, which form the peptide binding site. HLA Class II antigens (DR, DQ, DP) are heterodimers encoded by an a-chain and a b-chain gene (e.g. DRA/DRB1 or DQA1/DQB1) that co-localise at the centromeric part of the MHC (Figure 2) (1, 3). Essentially all of the polymorphism is located on exon 2 of b-chain genes, whereas the DRA gene is non polymorphic, and DQA1 and DPA1 loci exhibit a lower level of polymorphism (Figure 1). The HLA-DR sub-region presents an additional level of complexity since a second polymorphic DRB gene may be present, i.e. DRB3 in DR11/DR12/DR13/DR14/DR17/DR18 haplotypes, DRB4 in DR4/DR7/DR9 haplotypes, and DRB5 in DR15/DR16 haplotypes (Figure 2). Because of the codominant expression of HLA genes, a heterozygous individual may therefore express up to 12 different HLA antigens.
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Figure 2: HLA as a multigenic and polymorphic system
Schematic representation of the 12 HLA Class I and II loci in the MHC that comprises >200 genes on the short arm of chromosome 6. The corresponding number of antigens (as defined by serology) and alleles (as defined by nucleotide sequence) are indicated for each locus (3). About 10% of the alleles assigned by the HLA Nomenclature Committee are characterised by silent substitutions, and 2% are null alleles (3). Lower part of the figure: the HLA-DR subregion presents an additional level of complexity, with the presence of a second DRB gene in most haplotypes: DRB3, DRB4 or DRB5, which encode respectively the DR52, DR53, and DR51 antigens. For the nomenclature: see Appendix 1
4. Allelic polymorphism HLA genes are the most polymorphic loci of the human genome accounting for a total of 2584 alleles currently identified in worldwide populations (Figure 2) (3). Polymorphism of the HLA system was initially detected by serology, i.e. by using typing reagents derived from sera of multiparous women, or individuals who have been immunised by multiple transfusions. In the early 1980’s molecular cloning of the first HLA genes opened the way for a complete understanding of the molecular basis of HLA diversity and for the development of a variety of DNA-based typing techniques. Most of the serologically defined specificities are now subdivided into numerous alleles, and this number is still continuously growing (3). A regular update of the new alleles at the various HLA loci is available on the web (www.ebi.ac/imgt/hla) (Figure 2). Appendix 1 lists the HLA-A/B/C/DR/DQ serotypes and the corresponding groups of alleles detected by high resolution typing. The variability within a given serotype was recognised by cellular assays long before the DNA sequencing era. For example CTLs were shown to discriminate between two HAEMATOPOIETIC STEM CELL TRANSPLANTATION
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HLA-A2 serologically identical individuals. Figure 3 shows a schematic alignment of the a1/a2 domain sequences with unique residues shared by all A2 alleles (G62, T142-H145). These residues are recognised by the monospecific and/or polyspecific allo-antisera used as typing reagents. Each of the 103 different A2 alleles expressed at the cell surface differs by a very limited number of residues, usually 1–4, and such allele mismatches can be efficiently recognised by CTLs (4–7). Although many of the Class I and II antigens comprise a large number of alleles worldwide, a limited number of alleles are found in any given population at a gene frequency >0.1% (8, 9). The combination of HLA alleles inherited on the same segment of chromosome 6 is referred to as a haplotype, for example the A1-B8-DR3 or the DRB1*1501DQB1*0602 haplotypes. Because of linkage disequilibrium HLA disparities at a given locus will frequently be associated with incompatibilities at an adjacent locus, as observed in many instances for B-Cw and DRB1-DQB1 bi-locus groups.
Figure 3: Schematic representation of HLA-A2 alleles showing common residues on the a1/a2-domains that are recognised by alloantisera
Open boxes mark residues that differ between the 0201-0207, 9201, and 9215 alleles. For example 0201 and 0206 alleles differ at one single position (Phe9 vs. Tyr9). Such a serologically silent mismatch is recognised by CTLs (see Figure 4) 50
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5. HLA typing: Methods and resolution levels Serology (microlymphocytotoxicity) is still a method of choice for low resolution typing in most laboratories, at least for HLA-AB, due to its simplicity and low cost. However the lack of monospecific alloantisera, the low-resolution power of the method and the requirement for viable lymphocytes, all contributed to the development of genomic DNA typing techniques, initially for Class II, later on for Class I typing. DNA typing techniques are based on the nucleotide sequence information of the polymorphic DNA segments, using PCR technology. A number of HLA typing methods based on DNA sequence variations have been developed, mainly using PCR-SSP (sequence-specific primers) amplification, or reverse PCR-SSOP (sequence-specific oligonucleotide probes) hybridisation on solid support (e.g. microbead arrays), or direct sequencing (8, 10). Three different levels of resolution for HLA DNA typing are usually recognised (Table 1). Low-resolution, also referred to as generic typing or 2-digit typing, corresponds to the identification of broad families of alleles that cluster into serotypes (e.g. A*02), and is thus the equivalent of serological typing (A2). High resolution, or 4-digit
Table 1: HLA nomenclature and levels of resolution HLA (a)
Definition
Resolution
A2
Refers to the A2 antigen defined by monospecific/ polyspecific antisera Any of the A*02 alleles (A*0201-0299 and A*9201-9215) Either one of these 4 alleles, other DRB1*11 alleles are excluded Allele defined Allele with a substitution in the coding sequence that leads to a stop codon (null allele), this allele is not expressed 2 DRB1*0301 alleles that differ by a silent substitution in exon 2, this difference is functionally silent, but may influence DNA typing A*2402 allele with low expression due to a mutation in intron 2 sequence, this allele is expressed at a level that is undetectable by serological typing
low
A*02 DRB1*1102/1103/ 1111/1114 A*0201 A*0232N DRB1*030101 and DRB1*030102 A*24020102L
low intermediate high high high
high
(a) DNA nomenclature: the first 2 digits refer to the serotype (A*02), the 3rd and 4th digits define substitution(s) in the coding sequence (A*0201), the 5th and 6th digits describe synonymous substitution(s), and the 7th and 8th 2 digits refer to substitution(s) in intron or 5’ or 3’ sequences. N and L mark alleles with respectively no or low surface expression. The suffix S means an allele encoding a protein that is expressed as a secreted molecule only and the suffix Q means an allele with a mutation that has been shown previously to have a significant effect on cell surface expression but without confirmation, and therefore with a questionable expression status
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typing, allows the discrimination of the individual alleles within each serotype (e.g. A*0201). Intermediate resolution HLA typing of a potential donor for this patient might give as a result A*0205/08/22: that means that the donor can be either A*0205, or A*0208, or A*0222, but definitively not A*0201 or any of the other A2 alleles. This level of typing results from the fact that these 3 alleles share common sequence determinants and are therefore identified in the same hybridisation group pattern. It is obviously very practical for rapid donor selection. In the example given above, such a donor typed as A*0205/08/22 would be disregarded for further analysis, whereas a parallel donor with the HLA type A*0201/01L/09/43N/66/75 would be selected and tested further in order to disclose compatibility at the allele level with the A*0201-positive patient. The accreditation program for the histocompatibility laboratories set up by the European Federation for Immunogenetics (EFI) has defined minimal criteria for HLA typing in related and unrelated HSCT (www.efiweb.org/standards.html).
6. HLA matching in related HSCT The best donor is a HLA genotypically matched sibling as identified by family typing. The family study allows not only the identification of a potential related donor, but also the confirmation of the patient’s genotype, which is important if an unrelated donor search is initiated. HLA-A, B, DR low resolution typing (serology or 2-digit DNA typing) is able in most cases to determine the paternal and maternal haplotypes present in the patient and a potential sibling donor. Thus ABDR typing can confirm genotypic identity for the whole set of HLA genes, i.e. a 12/12 match (Figure 4). Because of weak linkage disequilibrium between DP and the DR/DQ loci, a low level of DP-mismatched sibling donors (1–2%) are identified due to recombination. An HLA-A/B or B/DRB1 recombination event is detected by routine HLA-A/B/DR typing in about 2% of families. For a given patient the probability of having a genotypically identical sibling donor is 25% for each sibling, whereas the probability of having a haploidentical donor (i.e. with one shared haplotype) is 50%. When the patient has a very frequent ABDR haplotype it may be worth searching for a phenotypically identical donor in the blood-related members of the extended family, particularly when information on consanguinity is provided, for example when there is sound information that there has been intermarrying between aunts/uncles on paternal and aunts/uncles on maternal side, so that cousins may be HLA genotypically identical. In some families, apparent homozygosity of one of the parents may prevent formal identification of genotypic identity. As illustrated in the example shown in Table 2, both siblings 1 and 2 are phenotypically identical to the patient, based on ABDR 52
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Figure 4: Matching criteria in related and unrelated HSCT
By definition an HLA-genotypically identical sibling donor is compatible at the allele level at all loci on both chromosomes (12/12 match). In unrelated HSCT, matching for A/B/C/DRB1/DQB1 loci is usually searched for (10/10 match). In addition to DRB1 compatibility, some centres also consider DRB3/DRB5 polymorphism. DRB3 mismatches occur frequently in DR13 haplotypes. Because of strong linkage disequilibrium with DRB1 (at least in Caucasoids), the DRB5 locus is usually not tested. In DR15/16 haplotypes, DRB5 mismatches usually co-occur with DRB1 disparities. Searching for a 12/12 match implies DPB1 typing. Donors with an 8/8 match (not shown) or a 6/6 match apply, respectively, when HLAC/DP, or HLA-C/DQ/DP are not tested
low resolution typing. However typing for HLA-C and HLA-DRB1 at the allele level shows that sibling 1 is actually compatible with the patient, whereas sibling 2 has inherited a different maternal haplotype with 2 mismatches. In case of mismatched related HSCT, the risk of GvHD (recipient alleles absent in the donor) and graft failure (donor alleles absent in the recipient) increase with the number of HLA disparities on the non-shared haplotype (reviewed in ref. 11). A differential effect of Class I and Class II mismatches has been described, in which aGvHD risk was associated with Class II disparities (12).
7. HLA matching in unrelated HSC transplantation When no HLA-genotypically identical sibling donor is available, transplantation with HSC from HLA-ABCDRB1/DQB1-allele matched unrelated donors can result in comparable disease-free survival rates, notably for good-risk patients (11). Donor
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Table 2: Low versus high resolution HLA typing in homozygous haplotypes Haplotype
Low resolution typing
High resolution typing
father
a b
A1-B8-DR3 A31-B18-DR15
A*0101-B*0801-Cw*0701-DRB1*0301 A*3101-B*1801-Cw*1203-DRB1*1501
mother
c d
A2-B51-DR11 A2-B51-DR11
A*0201-B*5101-Cw*0501-DRB1*1101 A*0201-B*5101-Cw*1203-DRB1*1104
patient
a c
A1-B8-DR3 A2-B51-DR11
A*0101-B*0801-Cw*0701-DRB1*0301 A*0201-B*5101-Cw*0501-DRB1*1101
sibling 1
a c
A1-B8-DR3 A2-B51-DR11
A*0101-B*0801-Cw*0701-DRB1*0301 A*0201-B*5101-Cw*0501-DRB1*1101
sibling 2
a d
A1-B8-DR3 A2-B51-DR11
A*0101-B*0801-Cw*0701-DRB1*0301 A*0201-B*5101-Cw*1203-DRB1*1104
High resolution typing may disclose hidden incompatibilities in families where the 4 parental genotypes cannot be unambiguously determined. In this example the mother is ABDR homozygous by serology, but in fact has 2 different haplotypes on the basis of high resolution typing
identification has been largely facilitated by the Bone Marrow Donor Worldwide (BMDW) Registry (www.bmdw.org) which provides access to >11 million HLA-typed donors and cord blood units available in the national registries. Compared to HSCT from HLA genotypically identical sibling donors, unrelated HSCT is associated with an increased frequency of post-transplant complications, which are mainly due to undisclosed HLA incompatibilities not detected by serology (8, 13). Allele mismatches usually involve difference(s) in the peptide-binding site (Figure 5) that influence
Figure 5: Examples of common serologically hidden HLA Class I incompatibilities that involve single amino acid mismatches recognised by CTLs
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T-cell recognition either by direct contact with TCR or indirectly by modulating the repertoire of peptides bound by the HLA molecule. 7.1. Matching criteria for unrelated donors Whereas genotypically identical siblings share by definition the same alleles at all loci, the degree of allele matching in two unrelated individuals strictly depends on how many loci are considered for high resolution typing. Figure 4 illustrates the three most common situations: the gold standard matching comprises the analysis of HLAA, -B, -C, -DRB1, and -DQB1 loci. When a patient and a donor share the same 5 alleles on both haplotypes the situation is referred to as a 10/10 match. If HLA-C or -DQB1 typing is omitted, this will be an 8/8 match, and if only the A/B/DRB1 loci are considered, it becomes a 6/6 match. Obviously because of the multiple possible BC and DRB1-DQB1 associations, a 6/6 match may in fact correspond to a 6/10 match, i.e. to an incompatible combination (Table 3). In the case of non-malignant diseases the graft versus leukaemia effect that may be mediated by DP incompatibilities is not necessary and DPB1 matching might be considered when several HLA-A/B/C/DR/DQ-compatible donors are available (12/12 match).
Table 3: Hidden incompatibilities in HLA-ABDRB1 allele-matched donors Matching
6/6
8/10
A*
B*
Cw*
DRB1*
DQB1*
patient
0201/1101
0801/3503
NT
0301/1101
NT
donor
0201/1101
0801/3503
NT
0301/1101
NT
patient
0201/1101
0801/3503
0701/0401
0301/1101
0201/0301
donor
0201/1101
0801/3503
0701/1203
0301/1101
0201/0501
7.2. Allele matching: Relative importance of individual loci In the early 1990’s the role of HLA matching was hampered by the poor resolution achieved by HLA typing, particularly for HLA Class I alleles. Based on high resolution typing methods recent studies (14–18) have reached the almost general consensus that allele-level matching does improve transplant outcome after both myeloablative and reduced-intensity conditioning regimen (reviewed in ref. 11). The HLA effect on transplant outcome is modulated by the disease risk with an effect of single disparities reported to be significant for low risk disease patients (16). However the relative importance of individual loci still remains an open issue. Whereas
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the effect of A/B/C/DRB1 mismatches have been well documented by most retrospective large scale studies, the role of DQ and DP incompatibilities appear more controversial (reviewed in ref. 11). The National Marrow Donor program (NMDP) Histocompatibility Committee has recommended allele-level typing for HLAA/B/C/DRB1 (8/8 match) (13). There is however some evidence suggesting a trend of an additive HLA-DQ effect in HSCT from donors with multiple mismatches (16). Recent data on T-cell-depleted unrelated HSCT have documented that HLA-DPB1 matching was associated with increased risk of relapse, irrespective of the compatibility status for the other loci (19). A comparison of serological versus allele class I mismatches in CML patients suggested that qualitative differences may influence the risk of graft failure, with a higher risk in serotype-mismatched patients (15, 20). Contrasting outcomes have been reported in studies with patients from different ethnic backgrounds: in the Japanese Marrow Donor Program (JMDP) study (14), HLAA/B/C/DRB1 mismatches were found to be significant risk factors for grades III–IV acute GvHD, whereas the U.S. National Marrow Donor program (NMDP) data revealed a DRB1 effect with no contribution of HLA-DQ/DP or HLA-B/C (15) mismatches. HLAA/B/C/DRB1, but not DQ/DP mismatches decreased survival in the NMDP study (15), whereas in the JMDP study only A/B/DRB1 disparities were associated with mortality (14). Differences between studies may involve selection criteria of each transplant centre, patients age or other pre-transplant risk factors, experience in treating GvHD, as well as the relevance of the GvL effect in CML patients. Also when comparing studies from varying population groups, major differences between mismatched allele combinations may contribute to contrasting clinical outcomes. Selection of cord blood units is generally based on HLA-A/B-low and -DRB1-high resolution matching, taking into account that cell dose is the most important parameter affecting outcome. Data on the effect of HLA matching in cord blood transplantation are difficult to interpret, since most reports lack HLA-C/DQ and high resolution HLA-AB typing results. Nevertheless cord blood appears less HLA restricted than adult HSCT, in other words HLA incompatibilities may be less detrimental (21).
8. Probability estimates of finding a matched unrelated donor Identification of an unrelated HLA allele-matched HSC donor is a costly and time consuming procedure. To improve search efficiency, an estimate of the probability of identifying a 10/10 HLA matched donor allows an early decision to transplant with HSC from an alternative donor (haploidentical, cord blood, autologous), or to accept varying degrees of allele-mismatches for searches with a low probability of success, depending on the clinical context and local guidelines. Based on the current 56
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knowledge of allele and haplotype frequencies in different populations the probability of identifying a 10/10 matched donor at the start of the search is highly predictable (22). The number of ABDR-matched (low resolution) donors available in the Registry for a given patient often reflects the chance of finding a donor compatible at the allele level. However, the following parameters have a negative impact on this probability: • presence of a rare allele in the patient (<5% within a given serotype, e.g. B*4405) • HLA-ABDR haplotype that does not belong to the 10 most frequent haplotypes in Caucasoids • unusual B-DR association (e.g. B65-DRB1*0101) • unusual DR-DQ association, e.g. DRB1*1501-DQB1*0603 • presence of an antigen that is split into >2 alleles occurring at a frequency >10% of a given serotype in the population, e.g. B35, B44, DR4, DR11, DR13 • presence of B*2705, B18, B*4402, B*4403, B51 (higher risk of HLA-C mismatch) • <3 donors available in the BMDW registry. Because of different distribution of alleles and HLA haplotypic associations, allele matching will also be more difficult when patients and potential donors belong to different ethnic groups. For European Caucasoid patients a 40–50% probability of identifying a 10/10 matched donor has been reported (22, 23).
9. Choice of donor based on HLA typing Vigorous T-cell responses are generated against allogeneic tissues when the graft and the host express different MHC molecules. Single amino acid differences in the antigen recognition site of a HLA molecule can initiate graft rejection or GvHD. The choice of the donor will therefore depend on the degree of HLA identity between donor and recipient. Looking for a related donor 1. HLA-A/B/DR typing (serology or low resolution DNA typing) of patient, the sibling(s) and both parents to determine the haplotypes. 2. If a sibling is geno-identical the transplant can be performed after a confirmatory typing on a second blood sample from patient and donor but without further HLA investigations. 3. If one sibling is a monozygotic twin this is a syngeneic transplant, this donor can be chosen but some of the GvL effect might be lost. 4. If both parents share a common haplotype or if one parent is HLA-ABDR homozygous, this is considered to be a pheno-identical transplant. High
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resolution Class I and II typing is recommended to define the exact degree of compatibility. 5. If a family donor with only one HLA difference (5/6 match) is found, the transplant can be performed despite the possibility of an increased risk of GvHD. This is a related one antigen mismatched donor transplant. High resolution Class I and II typing is recommended to define the exact degree of compatibility. 6. Parents and/or siblings sharing only one haplotype with the recipient are considered as a haploidentical-related donor transplant. Looking for an unrelated donor 1. One should obtain a complete Class I and Class II high resolution typing for the patient (HLA-A, -B, -C, -DRB1 and -DQB1). Several levels of compatibility are defined: 12 out of 12 (A/B/C/DRB1/DQB1/DPB1) 10 out of 10 (A/B/C/DRB1/DQB1) or 8 out of 8 (A/B/C/DRB1) (see section 7). The level of HLA incompatibility (low or high resolution typing) accepted can vary from centre to centre but the priority is to look for the highest degree of compatibility at the allele level. DPB1 typing should be included in case several 10/10 matched donors are available. 2. Look at the BMDW (Bone Marrow Donor Worldwide) database to evaluate the probability of finding a donor, and determine which registries have suitable donors. 3. Send a search request to the bone marrow donor registries which have suitable donors in order to perform a confirmatory typing (high resolution) to find the best HLA matched unrelated donor. 4. Search simultaneously for a cord blood unit through BMDW and Netcord in order to find an unrelated cord blood donor. If several HLA identical bone marrow donors are found, choose the male, ABO identical, and/or CMV-negative donor. If there is no well matched (10/10, 9/10, or possibly 8/10) unrelated donor or if the time frame is too short, choose a cord blood unit, as long as the number of nucleated cells is >2 x 107/kg and there are no more than 2 HLA mismatches (4/6).
10. Conclusions The HLA system, with 100 serologically defined specificities and over 2500 alleles, represents a major barrier to HSC transplantation. There is now a broad consensus that selection of unrelated HSC donor by high resolution molecular typing technology contributes to a better clinical outcome. However the relative importance of individual loci still remain to be better defined, and multicentre studies should contribute to resolving this issue. The difficulty in reaching clear consensus among 58
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the clinical studies, particularly with respect to the role of single locus mismatches, possibly results from subtle differences in patients groups with respect to patient and donor selection criteria, pre-transplant risk factors or GvHD prophylaxis. Additional immunogenetic factors such as minor histocompatibility antigens, cytokine and chemokine genes, or activating/inhibitory killer immunoglobulin-like receptor (KIR) genes, may play a role in determining transplant outcome. Donor HLA matching criteria should take into account parameters such as the time frame allowed by the patient’s disease and the probability of identifying a well matched donor based on the patient’s HLA phenotype.
References 1. Klein J, Sato A. The HLA system. First of two parts. New Engl J Med 2000; 343: 702-709. 2. Afzali B, Lechler RI, Hernandez-Fuentes MP. Allorecognition and the alloresponse: Clinical implications. Tissue Antigens 2007; 69: 545-556. 3. Marsh SGE, Albert ED, Bodmer WF, et al. Nomenclature for factors of the HLA system, 2002. Tissue Antigens 2002; 60: 407-464. 4. Fleischhauer K, Kernan NA, O'Reilly RJ, et al. Bone marrow-allograft rejection by T lymphocytes recognizing a single amino acid difference in HLA-B44. N Engl J Med 1990; 323: 1818-1822. 5. Rufer N, Tiercy J-M, Breur-Vriesendorp B, et al. Histoincompatibilities in ABDR-matched unrelated donor recipient combinations. Bone Marrow Transplantation 1995; 16: 641-646. 6. Oudshoorn M, Doxiadis II, van den Berg-Loonen PM, et al. Functional versus structural matching: Can the CTLp test be replaced by HLA allele typing? Hum Immunol 2002; 63: 176-184. 7. Scott I, O’Shea J, Bunce M, et al. Molecular typing reveals a high level of HLA class I incompatibility in serologically well matched donor/recipient pairs - Implications for unrelated bone marrow donor selection. Blood 1988; 92: 4864-4871. 8. Tiercy JM, Villard J, Roosnek E. Selection of unrelated bone marrow donors by serology, molecular typing and cellular assays. Transpl Immunol 2002; 10: 215-221. 9. Hurley CK, Fernandez-Vina M, Hildebrand WH, et al. A high degree of HLA disparity arises from limited allelic diversity: Analysis of 1775 unrelated bone marrow transplant donorrecipient pairs. Human Immunol 2007; 68: 30-40. 10.Little AM, Marsh SG, Madrigal JA. Current methodologies of human leukocyte antigen typing utilized for bone marrow donor selection. Curr Opin Hematol 1998; 5: 419-428. 11.Petersdorf EW. Risk assessment in hematopoietic stem cell transplantation. Best Pract Res Clin Haematol 2007; 20: 155-170. 12.Ottinger HD, Ferencik S, Beelen DW, et al. Hematopoietic stem cell transplantation: Contrasting the outcome of transplantations from HLA-identical siblings, partially HLAmismatched related donors, and HLA-matched unrelated donors. Blood 2003; 102: 11311137. 13.Hurley CK, Wagner JE, Setterholm MI, Confer DL. Advances in HLA: Practical implications for selecting adult donors and cord blood units. Biol Blood Marrow Transplant 2006; 12
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(1 Suppl 1): 28-33. 14.Morishima Y, Sasazuki T, Inoko H, et al. The clinical significance of human leukocyte antigen (HLA) allele compatibility in patients receiving a marrow transplant from serologically HLA-A, HLA-B, and HLA-DR matched unrelated donors. Blood 2002; 99: 4200-4206. 15.Flomenberg N, Baxter-Lowe LA, Confer D, et al. Impact of HLA class I and class II high resolution matching on outcomes of unrelated donor bone marrow transplantation. Blood 2004; 104: 1923-1930. 16.Petersdorf EW, Anasetti C, Martin PJ, et al. Limits of HLA mismatching in unrelated hematopoietic cell transplantation. Blood 2004; 104: 2976-2980. 17.Chalandon Y, Tiercy J-M, Schanz U, et al. Impact of high resolution matching in allogeneic unrelated donor stem cell transplantation in Switzerland. Bone Marrow Transplant 2006; 37: 906-916. 18.Carreras E, Jiminez M, Gomez-Garcia V, et al. Donor age and degree of HLA matching have a major impact on the outcome of unrelated donor hematopoietic cell transplantation for chronic myeloid leukemia. Bone Marrow Transplant 2006; 37: 33-40. 19.Shaw BE, Marsh SG, Mayor NP, et al. HLA-DPB1 matching status has significant implications for recipients of unrelated donor stem cell transplants. Blood 2006; 107: 1220-1226. 20.Petersdorf EW, Hansen JA, Martin PJ, et al. Major-histocompatibility-complex class I alleles and antigens in hematopoietic-cell transplantation. New Engl J Med 2001; 345: 17941800. 21.Gluckman E, Rocha V. Donor selection for unrelated cord blood transplants. Curr Op Immunol 2006; 18: 565-570. 22.Tiercy J-M, Nicoloso de Faveri G, Passweg J, et al. The probability to identify a 10/10 HLA allele-matched unrelated donor is highly predictable. Bone Marrow Transplantation, 2007; 40: 515-522. 23.Tiercy J-M, Bujan-Lose M, Chapuis B, et al. Bone marrow transplantation with unrelated donors: What is the probability of identifying an HLA-A/B/Cw/DRB1/B3/B5/DQB1matched donor? Bone Marrow Transpl 2000; 26: 437-441.
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Appendix 1. List of HLA-A, -B, -Cw, -DR and -DQ serotypes with their corresponding groups of alleles (June 2007) serotype A1 A2 A203 A210 A3 A11 A23 (9) A24 (9) A2403 A25 (10) A26 (10) A29 (19)
alleles A* 0101-0125 0201-0299, 9201-9215 0203 0210 0301-0329 1101-1130 2301-2315 2402-2476 2403, 2410 2501-2506 2601-2634 2901-2916
B7 B703 B8 B13 B14 B15 B18 B27 B2708 B35 B37 B38 (16) B39 (16) B3901 B3902 B40 B4005 B41 B42 B44 B45
alleles B* 0702-0754 0703 0801-0833 1301-1317 1401-1407N 1501-1599, 9501-9529 1801-1826 2701-2737 2708 3501-3575 3701-3712 3801-3816 3901-3941 3901 3902 4001-4074 4005 4101-4108 4201-4209 4402-4453 4501-4507, 5002
serotype A30 (19) A31 (19) A32 (19) A33 (19) A34 (10) A36 A43 (10) A66 (10) A68 (28) A69 (28) A74 (19) A80
B53 B54 (22) B55 (22) B56 (22) B57 (17) B58 (17) B60 (40) B61 (40) B62 (15)
B63 (15) B64 (14) B65 (14) B67 B70 (15) B71 (70)
alleles A* 3001-3021 3101-3117 3201-3215 3301-3310 3401-3408 3601-3604 4301 6601-6606 6801-6838 6901 7401-7412N 8001 alleles B* 5301-5312 5401-5411 5501-5526 5601-5620 5701-5712 5801-5815 4001,4007,4010,4014, 4031,4034,4048,4054 4002-4004,4006,4009, 4016,4027,4029 1501,1504-07,1515, 1520,1524,1525,1527, 1528,1530,1532,1535, 1539,1545,1548,1570, 1571,1573,1582,1584, 1516-1517 1401 1402 6701-6702 1509,1537,1551 1510,1518,1580,1593 HAEMATOPOIETIC STEM CELL TRANSPLANTATION
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B46 B47 B48 B49 (21) B50 (21) B51 (5) B5102 B5102 B52 (5)
4601-4610 4701-4705 4801-4816 4901-4905 5001-5002,5004 5101-5148 5102 5102 5201-5211
B72 (70) B73 B75 (15)
serotype Cw1 Cw2 Cw3 Cw4 Cw5 Cw6 Cw7 Cw8
alleles Cw* 0101-0118 0202-0218 0302-0340 0401-0427 0501-0516 0602-0616N 0701-0748 0801-0814
serotype Cw9 Cw10 Cwx Cwx Cwx Cwx
DR1 DR1 DR15 (2) DR16 (2) DR3 DR4 DR7 DR8
alleles DRB1* 0101-0110 0101-0116 1501-1522 1601-1611 0302-0335 0401-0464 0701, 0703-0712 0801-0832
DR52
DR53 blank
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DRB3*0101-01 DRB3*0201-0218 DRB3*0301-0303 DRB4*0101-0106 DRB4*01030102N, 0201N,0301N
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B76 (15) B77 (15) B78 B81 B82 B83
1503,1546 7301 1502,1508,1511,1521, 1531 1512,1514 1513 7801-7805 8101-8102 8201-8202 8301
Cwx Cwx
alleles Cw* Cw*0303 Cw*0302,0304 1202-1221 1402-1408 1502-1520 16011602,1604, 1606-1609 1701-1704 1801-1803
DR9 DR10 DR11 (5) DR12 (5) DR13 (6) DR14 (6)
alleles DRB1* 0901-0906 1001 1101-1162 1201-1215 1301-1379 1401-1466
DR51
DRB5*0101-0112 DRB5*0201-0205
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CHAPTER 3.1 • HLA and donor matching
DQ2 DQ3 DQ4 DQ5 (1)
alleles DQB1* 0201-0205 0301-0320 0401-0402 0501-0505
DQ6 (1) DQ7 (3) DQ8 (3) DQ9 (3)
alleles DQB1* 0601-0630 0301, 0304 0302, 0305, 0310 0303
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Multiple Choice Questionnaire To find the correct answer, go to http://www.esh.org/ebmt-handbook2008answers.htm 1. The biological function of HLA molecules is to present peptide antigens to T-cells. The peptide binding site of HLA Class I molecules is composed of: a) An a-chain and a b-chain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) The proximal part of the a-chain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) The distal part of the a-chain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) The a-chain and the b2-microglobulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. A heterozygous individual can express at the cell surface a maximum of: a) 10 different HLA Class I and Class II antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) 12 different HLA Class I and Class II antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) 13 different HLA Class I and Class II antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) 14 different HLA Class I and Class II antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. A HLA-ABDR haplotype is: a) A combination of alleles encoded on the same chromosome . . . . . . . . . . . . . . b) A combination of alleles shared by two unrelated individuals . . . . . . . . . . . . . c) A combination of alleles that differ between a patient and a partially incompatible sibling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) A combination of alleles that are frequent in a given population . . . . . . . . . 4. In which patient/donor combination would you expect a higher risk of HLA-C mismatches? a) Two HLA-ABDR phenotypically identical siblings. . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Two HLA-ABDR phenotypically identical unrelated individuals . . . . . . . . . . . . c) Two HLA-ABDR phenotypically identical cousins . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Two HLA-ABDR genotypically identical siblings . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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CHAPTER 3.1 • HLA and donor matching
5. The HLA-ABDR typing of a patient’s family leads to the identification of a potential sibling donor who differs from the patient by one single DR antigen. The father is homozygous for HLA-AB antigens and heterozygous for HLA-DR. The possible reason for the DR-incompatibility is: a) A recombination event between HLA-A and -B in one paternal haplotype. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) A recombination event between HLA-B and -DR in one paternal haplotye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Inheritance of 2 different paternal HLA-ABDR haplotypes . . . . . . . . . . . . . . . . . d) Either of the 3 possibilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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*
CHAPTER 3
Immunogenetics of allogeneic HSCT
*
3.2
KIR: Beneficial effects of natural killer cell alloreactivity in haploidentical HSCT A. Velardi, L. Ruggeri, A. Mancusi, F. Aversa, M. F. Martelli
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CHAPTER 3.2 • NK cells in haploidentical transplantation
1. Introduction Recent studies have shown natural killer (NK) cells influence the outcome of haploidentical haematopoietic transplantation in a remarkable way, with a favourable outcome where NK cells are alloreactive in the donor-recipient direction (1-4). NK cell activation is regulated by a balance between inhibitory and activating receptors, called killer-cell Ig-like receptors (KIRs). In humans, currently 16 inhibitory KIR genes and pseudo-genes are known, which codify for inhibitory and activating KIRs. Inhibitory KIRs recognise amino acids in the COOH-terminal portion of the MHC class I a1 helix (reviewed in refs. 3, 5, 6). They possess two (KIR2D) or three (KIR3D) extra-cellular C2-type Ig-like domains and a long cytoplasmic tail (L) containing immunoreceptor tyrosine-based inhibition motifs (ITIM) which recruit and activate SHP-1 and SHP-2 phosphatases for inhibitory signal transduction. KIR2DL1 recognises HLA-C alleles characterised by a Lys80 residue (HLACw4 and related, “Group 2” alleles). KIR2DL2 and KIR2DL3 (which are allele variants) recognise HLA-C with an Asn80 residue (HLA-Cw3 and related, “Group 1” alleles). KIR3DL1 is the receptor for HLA-B alleles sharing the Bw4 supertypic specificity (Table 1). Another type of human NK cell inhibitory receptor involved in HLA recognition is CD94-NKG2A. It binds to the non-conventional class I molecule HLA-E. Several HLA class I alleles provide signal sequence peptides that bind HLA-E and allow its expression at the cell surface. Consequently, it is expressed in every individual. Inhibitory KIRs, CD94/NKG2 and HLA-class I genes determine individual NK cell repertoires during development. As they are located on different chromosomes, receptors and ligands segregate independently in human pedigrees. The HLA class I genotype selects a self-tolerant repertoire by dictating which KIR and/or NKG2A
Table 1: HLA-class I allele specificity of the main KIRs expressed by human NK cells KIR gene
Encoded protein
HLA specificity
KIR2DL1
P58.1 receptor
HLA-C group 2 (e.g., -Cw2, -Cw4, -Cw5, -Cw6) (Lys80)
KIR2DL2/3
P58.2 receptor
HLA-C group 1 (e.g., -Cw1, -Cw3, -Cw7, -Cw8) (Asn80)
KIR3DL1
P70/NKB1 receptor
Bw4 alleles (e.g., HLA-B27)
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receptor combinations are to be used as inhibitory receptors for self HLA class I (7). Consequently, every functional NK cell in the mature repertoire expresses at least one inhibitory receptor for self HLA: co-expression of two or more receptors is less frequent.
2. Inhibitory KIRs and alloreactivity Since inhibitory KIRs recognise specific groups of HLA class I molecules, i.e., HLAC group 1, HLA-C group 2, HLA-Bw4 alleles, NK cells with the potential to exert alloreactions use KIRs as inhibitory receptors for self (1-6). NK cells which express, as their only inhibitory receptor for self, a KIR for the HLA class I group which is absent on allogeneic targets, sense the missing expression of the self class I KIR ligand and mediate alloreactions (“missing self” recognition). Importantly, most individuals possess a full complement of inhibitory KIR genes and can exert NK cell alloreactions (3, 4, 6). In particular, 100% of individuals possess the KIR2DL2 and/or KIR2DL3 receptors for HLA-C group 1 alleles. If they have HLA-C group 1 allele(s) in their HLA type, these individuals possess HLA-C1-specific NK cells which are alloreactive against cells from individuals who do not express HLA-C group 1 alleles. Ninety-seven percent of individuals possess the KIR2DL1 receptor for HLAC group 2. If they possess HLA-C group 2 allele(s) in their HLA type, these individuals have HLA-C2-specific NK cells which mediate alloreactions against cells from individuals who do not express HLA-C group 2 alleles. Finally, ~90% of individuals possess the KIR3DL1 receptor for HLA-Bw4 alleles. When they have HLABw4 allele(s) in their HLA type, these individuals may have HLA-Bw4-specific NK cells that are alloreactive against Bw4-negative cells. These KIR ligand mismatches often occur in haploidentical donor recipient transplant pairs.
3. Clinical effects of NK cell alloreactivity When exerted in the donor-versus-recipient direction, NK cell alloreactivity emerged as a crucial factor in improving outcomes of haploidentical transplantation (1-4). It reduced the risk of leukaemia relapse, did not cause graft versus host disease (GvHD) and markedly improved event-free survival (EFS) in a series of haploidentical transplants (57 acute myeloid leukaemia (AML) patients, 20 of whom were transplanted from NK alloreactive donors) (2). In an updated analysis (4), 112 highrisk AML patients received haploidentical transplants from NK alloreactive (n=51) or non-NK alloreactive donors (n=61). Transplantation from NK-alloreactive donors was associated with: - a significantly lower relapse rate in patients transplanted in any CR (3 vs. 47%) (p<0.003); 68
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- better EFS whether patients transplanted in relapse (34 vs. 6%, p=0.04) or in remission (67 vs. 18%, p=0.02); - overall reduced risk of relapse or death (relative risk vs. non-NK-alloreactive donor: 0.48 [95%CI: 0.29-0.78], p<0.001). The 67% probability of surviving event-free for acute myeloid leukaemia patients transplanted in any remission from NK alloreactive donors is in the range of best survival rates after transplantation from unrelated donors and cord blood units. The 34% EFS of patients transplanted in chemo-resistant relapse from NK alloreactive donors is also satisfactory. Transplantation from non-NK alloreactive haploidentical donors appears justified only for AML patients in remission and even in these patients it is associated with only an 18% EFS. Lack of an NK alloreactive donors is a contra-indication to transplant for patients in chemo-resistant relapse as very few survive. Several observations suggest alloreactive NK cells are responsible for favourable transplantation outcomes. Transfer of human alloreactive NK cells to NOD-SCID mice eradicated previously transplanted human AML cells (2). KIR ligand mismatches correlated with the ability of donor NK cell clones to kill cryopreserved haematopoietic recipient cells, including leukaemia cells (1, 2, 4). Most importantly, as depicted in Figure 1, upon transplantation from NK alloreactive donors the engrafted stem cells give rise to an NK cell repertoire which includes donor-vs-recipient alloreactive NK clones which kill cryopreserved haematopoietic recipient cells, including leukaemia cells (1). Donor versus recipient alloreactive NK clones are detectable in vivo in recipients for up to 1 year after transplant (4).
4. Selection of an NK alloreactive donor In the clinical studies above, NK alloreactive donors were found for ~50% of patients, which approaches the maximum because, in fact, 1/3 of the population expresses class I alleles belonging to all three class I groups recognised by KIRs and block NK cells from every donor. How is an NK alloreactive donor selected? Recipients who express alleles belonging to 1 or 2 of the 3 class I allele groups recognised by KIRs may find NK alloreactive donors. Donors who are HLA-C group mismatched with their recipients possess highfrequency NK clones which are alloreactive against recipients’ target cells (1-4). Thus, high-resolution HLA-C typing is a good predictor of NK cell alloreactivity. Since 3% of individuals do not possess the KIR2DL1 gene, the combination of a KIR2DL1negative donor and a recipient without HLA-C group 2 alleles could result in a 1.5% incidence of false positivity. KIR2DL1 gene typing of the donor may be necessary to assess the NK alloreactive potential of this combination.
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Figure 1 NK repertoire Host targets CD16 CD8 CD4
cells/cmm
600 400 200 0
High-intensity conditioning
0
50
100 days
150
200
KIR2DL1
HLA-C group 2 Cw2/Cw4
KIR2DL2/3
Missing group 1
KIR3DL1
HLA-B51
T cell
Stem Stem Stem Stem Stem Stem Stem Stem Stem Stem Stem
Mega-dose stem cells from “allo NK” donor No post-transplant immune suppression
Haematopoietic stem cell transplantation from a KIR ligand-mismatched donor who possesses donor versus recipient alloreactive NK clones in his/her repertoire gives rise to transient post-transplant generation of a repertoire which is identical to the donor’s, including the alloreactive clones. In fact, alloreactive clones can be isolated from the circulation of successfully engrafted recipients and can kill cryopreserved recipient targets, including leukaemic cells (1, 4)
In HLA-Bw4 mismatches, even when the KIR3DL1 gene is present in the donor (~90% of individuals), NK repertoire studies show alloreactive NK clones are non-detectable ~1/3 of individuals (4). In some allelic variants in the HLA-Bw4 inhibitory NK receptor gene KIR3DL1 may not allow full receptor expression at the cell membrane and affect NK cell inhibition by HLA-Bw4 ligand (8); others apparently express alloreactive NK clones in very low frequencies. Thus, for HLA-Bw4 mismatches, functional assessment of the donor NK repertoire appears necessary. As approximately half of unrelated donor transplants are mismatched for one or more HLA class I alleles, donor vs. recipient NK cell alloreactivity may also occur in this setting. However, some retrospective studies show no advantage in transplantation from KIR ligand-mismatched donors (9, 10). Unrelated donor transplant protocols are heterogeneous in conditioning regimens, patient populations and underlying diseases. They use T-cell-deplete bone marrow harvests (or, less frequently, peripheral blood progenitors) which contain ~4 log more T-cells and up to 1 log fewer stem cells than haploidentical grafts. Relatively few transplanted stem cells, combined with the high T-cell graft content and post-transplant immune suppression have been associated with poor reconstitution of potentially alloreactive, KIR-bearing NK cells (11). Nonetheless, other studies have observed an increased GvL effect (12–15). 70
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CHAPTER 3.2 • NK cells in haploidentical transplantation
A marked survival advantage was reported in patients who received anti-thymocyte globulins pre-transplant (which provided in vivo T-cell depletion) and a graft containing 2-3-fold more nucleated cells than usual in unrelated-donor transplants (12). Prospective studies are needed to determine whether strategies (high doses of stem cells, T-cell depletion, no post-transplant immune suppression), which in haploidentical transplantation harness donor-vs-recipient NK cell alloreactivity, can be implemented to improve outcome in unrelated donor transplants.
5. The “missing ligand” model Since the original report that NK cell alloreactivity in haploidentical transplantation rests upon KIR ligand mismatching and donor NK cell recognition of “missing self” on recipient targets (2), the “missing ligand” model has been proposed as a powerful algorithm for predicting favourable transplant outcomes not only in haploidentical transplants (16) but also in matched sibling (17) and in unrelated donor transplants (18). Under the perturbed conditions that exist after a haematopoietic stem-cell transplant, it hypothesises that NK alloreactions occur when KIR ligand-matched donors possess an “extra” KIR for which neither donor nor recipient have an HLA ligand. These donors may carry KIR-bearing NK cells in an anergic/regulated state which, upon transfer into the recipient, are hypothesised to become activated and exert a GvL effect. However, even though self-tolerant NK cells which do not express inhibitory receptors for self-MHC have been described (19) no studies have as yet determined whether tolerant NK cells acquire/resume cytotoxic effector function after transplant. When an adult series of AML patients who were transplanted from haploidentical donors was analysed according to the “missing ligand” algorithm, the “missing ligand” transplant recipients disappointingly had a worse prognosis than patients transplanted from NKalloreactive (KIR ligand mismatched) donors (4). While differences in diseases, age of patients and transplantation protocols, such as ATG vs. no ATG in the conditioning or peripheral blood CD34+ cells vs. bone marrow as a source of haematopoietic cells, may account for these conflicting results, the analysis shows that donor NK cell recognition of “missing self” on recipient targets is essential for triggering powerful NK cell alloreactions that impact beneficially on transplantation outcomes.
6. Effects of donor activating KIR genetics on donor vs. recipient NK cell alloreactivity Activating KIRs, which regulate NK and T-cell functions, are molecular homologues of the inhibitory KIRs with shorter cytoplasmic tails (S) (reviewed in refs. 3, 5, 6) and a charged residue in their transmembrane domain that allows association with
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ITAM containing signalling polypeptides. Knowledge of their ligand specificity is limited. Studies have reported a weak interaction between KIR2DS1 and Lys80 HLAC molecules, despite its homology to KIR2DL1, and an even weaker interaction between KIR2DS2 and Asn80 HLA-C molecules, despite its homology to KIR2DL2 and KIR2DL3. Unlike inhibitory KIRs, activating KIRs exhibit extensive variation in gene number and content, which leads to heterogeneity within the general population and diverse ethnic groups (reviewed in ref. 6). Indeed, activating KIRs may not even be present in approximately 25% of Caucasians who are homozygous for the so-called group A KIR gene haplotypes which contain inhibitory KIR genes and the KIR2DS4 activating KIR gene (encoding for a non-functional protein in 2/3 of individuals). On the other hand, 75% of Caucasians are either heterozygous or homozygous for B haplotypes which carry not only inhibitory KIR genes but also various combinations of activating KIR genes (KIR2DS1-2-3-5 and KIR3DS1).
7. Activating KIRs and results of haploidentical HSCT We, therefore, evaluated the role of donor activating KIR genetics in haploidentical haematopoietic transplantation (Mancusi et al., manuscript submitted for publication). In a series of 84 haploidentical transplants for AML, the impact of donor KIR genetics (group A vs. group B KIR gene haplotypes) was assessed separately in NK alloreactive and non-NK alloreactive transplants. Forty-seven recipients were transplanted from NK alloreactive donors (12 with group A KIR gene haplotypes vs. 35 with B haplotypes) and 37 recipients from non-NK alloreactive donors (8 with group A KIR gene haplotypes vs. 29 with B haplotypes). KIR gene haplotypes had no impact in non-NK alloreactive transplants. In transplants from NK alloreactive donors, presence of group B haplotype KIR genes in the donors was associated with reduced incidence of TRM (largely infection-related) (B vs. A haplotypes: 20 vs. 67% TRM, p<0.005). In multivariate analyses against disease status at transplant, age, patient and donor sex, conditioning regimens, and the number of CD34+ and CD3+ cells in the graft, it was the only significant variable predicting protection from TRM (RR: 0.24; 95%CI: 0.14-0.42; p<0.01) and resulted in a trend towards better EFS (60 vs. 33%, p<0.1). When the number of activating KIR genes in the donor was taken into account, donors carrying ≥ 3 activating KIR genes provided significant protection from TRM and significantly better EFS compared with A haplotype donors (TRM: 12 vs. 67%, p<0.003) (EFS: 71 vs. 33%, p=0.02). In multivariate analysis, transplantation from alloreactive donors carrying ≥3 group B haplotype activating KIR genes was the only variable predicting protection from TRM (RR: 0.40; 95%CI: 0.27-0.58; p<0.02) and significantly improved EFS (RR: 0.56; 95%CI: 0.320.98; p<0.05). Thus, while NK-alloreactive donors protect against leukaemia relapse, 72
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CHAPTER 3.2 • NK cells in haploidentical transplantation
those who also carry activating KIRs protect against infectious mortality and help improve survival. The chance of finding an NK alloreactive donor is ~ 50% of haploidentical transplants and the odds of finding an NK alloreactive donor carrying activating KIRs are ~ 30% of haploidentical transplants.
8. Activating KIRs and protection against infection Protection against infection may be mediated directly by NK cells or indirectly through other mechanisms. Activating KIRs could enhance NK cell cytokine secretion and cytotoxicity against pathogen infected cells in the context of missing self. A notable example of direct recognition of a pathogen by activating NK receptor is provided by murine CMV protein m157 and the murine Ly49H NK receptor (20). In humans, progression to AIDS is slower in patients who have both KIR3DS1 and the HLA-Bw4 allotype, the putative ligand of KIR3DS1 (21). More NK cells expressing NKG2C are present in CMV-exposed individuals, suggesting this activating NK cell receptor plays a role in the immune response to this infection (22). Therefore associations between activating NK receptors and enhanced immunity against infections have been documented. Activating KIRs could also help control infections indirectly through the interaction between NK cells and dendritic cells (DCs) (reviewed in ref. 23). NK cells regulate DC homeostasis and maturation. Mature DCs can, in turn, activate NK cells. In vivo NK/DC interactions in lymphoid organs or non-lymphoid tissues can lead to Th1 polarisation. NK cells in lymph nodes provide the early IFN-g production, which is essential for Th1 polarisation. Consequently, the interaction between NK cells and DCs influences the quality and the strength of adaptive immune response. The clinical data suggest either or both these mechanisms could operate in haploidentical transplants from NK-alloreactive donors who possess activating KIRs. Whatever the mechanisms, these studies have improved criteria for donor selection.
9. Conclusions Transplantation from a full HLA haplotype mismatched family member is nowadays a viable option for patients with acute leukaemia at high risk of relapse who urgently need a transplant and who do not have a matched donor (24). The mismatched transplant relies for its success on the combined action of: - high-intensity conditioning regimens to ensure the lowest possible residual leukaemia burden and optimal immunosuppression; - high doses of haematopoietic cells to ensure engraftment across the HLA barrier; - extensive T-cell depletion of the graft to prevent GvHD (with no post-transplant immunosuppression);
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- donor versus recipient NK cell alloreactivity to enhance anti-leukaemia effects; - NK-alloreactive donors who also carry activating KIRs to protect against infectious mortality.
References 1. Ruggeri L, Capanni M, Casucci M, et al. Role of natural killer cell alloreactivity in HLAmismatched hematopoietic stem cell transplantation. Blood 1999; 94: 333-339. 2. Ruggeri L, Capanni M, Urbani E, et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 2002; 295: 2097-2100. 3. Ruggeri L, Aversa F, Martelli MF, Velardi A. Haploidentical transplantation and natural killer cell recognition of missing self. Immunol Rev 2006; 214: 202-218. 4. Ruggeri L, Mancusi A, Capanni M, et al. Donor natural killer cell allorecognition of missing self in haploidentical hematopoietic transplantation for acute myeloid leukemia: Challenging its predictive value. Blood 2007; 110: 433-440. 5. Moretta L, Moretta A. Killer immunoglobulin-like receptors. Curr Opin Immunol 2004; 16: 626-633. 6. Parham P. MHC class I molecules and KIRs in human history, health and survival. Nat Rev Immunol 2005; 5: 201-214. 7. Yawata M, Yawata N, Draghi M, et al. Roles for HLA and KIR polymorphisms in natural killer cell repertoire selection and modulation of effector function. J Exp Med 2006; 203: 633-645. 8. Pando MJ, Gardiner CM, Gleimer M, et al. The protein made from a common allele of KIR3DL1 (3DL*004) is poorly expressed at cell surfaces due to substitution at position 86 in Ig domain 0 and 182 in Ig domain 1. J Immunol 2003; 171: 6640-6647. 9. Davies SM, Ruggieri L, DeFor T, et al. Evaluation of KIR ligand incompatibility in mismatched unrelated donor hematopoietic transplants. Blood 2002; 100: 3825-3827. 10.Farag SS, Bacigalupo A, Eapen M, et al. The effect of KIR ligand incompatibility on the outcome of unrelated donor transplantation: A report from the center for international blood and marrow transplant research, the European blood and marrow transplant registry, and the Dutch registry. Biol Blood Marrow Transplant 2006; 12: 876-884. 11.Cooley S, McCullar V, Wangen R, et al. KIR reconstitution is altered by T cells in the graft and correlates with clinical outcomes after unrelated donor transplantation. Blood 2005; 106: 4370-4376. 12. Giebel S, Locatelli F, Lamparelli T, et al. Survival advantage with KIR ligand incompatibility in hematopoietic stem cell transplantation from unrelated donors. Blood 2003; 102: 814-819. 13.Elmaagacli AH, Ottinger H, Koldehoff M, et al. Reduced risk for molecular disease in patients with chronic myeloid leukemia after transplantation from a KIR-mismatched donor. Transplantation 2005; 79: 1741-1747. 14.Beelen DW, Hottinger HD, Ferencic S, et al. Genotypic Inhibitory Killer Immunoglobulinlike Receptor Ligand Incompatibility Enhances the Long-term Antileukemic Effect of Unmodified Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Myeloid Leukemias. Blood 2005; 105: 2594-2600. 74
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CHAPTER 3.2 • NK cells in haploidentical transplantation
15.Dawson MA, Spencer A. Successful use of haploidentical stem-cell transplantation with KIR mismatch as initial therapy for poor-risk myelodysplastic syndrome. J Clin Oncol 2005; 23: 4473-4474. 16.Leung W, Iyengar R, Turner V, et al. Determinants of antileukemia effects of allogeneic NK cells. J Immunol 2004; 172: 644-650. 17.Hsu KC, Keever-Taylor CA, Wilton A, et al. Improved outcome in HLA-identical sibling hematopoietic stem-cell transplantation for acute myelogenous leukemia predicted by KIR and HLA genotypes. Blood 2005; 105: 4878-4884. 18.Hsu KC, Gooley T, Malkki M, et al. KIR ligands and prediction of relapse after unrelated donor hematopoietic cell transplantation for hematologic malignancy. Biol Blood Marrow Transpl 2006; 12: 828-836. 19.Fernandez NC, Treiner E, Vance RE, et al. A subset of natural killer cells achieves selftolerance without expressing inhibitory receptors specific for self-MHC molecules. Blood 2005; 105: 4416-4423. 20.Smith HRC, Heusel JW, Mehta IK, et al. Recognition of a virus-encoded ligand by a natural killer cell activation receptor. Proc Natl Acad Sci USA 2002; 99: 8826-8831. 21.Martin MP, et al. Epistatic interaction between KIR3DS1 and HLA-B delays the progression to AIDS. Nature Genetics 2002; 31: 429–434. 22.Guma M, et al. Imprint of human cytomegalovirus infection on the NK cell receptor repertoire. Blood 2004; 104: 3664–3671. 23.Degli Esposti MA, Smyth MJ. Close encounters of different kinds: Dendritic cells and NK cells take centre stage. Nature Reviews Immunology 2005; 5: 112-124. 24.Aversa F, Tabilio A, Velardi A, et al. Treatment of high risk acute leukemia with T-celldepleted stem cells from related donors with one fully mismatched HLA haplotype. N Engl J Med 1998; 339: 1186-1193.
Multiple Choice Questionnaire To find the correct answer, go to http://www.esh.org/ebmt-handbook2008answers.htm 1. Which molecules are recognised by inhibitory KIRs? a) HLA class II molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) HLA-E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) HLA class I molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) All of the above . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Which of the following statements is correct in pre-clinical models and in the clinical practice of haploidentical haematopoietic transplantation? a) Donor vs. recipient alloreactive NK cells cause GvHD. . . . . . . . . . . . . . . . . . . . . . .
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b) Donor vs. recipient alloreactive NK cells increase the incidence of leukaemia relapse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Donor vs. recipient alloreactive NK cells mediate a GvL effect . . . . . . . . . . . . d) Donor vs. recipient alloreactive NK cells cause rejection . . . . . . . . . . . . . . . . . . . 3. From the data in ref. 4 of this Chapter, the event-free survival of acute myeloid leukaemia patients transplanted in any remission from haploidentical NK alloreactive donors is: a) 30-40% . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) 40-50% . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) 50-60% . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) 60% . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. From the data in ref. 4 of this Chapter, the event-free survival for acute myeloid leukaemia patients transplanted in chemo-resistant relapse from NK alloreactive donors is: a) 10% . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) 10-20% . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) 20-30% . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) 30% . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. From the study by Mancusi et al. reported in this Chapter, transplantation from NK alloreactive donors who also possess activating KIR genes is associated with: a) Decreased incidence of leukaemia relapse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Increased incidence of GvHD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Decreased incidence of infectious mortality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Increased incidence of infectious mortality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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NOTES
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*
CHAPTER 3
Immunogenetics of allogeneic HSCT
*
3.3
Non-HLA immunogenetics and role in transplant outcome A.M. Dickinson
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CHAPTER 3.3 • Non-HLA immunogenetics
1. Introduction While HLA typing remains the central means of selecting donors and the major factor determining haematopoietic stem cell transplant (HSCT) outcome, the sequencing of the human genome has brought to light myriads of single nucleotide polymorphisms whose significance in determining an individual’s immunological phenotype and their possible role in influencing the outcome of an allogeneic stem cell transplant is only just beginning to be explored. This review discusses the field of non-HLA immunogenetics and the patient and/or donor genotypes which may influence transplant outcome, including occurrence and severity graft versus host disease (GvHD), infectious episodes and overall survival (OS). Results associated with polymorphisms of cytokine and immune-related genes, genes of uncertain function, and minor histocompatibility antigens will be summarised.
2. Cytokine gene polymorphisms (CGPs) Cytokine gene polymorphisms occurring within the 5’ or 3’ regulatory sequences of genes may alter the structure of the transcription factor binding sites within gene promoters and therefore alter the amount of cytokine produced, for example, upon allogeneic stimulation or infection. Many of the reported cytokine gene polymorphisms occur within apparent regulatory regions of the gene. Within normal populations high or low producers of cytokines naturally exist due to the inherited gene polymorphisms. Initial studies within the solid organ transplant setting demonstrated that patients with high-producer tumour necrosis factor (TNF) and low-producer interleukin 10 (IL-10) genotypes were more likely to reject their solid organ graft. These studies were extended to the HLA matched sibling and matched unrelated donor (MUD) HSCT. A number of cytokine gene polymorphisms have now been associated with GvHD and/or transplant outcome. The majority of the studies have been carried out in single centre series of HLA matched sibling transplants and some MUD transplant patients. 2.1. Influence of CGPs on GvHD and post-HSCT complications Acute graft versus host disease (aGvHD) incidence following allo-HSCT between HLA identical siblings is 30–80% and can be fatal in up to 50% of cases. GvHD is induced by release of pro-inflammatory cytokines IL-1, IL-6, IL-8, and TNF-a during the "cytokine storm" following both radiotherapy and chemotherapy conditioning regimens. This is amplified by the activation of transplanted donor T-cells via reaction to upregulated HLA and adhesion molecules on recipient target cells. Tissue damage then ensues from activated T-cells and NK cells and release of predominantly Th1 type cytokines (IL-2, IFN-g, TNF-a).
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Although T-cells are central to the initiation of GvHD, pro-inflammatory mediators such as IL-1 and TNF-a are capable alone of inducing the pathological changes of GvHD. Growing evidence suggests that inflammatory cytokines are also involved in transplant outcomes other than GvHD such as interstitial pneumonia and VOD. The impact of gene polymorphisms on the susceptibility to infection and drug metabolism or transport (pharmacogenomics) post-transplant is now being identified. Taken together it is now evident that the genetic make up of the recipient and the donor can strongly influence the success or failure of HSCT. The CGPs studied for association with outcome in allo-HSCT, to date, are summarised in Table 1.
Table 1: Non-HLA gene polymorphisms and their associations with GvHD and HSCT outcome CGP
Proposed function of polymorphism
Effect of recipient genotype (RG) or donor genotype(DG) on HSCT outcome
TNFd3 (a)
- Pro-inflammatory - Upregulates TNF-a production
TNF receptor TNFRII 196R (b)
- Unknown (TNFRII receptor stimulates T-cell proliferation and allo-immune responses)
IL-10-1064(12-15) (c) IL-10 haplotype
- Anti-inflammatory - Haplotype associated with decreased production of IL-10
IL-6-174 (d)
- Pro-inflammatory - G allele associated with increased IL-6 production - Pro-inflammatory - Lower in vitro IFN-g production - Pro-inflammatory
RG: Increases aGvHD in HLA matched sibling BMT. No association in CBT DG: More severe GvHD in MUD transplant RG acute/DG chronic Incidence of acute and chronic GvHD in HLA-matched siblings RG: Increase in GvHD in BMT RG & DG associated with aGvHD No association in CBT RG: Increases acute and chronic GvHD in HLA-matched sibling cohorts RG: Increased aGvHD in HLAmatched sibling cohorts
IFN-g (e) Intron 1 allele IL-1 gene family IL-1a 889; intron 6 VNTR (f) IL-1 Ra VNTR VNTR intron 2 (g)
- Anti-inflammatory - Down-regulates proinflammatory effects of IL-1
DG: associates with cGvHD in HLA-matched siblings Improved OS decreases TRM; DG or RG in MUD transplants DG decreases aGvHD and RG increases cGvHD in HLAmatched siblings continue
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CGP
Proposed function of polymorphism
Effect of recipient genotype (RG) or donor genotype(DG) on HSCT outcome
TGF-b TGF-509 (h)
- C allele at –509 associated with increased TGF-b production
RG: No association with TGF-b –509 with incidence of GvHD or outcome in HLA-matched sibling cohort. Also association with GvHD incidence and high TGF-b producers in paediatric HLA-matched sibling cohort
TGF-b1 codon 10 leucine/proline
Alteration in protein structure
Development of aGvHD in MUD paediatric transplants
NOD2/CARD15 SNP 8, 12, 13 (i)
Altered NFK-b function
Two or more variants associate with severe GvHD and TRM, HLA mismatched siblings and MUD
(a) (5, 6, 10, 25); (b) (7, 8); (c) (5, 6, 9, 10, 25, 26); (d) (6, 26); (e) (27); (f) (28, 29); (g) (30, 31); (h) (27, 32–34); (i) (17–19)
Both the patient and donor genotype are analysed with respect to transplant outcome, and the main findings are reviewed below and in recent reviews (1–4). 2.1.1. TNF genes The TNF-a gene is located within the class III region of the major histocompatibility complex (MHC). Therefore, in HLA-identical sibling HSCT recipient and donor genotype will be identical and may equally or additively affect TNF production and transplant outcome. Initial HSCT studies associated recipient genotype (d3/d3) of the TNF-d microsatellite with increased severity of aGvHD (grade III-IV) in CsA-alone treated HLA-matched sibling transplants (5). A larger cohort of transplants that received cyclosporine plus methotrexate prophylaxis demonstrated an association of recipient TNFd/d3 genotype with increased mortality (6). A Japanese MUD transplant study described an association of the TNF-863 and 857 polymorphisms in donors and/or recipients with a higher incidence of GvHD grade III-IV and a lower rate of relapse (7), however in HLA-A, -B and -DRb1 matched pairs only GvHD outcome remained significant. Studies have also investigated the role of the TNF-a (-308) and TNF-b (+1069) in HSCT and GvHD, but results have been inconsistent. A role of TNF-a-308 in HLA-matched
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sibling transplants was initially based on TNF-a secretion studies, not confirmed in larger cohorts. These polymorphisms, TNF-a and/or TNF-b, have been associated with toxic complications in both HLA matched sibling and MUD cohorts. TNF-a*2 (associated with higher TNF-a production) is associated with the TNF-b*1 allele contributing to more severe toxic complications than TNF-b* or TNF-a*1 homozygotes. Mullighan et al. (3) also analysed the TNF-488A allele in two independent cohorts of HLA matched sibling transplants, and this allele was associated with more severe aGvHD and cGvHD and early death. TNF-a polymorphisms are in linkage disequilibrium with HLA Class I and Class II genotypes within the MHC Class III region. Since HLA genotypes are known to influence GvHD development, associations with TNF-a genotype may interact or have additive effects with the HLA associations (3). A polymorphism (196 R/M) in the TNF receptor II gene in MUD HSCT found that recipients of TNFRII-196R-positive donors had a higher incidence of severe GvHD and a lower rate of relapse than from TNFRII 196M homozygous donors (7). Recipient TNFRII 196R allele was associated with aGvHD incidence but the TNFRII 196RR genotype in the donor was associated with (increased) incidence of extensive cGvHD in an HLA-matched sibling cohort (8). 2.1.2. IL-10 gene The SNPs and microsatellites of the IL-10 gene resolve into several conserved haplotypes. Three common haplotypes of the promoter region between -1082 and -592 represent high, intermediate, and low production of IL-10. The low producer (ACC) haplotype in the recipient associated with severe aGvHD grade III-IV in CsAalone (6) and CsA + MTx (methotrexate) (7) treated HLA-matched sibling cohorts. IL-10 low producer haplotype associations have recently been confirmed as playing a role in predicting outcome and GvHD (grades III-IV) HLA matched sibling transplants in two separate large (>400) cohorts (9). A recent study by Bettens et al. associated the IL-10-1064CA microsatellite with greater than 12 recipients and linked with the low producer IL-10-1082*A allele with worse survival (10). 2.1.3. IL-6 gene Possession of the G allele (IL-6-174 polymorphism) in the recipient (HLA-matched sibling transplant) associated with both acute and chronic GvHD, this allele has also been correlated with high serum IL-6 levels in normal individuals. 2.1.4. IFN-g gene Allele 2 of the CA repeat microsatellite in intron 1 has been associated with high 82
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in vitro IFN-g production, whereas allele 3 has been linked to lower IFN-g production. The possession of allele 2 within the 874A allele coincides with the NFK-b binding site which may give rise to changes in the IFN-g gene transcription. Lack of IFN-g can lead to accelerated acute GvHD in IFN-g knockout mouse models. Possession of allele 3 in the recipient genotype in HLA-matched sibling transplants was associated with development of aGvHD and increased risk of EBV infections. Other studies have shown association with lack of the 2/2 genotype with GvHD (11, 12). 2.1.5. IL-1 gene family IL-1 receptor antagonist (IL-1Ra) binds IL-1 and can lead to induction of other cytokines. HLA-matched siblings transplant studies have demonstrated an association of the IL-Ra VNTR (allele 2) (which downregulates IL-1 production) in the donor genotype with less severe aGvHD, and in the recipient genotype with cGvHD. Carriage of allele 2 (donor genotype) in either the VNTR or -889 polymorphisms of IL-1a gene associated with cGvHD. A study of paediatric MUD transplant group found that IL-1a-889 in either donor or recipient was associated with improved survival and decreased TRM, but not with GvHD. IL-1 polymorphisms and association with HSCT outcome has recently been reviewed by Cullup and Stark (13). 2.1.6. TGF-b gene Several studies have reported association of TGF-b genotype with GvHD. A polymorphism (-509 C/T) in the promoter region is associated with variation in plasma concentration of TGF-b (C allele with higher production), and amino acid substitutions at codons 10 (leuÆpro) and 25 (argÆpro), which alters protein structure. A small study on HLA-identical sibling BMT showed no association of the TGF-b-509 polymorphism with either GvHD or outcome. However a study in 67 paediatric patients showed an association with the TGF-b1 codon 10 polymorphism in the donor genotype and development of aGvHD. TGF-b1 receptor II polymorphism (1167 C/T) genotype in the recipients in the same study was also associated with the development of GvHD.
3. Minor histocompatibility antigens and transplant outcome GvHD occurring after HLA matched sibling transplants is initiated by T-cells recognising minor histocompatibility antigens (mHag) (peptides derived from intracellular proteins of restricted polymorphisms coded by autosomal or Y chromosome genes, presented by HLA molecules) and are polymorphic proteins with differences between patient and donor (reviewed in (14)). T-cell clones recognising mHags were initially isolated after BMT in patients developing GvHD or graft
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rejection. The male specific mHags encoded by the Y chromosome have been shown to be involved in HLA-matched sex mismatched HSCT. The tissue expression of some mHag (e.g., HA-1 and HA-2) is limited to the haematopoietic system whereas other mHag (e.g., H-Y, HA-3) are ubiquitously expressed on normal tissues. Mismatches between patient and donor for HA-1, HA-2, 4, and 5 are associated with increased GvHD incidence. The precise characterisation of the peptide sequence of haematopoietic tissue-restricted or cancer cell-restricted mHa make them ideal targets for immunotherapy mHag specific DLI has led to complete remission of relapsed leukaemia in 3 patients, mHag HA-1 is also expressed on epithelial cancer cells and not on normal epithelial cells giving rise to the concept of the use of mHag in more diverse cancer therapies.
4. Other non-HLA-encoded genes implicated in transplant related complications Other non-HLA encoded genes have also been implicated not only in GvHD but also in TRM, infectious episodes and outcome in HLA-matched sibling transplants. The steroid hormone receptor supergene family includes the estrogen receptor and the vitamin D receptor (VDR), both have marked effects on the development of the immune system and variations in VDR are associated with autoimmune and immune dysfunctional diseases. Vitamin D analogues can prolong graft survival and prevent GvHD in animal studies. Polymorphism in the VDR intron 8 region (15), and in intron 1 of the estrogen receptor alpha (ERa) genes have been associated with both occurrence of GvHD, post-transplant infection and likelihood of survival following allo-BMT. Donor and recipient genotype in genes that regulate the host response to microorganisms (myeloperoxidase (MPO), mannose binding lectin (MBL) and Fcg receptors (FcgRIIa, IIIa, IIIb)) have been associated with infections after BMT. First infection post-transplant has been associated with the R-131 allele in the patient FcgRIIa genotype and occurrence of severe bacterial infections increased when MPO donor genotype was AG or AA. TRM was influenced by FcgRIIIb genotype and donor MPO genotype (see Table 2). These studies further define and improve understanding of the mechanisms involved in host defence against infection during BMT. 4.1. Innate immunity Investigations into other non-cytokine genes, associated with innate immunity, have been studied including pattern recognition receptors (PRRs) which activate the innate and adaptive arms of the immune response against pathogens. PRRs include the transmembrane receptors, Toll-like receptors (TLRs), which have the ability to activate antigen presenting cells (APCs) (16, 17). Polymorphisms in the TLR4 gene, 84
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Table 2: Non-HLA genes associated with risk of infections and TRM Gene
Alleles
Effect
R or D genotype
MPO (a)
AG/AA
D
FcgRIIa (a)
R-131
FcgRIIIb (a)
HNA1a/HNA1a
Increased bacterial infection Increased risk of non-leukaemic death Increased first overall infections (infections occurring before d 80) Delayed neutrophil recovery Severe infection Increased risk of non-leukaemic death Increased TRM rates Quick neutrophil recovery Decreased infection Increased risk of major infection
MBL (b)
HNA1b/HNA1b HNA1a/HNA1b HYA haplotype MBL2
R
D
D D/R D/R
(a) (31); (b) (35). MPO: myeloperoxidase, an enzyme found primarily in the lysosomes of neutrophils. A single base substitution (G > A) in the promoter region of the MPO gene (–463) decreases expression and alters binding site for the transcription factor; FcgRIIa: Fcg receptor, polymorphisms of the receptor affect receptor affinity and specificity; FcgRIIIb: Fcg receptor HNA1a and HNA1b, isoforms affect different ligand binding of antibodies; MBL: mannose-binding lectin
for example, have been studied and associated with hypo-responsiveness to lipopolysaccharides with a reduced risk of aGvHD, but increased risk of Gramnegative bacteraemia. Polymorphisms in the TLR1 and TLR6 genes have been associated with invasive aspergillosis following HSCT; patient genotype being associated with increased risk of infection. Intracellular PRRs associated with inflammatory bowel disease, such as the intracellular group of NOD-like receptors (nucleotide-binding oligomerisation domain containing receptors) have also been studied in cohorts of HSCT patients. There are three SNPs in NOD2/CARD15 (SNP8, SNP12, SNP13) gene, involved in defective NF-kB responses and possession of two or more NOD2/CARD15 variants has been associated with more severe GvHD and increased transplant-related mortality (TRM) (18). Further studies showed that in HLA-matched siblings the genetic risk could be modulated depending on the type of gastrointestinal decontamination pre-transplantation (17). In T-cell-depleted transplants, possession of NOD2/CARD15 SNPs associated with lower disease-free survival (19) and the results illustrate that altered immune responses associated with NOD gene variants may be dependent on type of immunomodulation, e.g. bacterial decontamination and by the type of immunosuppression (e.g. T-cell depletion).
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5. Pharmacogenomics in HSCT Knowledge of an individual’s drug response has the potential to improve HSCT outcome, and several polymorphisms of genes involved in the metabolism of drugs employed in conditioning regimens and prophylaxis strategies have been associated with GvHD, survival, toxicities and platelet recovery following HSCT (3). A SNP (C677T) associated with lower methylenetetrahydrofolate reductase (MTHFR) activity was linked to increased mucositis and delayed platelet engraftment in HSCT recipients receiving methotrexate, but results have not been consistent. These findings were confirmed by Robien et al. (20), but, Kalayoglu-Besisik et al. (21) found no associations with HSCT outcome. Other studies have involved HLA matched siblings and MUD transplant cohorts with differing results.
6. Gene associations with transplant outcome – Problems of interpretation The cytokine gene polymorphisms examined in our initial studies were selected on the basis of their association with altered cytokine production in vivo or their demonstrated association with transplant-related pathology, and or their association with immune dysfunction/autoimmune diseases. Typically, alleles linked with increased expression of pro-inflammatory cytokines such as TNF-a, IL-6, and IFNg show associations with complications such as GvHD, whereas alleles associated with increased expression of anti-inflammatory cytokines (IL-10 and IL-1Ra) show protection effects (see Table 1). However, as more studies are reported this model appears too simple to account for all the findings. Several problems surround the interpretation of the role of cytokine variations in transplant outcome. HLA association: In HLA matched sibling transplants HLA-A3 is associated with a higher and DR1 with a lower risk of GvHD. These associations may represent HLA types best able to present specific antigens relevant to disease processes. Alternatively, the effect on GvHD may reflect Class III region TNF polymorphisms forming an extended haplotype with the HLA Class I and Class II region. Determining what precisely occurs at this locus is therefore complex. In the MUD transplant setting the role of HLA will be of a paramount importance in interpretation of non-HLA immunogenetic data. High resolution tissue typing and standardisation of results is necessary across BMT centres for comparative studies. GvHD increases proportionally with the degree of HLA Class I and II disparity between patient and donor. Mismatching for HLA-C alleles increases the risk of graft failure, GvHD and mortality. Disparities between HLA sequence polymorphisms detected by serology are termed antigen mismatches; those identified only by DNA-based typing are termed allele mismatches. The risk of mortality after MUD transplants is increased by mismatches at a single allele at HLA A B C or DRB1. The significance of these mismatches must 86
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be taken into account prior to further analysis of the data for the role of non-HLA immunogenetics in the MUD transplant setting. 6.1. Population differences in gene frequencies In vivo disease association may not necessarily correlate with in vitro cytokine expression or production. This could be due to genetic variation in allele frequencies between different ethnic populations resulting in different associations being found. Therefore, comparisons between populations are difficult unless local population allele frequency is simultaneously assessed and are taken into account. For example, IL-10 GCC haplotype in Japan is of a very low frequency compared to Europe. One study compared the incidence of aGvHD and TRM in HLA identical sibling transplants within the Japanese, white and African Americans, Scandinavians and the Irish (22), and showed differences between the American groups and the Irish compared to the Japanese or Scandinavians. The results indicate that genetic differences other than HLA may affect transplant outcome, and several studies on allele frequencies in transplant populations worldwide are currently being undertaken. 6.2. Different transplant procedures Analysis of the complex data emerging on the role of non-HLA immunogenetics on stem cell transplant outcome is compounded by a number of factors including the heterogeneity of the diagnoses and clinical state of the recipients, GvHD prophylaxis regimes, and conditioning strategies. Most of the research has been carried out within the HLA-matched sibling setting. Larger studies are now needed together with multivariate analysis in both HLA-matched siblings and matched unrelated donor transplants, especially those undergoing homogeneous clinical trials.
7. Future developments Current clinical studies are aimed at the development of a clinical and genetic risk score which will extend the predictive power of genetic polymorphisms. To this aim large cohorts of HLA-matched and MUD transplants need to be complied to fully investigate the impact of non-HLA immunogenetics on transplant related complications, and this is underway both at the European and International level including involvement of the International Bone Marrow Transplant Registry. Combined analysis of CGPs suggest that a risk index for GvHD could be developed alongside clinical risk factors such as gender, age, CMV status, and minor histocompatibility mismatches. Combining the EBMT risk score for CML with genetic factors has led to a scoring system which can further subdivide the patients for good, intermediate and poor prognosis (23). Further research into the possible
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role of pharmacogenomics (24) in determining outcome may allow risk prediction to tailor prophylaxis and therapy for GvHD on a disease specific or individual patient basis.
Acknowledgments Funding for current research on non-HLA gene polymorphisms is from European Commission grants TRANS-NET (MRCN-CT-2004-512253) and StemDiagnostics (LSHB-CT-2007-037703), the Leukaemia Research Fund and the Tyneside Leukaemia Research Association.
References 1. Dickinson AM. Risk assessment in haematopoietic stem cell transplantation: pre-transplant patient and donor factors: non-HLA genetics. Best Pract Res Clin Haematol 2007; 20: 189207. 2. Dickinson AM, Charron D. Non-HLA immunogenetics in hematopoietic stem cell transplantation. Curr Opin Immunol 2005; 17: 517-525. 3. Mullighan CG, Bardy PG. Advances in the genomics of allogeneic haemopoietic stem cell transplantation. Drug Development Research 2004; 62: 273-292. 4. Dickinson AM, Harrold J, Cullup H. Haematopoietic stem cell transplantation: Can our genes predict clinical outcome? Expert Rev Mol Med 2007; 9: 1–19. 5. Middleton PG, Taylor PRA, Jackson G, et al. Cytokine Gene polymorphisms associating with severe acute graft-versus-host disease in HLA-identical sibling transplants. Blood 1998; 92: 3943-3948. 6. Cavet J, Middleton PG, Segall M, et al. Recipient tumour necrosis factor-alpha and interleukin-10 gene polymorphisms associate with early mortality and acute graft-versushost disease severity in HLA matched sibling bone marrow transplants. Blood 1999; 94: 3941-3946. 7. Ishikawa Y, Kashiwase K, Akaza T, et al. Polymorphisms in TNFA and TNFR2 affect outcome of unrelated bone marrow transplantation. Bone Marrow Transplant 2002; 29: 569-575. 8. Stark GL, Dickinson AM, Jackson GH, et al. Tumour necrosis factor receptor type II 196M/R genotype correlates with circulating soluble receptor levels in normal subjects and with graft-versus-host disease after sibling allogeneic bone marrow transplantation. Transplantation 2003; 76: 1742-1749. 9. Lin MT, Storer B, Martin PJ, et al. Relation of an interleukin-10 promoter polymorphism to graft-versus-host disease and survival after hematopoietic-cell transplantation. N Engl J Med 2003; 349: 2201-2210. 10.Bettens F, Passweg J, Gratwohl A, et al. Association of TNFd and IL-10 polymorphisms with mortality in unrelated hematopoietic stem cell transplantation. Transplantation 2006; 81: 1261-1267. 88
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11.Bogunia-Kubik K, Mlynarczewska A, Wysoczanska B, et al. Recipient interferon-gamma 3/3 genotype contributes to the development of chronic graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Haematologica 2005; 90: 425-426. 12.Mlynarczewska A, Wysoczanska B, Karabon L, et al. Lack of IFN-gamma 2/2 homozygous genotype independently of recipient age and intensity of conditioning regimen influences the risk of aGVHD manifestation after HLA-matched sibling haematopoietic stem cell transplantation. Bone Marrow Transplant 2004; 34: 339-344. 13.Cullup H, Stark G. Interleukin-1 polymorphisms and Graft versus Host Disease. Leuk Lymphoma 2005; 46: 517-523. 14.Goulmy E. Human minor histocompatibility antigens. Curr Opin Immunol 1996; 8: 75-81. 15.Middleton PG, Cullup H, Dickinson AM, et al. Vitamin D receptor gene polymorphism associates with graft-versus-host disease and survival in HLA-matched sibling allogeneic bone marrow transplantation. Bone Marrow Transplant 2002; 30: 223-228. 16.Napolitani G, Rinaldi A, Bertoni F, et al. Selected Toll-like receptor agonist combinations synergistically trigger a T helper type 1-polarizing program in dendritic cells. Nature Immunol 2005; 6: 769-776. 17.Holler E, Rogler G, Brenmoehl J, et al. Prognostic significance of NOD2/CARD15 variants in HLA-identical sibling hematopoietic stem cell transplantation: Effect on long term outcome is confirmed in 2 independent cohorts and may be modulated by the type of gastrointestinal decontamination. Blood 2006; 107: 4189-4193. 18.Holler E, Rogler G, Herfarth H, et al. Both donor and recipient NOD2/CARD15 mutations associate with transplant-related mortality and GvHD following allogeneic stem cell transplantation. Blood 2004; 104: 889-894. 19.Granell M, Urbano-Ispizua A, Arostegui JI, et al. Detrimental effect of NOD2/CARD15 polymorphisms in T-cell depleted allogeneic stem cell transplantation. Bone Marrow Transplant 2006; 37: S79; P431. 20.Robien K, Ulrich CM, Bigler J, et al. Methylenetetrahydrofolate reductase genotype affects risk of relapse after hematopoietic cell transplantation for chronic myelogenous leukemia. Clinical Cancer Res 2004; 10: 7592-7598. 21.Kalayoglu-Besisik S, Caliskan Y, Sargin D, et al. Methylenetetrahydrofolate reductase C677T polymorphism and toxicity in allogeneic hematopoietic cell transplantation. Transplantation 2003; 76: 1775-1777. 22.Oh H, Loberiza FR, Jr., Zhang MJ, et al. Comparison of graft-versus-host-disease and survival after HLA-identical sibling bone marrow transplantation in ethnic populations. Blood 2005; 105: 1408-1416. 23.Dickinson AM. A Distinct Pattern of Non-HLA Polymorphisms Predicts an Increased Risk for GvHD without Benefit of GvL in HLA Matched Sibling Transplants for Chronic Myeloid Leukemia (CML). BMT Tandem meeting, Keystone, Colorado 2007 (Abstract). 24.Leather HL. Drug interactions in the hematopoietic stem cell transplant (HSCT) recipient: what every transplanter needs to know. Bone Marrow Transplant 2004; 33: 137-152. 25.Kogler G, Middleton PG, Wilke M, et al. Recipient cytokine genotypes for TNF-alpha and
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IL-10 and the minor histocompatibility antigens HY and CD31 codon 125 are not associated with occurrence or severity of acute GVHD in unrelated cord blood transplantation: a retrospective analysis. Transplantation 2002; 74: 1167-1175. 26.Socié G, Loiseau P, Tamouza R, et al. Both genetic and clinical factors predict the development of graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Transplantation 2001; 72: 699-706. 27.Cavet J, Dickinson AM, Norden J, et al. Interferon-gamma and interleukin-6 gene polymorphisms associate with graft-versus-host disease in HLA-matched sibling bone marrow transplantation. Blood 2001; 98: 1594-1600. 28.Cullup H, Dickinson AM, Cavet J, et al. Polymorphisms of interleukin-1alpha constitute independent risk factors for chronic graft-versus-host disease after allogeneic bone marrow transplantation. Br J Haematol 2003; 122: 778-787. 29.MacMillan ML, Radloff GA, DeFor TE, et al. Interleukin-1 genotype and outcome of unrelated donor bone marrow transplantation. Br J Haematol 2003; 121: 597-604. 30.Cullup H, Dickinson AM, Jackson GH, et al. Donor interleukin 1 receptor antagonist genotype associated with acute graft-versus-host disease in human leucocyte antigen-matched sibling allogeneic transplants. Br J Haematol 2001; 113: 807-813. 31.Rocha V, Franco RF, Porcher R, et al. Host defense and inflammatory gene polymorphisms are associated with outcomes after HLA-identical sibling bone marrow transplantation. Blood 2002; 100: 3908-3918. 32.Grainger DJ, Heathcote K, Chiano M, et al. Genetic control of the circulating concentration of transforming growth factor type beta 1. Human Mol Gen 1999; 8: 93-97. 33.Tambur AR, Yaniv I, Stein J, et al. Cytokine gene polymorphism in patients with graftversus-host disease. Transplant Proc 2001; 33: 502-503. 34.Hattori H, Matsuzaki A, Suminoe A, et al. Polymorphisms of transforming growth factorbeta1 and transforming growth factor-beta1 type II receptor genes are associated with acute graft-versus-host disease in children with HLA-matched sibling bone marrow transplantation. Bone Marrow Transplant 2002; 30: 665-671. 35.Mullighan CG, Heatley S, Doherty K, et al. Mannose-binding lectin gene polymorphisms are associated with major infection following allogeneic hemopoietic stem cell transplantation. Blood 2002; 99: 3524-3529.
Multiple Choice Questionnaire To find the correct answer, go to http://www.esh.org/ebmt-handbook2008answers.htm 1. Which cytokine gene polymorphism has most consistently been associated with GvHD? a) TNF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) IL-10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
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c) IL-1 gene family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) TGF-b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Identify genes of the innate immune system a) HA-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) TGF-b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) NOD2/CARD15 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) HLA-A3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. What outcome of the transplant procedure may be influenced by the non-HLA genetics of the patient and donor? a) Transplant related mortality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Incidence and severity of GvHD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Survival . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) All three . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. What are the potential future developments of studying non-HLA genetics? a) Improve donor selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Aid in design of new therapeutic regimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Development of a prognostic risk score for individual patients . . . . . . . . . . . . d) All of a), b) & c) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. In MUD and sibling transplants what are the considerations to be taken into account in studying non-HLA immunogenetics? a) HLA matching and linkage disequilibrium between HLA and cytokine genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Clinical risk factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Population genetics and allele frequency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) All three . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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*
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JACIE accreditation of transplant centres
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1. Introduction JACIE (The Joint Accreditation Committee of ISCT-Europe and EBMT) is a non-profit body established for the purposes of assessment and accreditation in the field of haematopoietic stem cell transplantation (HSCT). The Committee was founded in 1998 by the European Group for Blood and Marrow Transplantation (EBMT) and the International Society for Cellular Therapy (ISCT), the two leading scientific organisations involved with HSCT in Europe (1). JACIE modelled itself on the USbased Foundation for the Accreditation of Cellular Therapy (FACT), established in 1996 by the ISCT and the American Society for Blood and Marrow Transplantation (ASBMT). JACIE actively collaborates with FACT in establishing standards for the provision of quality medical and laboratory practice in HSCT, and the two organisations now issue joint FACT-JACIE standards, which are applicable internationally (2, 3). The primary aim of JACIE is to improve the quality of HSCT in Europe by providing a means whereby transplant centres, HSC collection facilities and processing facilities can demonstrate high quality practice. This is achieved through external inspection of facilities to ensure compliance with the FACT-JACIE standards. An additional and wider aim is to ensure harmonisation between FACT-JACIE standards and other national/international standards, including the EU Tissues & Cells Directive (Directive 2004/23/EC) and the related implementing Commission Directives 2006/17/EC and 2006/86/EC (4–6). JACIE accreditation is voluntary, but provides a means whereby transplant facilities can demonstrate that they are working within a quality system covering all aspects of the transplantation process and thus show compliance with the requirements of insurance companies or national and/or international regulatory authorities. The current organisation of JACIE is shown in Figure 1. The structure ensures wide consultation with over 20 European countries now represented on the Board in addition to nursing, paediatrics and cord-blood representatives. In 2007 an Accreditation Committee was established which reviews all inspection reports and advises the JACIE Board. Accreditation of HSC transplant facilities is through on-line submission of documentation and an on-site visit by a team of trained inspectors. Centres may apply for accreditation as complete programmes comprising clinical programme, collection facility and a processing laboratory or, for example, as a single collection or processing facility which may serve a number of clinical programmes.
2. The FACT-JACIE standards The FACT-JACIE standards cover all aspects of clinical transplant programmes, collection facilities (BM collection and PBPC collection) and processing of HSC, as
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Figure 1: Structure of JACIE JACIE Board Executive Committee
JACIE Office
EBMT - ISCT
Accreditation Committee
JACIE BOARD Officers (JACIE Executive Committee) President; Vice-President; Medical Director; Accreditation Committee Chairman; Executive Officer Sector Representatives NETCORD; Nursing; Paediatric National Representatives Currently: Austria; Belgium; Czech Republic; Denmark; Estonia; Finland; France; Germany; Greece; Hungary; Italy; The Netherlands; Norway; Poland; Slovakia; Slovenia; Spain; Sweden; Switzerland; Turkey; United Kingdom (Elected by National Transplant Societies) JACIE ACCREDITATION COMMITTEE Accreditation Committee Chairman; Medical Director; 1-2 Clinical Representatives; 1-2 Collection Representatives; 1-2 Processing Representatives. (Appointed by the Executive Committee from a pool of experienced inspectors) JACIE OFFICE Executive Officer; Administrative Officer
shown in Table 1. The standards also apply to the use of therapeutic cells (TC) derived from blood or marrow, including donor lymphocytes and MSCs. The standards cover the clinical use of Cord Blood HSC by clinical programmes but not the collection of banking of cord blood, which are covered by the related Netcord-FACT standards and inspected/accredited by FACT-Netcord (7, 8). Within each subsection of the standards are detailed lists of specific requirements; for example the standard on donor evaluation and selection contains over 35 specific items relating to clinical evaluation, laboratory testing, informed consent etc. The current edition of the standards is the 3rd edition, dated 2007. The 4th edition is currently in preparation. 94
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Table 1: Contents of the FACT-JACIE standards (3rd edition 2007) Clinical programme
Collection (BM and PBPC)
Processing
General - including programme organisation and transplant activity
General
General
Facilities - inpatient and outpatient
Facilities – theatre/apheresis facilities
Facilities – laboratory size and organisation
Staffing – qualifications and training – medical, nursing and other staff
Staffing – qualifications and training
Staffing – qualifications and training
Quality Management Programme – including adverse event reporting, audit and outcome review
Quality Management Programme – including adverse event reporting, audit, validation of procedures and equipment, and outcome review
Quality Management Programme – including adverse event reporting, audit, validation of procedures and equipment, and outcome review
Policies and Procedures (SOPs) – requirements for SOPs including format, document control, lists of specific areas to be covered
Policies and Procedures – as left
Policies and Procedures – as left
Donor evaluation, selection and Donor evaluation/care at time testing – allogeneic and of collection autologous
Process Controls – processing techniques, testing, assays, validation
Administration of high dose therapy and infusion of HSC
Labelling-specific requirements Labelling-specific requirements for labelling and documentation for labelling and documentation during and after collection during and after processing and at distribution
Clinical research
Collection procedure (BM or PBPC) – including paediatrics donors
Distribution and Release – review of products prior to release, documentation to be sent with product
Data management – including Storage – conditions for accuracy of recording transplant storage data
Storage – conditions for storage including temperature control, alarm systems
Records – departmental and medical
Transport – within facility and/or to external facility
Receipt and transportation – acceptance, quarantine, transport of fresh/frozen products
Records – including electronic records
Disposal – including consent, documentation
Direct Distribution to Clinical Programme – requirements for record keeping
Records – including electronic records
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The standards are accompanied by a manual which contains the standards together with detailed guidance on the interpretation and measures required to demonstrate compliance. Each standard is followed by specific questions relating to that standard and these questions form the basis of the inspection checklist, which must be completed prior to inspection by the applicant centre and verified by the inspector during the inspection (Figure 2). The complete standards and the accompanying guidance manual are available on the JACIE website, together with other useful information relating to inspection and accreditation (3).
Figure 2: Example from checklist INSPECTION CHECKLIST
APPLICANT
B6.000 D O N O R E V A L U A T I O N , SELECTION AND MANAGEMENT
Y
N
INSPECTOR
N/A
Y
N
N/A
B6.100 Are there donor evaluation procedures in place to protect the safety of the haematopoietic progenitor cell donor D* and recipient? Do these evaluation procedures assess the potential for disease transmission D from the donor to the recipient? Do these evaluation procedures assess the risks to the donor from the D collection procedure? Are donor evaluation and selection test results documented?
D
B6.110 Are there written criteria for donor evaluation and selection?
D
*D: deficiency in compliance if answer is N
2.1. General aspects of the standards Although the standards are very specific in certain areas, e.g. labelling requirements, some essential principles apply throughout. The first of these is a requirement for documentation of policies, procedures, actions, requests and so on. This does not just apply to the need to have written policies and procedures, but extends to all 96
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aspects of transplant activity. For example, the initial diagnosis of a patient must be documented by source material or reports. A request from the clinical unit to the laboratory for issue of cells must be made in writing. A potential donor must not only be properly evaluated for eligibility, but the programme must have clear written criteria for what constitutes an eligible donor and must clearly document whether the donor meets these criteria. The second is a requirement that personnel must not only be appropriately qualified, they must be trained in the procedures they regularly perform and their competency to perform the task after training must be assessed and documented. Similarly there is a requirement for validation of all equipment and procedures. Validation is a term used to describe the activity required to prove that any procedure, process, equipment, material, activity or system actually leads to the expected results. For example a new apheresis machine must be shown to produce the expected results in terms of cell yields. Also important is the requirement for close cooperation and interaction between the different parts of the programme, especially important where a clinical programme may use a distant collection and/or processing facility. Mechanisms for transfer of information e.g. regarding donor assessment and consent, results of collection, request for product issue, adverse events etc must be clearly established and often require transfer of written information. The Quality Management Programme is essential to ensure that all these aspects of the programme run smoothly. 2.2. Quality management (QM) An active quality management programme (QMP) is essential to the FACT-JACIE standards. A QMP is a mechanism to ensure that procedures are being carried out by all staff members in line with agreed standards. In a transplant programme, this ensures that the clinical, collection and laboratory units are all working together to achieve good communication, effective common work practices and increased guarantees for patients. It is a means of rapidly identifying errors or accidents and resolving them so that the possibility of repetition is minimised. It assists in training and clearly identifies the roles and responsibilities of all staff. Once the required level of quality has been achieved, the remaining challenge is to maintain this standard of practice. With a working quality management system in place and adequate resources, the fundamental elements necessary to sustain the programme are continued staff commitment and vigilance. The culture and systems for quality management are well-established in laboratories but are relatively new in clinical units and many programmes have experienced difficulty setting up a QMP to cover the clinical programme and collection facility.
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It is recommended that a programme have a dedicated quality manager, ideally with specific training in QM, not only to develop the QMP to meet the initial requirements for accreditation but to ensure continued adherence to standards. To assist centres in developing a QM programme JACIE has recently published “A practical guide to implementing quality management in a stem cell transplantation (SCT) programme”, a copy of which will be distributed to all EBMT centres, and which will be available via the JACIE website (3). This contains detailed explanation of different aspects of QM together with example scenarios and documents. The QMP must address at a minimum: - Organisational structure - Third party agreements - Process developments and review - Personnel requirements, training and competency - Outcome analysis - Audits - Management of products with positive anti-microbial culture - Detection and reporting or errors, accidents and adverse events - Record review and document control - Validation/qualification of equipment, reagents and procedures - Inventory control - Product tracking - Process controls Within a programme there can be one QM system covering all areas, i.e. clinical, collection and processing, or the laboratory may have a separate system, and other combinations are possible. If there is more than one system then there should be clear interaction between them. 2.3. Policies and procedures There are specific requirements for how policies and procedures should be written, to ensure that all essential aspects are covered. They must be available to all relevant staff at all times. Electronic access is perfectly acceptable but there must be at least one hard copy available at all times and staff must know where to access this. For each section – clinical, collection and processing – there is a list of specific policies and procedures that must be covered. These are shown in Table 2. All SOPs must be reviewed annually. 2.4. Clinical programme, collection facility and processing facility standards The following paragraphs will comments on some particular aspects of the standards, but for full details the reader should refer to the standards and manual. 98
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Table 2: List of required policies and procedures All sections
-
Donor and patient confidentiality Emergency and safety procedures Infection control, biological, chemical and radiological safety Quality Management and improvement Personnel training Competency assessment Errors, accidents and adverse events Outcome analysis Audits Corrective actions Biological product deviations Product tracking Facility maintenance and monitoring Disposal of medical and biohazard waste Disaster response
Clinical programme
-
Donor and patient evaluation, selection and treatment Donor and patient consent Infection prevention and control (clinical unit) Administration of the preparative regimen Administration of HSC Blood product transfusion
Collection facility
-
Donor treatment, screening and consent Management of paediatric donors, if applicable Product collection (BM and/or PBSC) Labelling (including forms and samples) Expiration dates Storage Release and exceptional release Transportation Reagent and supply management Equipment maintenance and monitoring
Processing facility
-
Product receipt Processing and process control Prevention of cross-contamination RBC compatibility testing Processing of ABO incompatible products Cryopreservation and thawing Labelling (including forms and samples) Expiration dates Storage Release and exceptional release Product recall Transportation Reagent and supply management Equipment maintenance and monitoring Environmental control Hygiene and use of personal protective attire
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2.4.1. Definition of a programme The standards define a Clinical Transplantation Programme as an integrated medical team housed in geographically contiguous or proximate space with a single Programme Director and common staff training programs, protocols, and quality management systems. Programmes that include non-contiguous institutions in the same area must share common protocols, staff training procedures, quality management systems, and review of clinical results and evidence of regular interaction. This means that two separate units should not combine together for the purposes of JACIE accreditation unless they are truly working together in everyday practice. Where two separate units are working together, they must be within one hours travelling time to ensure close and regular interaction. 2.4.2. Clinical programme activity To ensure continuing proficiency in a transplant programme, a minimum volume of patients must be treated per year. The current minimum activity requirements for each 12-month period are as follows: - For allogeneic transplantation: at least 10 new patients - For autologous transplantation only: at least 5 new patients - For allogeneic and autologous transplantation there is no minimum requirement for autograft numbers - For combined adult and paediatric programmes, a minimum of 5 new adult patients and 5 new paediatric patients - For programs utilising more than one clinical site for transplantation, a minimum of 5 new patients must be transplanted per site. 2.4.3. Donor evaluation This section covers information, consent, and requirements for evaluation including medical history and testing for infectious disease markers. The medical history for allogeneic donors must include a number of specific items relating to possible transmission of infections and non-infections disease. The use of a standard donor history questionnaire is recommended. Testing for infection disease markers must include: HIV-II, hepatitis B virus, hepatitis C virus and Treponema pallidum. HTLV1 and 2 testing only required as per governmental regulations, which in the EU requires that donors are tested if they are in a risk-group for infection. These tests are required for all donors, allogeneic and autologous. CMV testing is also required for allogeneic donors. The tests must be performed within 30 days prior to collection of cells. If more than one collection is performed the tests will need to be repeated if they are out of this time period. 100
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Other required tests include at least: ABO group and Rh type and pregnancy assessment for all female donors of childbearing potential. For allogeneic donors HLA-A, B, DR typing must be performed by an EFI-accredited laboratory. 2.4.4. Data collection The Programme must collect all the data contained in the EBMT MED-A forms. Prior to inspection the team must submit records of this data for 10 consecutive patients and on the day of inspection the inspectors verify the data against the source material. It is important to have records of the tests used to confirm the original diagnosis as well as of all the peri-transplant data. 2.4.5. Bone marrow collection If a programme that regularly uses bone marrow (BM), this must be collected in a facility that meets the FACT-JACIE standards. This means that the BM collection facility must also be inspected and accredited. The minimum activity required to apply for accreditation is one collection per year, with 3 collections during the 3year period of each accreditation cycle. Even where only one harvest is performed per year, all requirements of the standards must be met, e.g. relevant SOPs must be in place and regularly reviewed, with evidence of continued training of staff and review of procedures. 2.4.6. Apheresis collection The minimum activity requirement for an apheresis collection facility (CF) is 10 HSC collection procedures per year, with 30 during the 3-year period of each accreditation cycle. A CF must have a Director and a Medical Director. These can be the same person, but the Director does not have to be medically qualified. 2.4.7. Processing facility standards A CF must have a Director and a Medical Director. These can be the same person, but the Director does not have to be medically qualified. There must be appropriate and validated assays and test procedures for the evaluation of cellular therapy products. Procedures must be demonstrated to result in acceptable target cell viability and recovery. Where processing includes exposure to the environment this must take place in an environment with specified air quality and cleanliness. This means that the Director should establish through validation and testing what air quality is appropriate. The FACT-JACIE standards do not specify any defined level of air quality. However in EU member states the laboratory must comply with the requirements of the EU Directive on air quality.
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3. The inspection and accreditation process 3.1. Initial application The centre implements measures as described in the JACIE accreditation manual, and then applies for inspection by submitting basic information about the programme/facility and a number of supporting documents including a selfassessment checklist (Figure 2). The application information and checklist must be submitted in English but all other documentation, including Standard Operating Procedures (SOPs) is accepted in the language of the centre. 3.2. Inspection An on-site visit is carried out by a team of trained inspectors, usually one per facility (clinical/collection/processing). Inspectors are medical, scientific or other professional persons working in HSCT, with specific qualifications and experience for inspecting clinical, collection and/or processing facilities (9). Inspectors must attend a JACIEsponsored training course and pass an examination. Where a clinical programme performs adult and paediatric transplants, an adult and a paediatric inspector will attend. Inspectors may also be from another country but should be either native or fluent speakers of the relevant language. In countries where it is not possible to assign an inspector who speaks the language, a local expert is requested to assist with translation of interviews and documents as necessary. An inspection visit generally lasts 1.5 days and involves discussion with staff during their work, review of documents/records and completion of a detailed checklist relating to the standards. The inspectors write a report in English, noting any areas of non-compliance with the standards, which is reviewed by the JACIE Office, the medical director and the accreditation committee. 3.3. Report and correction phase Based on the inspectors’ findings, a summary report is prepared making specific recommendations for corrections and improvements. Between 3 and 12 months is allowed for the centre to correct deficiencies, depending on the amount of work required. The centre must indicate acceptance of the findings and then in due course submit documentary evidence to confirm corrections or amendments. The original inspectors review the documentation. Review by the inspectors rather than by the Accreditation Committee, MD or JACIE office alone is required because of language issues. In some cases a limited revisit may be the best way to show that deficiencies have been remedied. The inspectors confirm to JACIE that all necessary corrections have been made or indicate that there are still outstanding areas for completion. 102
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CHAPTER 4 • JACIE accreditation
3.4. Accreditation The Accreditation Committee reviews all the reports and relevant documentation and if satisfied that all previous deficiencies have been corrected, makes a recommendation to the JACIE Board that the centre be awarded accreditation. If approved, accreditation is awarded, valid for 3 years, subject to an annual report from the centre noting any significant changes in personnel or procedures and including annual activity figures. After 3 years reaccreditation is necessary, which follows exactly the same process as the initial inspection.
4. Outcome of inspections 4.1. Inspections to date To date, over 125 facilities have formally applied for accreditation. Between January 2004 and January 2008, 86 centres were inspected. Of these, the majority (70 centres) applied for accreditation for a combination of clinical, collection and processing facilities. Seven centres applied for clinical and collection only, 5 centres applied for collection and processing only, 3 for apheresis collection only, 29 for the clinical programme only, and 11 for processing only. Almost all were found to be functioning at a high level of excellence. However some deficiencies in compliance were found in all programmes inspected so far, varying from minor non-compliances, e.g. in formatting of SOPs, to major deficiencies e.g. lack of reliable alarm systems for products stored in liquid nitrogen. In almost all cases correction of deficiencies was verified by submission of documentation by the centre. The time taken for correction of deficiencies has varied from 3 to 16 months. In 3 cases a limited reinspection was required to demonstrate correction of deficiencies. At the time of reporting 45 inspected centres have completed correction of all deficiencies and achieved accreditation. These are listed on the JACIE website (3). 4.2. Common deficiencies The most common deficiencies are in documentation, labelling and in the quality management programme (Figure 3). This is consistent with the initial experience of the FACT accreditation programme in the United States (10). 4.2.1. Quality management programme (QMP) Deficiencies in QPM were by far the most common cause of failure of compliance with the standards and, including problems with policies and procedures (SOPs), accounted for 35% (201 of 570) of total cited deficiencies in the initial 2 years of inspections (11). HAEMATOPOIETIC STEM CELL TRANSPLANTATION
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Figure 3: Common Deficiencies at Inspection Facilities Communication Safety Staffing Training QM general SOP format/review Missing SOP(s) Donor selection policy Donor evaluation Donor consent Testing
Lab accreditation Therapy Clinical-other Procedures (collection) Equipment, supplies & reagents Procedures (processing) Labelling Storage Transport Issue/disposal Records 0%
2%
4%
6%
8%
10% 12% 14% 16% 18% 20%
Areas of deficiencies as % of total deficiencies. Based on analysis of 570 deficiencies encountered in 35 inspections
Deficiencies in QMP included: - Problems with the formatting and content of SOPs, e.g. • missing examples of worksheets/forms/labels 104
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CHAPTER 4 • JACIE accreditation
missing references (where relevant) failure to include range of expected results (where relevant) Lack of procedure for documenting deviations from SOPs Lack of regular review process for SOPs No SOPs for critical procedures e.g. bone marrow collection Inadequate document control procedure Lack of validation of equipment/procedures in collection and processing facilities Inadequate audit activity e.g. • no SOP for audit • no written programme for planned audits • no documentation of results of audits • no formal process for disseminating results Inadequate adverse event (AE) reporting/reviewing. Centres often used a hospitalbased incident reporting system, but in many cases it appeared from the number of reported AEs that this was not adequate to meet the needs of the HSCT programme. Often it was not clear that all AEs were reviewed by the programme director and/or that a report was issued to the patient’s physician. • •
-
-
4.2.2. Patient/donor issues Deficiencies in documentation were frequent: - No verification of patient’s initial diagnosis from primary records - No formally documented criteria for defining a suitable donor - Failure to document vaccination history, transfusion history or travel history - Pregnancy not always assessed in female donors of childbearing age 4.2.3. Labelling of components JACIE standards for labelling of components require that essential information including a unique alphanumeric identifier, name of the product and the recipients name are shown. The label must be securely attached, of a size that allows the contents of the pack to be inspected and clearly labelled using indelible ink. In the majority of centres it was found that labelling of products during collection and processing was not compliant with the standards in a variety of ways and this accounted for 15% of all cited deficiencies (83 of 570), e.g. - time of end of collection/processing missing - volume of anticoagulant missing - unique alphanumeric identifier not used at collection. Many centres have a common problem in designing JACIE-compliant labels. This has been addressed by an international collaboration – the Cellular Therapy Coding and Labeling Advisory Group (CTCLAG) – which has designed HPC and TC component labels
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based on the ISBT 128 system for stem cell component identification (7). Their proposals are currently being discussed by a workshop of European Union member states so that a single coding system for cellular therapy products as required by the ED Directive on Tissues and cells (2004/23/EC) can be agreed. A date of March 2008 has been agreed as the date by which to complete its final documents and recommendations to the EU. Further information on the work of the CTCLAG and EU workshop is available on the ICCBBA and CEN websites (12, 13). 4.3. Experience of centres implementing JACIE It was anticipated that implementation of the JACIE standards would pose some difficulties for applicant centres, particularly in relation to establishing a Quality Management system (QMS). It was also anticipated that there would be resource implications in terms of staff time because of the amount of detailed documentation that is required to demonstrate compliance with the standards. An initial survey was designed to assess the difficulties experienced by centres in preparing for accreditation. The results showed that the most difficult part of preparation was implementing the QM system, adverse event reporting system and other documentation. Lack of a culture of QM was cited as an important problem. The extra resources most frequently required were a quality manager (62% centres) and a data manager (35%). Only 19% needed to improve their physical facilities. There is clearly an important need for training of clinical staff (doctors and nurses) in quality management. It is also important for centres to have a designated quality manager who has appropriate experience in quality management systems. A second survey was designed to assess the effect of implementation of JACIE. Centres were asked to complete this survey 6 months after inspection. All responding centres indicated that they had benefited from implementing the JACIE standards. As shown in Table 3, the areas of greatest perceived benefit were in procedure and practices, staff motivation, control of adverse events, and co-ordination between different areas of the programme. Significant benefits were also perceived in patient satisfaction, facilities, patient care and safety and training of new and existing staff. Improvements clearly depend on the level of existing services, so that failure to demonstrate improvement in, for example, facilities or data management may reflect good pre-existing resources. In other areas, e.g. adverse event reporting, the systems for monitoring performance were only set up as part of implementing JACIE, so that it is difficult to monitor improvements without an established baseline for comparison. Indeed implementation of JACIE may have the paradoxical effect of seeming to increase adverse events because these were not previously adequately reported. 106
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CHAPTER 4 • JACIE accreditation
Table 3: Effect of implementation of JACIE Level of improvement
High
Medium
Low
None
N/A
Control of incidents, events and adverse reactions
41%
59%
-
-
-
Data management
23%
64%
9%
-
5%
Internal coordination
50%
32%
9%
-
9%
Patient satisfaction
23%
36%
18%
14%
9%
Staff motivation
55%
45%
-
-
-
Costs
9%
18%
23%
45%
5%
Facilities
-
50%
18%
32%
Patient safety and care
27%
55%
9%
5%
5%
Procedures and practices
50%
45%
5%
-
-
Training of new/existing staff
41%
45%
9%
-
5%
Compliance with health insurers'/social security demands
14%
9%
23%
45%
9%
Government recognition
18%
27%
27%
23%
5%
Level of improvements in programme functioning experienced by centres, as reported in Survey 2. Results are expressed as percentage of responding centres. N/A: this question not answered
Eighty-one percent of the centres reported that implementation of the QM system had highlighted a need for changes in the running of the transplant programme. The most common areas cited were a need for improved co-ordination between the different facilities, i.e. clinical, collection and processing, and a need for more systematic audit and adverse event reporting. Other items were a need to improve patient medical records and to improve donor evaluation procedures. All centres felt that accreditation was worth the effort invested. In addition, with the implementation of the EU Directive on Safety of Tissues and Cells (Directive 2004/23/EC) it is likely that collection and processing facilities will increasingly view compliance with JACIE standards as important in providing evidence that they are complying with the requirements of the directive.
5. Conclusion The JACIE accreditation system is now firmly established in Europe and the experience of centres that have been inspected is that implementation of the JACIE standards has led to significant improvements in different aspects of their transplant
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programmes. JACIE has further assisted with a number of training courses for preparing centres for accreditation and is currently working on a practical guide to quality management in HPC units. JACIE has also developed a close working relationship with other organisations involved in cellular therapy, which will form the basis for a new global approach to harmonisation of standards and accreditation systems worldwide. This collaboration represents an innovative and proactive approach to solving the problems of international exchange of tissues and cells as these relate to the stem cell transplant community.
References 1. Kvalheim G. EBMT and ISHAGE-Europe create a foundation for inspection and accreditation in Europe. Editorial. Cytotherapy 1999; 1: 363-364. 2. FACT: www.factwebsite.org 3. JACIE: www.jacie.org 4. Directive 2004/23/EC of the European Parliament and of the Council of 31 March 2004 on setting standards of quality and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells: http://ec.europa.eu/health/ph_threats/human_substance/legal_tissues_cells_en.htm 5. Commission Directive 2006/17/EC implementing Directive 2004/23/EC of the European Parliament and of the Council as regards certain technical requirements for the donation, procurement and testing of human tissues and cells: http://ec.europa.eu/health/ ph_threats/human_substance/legal_tissues_cells_en.htm 6. Commission Directive 2006/86/EC implementing Directive 2004/23/EC of the European Parliament and of the Council as regards traceability requirements, notification of serious adverse reactions and events, and certain technical requirements for the coding, processing, preservation, storage and distribution of human tissues and cells: http://ec.europa.eu/health/ph_threats/human_substance/legal_tissues_cells_en.htm 7. NETCORD: www.Netcord.org 8. FACT-Netcord: Details of Cord Blood Bank accreditation process: http://www.factwebsite.org/ main.aspx?id=100 9. JACIE: Requirements for inspectors: www.jacie.org/portal/jacie/inspectors 10.Warkentin PI, Nick L, Shpall EJ. FAHCT accreditation: Common deficiencies during on-site inspections. Cytotherapy 2000; 2: 213-220. 11.Samson D, Slaper-Cortenbach I, Pamphilon D, et al. Current status of JACIE accreditation in Europe: a special report from the Joint Accreditation Committee of the ISCT and the EBMT (JACIE). Bone Marrow Transplantation 2007; 39: 133-140. 12.International Cell Therapy Coding and Label Advisory Group: www.iccbba.org 13.CEN workshop: www.cen.eu/cenorm/businessdomains/technicalcommitteesworkshops/workshops/guideline s_cen_workshops.asp 14.AHCTA: www.ahcta.org
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Multiple Choice Questionnaire To find the correct answer, go to http://www.esh.org/ebmt-handbook2008answers.htm 1. Which of the following statements about JACIE accreditation is not correct? a) JACIE accreditation is voluntary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) JACIE accredits Collection and Processing facilities . . . . . . . . . . . . . . . . . . . . . . . . c) JACIE accredits Cord Blood Banks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) An inspection visit usually lasts 1.5 days . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Which of the following statements about required clinical activity is correct? a) Programmes performing allogeneic and autologous transplants must perform 10 transplants of each type every year . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Programmes performing only autologous transplants must transplant 10 patients every year. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Programmes performing allogeneic and autologous transplants must perform 10 allogeneic transplants but there is no minimum requirement for autograft numbers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Programmes performing transplants on 2 sites must transplant 10 patients at each site every year . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Which of the following statements about donor evaluation is correct? a) There must be written criteria for donor eligibility . . . . . . . . . . . . . . . . . . . . . . . . . b) HTLV 1 and 2 testing is mandatory for all donors . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Pregnancy assessment is not required for autologous donors . . . . . . . . . . . . . . d) Infectious disease markers must be tested within 10 days prior to collection of cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Which of the following statements about collection and processing facilities is correct? a) The minimum number of marrow harvests that must be carried out each year by a marrow collection facility is 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) A collection facility must have SOPs in place covering transport of components only if products are transported outside of the facility . . . . . .
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c) All products must be processed in an environment providing Grade A quality air in a grade B background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Every individual donation must be identified with a unique alphanumeric number . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Which of the following statements about JACIE inspection is correct? a) All deficiencies found at inspection must be corrected within 6 months . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Accreditation is valid for 3 years . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Reaccreditation does not necessarily require a reinspection visit . . . . . . . . . d) Accreditation is awarded by the inspectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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NOTES
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*
CHAPTER 5
Sources and procurement of stem cells
J. Larghero, J. Garcia, E. Gluckman
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CHAPTER 5 • Stem cell procurement
1. Introduction The source and procurement of haematopoietic stem cells (HSC) has varied and diversified over time. Nowadays, there are three possibilities: bone marrow (BM) HSC, granulocyte-colony stimulating factors (G-CSF) mobilised peripheral blood HSC and cord blood (CB). Each of them seems to give similar clinical results although the use varies according to age of the donor and the recipient, the indication and the preference of the centres and the donors. Usually BM collection is preferred in children, for adults it can be either BM or PBSC, CB depends on the availability of a suitable unit in banks and the absence of an HLA identical donor.
2. Use of different cell sources for HSCT in Europe An EBMT survey on HSC transplantation (HSCT) activity in 2005 reported that 24,168 first HSCT were performed in Europe by 597 centres (1). Among these transplants, 15,278 were autologous and 8,890 allogeneic. Of the 8,890 allo-HSCT, 4,702 were provided by an HLA identical sibling donor, 514 by an HLA-mismatched family donor, and 57 by a homozygous (syngeneic) twin. An UD was used in 3617 patients (41%). The main indications were leukaemia (6,107 patients), lymphoproliferative diseases (1,520 patients), solid tumours (130 patients) and nonmalignant diseases (1,048 patients). The number of allo-HSCT increased by 20% from 2004 to 2005, whereas numbers of auto-HSCT remained stable (1).
3. Choice of stem cell source Traditionally, HSC were harvested from the iliac crests under general anaesthesia. Thereafter, mobilised PBSC have been increasingly used in both auto- and allo-HSCT. Mobilisation of HSC in sufficient number in peripheral blood can be achieved by the classical administration of growth factor such as G-CSF (allo-HSCT) and/or myelosuppressive chemotherapy (auto-HSCT). In the 1990s, unmanipulated CB cells collected and cryopreserved at birth have been used both in related and unrelated HLA matched and mismatched allogeneic transplants in children, and more recently in adults. In 2005, the stem cell source for auto-HSCT was from peripheral blood in 98% of the 15,278 transplants and from BM in 2%. In the allogeneic setting, BM was used in 2,331 of 8,890 transplants (21%) and PBSC in 74%, confirming the increasing use of this stem cell source. CB was used in 395 allogeneic HSCT compared to 281 in 2004. It has become evident that there are many differences, both quantitative and qualitative, between these cell sources (1). The main differences between cell sources are:
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Bone marrow
Collection under general anaesthesia Limited number of haematopoietic stem cells Median number of nucleated cells: 2 x 108/kg Median number of CD34+ cells: 2.8 x 106/kg Median number of T-cells: 2.2 x 107/kg
G-CSF mobilised PBSC
Collection easy No requirement for general anaesthesia Side effects of G-CSF High number of cells Median number of nucleated cells: 9 x 108/kg Median number of CD34+ cells: 7 x 106/kg Median number of T-cells: 27 x 107/kg
Cord blood
Collection easy and harmless Immediate availability of cryopreserved units and lower risk of transmissible diseases Acceptable partial HLA mismatches Number of cells is the limiting factor Median number of nucleated cells: 0.3 x 108/kg Median number of CD34+ cells: 0.2 x 106/kg Median number of T-cells: 0.4 x 107/kg
4. Donor work-up (2–5) (Tables 1–3) 4.1. Donor risk from the procedure The risks for the healthy donor must be minimised. A careful inquiry before donation must be performed. It is recommended that two separate physicians examine the donor prior to the procedure. Detailed information must be given and signed consent must be obtained from the donor and also from both parents in the case of donors under the age of legal consent. In some European countries, when the donor is <16 years old, the consent must be signed in front of a judge and both the family and the child must be seen by a committee of 3 independent experts. The presence of any risk from general anaesthesia for BM aspiration or from cardiac disease for PBSC collection is an absolute contraindication to the donation. The situation is of course completely different for CB, which represents a harmless and easily accessible source of haematopoietic stem cells without any risk to donors.
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CHAPTER 5 • Stem cell procurement
Table 1: Potential transmission of donor diseases to recipients
Infectious diseases
Transmission of
Donor disease
Viruses
HIV / Hepatitis B and C / HTLV-1 / CMV / EBV / Parvovirus B19 / West Nile virus
Bacteria
Contaminants(a)/ Brucellosis
Parasites
Toxoplasmosis / Malaria / Leishmania / Babesia
Fungi
Candida / Aspergillus
Prions
Creutzfeld-Jakob disease(b)
Congenital disorders
Enzyme deficiencies
Gaucher’s disease
Haemoglobinopathies
Thalassemia / Sickle cell anaemia
Acquired disorders
Autoimmune diseases
Myasthenia gravis / Atopy / Lupus erythematosus / Thyrotoxicosis / Diabetes mellitus type I / Sarcoidosis / Coeliac disease / Autoimmune thrombocytopenia
Haematological malignancies
AML / CML / T-cell lymphoma
Non-haematological malignancies (c)
Small-cell lung cancer from renal transplantation / Glioblastoma multiforme from liver transplantation
(a) Contamination rates of 1 in 3000 units in platelet concentrates. No information available on stem cell grafts. (b) No known cases in HSCT and blood transfusions. (c) Not reported in HSCT
Table 2: Recommendations for donor work-up Test
WMDA
NMDP (a)
JACIE (b)
Proposal
Medical history
Yes
Infectious (including risk for infections), pregnancy, blood donation history
Vaccination, travel, blood transfusion history, questions to identify persons at high risk of trasmitting infectious, inherited, haematological or immunological disease
Family history, travel, vaccination, smoking, blood transfusion and donation, infectious, pregnancy, aller-gy, autoimmune, vaccination and tumour history
Physical examination
Yes
Yes
Yes
Particular attention to cardiovascular, bleeding and malignant diseases
ECG
Yes
Yes
Not specified
Yes
Chest X-ray
Yes
Yes
Not specified
Yes
continue
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Test
WMDA
NMDP (a)
Blood counts
Full blood count Complete blood count and differential w. blood film
Blood group
Yes with antibody screen
Coagulation screen
JACIE (b)
Proposal
Not specified
Complete blood count, manual differential on blood film
Yes
ABO group and Rh type ABO Rh type and appropriate red cell compatibility with the recipient No No
ABO group Rh type and appropriate red cell compatibility with the recipient PT, PTT, fibrinogen
HLA-typing
Yes
Yes
Yes
A, B, C, DRB1, DQB1
Biochemical profile
Urea, electrolytes, creatinine, liver function tests, blood sugar
Urea, electrolytes, creatinine, bilirubin, serum protein electrophoresis, Hb S for PBSC donors only
Not specified in detail
Urea, electrolytes, creatinine, liver function tests (bilirubin, AST, ALT, AP, g-GT), LDH, blood sugar, protein electrophoresis, urine analysis
Infectious disease markers (within 30 days prior to collection)
Syphilis, HBsAg, HIV Ag + Ab, HB core Ag, HCV, HTLV-1, HSV, CMV, VZV, EBV serology
HIV 1 and 2, HBV, HCV, HTLV 1 and 2, Treponema pallidum (syphilis), CMV
HIV 1 and 2, HBV, HCV, HTLV 1 and 2, Treponema pallidum and CMV
HIV 1 and 2, HBV (HBs Ag, HBsAb, and HBcAb), HCV (HCVAb), HTLV type II, Treponema pallidum, CMV (IgG, IgM), Toxoplasmosis (IgG, IgM) and EBV
Yes
Yes
Gynaecological visit + pregnancy test + physical breast exam
Pregnancy test Yes (when indicated)
Other proposals: - Check for malignant diseases in donors >55 yrs (applies to related HSCT) Æ PSA in males, physical examination, occult blood in stool. BM aspiration if medical history or tests are abnormal. CT-chest scans in case of a long smoking history. - Congenital disorders Æ Testing for congenital disorders of planned recipient within family donors; testing for other congenital disorders of the family.
(a) 18th Edition Standards/ September 2002. (b) www.jacie.org
Table 3: Donor selection in case of donors with abnormal findings Family Donors Abnormal finding
Absolute contraindications
Relative contraindications
Specific consideration
Infectious
HIV HTLV-1
HBV, HCV, malaria within past 3 yrs, parvovirus B19
EBV, CMV, toxoplasmosis,
Congenital
Thalassaemia major Thalassaemia minor Combined immune deficiency Gaucher’s disease Congenital disease with severely reduced life expectancy
Malignancies
Any malignancy except in situ cancer
Targeted CB should be tested for congenital disease of planned recipient
Skin cancer removed in toto
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CHAPTER 5 • Stem cell procurement
Family Donors Abnormal finding
Absolute contraindications
Pregnancy
Relative contraindications
Specific consideration
BM donation and G-CSF stimulated or unstimulated apheresis
Unrelated Donors (a) Infectious
As for blood donation
Congenital
As for blood donation
Malignancies
Any malignancy except in situ cancer
Pregnancy
Any donation
Parvovirus B19, if known after collection: Gram-positive or Gramnegative bacterial graft infection
EBV, CMV, toxoplasmosis
Skin cancer removed in toto
Unrelated Cord Blood (b) Infectious
As for blood donation. Gram-positive or Gramnegative contamination
Congenital
As for blood donation Exclude, if congenital diseases known in family
Malignancies
Any, in child
EBV, CMV, toxoplasmosis
(a) Not enough information is available for West Nile virus. Contamination of stem cell graft with epidermal bacteria might be a relative contraindication
4.2. Recipient risk from a particular donor (2) Potentially transmittable diseases from donors include infections, congenital disorders, and acquired diseases such as malignancies or autoimmune diseases.
5. Comparison of BMT and PBSCT (5–12) In 1995, 3 pivotal studies demonstrated the safety and feasibility of using G-CSF mobilised PB allografts (Table 4). Patients experienced prompt engraftment with an incidence of GvHD similar to that of BM recipients (Table 5). In addition, no serious short-term complications of G-CSF mobilised PB harvesting were observed in the donors. Direct comparison of PB and BM in allogeneic sibling donor transplantation has been reported in at least 8 randomised trials. Most of them did not show a benefit in overall
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Table 4: Collection protocols for PBSC allografts in 4 randomised trials Study
G-CSF dose µg/kg
Days G-CSF
No of aphereses
106/kg 106/kg 106/kg Ttarget actual cell dose CD34+ dose CD34+ dose
Schmitz Blood 108: 4288, 2006
10
4
1 (1-3)
4
5.8 (1.5-68)
300 (16-2123)
Couban BBMT 10: 624, 2004
5
4
2 (1-2)
2.5
6.4 (0.7-32)
370 (120-3080)
Bensinger 16 NEJM 344: 175, 2001
5
1 (1-4)
5
7.3 (1-30)
279 (143-788)
Blaise JCO 18: 537, 2000
5
2 (1-3)
4
6.6 (1.5-19)
356 (131-754)
10
Table 5: Neutrophil and platelet recovery and incidence of aGvHD and cGvHD after allo-PBSCT compared to allo-BMT in different randomised studies Reference
Source
n
Neutrophils (median days)
Platelets (median days)
aGvHD (%)
Extensive cGvHD (%)
Bensinger
BM PBSC
91 81
21 16
19 13
57 64
35 46 p=ns
Blaise
BM PBSC
52 48
21 15
21 13
42 44
7.5 31 p=0.03
Schmitz
BM PBSC
166 163
15 12
20 15
37 50 p=0.01
11.5 25 p=0.0009
Table 6: Relapse incidence (RI) and survival after allo-PBSCT compared to alloBMT in different randomised studies Reference
Source
n
RI
Survival
Bensinger
BM PBSC
91 81
25% 14% p=0.04
54% 66% p=0.06
Blaise
BM PBSC
52 48
ns
65% 67% p=ns
BM PBSC
166 163
ns
ns
Schmitz
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- Early status: 72 vs. 75% p=ns - Advanced: 33 vs. 57% p=0.04
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survival (OS). The incidence of aGvHD was the same in all but one of the studies, but an increase (statistically significant or a trend) in the incidence of overall and extensive cGvHD was demonstrated in recipients of PB allografts (Table 6). The magnitude of this observation and its effect on relapse, OS and recipient quality of life is less clear. In unrelated transplant recipients, matched cohort comparisons of unrelated (UD) BMT and PBSCT reported faster haematological recovery among PB recipients with no difference in either acute or chronic GvHD. While results of randomised studies are pending, the use of PB allografts in UD HSCT has varied among transplant centres and countries. Some unrelated Donor Registries have permitted the collection of allografts from PB whereas others have not. Transplant centres may request a PB or a BM graft but the collection centre and wishes of the donor also determine which product is ultimately collected. Because of the absence of definitive data comparing both sources of cells, there is no indication to prefer either source of cells except perhaps in patients with advanced disease where chronic GvH and subsequent GvL might decrease relapse and improve OS or, in a situation where a high number of cells is necessary for engraftment, for example after non-myeloablative conditioning or if TCD is planned for a HLA mismatched transplant.
6. Cord blood collection and banking The first allogeneic CB transplantation (CBT) was successfully performed in 1988 in a child with Fanconi’s anaemia; the CB donor was his HLA identical sister. Fifteen years later, this patient is doing well with full donor haematopoietic and lymphoid reconstitution. This first success showed that a single CB unit contained enough HSC to reconstitute definitely the host lympho-haematopoietic compartment, that a CB unit could be collected at birth without any harm to the new-born infant and finally that CB HSC could be cryopreserved and transplanted in a myeloablated host after thawing without losing their repopulating abilities. CB has many theoretical advantages due to the immaturity of newborn cells. HSC from CB are enriched in primitive haematopoietic stem cells, which are able to produce in vivo long-term repopulating SC. The properties of CB cells should compensate for the relatively low number of cells contained in a single CB unit and, through rapid expansion reconstitute myeloablated patients. In spite of the capacity of CB cell expansion, clinical results showed that haematopoietic recovery is delayed after CBT engraftment but improves if a higher number of nucleated and CD34+ cells are infused. In the long term, a higher frequency of reconstitution of early and committed haematopoietic progenitors was observed in children receiving a CBT compared to BMT suggesting that the delay in engraftment may reflect the difficulty
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of CB progenitors to reprogram themselves toward differentiation. The second advantage of CBT relates to the immaturity of the immune system at birth. This property should decrease the alloreactive potential of lymphocytes and as a consequence reduce the incidence and severity of GvHD after an HLA matched or mismatched transplant. These properties should lead to less stringent criteria for HLA donor recipient selection (Table 7). Recent studies have shown that the cell dose is the most important factor for donor selection: a minimum cell dose of 3 x 107 nucleated cells/kg is required for successful engraftment. More recently the use of two CB units seems to improve the engraftment rate.
Table 7: Advantages and disadvantages in the search and identification process of BM and CB unrelated donor
Information on A+B+DRB1(DNA) type Median search time Donors identified but not available Rare haplotypes represented (a) Major limiting factors to graft acquisition Ease of arranging date of cell infusion Potential for second HSC graft or DLI Potential - for viral transmission - for congenital diseases Risk to donor
Bone marrow
Cord blood
16–56% 3–6 months 30% 2% HLA match Difficult Yes Yes No Yes
50–80% <1month < 1% 29% Cell dose Easy No from the same donor No Yes No
(a) Data of the ethnic distribution of CB (n=4577) and BM donors registered (n=68487) in the British Bone marrow Registry (Cristina Navarrete, personal communication)
7. Cord blood banking for unrelated CBT In order to organise the development of CB banks, Netcord, an International Foundation promoting high quality cord blood banking, currently includes more than twenty experienced large CB banks in the USA, Europe, Japan and Australia. Netcord has developed a detailed set of standards for CB banking, the third edition of which has been recently released. These include respective national and international regulatory aspects and, with the collaboration of the Foundation for Accreditation of Hemopoietic Cell Therapy (FAHCT) (USA), an international accreditation program was started in 2002, with more than 15 accredited CBBs up to date. Furthermore, a joint allocation system employing most recent Internet technology has been implemented to facilitate the rapid allocation of CB units 120
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according to histocompatibility and number of nucleated cells within an average time of 48 hours. The number of CB transplants from UD has increased dramatically, and more than 5,800 patients have undergone CBT from UD thus far (www.netcord.org). 7.1. Practical aspects of cord blood banking 7.1.1 Informed consent CB is a discarded product that, up to some years ago, could be used without permission; however, today it is considered a transplantable product (cells, tissue or pharmaceutical drug according to the regulations in different countries) and informed consent must be obtained for its donation and for carrying out tests on the mother and the CB. Careful questioning about medical history allows the opportunity of excluding donors who have a high risk of transmitting any infectious or genetic diseases. The families must be informed about the genetic and infectious tests performed. 7.1.2. Collection techniques Two techniques are used to collect CB: - One is collection in the delivery room while the placenta is still in utero, and - The other is collection of the CB in an adjacent room after the delivery. In the first instance, the collection can be done in the delivery room by an obstetrician or a midwife. The advantage of this procedure is that the volume of cells collected is usually higher if the cord is clamped early and the collection is begun immediately; however, this can disrupt the normal process of delivery and is not always feasible. The collection of CB after delivery is easier and it can be performed by designated personnel; however, fewer cells may be collected and there may be an increase in the risk of bacterial contamination or clotting. 7.1.3. Infectious disease testing Tests for syphilis and for viral infections (including those for HIV, HBV and HCV, and CMV) are performed on the mother’s blood. In some countries, the mother is also tested for HTLV-I/II and toxoplasmosis or other transmissible diseases according to local prevalence. Most often, virology tests are not performed immediately; rather, a separate frozen aliquot is kept so that these tests can be performed before a transplant procedure. CB should be quarantined until a confirmatory test is performed on the mother 3–6 months after delivery. This should decrease the risk of virus transmission considerably. These risk estimates may be higher in CB donors because, unlike regular whole blood
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donors, CB donors are recruited only once. Bacterial infection is also a major issue, but it seems that the incidence of bacterial contamination diminishes with the expertise of the staff in charge of the collection. In all cases, a bacterial culture for anaerobic and aerobic bacteria must be performed. 7.1.4. Genetic disease testing The decision to perform tests for genetic diseases should be directed by the family medical history, its ethnic background and the follow-up of the donor. Tests on cord blood are expensive and there is no real consensus on the type and number of tests that should be performed. There are also some concerns about notifying the family of the test results. 7.1.5. HLA typing HLA typing is performed on an aliquot of CB. In collaboration with the Eurobank project, Eurocord/Netcord collects DNA from the donor and recipient to perform molecular typing for HLA-C, -DQB1, -DPB1, some HLA-A and -B antigens, and other markers. Some banks type routinely the mother for HLA in order to have information on the haplotype and to control the accuracy of CB typing. 7.1.6. Cell processing Because of the small volume of CB collected, which usually ranges from 40–150 mL, there is some concern that any attempt at cell manipulation and concentration may result in a considerable cell loss that could impair engraftment. Many banks, specially the larger ones, use advanced GMP procedures for CB volume reduction, freezing and storage, which have minimised cell loss. The thawing technique is well established and aims at removing red residual cells and cryoprotectants. Evaluation of HSC content is very important. Several studies have shown that the number of nucleated cells infused correlates with engraftment. A correlation has also been demonstrated between placental weight, time of clamping, speed of processing, volume collected, and progenitor cell content. Quantification of the cellular content of cord blood is not always easy. Most studies refer to nucleated or mononuclear cells infused per kilogram of body weight before and after thawing of cells. Enumeration of CD34+ cells by flow cytometric analysis is performed routinely by most laboratories, but these results are not always comparable. Others count the number of CFU-GM in clonogenic assays. There is a large variation of methodology among laboratories, which explains why quantification has been a problem. In the European study, a correlation was found between the number of nucleated cells infused and engraftment, but this correlation was not found when the number of CD34+ cells and CFU-GM were evaluated.
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7.1.7. Ethical and legal aspects CB can be collected for infusion into an unrelated or a related recipient and, more hypothetically, for autologous use. For unrelated CBT, the mother must be aware that the donation is anonymous and free, and that there will be no guarantee that it will be possible to retrieve the cord blood if it is needed later for family or autologous use.
8. Comparison of UD CBT and UD BMT 8.1. Children with acute leukaemia In 2001, a comparison between UD CBT and UD BMT was carried out by Eurocord and EBMT in 515 children with AL, transplanted either with an UD CBT (n=99) or an UD BMT (n=416) (13). Recipients of CB were younger and had more pre-transplant adverse disease factors. Most of the BM donors were HLA matched or had a maximum of 1 HLA difference while most of the CB donors had 2 to 3 HLA differences. The median number of nucleated cells infused was 4 x 108/kg in UD BMT and 0.38 x 108/kg in UD CBT. In unrelated BMT, 262 children received an unmanipulated BMT and 180 a TCD BMT using Campath-1M as the most frequent mean of TCD. The median followup was 29 months. During the first 100 days after HSCT, while unmanipulated and TCD BMT did not differ in haematopoietic recovery and TRM, the main findings that emerged from these adjusted comparisons were the poor results of CBT in relation to neutrophil and platelet recovery and early TRM. In contrast CBT and TCD BMT gave less aGvHD than the unmanipulated BMT group. Finally the unmanipulated BMT and CBT groups had less leukaemic relapse than the T-cell depleted group. In the long term, the unmanipulated BMT group had more cGvHD than the TCD BMT and the CBT groups. Furthermore, while the outcome of the 3 groups were comparable in terms of long term relapse, mortality after day 100 was increased in the TCD BMT group, mainly because of the occurrence during the first 100 days after BMT of early relapse, GvHD and lack of engraftment. In summary, the main differences in adjusted outcomes between the three unrelated transplant groups appeared in the first 100 days post transplant but without giving an advantage to any group. Indeed, the delay of engraftment and increased TRM observed after UD CBT must be balanced against the higher risk of aGvHD after unmanipulated UD BMT and to the higher risk of relapse after TCD. In contrast, after day 100 posttransplant, the 3 groups achieved similar results in terms of relapse but cGvHD occurred more frequently with unmanipulated UD BMT and death with TCD UD BMT. Recently, these results have been reinforced by the report of CIBMTR and New York cord blood bank, comparing the outcomes of 503 children with acute leukaemia transplanted with UD mismatched CB with 282 UD BM recipients (of which 116 were
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8/8 antigen HLA matched) (14). While they showed that TRM was increased in children receiving a low CB cell dose (less than 3 x 107/kg) and one HLA-mismatched CB, or two HLA-mismatched CBT independently of cell dose, this study also demonstrated that leukaemia-free survival (LFS) was not statistically different from one or two HLA-mismatched CBT compared with a HLA-allele-matched UD BMT. These results justify the simultaneous search of UD CB donors and UD BM in children with AL. The decision to perform CBT will be based on the cell content of the graft the number of HLA disparities and the urgency of the transplant. 8.2. Adults with acute leukaemia (14, 15) In 2004, two groups reported the transplant outcomes of adults with leukaemia transplanted with either UD CB or UD BM (15, 16). In both studies, CB recipients were younger and had more advanced disease at the time of transplantation. In the study comparing CB with HLA-matched UD BM, multivariate analysis showed lower acute GvHD ≥ grade II after CB transplantation but delayed neutrophil recovery. The 2-yrs cumulative incidence of cGvHD, TRM, leukaemia relapse, and the 2-yrs probability of OS and LFS were not significantly different. The second report compared transplant outcomes between patients receiving UD CB (one or two mismatch) with patients grafted with UD BM (HLA-matched or one mismatch). UD CB and mismatched BM transplantation were associated with a higher risk of cGvHD and aGvHD, respectively. Interestingly, the incidence of TRM, treatment failure, and overall mortality were similar in patients who received mismatched BM or mismatched CB transplants. These data suggest that, despite increased HLA disparity, CB from UD offers comparable results to matched UD BM in adults with AL leading to the conclusion that the donor search process for BM and CB from UD should be started simultaneously in adults especially in patients with AL where the time factor is very important.
9. Algorithm for donor search High resolution HLA typing of patient and family No donor: Alternative donor search
124
BM donor registries
Cord blood banks
HLA identical A, B, C, DR, DQ 10 /10 or 9/10
Cell dose >2 x 108/kg 1 or 2 HLA mismatches A, B, DR
Transplant
Transplant
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CHAPTER 5 • Stem cell procurement
10. HSC and regenerative medicine The need for an alternative source of pluripotent stem cells for tissue engineering that does not require the use of human embryos or therapeutic cloning has resulted in increased interest in the use of adult stem cells, although they are likely more limited in potential. However, stem cells (haematopoietic, mesenchymal, endothelial, etc.) isolated from either BM or CB represent an ethically uncontroversial source of cells for clinical applications. The experimental and first clinical data reported with such stem cells herald new applications in cellular therapy and tissue regeneration, ranging from neurodegenerative, muscular, brain or bone diseases to myocardial infarction, liver cirrhosis, or vascular disorders.
References 1. Gratwohl A, Baldomero H, Frauendorfer K, et al. Results of the EBMT activity survey 2005 on haematopoietic stem cell transplantation: Focus on increasing use of unrelated donors. Bone Marrow Transplant 2007; 39: 71-87. 2. Niederwieser D, Gentilini C, Hegenbart U, et al. Transmission of donor illness by stem cell transplantation: Should screening be different in older donors? Bone Marrow Transplant 2004; 34: 657-665. 3. Cleaver SA, Warren P, Kern M, et al. Donor work-up and transport of bone marrowrecommendations and requirements for a standardized practice throughout the world from the Donor Registries and Quality Assurance Working Groups of the World Marrow Donor Association (WMDA). Bone Marrow Transplant 1997; 20: 621-629. 4. Favre G, Beksac M, Bacigalupo A, et al. for the European Group for Blood and Marrow Transplantation (EBMT). Differences between graft product and donor side effects following bone marrow or stem cell donation. Bone Marrow Transplant 2003; 32: 873-880. 5. Couban S, Barnett M. The source of cells for allografting. Biol Blood Marrow Transplant 2003; 9: 669-673. 6. Champlin RE, Schmitz N, Horowitz MM, et al. Blood stem cells compared with bone marrow as a source of hematopoietic cells for allogeneic transplantation. Blood 2000; 95: 37023709. 7. Bensinger WI, Martin PJ, Storer B, et al. Transplantation of bone marrow as compared with peripheral blood from HLA identical relatives in patients with hematologic cancers. N Engl J Med 2001; 344: 175-181. 8. Blaise D, Kuentz M, Fournier C, et al. Randomized trial of bone marrow versus lenogastrimprimed blood cell allogeneic transplantation in patients with early stage leukemia: A report from the Société Française de Greffe de Moelle. J Clin Oncol 2000; 18: 537-546. 9. Gorin NC, Labopin M, Rocha V, et al. for the Acute Leukemia Working Party (ALWP) of the European Cooperative group for Blood and Marrow Transplantation (EBMT). Marrow versus peripheral blood for geno-identical allogeneic stem cell transplantation in acute myelocytic leukemia: Influence of dose and stem cell source shows better outcome with rich marrow. Blood 2003; 102: 3043-3051.
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10.Ringden O, Remberger M, Runde V. Peripheral blood stem cell transplantation from unrelated donors: A comparison with marrow transplantation. Blood 1999; 94: 455-464. 11.Remberger M, Ringden O, Blau IW, et al. No difference in graft versus host disease, relapse and survival comparing peripheral blood stem cells to bone marrow using unrelated donors. Blood 2001; 98: 1739-1745. 12.Ringden O, Labopin M, Bacigalupo A, et al. Transplantation of peripheral blood stem cells as compared with bone marrow from HLA identical siblings in adult patients with acute myeloid leukemia and acute lymphoblastic leukemia. J Clin Oncol 2002; 20: 4655-4664. 13.Rocha V, Cornish J, Sievers EL, et al. Comparison of outcome of unrelated bone marrow and umbilical cord blood transplants in children with acute leukemia. Blood 2001; 97: 2962-2971. 14.Eapen M, Rubinstein P, Zhang MJ, et al. Outcomes of transplantation of unrelated donor umbilical cord blood and bone marrow in children with acute leukaemia: A comparison study. Lancet 2007; 369: 1947-1954. 15.Laughlin MJ, Eapen M, Rubinstein P, et al. Outcomes after transplantation of cord blood or bone marrow from unrelated donors in adults with leukaemia. N Engl J Med 2004; 351: 2265-2275. 16.Rocha V, Labopin M, Sanz G, et al. Transplants of umbilical-cord blood or bone marrow from unrelated donors in adults with acute leukaemia. N Engl J Med 2004; 351: 22762285.
Mutiple Choice Questionnaire To find the correct answer, go to http://www.esh.org/ebmt-handbook2008answers.htm 1. Compared to allogeneic bone marrow cells, peripheral blood HSC give: a) Less GvHD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Accelerated neutrophil recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Higher relapse rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Better overall survival . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. The best criterion for cord blood search is: a) Number of nucleated cell dose >3 x 107/kg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) ABO compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) HLA identity determined by allele typing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Donor sex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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3. Quality of unrelated matched HSCT collection relies on which of the following criteria? a) Total volume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Number of CD34 cells/kg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Number of viable cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Number of CFU-GM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Which of the following viral infections in the unrelated donor does contraindicate the use of the donor? a) HIV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) CMV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Hepatitis B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Hepatitis C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. The main complication of collection of PBSC is: a) Hypotension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Bleeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Flu like symptoms due to G-CSF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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*
CHAPTER 6
Principles of conditioning
A. Gratwohl
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CHAPTER 6 • Principles of conditioning
1. Introduction Conditioning plays a central part in HSCT. It is the source of the inherent dilemma in HSCT: how to get rid of the disease but without toxicity. Conditioning is a key cause of early mortality but holds out the prospect of long-term disease control or even cure. Errors in its application can have serious, even immediately fatal consequences. Quality management issues (see below) therefore have a central role in all aspects of conditioning. It is of importance to be familiar with all aspects of conditioning. The term “conditioning” in HSCT means to “condition”, e.g. to prepare the patient for its transplant. Conditioning is given with three main objectives: "creation of space", immunosuppression and disease eradication. 1.1. Creation of space This is a somewhat controversial concept, which originated from the belief that immature progenitor cells occupy defined niches within the marrow stroma in order to obtain the necessary support for proliferation and differentiation. According to this theory, existing host stem cell cells must be eradicated in order for donor stem cells to obtain access to these niches and for engraftment to occur. Experimental and clinical data are controversial about the need to create space. There are indications that engraftment after RIC HSCT is more rapid in patients with an “empty” bone marrow compared to a “full” bone marrow. 1.2. Immunosuppression to prevent a host-versus-graft reaction Immunosuppression is required to prevent rejection of the incoming donor cells by host immune cells. This is clearly not required in autologous or syngeneic HSCT. The need for immunosuppression increases with the increase of disparity in major histocompatibility antigens (HLA). The risk of rejection is increased in situations where the recipient has been "pre-sensitised" against minor histocompatibility antigens, e.g. by the administration of multiple blood products prior to HSCT. Rejection is also increased in TCD HSCT. Vice versa, graft acceptance is increased by high stem cell dose and high T-cell dose. 1.3. Disease eradication The key goal of the conditioning regimen is long-term disease control. This is the main objective for patients with malignancies, but it is also of vital importance in diseases characterised by hyperplastic marrows, e.g. thalassaemia. Partial engraftment may be sufficient in situations where only a "specific product" is required, e.g. Bcells in some immunodeficiency states or when an enzyme produced by haematopoietic cells is required, e.g. in mitochondrial neurogastrointestinal encephalopathy.
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2. Historical perspective In order to understand the diversity and multitude of today's conditioning regimens, they have to be seen in their historical context. Bone marrow transplantation was initially investigated as a tool to protect survivors of accidental high dose irradiation due to nuclear accident or atomic bomb exposure. At the same time, it was realised that TBI could be used as a tool to eliminate a leukaemic haematopoietic system without causing irreversible damage to other organs, similar to surgical removal of a diseased organ in solid organ transplantation. “Condition” meant the preparation of the recipient to accept a new organ in place of the diseased and eradicated haematopoietic system. TBI proved to be sufficient for engraftment but insufficient for long-term disease control when used on its own. The addition of cyclophosphamide (Cy) to TBI and transplantation at an early stage of the disease were the main elements for successful BMT in the late seventies. At that time attempts were already being made to replace TBI with “radiomimetic” drugs or high dose “leukaemia specific” chemotherapy, using drugs including Cy, busulfan (Bu), etoposide (VP16), cytosine arabinoside (ARA-C), carmustine (BCNU) and melphalan (MEL). The combinations Cy/TBI, Bu/Cy alone or BACT (BCNU, ARA-C, VP16 and 6TG) were the main conditioning regimens for HSCT at this time. In the 1980’s, emphasis was on dose intensification and exploring alternatives to Cy in combination with TBI. New conditioning regimens were designed with the aim of reducing the risk of relapse and rejection. A number of studies defined maximum tolerable doses for single drugs in combination with TBI. Dose escalation studies suggested that up to 60 mg/kg of VP16, 110-180 mg/m2 of MEL and 36 g/m2 of ARA-C could be combined with TBI. Dose limiting toxicities were interstitial pneumonitis (IP) for TBI, stomatitis and veno-occlusive disease (VOD) for MEL/TBI and VP16/TBI, and CNS and skin toxicity for ARA-C/TBI. Several centres evaluated the use of TBI plus more than one drug, but no consensus was reached regarding the maximal tolerated doses. None of these regimens made any significant improvements to clinical outcome. Any reduction in relapse or rejection risk was usually accompanied by an increase in TRM. OS and DFS remained unchanged or worsened. Similarly, no study with “leukaemia specific agents” such as ARA-C or VP16, showed superiority to Cy, a drug that is not used in the conventional treatment of leukaemia. So far, no single study has shown any conditioning regimen to provide better long-term survival than Cy/TBI or Bu/Cy. In the 1990’s the emphasis switched to concepts focused on reducing TRM and morbidity with improvements in QoL. Better understanding of GvL effects, the recognition of the capacity of DLI to shift the balance between donor and recipient in a predictable way and preclinical experiments which defined the minimal requirements for stable engraftment, led to the introduction of the concept: 130
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“reduced intensity conditioning”. Most of the conditioning regimens (standard, intensified or reduced) were developed as phase I/II trials and then adopted by individual institutions without formal phase III studies. There are a few exceptions. Standard conditioning with Cy/TBI was compared with Bu/Cy in several studies in haematological malignancies. There were substantial differences in toxicities (more VOD, more permanent alopecia with Bu/Cy) but little differences in long-term survival. In ALL there is probably an advantage at long term with TBI containing regimens. Current strategies focus on a new concept. Until recently, most patients were given the same conditioning regimen. Today, it is believed that patients with high risks for TRM and low disease risk should receive a different conditioning regimen from patients with low risk for TRM and high-risk disease. The approach should integrate conditioning, graft product and GvHD prophylaxis and treatment from the very beginning (see Tables 1 and 2).
3. RIC HSCT and the concept of “immunoablation versus myeloablation” The concept of RIC HSCT is based on the idea of circumventing the high morbidity and mortality associated with standard conditioning in patients with advanced age or comorbidities. It evolved from preclinical studies which defined the minimal requirements for engraftment. They also made use of the potential of DLI to shift mixed chimerism to full chimerism. The goal of RIC HSCT is not tumour eradication or destruction of host haematopoiesis by cytotoxic therapy but via immune mediated effects. The graft-versus-tumour potential of the approach is based on several components: initial conditioning, graft composition, prevention of post-transplant rejection and use of DLI in case of incomplete chimerism at specified time points. Many of these regimens include double immunosuppression with both CsA and mycophenolate mofetil (MMF) post-transplant. Initially, emphasis was given to the idea that “immunoablative” drugs, e.g. fludarabine or anti-T-cell antibodies, were more important than “myeloablative” drugs, such as MEL or Bu. This idea has been largely abandoned. Most drugs as well as TBI have profound effects on lymphoid as well as myeloid elements and the total doses of drugs and TBI appear to have more influence on toxic side effects than on immunosuppression. Isolated antilymphoid antibodies might be the only exception. RIC HSCT are an established component of transplant strategies today with emphasis on patients with advanced age. Despite many studies, none has shown unequivocal benefit in long term survival compared to standard conditioning. Early organ toxicity can be reduced but impact on GvHD and infectious complications is much less and RIC HSCT is always associated with an increased risk of relapse during follow-up (see Figure 1).
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Table 1: Conditioning regimens Total Dose Regimen • Conventional “old” regimens Cy/TBI 120 mg/kg Cyclophosphamide 12–14.4 Gy Total Body Irradiation Bu/Cy 16 mg/kg Busulfan 200 mg/kg Cyclophosphamide BACT 200 mg/m2 BCNU ARA-C 800 mg/m2 Cyclophosphamide 200 mg/kg 6-Thioguanine 800 mg/m2 • Alternative “standard” regimens TBI/VP Total Body Irradiation 12–13.2 Gy Etoposide 60 mg/kg AC/TBI ARA-C 36 g/m2 Total Body Irradiation 12 Gy MEL/TBI Melphalan 110–140 mg/m2 Total Body Irradiation 10–14.85 Gy Bu/Cy Busulfan 16 mg/kg Cyclophosphamide 120 mg/kg Bu/MEL Busulfan 16 mg/kg Melphalan 140 mg/m2 • Intensified regimens Cy/VP/TBI Cyclophosphamide 120 mg/kg Etoposide 30–60 mg/kg Total Body Irradiation 12–13.75 Gy TBI/TT/Cy/ATG Total Body Irradiation 13.75 Gy Thiotepa 10 mg/kg Cyclophosphamide 120 mg/kg ATG** 120 mg/kg Bu/Cy/MEL Busulfan 16 mg/kg Cyclophosphamide 120 mg/kg Melphalan 140 mg/m2 • Reduced intensity regimens TBI/Fluda Total Body Irradiation 2 Gy Fludarabine 90 mg/m2 Fluda/Bu/ATG Fludarabine 180 mg/m2 Busulfan 8 mg/kg ± ATG** 40 mg/kg
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Daily Dose
Administration
Days
60 mg/kg 2–2.4 Gy (2x/day)
IV in 1 hour
-6, -5 -3, -2, -1
4 mg/kg* 50 mg/kg**
p.o. q 6 hour IV in 1 hour
-9, -8, -7, -6 -5, -4, -3, -2
200 mg/m2 200 mg/m2 50 mg/kg 200 mg/m2
IV in 2 hours IV in 2 hours IV in 1 hour p.o
-6 -5, -4, -3, -2 -5, -4, -3, -2 -5, -4, -3, -2
2–2.5 Gy (2x/day) 60 mg/kg
IV in 2 hours
-7, -6, -5, -4 -3
3 g/m2 2 Gy (2x/day)
IV q 12 hours in 2 h -9, -8, -7, -6, -5, -4 -3, -2, -1
110–140 mg/m2 2 Gy (2x/day)
IV in 1 hour
-3 -2, -1, 0
4 mg/kg* 60 mg/kg
p.o. q 6 hours IV in 1 hour
-7, -6, -5, -4 -3, -2
4 mg/kg* 140 mg/m2
p.o. q 6 hours IV in 1 hour
-5, -4, -3, -2 -1
IV in 1hour 60 mg/kg IV in 2 hours 30–60 mg/kg 2–2.25 Gy (2x/day)
-6, -5 -4 -3, -2, -1
1.25 Gy (3x/day) 5 mg/kg 60 mg/kg 30 mg/kg
p.o. q 6 hours IV in 1 hour IV in 5–6 hours
-9, -8, -7, -6 -5, -4 -3, -2 -5, -4, -3, -2
4 mg/kg* 60 mg/kg 140 mg/m2
orally every q 6 hours -7, -6, -5, -4 -3, -2 IV in 1 hour -1 IV in 1 hour
2 Gy 30 mg/m2
IV in 30 min
0 -4, -3, -2
30 mg/m2 4 mg/kg* 10 mg/kg
IV in 30 min p.o. q 6 hours IV in 8–10 hours
-10 to -5 -6, -5 -4, -3, -2, -1
* Aternatively 3.2 mg/kg as IV formulation (Busulfex®); **ATG: specific dose depends on brand of ATG
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Table 2: Conditioning regimen by diseases Total Dose Regimen • Severe Aplastic Anaemia Cy 200 mg/kg Cyclophosphamide Cy/ATG 200 mg/kg Cyclophosphamide 90 mg/kg ATG** • Lymphoma BEAM 300 mg/m2 BCNU Etoposide 400–800 mg/m2 ARA-C 800–1600 mg/m2 Melphalan 140 mg/m2 CBV BCNU 300–600 mg/m2 Etoposide 750–2400 mg/m2 Cyclophosphamide 4.8–7.2 g/m2 • Solid Tumours LACE CCNU 200 mg/m2 Etoposide 1 g/m2 ARA-C 4 g/m2 Cyclophosphamide 5.4 g/m2 CCB Cyclophosphamide 5625 mg/m2 Cisplatin 165 mg/m2 BCNU 600 mg/m2 CTCb (Stamp) Thiotepa 500 mg/m2 Cyclophosphamide 6 g/m2 Carboplatin 800 mg/m2 ICE (high dose) Ifosfamide 16 g/m2 Carboplatin 1.8 g/m2 Etoposide 1.5 g/m2 Cy/Fluda/ATG Cyclophosphamide 120 mg/kg Fludarabine 125 mg/m2 ATG** (ATGAMR) 200 mg/kg • Fanconi Fluda/Bu/Cy/ATG Fludarabine 100 mg/m2 Busulfan 6 mg/kg Cyclophosphamide 40 mg/kg ATG** 6 mg/kg • Thalassaemia Bu/Cy Busulfan 14–16 mg/kg Cyclophosphamide 200 mg/kg
Daily Dose
Administration
Days
50 mg/kg
IV in 1 hour
-5, -4, -3, -2
50 mg/kg 30 mg/kg
IV in 1 hour IV in 8–10 hours
-5, -4, -3, -2 -5, -4, -3
300 mg/m2 150–200 mg/m2 200–400 mg/m2 140 mg/m2
IV in 2 hours IV in 2 hours IV in 2 hours IV in 1 hour
-6 -5, -4, -3, -2 -5, -4, -3, -2 -1
100-200 mg/m2 250-800 mg/m2 1.2–1.8 g/m2
IV in 2 hours IV in 2 hours IV in 1 hour
-8, -7, -6 -8, -7, -6 -5, -4, -3, -2
200 mg/m2 1 g/m2 2 g/m2 1.8 g/m2
p.o. IV in 2 hours IV in 2 hours IV in 1 hour
-7 -7 -6, -5 -4, -3, -2
1406.3 mg/m2 41.2 mg/m2 600 mg/m2
IV in 1 hour IV in continuous inf. IV in 2 hours
-6, -5, -4, -3 -6, -5, -4, -3 -3
125 mg/m2 1.5 g/m2 200 mg/m2
IV in continuous inf. IV in continuous inf. IV in continuous inf.
-7, -6, -5, -4 -7, -6, -5, -4 -7, -6, -5, -4
4 g/m2 600 mg/m2 500 mg/m2
-6, -5, -4, -3 IV in 2 hours IV in continuous inf. -6, -5, -4 IV in 2 hours (2x day) -6, -5, -4
60 mg/kg 25 mg/m2 40 mg/kg
IV in 1 hour IV 30 min IV in 8–10 hours
-7, -6 -5, -4, -3, -2 -5, -4, -3, -2
25 mg/m2 1.5 mg/kg* 10 mg/kg 1.5 mg/kg
IV 30 min p.o. q 6 hours IV in 1 hour IV in 8–10 hours
-5, -4, -3, -2 -9, -8, -7, -6 -5, -4, -3, -2 -4, -3, -2, -1
3.5–4 mg/kg 50 mg/kg
p.o. q 6 hours IV in 1 hour
-9, -8, -7, -6 -5, -4, -3, -2
* Aternatively 0.8 mg/kg as IV formulation (Busulfex®); ** ATG: specific dose depends on brand of ATG HAEMATOPOIETIC STEM CELL TRANSPLANTATION
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Figure 1: Survival and cause of death after HSCT for early stage leukaemia with standard conditioning 1a: Overall survival and cumulative incidence of main cause of death Other causes 0.10 Infections 0.20 GvHD 0.30 Cumulative incidence
Relapse 0.40 0.50 0.60 0.70 0.80 0.90
12.00 24.00 36.00 48.00 60.00 72.00 84.00 96.00 108.00 Months since transplantation
1b: Relative contribution in cumulative incidence of main cause of death. Cause of death over time is allocated for deceased patients only
Relative contribution to cumulative incidence
100%
Relapse 75%
GvHD 50%
Infections 25% Other causes 0% 12.00 24.00 36.00 48.00 60.00 72.00 84.00 96.00 108.00 Months since transplantation
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4. Special considerations for specific conditioning regimens 4.1. TBI TBI requires specific knowledge and should only be applied at an experienced institution under the guidance of a specialist radiophysicist or radiation oncologist. It has the specific advantage of having access to so-called "sanctuary sites" of malignancies, e.g. CNS or gonads, where some drugs may not penetrate. TBI is most commonly delivered to the patient using a linear accelerator. Patients are placed within a single irradiation field and lie on their side or in lateral position at a specified distance from the radiation source. Irradiation is given with horizontal beams using the anterior-posterior or lateral-lateral technique. Effects of TBI depend on total dose, dose rate and fractionation. TBI is usually prescribed in one of three ways: - as a single dose (sTBI) from 1 or 2 Gy up to 7.5–8 Gy total dose, - as a fractionated dose (fTBI) in 5–6 fractions over 3 days to a total of 10–14 Gy or - as "hyperfractionation" TBI consisting of 10–12 fractions over 4 days, usually to a total of 14–15 Gy. Dose rates differ considerably between machines and have different radiobiological effects. Lower total doses must be applied when higher dose rates are used. Fractionation reduces the incidence and severity of acute and late complications of normal tissue. As such TBI is similar to conditioning with chemotherapy. Low total doses, e.g. 6 Gy, may decrease toxicity but increase the risks of graft failure and disease recurrence. Very high doses, e.g. 15.75 Gy, may reduce the incidences of graft rejection and disease relapse, but at the expense of increased morbidity and mortality. Radiation dose is normally calculated as midline tissue dose at the level of the umbilicus but other strategies, e.g. targeting maximum lung dose, are possible. The received radiation doses to various parts of the body are monitored by in vivo dosimetry using diodes and thermoluminescent detectors placed anteriorly and posteriorly on various parts of the body. The doses given using standard techniques are relatively uniform and most parts of the body receive within ±10% of the dose delivered to the abdomen. In some institutions parts of the body, usually the lungs or the eyes, are protected with lead blocks during part of the application to "shield" this organ and to reduce organ specific toxicity. Other centres avoid shielding since leukaemic cells could also be protected by the shields. Dosimetry is still a difficult undertaking and measured dose is less exact than calculated delivered dose. Monitoring during TBI is essentially required to detect major
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technical errors, e.g. too high or too low an applied dose. Immediate effects of TBI include a transient increase in granulocyte numbers. Simultaneously, lymphocyte numbers fall. Changes in the serum concentration of various hormones are observed. Cortisol, adrenocorticotropic and corticotropin releasing factor levels increase a few hours after the completion of TBI with a concomitant increase in IL-6 and TNF levels. Some of these findings may be explained by activation of the hypothalamic-pituitary-adrenal axis. The mechanism by which TBI kills leukaemic (and normal) cells is still a matter of debate. TBI induces DNA and cell membrane damage, which triggers apoptosis. Data suggest that there may be a correlation between the magnitude of the immediate apoptotic response and "radiocurability". Similarly, "survival factors" for specific cells, e.g. cytokines, such as flt-3 ligand can protect stem cells from the toxic effects of TBI by their anti-apoptotic effects. Immediate side effects of TBI are nausea, vomiting and, typically, parotid swelling. Adequate preventive measures are recommended. Pilocarpin p.o. is used to reduce parotid swelling. 4.2. TCD and conditioning T-cells are an essential component of the graft product and for engraftment. Graft facilitating cells in the T-cell fraction enhance engraftment via soluble factors and by their direct effect on residual host T-cells. Simultaneously, T-cells are responsible for acute and chronic GvHD and for graft versus leukaemia (GvL) effects. Therefore, TCD of the stem cells prior to infusion is highly effective in the prevention of acute and chronic GvHD, but at the expense of an increased risk of graft rejection and disease recurrence. TCD is essential in haploidentical transplants to prevent lethal GvHD. The incidence of graft failure and graft rejection in TCD HLA-identical sibling HSCT is in the order of 10–20% with standard conditioning, and is higher in haploidentical, UD HSCT or RIC HSCT. Several approaches have been used to overcome the increase in graft failure associated with TCD. Some centres have intensified CT/RT by increasing dosages or adding TLI or TAI (total lymphoid/abdominal irradiation). This approach can effectively overcome graft failure but is associated with increased morbidity and mortality, resulting in similar OS rates. Because TCD is so effective at preventing GvHD, no further GvHD prevention e.g. with CsA and/or MTX is given after HSCT. The re-introduction of CsA to these protocols can reduce the rate of graft failure/rejection. Other centres combine TCD with additional immunosuppressive therapy for patients prior to stem cell infusion, e.g. in vivo Campath-1H, ATG or MoAb to OKT-3 for several days before HSCT. It may also be possible to overcome graft failure by increasing the SC dose as has 136
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been shown not only in murine models of transplant but also in haplo-identical family member transplants for acute leukaemia. Addition of T-cells which are depleted of alloreactive T-cells or selected NK cells are novel approaches under current investigation. 4.3. Paediatric transplants There are two main differences between adult and paediatric patients in relation to conditioning. Children in general tolerate side effects better than older patients and higher total doses may be applied. In contrast, conditioning affects growth and endocrine development. Retarded growth and failed or retarded puberty are main late sequelae in paediatric HSCT patients. There is no general consensus between the various paediatric groups about the best conditioning. This reflects the uncertainty about the optimal regimen. Nevertheless, a general outline of the strategies used in children can be given. Since many reports comparing TBI containing conditioning regimens with those containing only CT show similar outcomes, TBI should be avoided in small children, whenever possible. It should probably never be given to children below 2 years of age. TBI (2 doses per day) in combination with Cy and/or VP16 is commonly used in children undergoing HSCT for ALL. These children have usually already received all the drugs useful in this disease and TBI is thought to have an additional antileukaemic effect in ALL. Probably the most frequently used conditioning protocols in paediatric HSCT are: 1. Fractionated TBI + Cy and/or VP in ALL (>2 years of age) 2. Cy + Bu (+ MEL) in AML, CML and MDS.
5. Disease specific aspects 5.1. Inborn errors of the lymphohaematopoietic system In patients with profound deficiencies of the lymphoid immune system, such as SCID, the capacity for graft rejection is extremely low or absent. Preparative regimens are therefore unnecessary but HSCT results in an unusual form of lympho-haematopoietic chimerism. Donor cell engraftment after HLA identical sibling HSCT is usually restricted to the lymphocytes, whilst the myeloid system remains of host cell origin. The same approach in TCD HLA-mismatched HSCT has led to more variable results. Here, complete or partial graft failure, in particular failure of B-cell reconstitution, has been observed. This experience has resulted in the use of CT based preparative
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regimens in patients transplanted for SCID from HLA-mismatched donors. The regimen most frequently employed is that of Bu 2 mg/kg/day for 4 d + Cy 50 mg/kg/d for 4 d. In the presence of serious clinical conditions, e.g. pulmonary infections prior to the transplant, the use of chemotherapy may greatly increase the morbidity and mortality of the procedure. In these cases, HSCT may be performed without conditioning, as even partial immunological reconstitution may be of therapeutic benefit to the child. In less severe variants of lymphoid deficiencies and in all other inborn errors, conditioning is required for myelo-ablation and to prevent graft rejection and disease recurrence. Some young patients come to transplant with a number of complications related to their underlying disease, in particular poorly controlled chronic infections. The use of non-CT based conditioning protocols which allow stable engraftment remains a long-term goal. 5.2 Severe aplastic anaemia The marrow is empty in patients with SAA and the sole aim of the conditioning protocol is to provide immunosuppression is. Cy 50 mg/kg for 4 successive days is an appropriate conditioning regimen. It is very immunosuppressive and its use involves few serious long-term side-effects. Rejection remains a concern, especially in patients sensitised to their donors via blood products. The addition of ATG has reduced risk of rejection in one pilot study but prospective randomised and retrospective multicentre analyses have failed to confirm its value in nonsensitised recipients. Still, it is the most frequently used approach at the present time. The optimal conditioning regimen for patients with SAA and alternative donor transplants still needs defining. The addition of Fluda or low dose TBI (2 Gy) is under current investigation. All long-term follow up studies have shown a higher incidence of late malignancies in SAA patients given TBI or TAI during conditioning. 5.3. Fanconi’s anaemia Fanconi’s anaemia is a genetic disorder associated with diverse congenital abnormalities, progressive BM failure and increased risk of leukaemia and other cancers. HSCT is an effective treatment. The underlying molecular defects are heterogeneous but all result in defective DNA repair mechanisms. These patients are therefore extremely susceptible to the effects of CT/RT, in particular to irradiation and alkylating agents. Conditioning regimens must be reduced in intensity. Most regimens contain Cy 5–10 mg/kg/d for 4 days. Some centres add low dose TBI (400–450 cGy) or TAI or TLI (500 cGy) to overcome graft rejection. More recent approaches investigate the use of Bu or the addition of Fluda. 138
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However, there is concern that the addition of TBI may increase the incidence of secondary malignancies in a group of patients already susceptible to further malignant disease. 5.4. Lymphoma The conditioning regimens for high dose therapy in lymphoma have been based on custom and practice rather than proof or confirmation by randomised trials. Most frequently used regimens avoid inclusion of TBI. Auto-HSCT: BEAM (BCNU, VP, ARA-C and MEL) has been the most commonly used regimen both for NHL and HD. Originally described jointly by groups in Lyon and London, it has become standard treatment both in Europe and, to a lesser extent, in the United States. The standard BEAM uses a total dose of VP of 800 mg/m2/d for 4 days. Several groups have attempted to change the individual components. None has proved superior and there is no evidence that any other regimen has more anti-tumour effect than BEAM. Allo-HSCT: BEAM and CBV have both been used in allografting but neither regimen is fully myeloablative. Fluda based RIC HSCT have been proposed. TBI plus CT is considered as the standard preparative regimen for allo-HSCT. The incidence of TRM is clearly associated with the status of the patients at the time of transplant. If transplant is performed for refractory HD or NHL, the TRM reaches 30–40%. Bu and Cy have also been used but Bu is not in common use in the treatment of lymphoma. There are no comparative data for Bu/Cy versus TBI containing regimens or for RIC versus standard conditioning HSCT. 5.5. Myeloma Relatively clear data are available concerning the best conditioning regimen for MM. Auto-HSCT: retrospective data on several thousand autologous HSCT for MM show clear results. Survival is best with a conditioning using MEL 200 mg/m2. No other regimen has shown better results. Allo-HSCT: standard conditioning with Cy TBI shows best long-term survival. RIC HSCT reduces early mortality but retrospective comparative data show inferior survival at +5 years. RIC HSCT may be used in planned double autologous allogeneic HSCT programs. The best approach is not known. 5.6. Solid tumours Dose intensification was the basic concept when auto-HSCT became an investigational tool for treatment of solid tumours. Initial results appeared promising and there was huge interest in the mid-nineties. Conditioning regimens were developed with "tumour specific" drugs in mind and a multitude of regimens appeared. None has
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proved to be superior to any other and HSCT in solid tumours with very few exceptions is still considered investigational. Conditioning regimens should follow those specified in the clinical protocols. They are discussed in the Chapter on solid tumours. Allo-HSCT is being investigated in more recent years as a tool for promoting graft vs. tumour effects. Hence, primarily RIC regimens are chosen with the aim of establishing donor type engraftment. Any RIC regimen can be used without a need for tumour specific therapy. RIC HSCT for solid tumours is still to be considered investigational.
6. Conditioning induced organ damage The desirable effects of conditioning regimens are offset by their highly predictable toxicities, which are responsible for considerable morbidity and mortality (see Table 3). These involve the GI, renal, hepatic, pulmonary and cardiac systems and include cardiac toxicity (Cy >1.5 g/m2/d; BCNU), pulmonary toxicity (TBI >8 Gy), mucositis, hepatic toxicity, bladder toxicity, renal toxicity and neurological toxicity. Prevention and treatment of early and late complications are discussed in detail in Chapters 9 and 12.
7. Quality management issues Application of high dose therapy in the context of HSCT is a complex and challenging therapy. It is prone to errors and all strategies should be undertaken to minimise such risks. This includes specifically the following points, as indicated in the JACIE standards and as outlined in Chapter 4. Each institution administering high dose CT in the context of HSCT must establish a quality management system for administration of this therapy. Such a system must be established in close cooperation with the institutional pharmacy and the nursing team. Conditioning has to be based on pre-printed orders. Regular check points at different levels and immediately prior to administration have to safeguard that the right patient is given the specified drugs at the correct dose and appropriate timing. All check points include the “4 eyes” principle. Similar considerations apply to the administration of TBI.
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CHAPTER 6 • Principles of conditioning
Table 3: Factors influencing the outcome of HSCT* Disease factors Stage: increasing stage Chronic phase patients only: Basophil count (>3%) Sokal/Hasford score (higher) Leukocyte count (higher) Time interval (> 12 m) Patient-related factors Age (higher) Sex (male) Race Viral status (CMV positivity) Donor-related factors Histocompatibility (vs. HLA-id sibling) Identical twin Unrelated Mismatched Sex (female D for male R) Viral status (CMV positivity) Peri-transplant factors Conditioning (intensified) (reduced) GvHD prevention (intensified) (reduced) Cell content (CD34) high low Stem cell source: BM vs. PB Post-transplant factors Acute GvHD (increasing grade) Chronic GvHD (none vs. any)
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LFS
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D: donor; R: recipient * These factors are confirmed for CML but apply in principle to most other situations
References 1. Thomas et al. A history of bone marrow transplantation. In: Thomas' Hematopoietic Cell Transplantation, 3rd Edition (Blume KG, Forman SJ, Appelbaum FR Eds.), Blackwell Publishing Oxford, Malden, Victoria, 2004, pp 4-8. 2. Storb RF, Lucarelli G, McSweeney, Childs PW. Hematopoietic stem cell transplantation for benign hematological disorders and solid tumors. Hematology 2003; 372-397. 3. Santos GW, Tutschka PJ, Brookmeyer R. Marrow transplantation for acute non-lymphocytic
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leukemia after treatment with busulfan and cyclophosphamide. N Engl J Med 1983; 309: 1347-1353. 4. Ridell S, Appelbaum FR, Buckher CD, et al. High-dose cytarabine and total body irradiation with or without cyclophosphamide as a preparative regimen for bone marrow transplantation for acute leukemia. J Clin Oncol 1988; 6: 576-582. 5. Gore ME, Selby PJ, Viner C, et al. Intensive treatment of multiple myeloma and criteria for complete remission. Lancet 1989; ii: 879-882. 6. Philips G, Shepherd J, Bernett M, et al. Busulfan, cyclophosphamide and melphalan as a conditioning regimen for bone marrow transplantation in children with myelodysplastic syndrome. Leukemia 1994; 8: 1880-1888. 7. Slavin S, Nagler A, Naparstek E, et al. Non-myeloablative stem cell transplantation and cell therapy as an alternative to conventional bone marrow transplantation with lethal cytoreduction for the treatment of malignant and non-malignant hematologic diseases. Blood 1998; 3: 756-763. 8. Giralt S, Estey E, Albitar M, et al. Engraftment of allogeneic hematopoietic progenitor cells with purine analog-containing chemotherapy: Harnessing graft-versus-leukemia without myeloablative therapy. Blood 1997; 89: 4531-4536. 9. Copelan EA. Hematopoietic stem-cell transplantation. N Engl J Med 2006; 354: 1813-1826. 10.Appelbaum FR. Hematopoietic-cell transplantation at 50. N Engl J Med 2007; 357: 14721475. 11.Armand P, Antin JH. Allogeneic stem cell transplantation for aplastic anemia. Biol Blood Marrow Transplant 2007; 13: 505-516. 12.Blaise D, Vey N, Faucher C, Mohty M. Current status of reduced-intensity-conditioning allogeneic stem cell transplantation for acute myeloid leukemia. Haematologica 2007; 92: 533-541. 13.Attal M, Moreau P, Avet-Loiseau H, Harousseau JL. Stem cell transplantation in multiple myeloma. Hematology (Am Soc Hematol Educ Program) 2007; 311-316. 14.Hegenbart U, Niederwieser D, Sandmaier BM, et al. Treatment for acute myelogenous leukemia by low-dose, total-body, irradiation-based conditioning and hematopoietic cell transplantation from related and unrelated donors. J Clin Oncol 2006; 24: 444-453. 15.Secondino S, Carrabba MG, Pedrazzoli P, et al.; European Group for Blood and Marrow Transplantation Solid Tumors Working Party. Reduced intensity stem cell transplantation for advanced soft tissue sarcomas in adults: A retrospective analysis of the European Group for Blood and Marrow Transplantation. Haematologica 2007; 92: 418-420. 16.Satwani P, Cooper N, Rao K, et al. Reduced intensity conditioning and allogeneic stem cell transplantation in childhood malignant and nonmalignant diseases. Bone Marrow Transplant 2007; 26: in press. 17.Barrett AJ, Savani BN. Stem cell transplantation with reduced-intensity conditioning regimens: A review of ten years experience with new transplant concepts and new therapeutic agents. Leukemia 2006; 20: 1661-1672.
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CHAPTER 6 • Principles of conditioning
Mutiple Choice Questionnaire To find the correct answer, go to http://www.esh.org/ebmt-handbook2008answers.htm 1. Total body irradiation (TBI) is frequently used for conditioning in HSCT. Its biological effects depend primarily on: a) Radiation source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Total radiation dose applied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Combination of dose rate and fractions of doses . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Combination of total dose, dose rate and fractions of doses . . . . . . . . . . . . . . 2. The type of conditioning regimen is more important in autologous than in allogeneic HSCT, because: a) Graft versus tumour reaction is the only element in allogeneic HSCT for tumour control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Dose intensification of tumour specific therapy is the key element of autologous HSCT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) There is no specific chemotherapy in allogeneic HSCT. . . . . . . . . . . . . . . . . . . . . . d) Monoclonal antibodies, such as CD20+ antibodies can only be integrated in the conditioning in autologous HSCT . . . . . . . . . . . . . . . . . . . . . . . . 3. Which of the following statements about reduced intensity conditioning (RIC) is correct? a) RIC has a specific advantage in patients with advanced malignancies . . . b) RIC has a specific advantage in patients with non-malignant conditions where no graft-versus-disease effect is required . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) RIC has a specific advantage in CMV positive patients with CMV negative donors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Peripheral blood as a stem cell source is associated with less rejection than bone marrow in patients with RIC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. There is an inherent dilemma in allogeneic HSCT: Increasing conditioning decreases risk of rejection and risk of relapse at the expense of increased toxicity; vice versa, decreased conditioning decreases direct regimen related toxicity at the expense of increased risk of rejection and relapse. Which of the following statements about this dilemma is true?
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a) Different regimens should be employed for patients with high-risk disease and low transplant risk compared to patients with low-risk disease but high transplant risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) T-cell depletion can abolish this dilemma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) The statement does not hold true for unrelated cord blood transplants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) The statement is not correct for non-malignant disorders . . . . . . . . . . . . . . . . . 5. The following conditioning regimens can be generally accepted as “established”; e.g., novel regimens need to be evaluated against this standard: a) Cyclophosphamide +/- ATG for severe aplastic anaemia . . . . . . . . . . . . . . . . . . . . b) Cyclophosphamide + busulfan for Hodgkin’s lymphoma . . . . . . . . . . . . . . . . . . . . c) Cyclophosphamide + TBI for multiple myeloma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Fludarabine for myeloid malignancies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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NOTES
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*
CHAPTER 7
Transfusion policy
D.H. Pamphilon
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CHAPTER 7 • Transfusion policy
1. Introduction Haematopoietic stem cell transplant (HSCT) patients often require intensive blood component support. Transfusion may be complicated by transfusion transmitted infection (TTI) – both viral and bacterial, transfusion-associated (TA)-GvHD, febrile non-haemolytic transfusion reactions (FNHTR) and transfusion-related acute lung injury (TRALI). Alloimmunisation (AI) to red cell antigens may cause difficulties in selecting compatible blood whilst AI to the human leukocyte antigens (HLA) present on platelets may cause refractoriness to subsequent transfusions of randomly-selected platelets. It is therefore essential to define robust transfusion policies and procedures and these should be regularly audited. This Chapter describes the blood components available for transfusion including granulocytes and their clinical use in the setting of HSCT. The impact of reduced-intensity conditioning (RIC) transplantation on transfusion requirements is highlighted. Amongst infectious agents transmissible by blood components, cytomegalovirus (CMV) is particularly important in BMT patients and strategies to minimise CMV transmission in susceptible recipients are described.
2. General policies for the selection of high quality, appropriate transfusions The European Union Directive 2002/98/EC sets standards for the collection, testing, processing, storage and distribution of human blood and blood components (1). It requires that Blood Establishments should be licensed and this is of importance for both Blood Centres in EU countries that undertake these activities as well as hospitals that collect and issue e.g. granulocytes for transfusion. The most important aspects of the Directive are: • The fate of each unit of all blood components should be recorded and this record kept for 30 years, i.e. donor to recipient traceability • Robust Quality Systems should be in place • The processing of blood and blood components should be undertaken by licensed blood establishments (see above) • Training should be provided for hospital transfusion laboratory staff • Haemovigilance systems should be established to include the reporting of adverse events. Establishments are licensed by the Competent Authorities in EU Member States following inspection by a regulatory body – in the UK this is the Medicines and Healthcare Products Regulatory Authority (MHRA). Reports of compliance must be submitted.
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2.1. Testing of donated blood for infectious disease markers (IDM) A number of microbial agents may be transmitted by blood transfusion. These include hepatitis B and C, HIV-1 and -2, HTLV-1 and 2, CMV and syphilis. Blood Services routinely test blood for the following: • Hepatitis B: Hepatitis B surface antigen (HbsAg)* • Hepatitis C: Hepatitis C antibodies (anti-HCV)* • Human immunodeficiency virus 1 and 2: HIV-1 + 2 antibodies (anti-HIV 1+2)* • Human T-lymphotropic virus 1 and 2: HTLV-1 + 2 antibodies (anti-HTLV-1+2) • Syphilis. * These tests are mandated by the EU Blood Directive (2002/98/EC) (1) In addition, donations may be tested for: • anti-HBc, i.e. anti-hepatitis B core antigen • alanine aminotransferase (ALT): A surrogate marker of hepatitis C • HCV-RNA by PCR for hepatitis C • p 24 antigen for HIV-1. There is a variation from country to country in the number of tests performed on each blood donation. Testing for anti-CMV antibody to identify CMV seronegative donors is done on a proportion of blood donations, sufficient to identify enough CMV seronegative components for transfusion to those patients for whom it is appropriate. Bacterial contamination is a relatively common occurrence with an incidence estimated at 0.05–0.5% of components (2). The sources of bacteria are donor bacteraemia and contamination with bacteria present on the skin at the time of donation or present in blood packs. The organisms that most frequently contaminate red cell and platelet transfusions are shown in Table 1. Screening tests for bacteria in platelet concentrates (PCs) using automated blood culture systems e.g. BacT/ALERT have been evaluated (2) and are now used routinely by some transfusion services. PCs are not issued until at least 48 hours
Table 1: Bacteria that most frequently contaminate blood components Red cells Yersinia enterocolitica Pseudomonas fluorescens Other species
51% 27% 22%
Platelets Staph. epidermidis Staphylococcus aureus Salmonella choleraesuis Serratia marcescens Bacillus cereus Others
Figures are percentage of total contaminants for red cells and platelets respectively 148
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25% 6% 14% 10% 6% 39%
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CHAPTER 7 • Transfusion policy
after collection but the storage period may be extended to 7 days once sterility has been evaluated. The risk of bacterial transmission is also minimised by careful donor selection, meticulous attention to sterility during venepuncture, diversion of the first 30 mL of blood collected (contains most of the bacteria) away from the primary collection pack and sterility during preparation of blood components. Bacterial contamination should be suspected in any patient who develops a febrile reaction characterised by fever, chills ± hypotension. Microbiological testing does not completely remove the risk of TTI, although the chance of infection in the UK after transfusion of screened blood components from known/previously tested donors is estimated to be less than 1 in 2 x 106 for HIV1, HBV and HCV. This risk will vary somewhat according to the donor selection and testing policies that are operative within a Blood Service. 2.2. Blood grouping and antibody testing The ABO and Rhesus D types of all donated blood are determined by standard techniques. This is a requirement of the EU Blood Directive (1). All donations are tested to exclude the presence of immune IgG antibodies that are reactive with common blood groups and which occur after an immunising stimulus such as pregnancy or transfusion. Selected units of red cells may be more extensively phenotyped (Kell, Duffy, Kidd, MNSs antigens) for patients who develop red cell alloantibodies. 2.3. Prevention of CMV transmission A proportion of HSCT patients are CMV seropositive pre-transplant or have seropositive donors. They require regular screening by PCR and antigenaemia testing together with ganciclovir therapy where appropriate to minimise the impact of virus reactivation and prevent clinical infection post-transplant. All CMV seronegative HSCT patients with CMV seronegative donors (neg/neg) and CMV seronegative patients with haematological and other disorders who are likely to proceed to a transplant should receive blood components that have a minimal risk of causing CMV acquisition (3). Studies show that the use of CMV seronegative components is associated with a less than 3% incidence of CMV infection and/or disease in neg/neg HSCT. CMV is transmitted via leukocytes, and leukodepletion also minimises the risk of CMV transmission. CMV seronegative and leukodepleted blood components are probably of equivalent efficacy but this view is not generally accepted (4, 5). Further evidence from prospective randomised controlled studies (PRCT) using pre-storage leukodepleted blood components is required. Centres must establish their own policies.
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2.4. Leukodepleted blood components Transfused leukocytes cause alloimmunisation (AI) to HLA Class 1 antigens (HLA AI) in a proportion of patients. This may be manifested clinically as FNHTRs, although these may also be caused by antibodies to neutrophils, platelets or plasma proteins and by cytokines such as interleukin (IL)-1, IL-6, IL-8 and tumour necrosis factor (TNF)-a which accumulate in stored blood components, especially PCs. HLA AI may cause accelerated destruction of transfused platelets that are HLA incompatible. This is clinically manifest as a failure to achieve a satisfactory increment after platelet transfusion (refractoriness). A summary of the adverse effects of transfused leukocytes is shown in Table 2. Donor dendritic cells (DC) which are present in red cell and platelet transfusions appear to be responsible for sensitisation to HLA. Studies show that removal of leukocytes to less than 5 x 106 per blood component prevents primary HLA AI in >97% of patients with haematological malignancies. The use of leukodepleted components also reduces secondary AI and refractoriness to platelet transfusion. Refractoriness is not always prevented since in >50% of cases it results from increased platelet destruction due to non-immune causes which include fever, splenomegaly, DIC and amphotericin therapy. AI is also associated with a higher incidence of graft failure in patients with severe aplastic anaemia. Filtration of blood or its components is best performed in Blood Centres and hospital blood banks. Data from studies where leukocytes were filtered from blood components at the bedside show that this may not be effective in preventing or reducing FNHTR, AI and refractoriness.
Table 2: Adverse effects of transfused leukocytes
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HLA alloimmunisation causing
- FNHTR - Refractoriness to random donor platelets - Graft rejection - Shortened red cell survival
Transmission of microorganisms
- CMV - HTLV-1/11 - Toxoplasma gondii - Yersinia enterocolitica
Immunomodulation
- GvHD - Activation of viruses in host cells e.g. HIV-1 - Immune suppression of T- and NK-cell functions
Affecting the quality of stored blood
- Microaggregate formation - Metabolic deterioration during storage
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2.4.1. Indications for leukodepleted blood components (6) • Pre-HSCT in patients with SAA to reduce the likelihood of graft failure; • Pre- and post-HSCT to prevent recurrent FNHTR; • Pre-and post-HSCT to minimise HLA AI and platelet refractoriness. This is optional since there is no evidence of a significant impact on important clinical outcome measures such as survival post-HSCT except in patients with SAA. Nonetheless many Blood Services have implemented leukodepletion of a large proportion or, in some cases, all of their blood components. In the UK universal leukodepletion was implemented in 1999 with the aim of minimising the risk of transfusionassociated transmission of the causative agent of variant Creutzfeld-Jakob disease (vCJD); • As an alternative to CMV seronegative components. 2.5. Gamma-irradiation of blood components and TA-GvHD HLA incompatible third party leukocytes contained in donated blood components can engraft and initiate an alloreactive response after transfusion. This can cause TA-GvHD, manifest clinically by fever, rash, diarrhoea, jaundice and pancytopenia, and this is fatal in >90% of cases, so prevention is essential. Donor leukocytes are inactivated by gamma-irradiation of 2500 cGy and all components for HSCT recipients should be irradiated from the time that conditioning therapy is started and continued until 6 months post-transplant or until the lymphocyte count is 1 x 109/L in the absence of chronic GvHD. In addition, HLA matched PCs should be irradiated, as should those from family members, since HLA haplotype sharing may result in TA-GvHD even in immunocompetent patients. A summary of the indications for blood component irradiation is shown in Table 3. Platelets show normal functional characteristics through 5 days storage after irradiation with doses up to 5000 cGy. Red cells leak potassium during storage and
Table 3: Indications for irradiated blood components • • • • • • •
Allo-HSC recipients from time of conditioning therapy for 6 months or until the lymphocyte count is 1 x 109/L in the absence of cGvHD Allo-HSC donors Auto-HSC recipients (from 7 days before harvest until 3 months post transplant) All donations from HLA-matched donors or 1st or 2nd degree relatives All patients with Hodgkin disease at any stage of therapy All patients treated with purine analogues e.g. fludarabine All patients with congenital immunodeficiency states
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this is worsened by irradiation. Therefore, storage is limited to 14 days after irradiation with 2500 cGy. TA-GvHD has been shown to occur after 1500–2000 cGy and this dose range is not recommended (7).
3. Pre-transplant transfusions The following provisions apply: • Red cell transfusions for patients with sickle cell disease are initially matched for ABO, Rhesus D and Kell antigens but additional matching for the Rhesus CcEe and for Duffy (Fya Fyb), Kell (Kk), Kidd (Jka Jkb) and MNSs antigens may be required if the patient has developed alloantibodies; • Leukodepleted blood components should be transfused to all patients with aplastic anaemia (6); • Either CMV seronegative or leukodepleted blood components should be transfused to susceptible patients to prevent CMV acquisition (3); • Blood components should be gamma-irradiated for PBSC transplant patients during stem cell mobilisation and collection since transfused leukocytes might be captured in the PBSC harvest and subsequently induce TA-GvHD (7); • Blood components transfused to allogeneic marrow donors immediately pre- or intra-operatively should also be irradiated (7).
4. Blood component transfusions The following definitions for red cells, PCs, FFP, cryoprecipitate and granulocyte products were derived from the Guidelines for UK Blood Services. The requirement for red cell and platelet transfusions is decreased in the setting of RIC transplantation (8). 4.1. Red cells Red cells, usually suspended in an optimal additive solution (OAS) are transfused to correct anaemia due to marrow failure, haemorrhage or haemolysis, aiming to keep the haemoglobin or packed cell volume (PCV) above predefined levels to ensure good tissue oxygenation. Reduced intensity conditioning (RIC) transplants require fewer red cell transfusions. Transfusions may be: • Suspended in OAS, usually a combination of saline, adenine, glucose and mannitol (SAG-M): PCV 50–70%; volume 220–420 mL. This is the product of choice; • Derived from whole blood from which a proportion of the plasma has been removed – plasma reduced blood (PRB): PCV 50–60%; volume 200–450 mL; • Unmodified whole blood: volume 420–520 mL. This last term is misleading since platelets and labile coagulation factors deteriorate rapidly in stored blood. 152
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The storage period is 35–42 days at 4 ± 2ºC. Transfusion policy • Red cells should be matched for ABO and Rhesus D type (1). • Extended phenotyping may be necessary in patients, e.g. those with sickle cell disease, who have formed red cell alloantibodies after previous transfusions. • Red cells should be cross-matched against the patient’s serum by standard techniques prior to transfusion. • Thresholds should be defined for haemoglobin and PCV below which red cell transfusions are always given. Suggested arbitrary cut off points are Hb less than 8.0 g/dL and PCV less than 25%. • In adults 1 unit of red cells raises the Hb by 1.0 g/dL whereas in children the volume of blood to be transfused is derived from the formula: Volume = Increase in Hb (g/dL) required x 4 x weight (kg) 4.2. Platelet transfusions 4.2.1. Manufacture Platelet concentrates (PCs) are made: • From whole blood by centrifuging units in a “top – top” pack format to obtain platelet rich plasma (PRP), which is then further concentrated to give a PC. PRPPCs may be transfused individually or pooled in multiples – usually 6; • From whole blood by centrifuging units in a “bottom & top“ pack format to separate the buffy coat (BC), pooling 4 BCs and recentrifuging to separate PRP which is then expressed into a secondary storage container for PC preparation; • By collecting PCs directly on a cell separator. Dual arm, continuous flow apheresis is preferred and some cell separators collect PCs with an inherently low WBC content (Table 4) (9).
Table 4: Platelet content and WBC contamination of different types of platelet concentrates PRP-PC
BC-PC
Apheresis PC
Mean platelet Content x 1011/unit
3.4
3.2
3.15
Mean WBC Contamination x 106/unit
365
5.7
0.3
PRP-PC: platelet concentrate prepared from whole blood; BC-PC: platelet concentrate prepared from buffy coat; Apheresis PC: platelet concentrate obtained directly by apheresis. Data from the National Blood Service, Bristol Centre (9)
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The storage period is 5 days at 22 ± 2ºC unless bacterial screening has been carried out in which case it may be extended to 7 days. Transfusion policy Current practice, based on the results of randomised studies, is to transfuse PC prophylactically when the platelet count is less than 10 x 109/L. A recent Cochrane Systematic Review concluded that, whilst there is no reason to change current practice, blood products may become scarcer and further trials should be undertaken to compare prophylactic versus therapeutic platelet transfusion – i.e. PC given only when there is clinical bleeding (10). In autologous PB HSCT this has been found to be safe (11). Fewer PC transfusions are required in RIC allografted patients compared to those who receive full myeloablative conditioning (8). Best current practice is that: • PCs should be ABO and Rh compatible wherever possible since ABO incompatibility may reduce the expected count increment (CI) by 10–30%; • Group O PCs should be tested for high titre anti-A, B and if positive should only be transfused to group O recipients to avoid haemolysis caused by passive administration of antibody; • If Rh D positive platelets are given to an Rh D negative patient then give 250 IU polyclonal anti-Rh (D) immunoglobulin. Since the chance of Rh immunisation is probably less than 5% this may be omitted and the patients serum screened for immune red cell antibodies, or prior to a red cell transfusion; • Studies show that a threshold of 10 x 109/L in stable thrombocytopenic patients is optimal for prophylactic platelet transfusion; • A higher threshold of 20 x 109/L should be used in patients with fever, sepsis, splenomegaly and other well-established causes of increased platelet consumption; • If an invasive procedure is planned, e.g. central line insertion, the platelet count should be >50 x 109/L; • PCs should be transfused when there is significant clinical bleeding, irrespective of the platelet count; • PCs are contraindicated in patients with TTP; • In adults the usual dose of platelets is 3 x 1011 (an adult therapeutic dose – ATD) in a volume of 200–300 mL; • Children >30 kg receive one ATD. Children <30 kg are given 10 mL/kg; • Rate of transfusion: adults 1 ATD is given in less than 60 minutes children e.g. 2–5 mL/kg/hr. The outcome of platelet transfusions can be monitored by: • Looking for cessation of bleeding; • Measuring the platelet count the following day. A persistent value <20 x 109L suggests refractoriness; 154
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•
Measuring the platelet count at between 10–60 minutes post-transfusion – the CI. A corrected (C) CI is calculated as follows: CCI =
CI x 109/L x surface area (m2) Platelets transfused x 1011
The CCI should be more than 7.5. If the patient is refractory to transfusion of PCs, samples should be taken to test for HLA antibodies. If these are detected, HLA-matched platelets collected by apheresis of HLA-typed donors should be used in these patients. If the CCI is less than 7.5 following transfusion of HLA matched PCs and the patient is not bleeding then withhold platelet transfusions. If the CCI using well-HLA-matched PCs is less than 7.5 and/or bleeding persists then: • Check for non-immune causes of refractoriness. If refractoriness is due to nonimmune causes, particularly if there is significant clinical bleeding then either give 2 or 3 ATD or give 1 ATD twice or three times daily; • Look for platelet-specific antibodies – this is a rare cause of refractoriness in HSCT patients; • Consider using cross-matched platelets. HLA-typed or random units or platelets are cross-matched against the patient’s serum usually by an immunofluorescent technique and non-reactive units selected if possible. 4.3. FFP and cryoprecipitate transfusion Fresh frozen plasma (FFP) may be given to correct the abnormalities of coagulation that are observed where there is, for example, liver disease resulting from graft versus host disease (GvHD) or veno-occlusive disease (VOD), thrombotic thrombocytopenic purpura (TTP) or disseminated intravascular coagulation (DIC). • FFP is made by centrifuging whole blood and freezing separated plasma within 6 hours of collection. The volume is 200–340 mL and the Factor VIII level should be greater than 70 IU/mL. • Cryoprecipitate is made by thawing FFP at 4ºC and collecting the precipitate that forms by further centrifugation in a volume of approximately 20–30 mL. This is then refrozen. The fibrinogen content should be greater than 140 mg/dL and the Factor VIII level greater than 70 IU/mL. FFP and cryoprecipitate have a storage period of 12 months at –30ºC. Transfusion policy FFP transfusion (at a volume of 10–15 mL/kg) is indicated after HSCT: • As replacement fluid in TTP where plasma exchange is undertaken;
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In the presence of liver disease causing significant defects of coagulation factors; In severe DIC. FFP transfusion may also be indicated after HSCT where a large volume blood transfusion, e.g. after haemorrhage, has caused a dilutional coagulopathy. Cryoprecipitate transfusion is indicated in severe DIC when the fibrinogen is <100 mg/dL. The outcome of FFP and cryoprecipitate transfusion should be monitored by measuring the prothrombin time (PT) and activated partial thromboplastin time (APTT). The ratios compared to control should correct to less than 1.5. In DIC the fibrinogen should be greater than 100 mg/dL. •
•
4.4. Granulocyte transfusions Granulocyte transfusions (GT) are prepared by pooling buffy coats from e.g. 10–20 whole blood donations and then reducing the red cell content further by sedimentation using starch or dextran. They may also be collected by the apheresis of steady state healthy donors who may be family members or unrelated volunteers. The granulocyte content is in the range 5–10 x 109/unit for both these preparations. Mobilised granulocytes are collected from donors who receive G-CSF (5–10 micrograms/kg) and/or dexamethasone (8 mg) – both given 12–24 hours before to increase the number that can be collected during a standard apheresis procedure. This strategy gives a granulocyte yield of between 10–100 x 109 per unit and data available so far indicates that significant granulocyte increments e.g. 1–2 x 109/L can be obtained. By contrast it is unusual to observe such increments with buffy coat or unmobilised granulocytes. All granulocyte products must be irradiated prior to transfusion to prevent TA-GvHD. Cross-matching is also required. A recent Cochrane Systematic Review indicated that there is currently inconclusive evidence from PRCTs to support or refute the use of GT in neutropenic patients. Further PRCT are required before definitive recommendations can be made (12). Transfusion policy • There is recent anecdotal evidence that prophylactic administration of granulocytes may reduce the incidence of severe fungal infections after BMT but currently few centres use such transfusions and further studies are needed (13). Furthermore, granulocyte transfusions increase the likelihood of HLA immunisation and platelet refractoriness. • Granulocyte transfusions are probably best reserved for patients with granulocyte counts less than 0.2 x 109/L and documented bacterial or fungal infections not responding to at least 3 days of appropriate antimicrobial therapy, in situations where the granulocyte count is not expected to recover within 7 days. 156
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5. Donor/recipient ABO incompatibility and transfusion support 5.1. Background Approximately 15–25% of HLA identical sibling donor/recipient pairs are ABO incompatible. The figure is higher in alternative donor transplants. In myeloablative transplants ABO incompatibility is associated with an increased risk of delayed red cell engraftment, pure red cell aplasia (PRCA), haemolysis and increased transfusion requirements. There are some reports of increased platelet transfusion requirements. ABO mismatch does not affect neutrophil engraftment, the incidence of graft rejection, GvHD, disease progression or overall survival (14). In RIC transplants it was demonstrated by chimerism studies that early erythroid progenitors engrafted as promptly as myeloid progenitors (15). However, as with myeloablative HSCT, engraftment of mature red cells is delayed, cases of PRCA have also been reported and ABO mismatch is associated with increased red cell transfusion requirements (16). Recipient plasma cells produce anti-donor ABO alloagglutinins and after RIC HSCT the rate of decline of anti-donor alloagglutinins takes twice as long as after myeloablative conditioning, and this can lead to more haemolysis (17). In one report of 40 patients who had RIC HSCT, ABO mismatch was associated with one death due to haemolysis, 3 cases of PRCA, 6 cases of thrombotic microangiopathy (3 fatal), an increase in rehospitalisation days, relapse or disease progression and higher TRM (5, 18). By contrast other reports do not show an inferior outcome (15–17). The same authors also reported in 2007 on 32 RIC HSCT; in 10 cases there was a minor or major plus minor (bidirectional) mismatch and in 5 this caused severe haemolysis leading to death in 3 patients (19) (see also 5.3 below). 5.2. Definitions • Major ABO incompatibility is defined as the presence in the recipient's plasma of anti-A, -B or -A,B alloagglutinins reactive with the donor’s red cells, e.g. donor group A and recipient group O. • Minor ABO incompatibility is defined as the presence of anti-A, -B or -A,B alloagglutinins in the donors plasma reactive with the recipient’s red cells, e.g. donor group O and recipient group A. • Major plus minor ABO (bidirectional) incompatibility is defined as the presence in both the donor and recipients plasma of anti-A, -B or -A,B alloagglutinins reactive with recipient and donor cells respectively, e.g. donor group A and recipient group B. 5.3. Incompatible stem cell graft infusion If the alloagglutinin titre is less than 1:64 unmodified bone marrow or PBPC grafts
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may be infused. At higher titres red cells should be removed from the graft. Marrow processing or PBPC collection on certain apheresis machines e.g. the GAMBRO Spectra, usually results in red cell contamination of less than 5 mL and ABO incompatibility may be ignored. Likewise, stem cells that are separated on density gradients and washed can also be infused without regard to ABO incompatibility. Plasma may be removed from the transplant in cases of minor ABO mismatch where the e.g. anti-A titre is high to avoid acute haemolysis in the recipient. Delayed haemolytic transfusion reactions may follow the infusion of donor HSC where there is a minor ABO mismatch. This is called Passenger Lymphocyte Syndrome (PLS) and occurs because of a secondary (anamnestic) immune response mediated via memory B-cells in the graft against recipient ABO antigens. A rise in anti-A, -B or -A,B titre is seen together with anaemia and jaundice. This phenomenon is rarely, if ever, seen when bone marrows are depleted of alloreactive T-lymphocytes using strategies such as alemtuzumab (Campath-1) antibody or CD34 positive cell selection since B-cells are also depleted. In RIC HSCT minor or bidirectional ABO mismatch can be associated with severe and even fatal haemolysis and in one study recipient red cell exchange was performed pre-transplant. This reduced TRM from 53 to 16% and overall survival was improved (65 vs. 40%; both p<0.05) (19). 5.4. Blood groups used for transfusion support Pre-transplant, recipient-type red cells and platelets should be given. Post-transplant (see Figure 1): • For major ABO mismatch use group O red cell products, irrespective of ABO group of recipient or donor until recipient ABO antibodies are undetectable and the antiglobulin test is negative. Give platelets and plasma from donors of the recipient’s ABO type until recipient red cells are no longer detected; • For minor ABO mismatch use red cells of the donor type, i.e. group O throughout. Give platelets and plasma of recipient type until recipient-type red cells are no longer detected; • For major and minor ABO mismatch use group O red cells until recipient ABO antibodies are undetectable and the antiglobulin test is negative and then switch to donor type. For platelets and plasma use group AB until recipient red cells are undetectable; • Following graft rejection, revert to recipient-type red cells and platelets.
6. Conclusion Transfusion support in BMT patients requires special consideration and carefully defined policies. The use of high quality blood components which have a high degree 158
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Figure 1: Strategy for the provision of blood components in ABO mismatched HSCT Recipient group
Group 0
Group AB
Donor group
Major ABO incompatibility
Red cells Plasma/platelets Minor ABO incompatibility
Red cells Plasma/platelets Major and minor ABO incompatibility
Red cells Plasma/platelets 1 1 2 3
2
3
HSCT ABO antibodies to donor RBC not detected. Direct antiglobulin test negative RBC of recipient group no longer detected
of microbiological safety and which are also gamma-irradiated and, in addition, may be CMV seronegative and leukodepleted provides optimum transfusion support and minimises the chance of adverse effects.
References 1. European Union Directive 2002/98/EC http://eur-lex.europa.eu/LexUriServ/site/en/oj/2003/l_033/l_03320030208en00300040.pdf 2. Ramirez-Arcos S, Jenkins C, Dion J, et al. Canadian experience with detection of bacterial contamination in apheresis platelets. Transfusion 2007; 47: 421-429. 3. Pamphilon DH, Rider JR, Barbara JA, Williamson LMP. Prevention of transfusion-transmitted cytomegalovirus infection. Transf Med 1999; 9: 115-123. 4. Narvios AB, de Lima M, Shah H, et al. Transfusion of leukoreduced cellular blood components from cytomegalovirus-unscreened donors in allogeneic hematopoietic transplant recipients: Analysis of 72 recipients. Bone Marrow Transplantation 2005; 36: 499-501. 5. Nichols WG, Price TH, Gooley T, et al. Transfusion-transmitted cytomegalovirus infection after receipt of leukoreduced blood products. Blood 2003; 101: 4195-4200. 6. BCSH Blood Transfusion Task Force. Guidelines on the clinical use of leucocyte-depleted
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blood components. Transf Med 1998; 8: 59-71. 7. BCSH Blood Transfusion Task Force. Guidelines on gamma irradiation of blood components for the prevention of transfusion-associated graft-versus-host disease. Transf Med 1996; 6: 261-271. 8. Weissinger F, Sandmaier BM, Maloney DG, et al. Decreased transfusion requirements for patients receiving nonmyeloablative compared with conventional peripheral blood stem cell transplants from HLA-identical siblings. Blood 2001; 98: 3584-3588. 9. Anderson NA, Gray S, Copplestone JA, et al. A prospective randomised study of three types of platelet concentrates in patients with haematological malignancy: Corrected platelet count increments and frequency of nonhaemolytic febrile transfusion reactions. Transf Med 1996; 7: 33-39. 10.Stanworth SJ, Hyde C, Heddle N, et al. Prophylactic platelet transfusion for haemorrhage after chemotherapy and stem cell transplantation. Cochrane Database Syst. Rev. 2004 Oct 18; (4): CD004269. 11.Wandt H, Schaefer-Eckart K, Frank M, et al. A therapeutic platelet transfusion strategy is safe and feasible in patients after autologous peripheral blood stem cell transplantation. Bone Marrow Transplantation 2006; 37: 387-392. 12.Stanworth SJ, Massey E, Hyde C, et al. Granulocyte transfusions for treating infections in patients with neutropenia or neutrophil dysfunction. Cochrane Database Syst. Rev. 2005 Jul 20; (3): CD005339. 13.Kerr JP, Liakopoulou E, Brown J, et al. The use of stimulated granulocyte transfusions to prevent recurrence of past severe infections after allogeneic stem cell transplantation. British Journal of Haematology 2003; 123: 114-118. 14.Helbig G, Stella-Holowiecka B, Wojnar J, et al. Pure red-cell aplasia following major and bi-directional ABO-incompatible allogeneic stem cell transplantation: Recovery of donorderived erythropoiesis after long-term treatment using different therapeutic strategies. Ann Hematol 2007 May 8 (Epub ahead of print). 15.Maciej Zaucha J, Mielcarek M, Takatu A, et al. Engraftment of early erythroid progenitors is not delayed after non-myeloablative major ABO-incompatible haematopoietic stem cell transplantation. British Journal of Haematology 2002; 119: 740-750. 16.Canals C, Muniz-Diaz E, Martinez C, et al. Impact of ABO incompatibility on allogeneic peripheral blood progenitor cell transplantation after reduced intensity conditioning. Transfusion 2004; 44: 1603-1611. 17.Griffith LM, McCoy JP, Bolan CD, et al. Persistence of recipient plasma cells and anti-donor isohaemagglutinins in patients with delayed donor erythropoiesis after major ABO incompatible non-myeloablative haematopoietic cell transplantation. British Journal of Haematology 2005; 128: 668-675. 18.Worel N, Kalhs P, Keil F, et al. ABO mismatch increases transplant-related morbidity and mortality in patients given nonmyeloablative allogeneic HPC transplantation. Transfusion 2003; 43: 1153-1161. 19.Worel N, Greinix HT, Supper V, et al. Prophylactic red cell exchange for prevention of severe immune hemolysis in minor ABO – mismatched allogeneic peripheral blood progenitor cell transplantation after reduced-intensity conditioning. Transfusion 2007; 47: 1494-1502.
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Mutiple Choice Questionnaire To find the correct answer, go to http://www.esh.org/ebmt-handbook2008answers.htm 1. The European Union (EU) Blood Directive (2002/98/EC) requires that all hospital blood banks should: a) Keep a record of all blood components for 10 years to ensure donor-recipient traceability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Be licensed by EU competent authority following inspection or completion of a compliance report every 3 years . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Establish a haemovigilance system which includes optional reporting of adverse events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Provide training for all hospital transfusion laboratory staff . . . . . . . . . . . . . . 2. The EU Blood Directive (2002/98/EC) requires that all donated blood for transfusion should be tested for all of the following except one. Which one? a) Hepatitis B (HbsAg) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Hepatitis C (Anti-HCV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) HIV 1 and 2 (Anti-HIV 1 + 2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Syphilis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Which of the following statements about leukodepleted blood components is correct? a) Routinely contain less than 5 x 106 leuckocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Their use in transplant patients prevents the development of platelet transfusion refractoriness in >80% of the patients who receive them . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Their use in transplant patients prevents CMV acquisition in all allogeneic SCT where both the donor and recipient are CMV negative . . . . d) Leukodepletion is best done at the patient’s bedside . . . . . . . . . . . . . . . . . . . . . . 4. Gamma irradiation of blood components is indicated: a) For all autologous SCT patients until 1 year post-transplant . . . . . . . . . . . . . . b) After allogeneic SCT until the lymphocyte count is >0.5 x 109/L in the absence of chronic GvHD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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c) In all patients with non-Hodgkin’s lymphoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) In all patients treated with purine analogues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Platelet transfusions given to allograft recipients should always be: a) ABO and Rh compatible . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Given when the platelet count falls below 20 x 109/L . . . . . . . . . . . . . . . . . . . . . c) Tested for high titre anti-A,B if they are to be transfused to non-group O recipients. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Transfused to patients with TTP when the platelet count falls below 50 x 109/L . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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NOTES
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*
CHAPTER 8
Supportive care
T. Masszi
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CHAPTER 8 • Supportive care
1. Introduction Supportive care in HSCT is essential in optimising the outcome of the treatment process. This Chapter will focus on nutritional support, central venous catheter related problems, the use of haematopoietic growth factors, the management of mucosal injury and anti-emesis strategies.
2. Nutritional support Patients with haematological malignancies can be well nourished at the time of transplantation, whereas malnutrition is more frequent in patients with solid tumours. Impaired nutritional status before transplantation is a negative prognostic factor for outcome after HSCT and better-nourished patients have a shorter time to engraftment. Irrespective of nutritional status, however, parenteral nutritional support is commonly administered prophylactically after HSCT until patients are able to maintain an adequate oral nutritional intake, usually following BM recovery. Although enteral nutrition is theoretically possible, total parenteral nutrition (TPN) is largely favoured in HSCT patients mainly because nausea, vomiting and orooesophageal mucositis prevent the insertion and subsequent tolerability of nasogastric tubes. Moreover virtually all patients undergoing HSCT have a CVC in situ through which TPN can be easily administered. Finally, parenteral nutrition probably allows better modulation of fluid, electrolyte, and nutrient administration which can be of critical importance when complications such as GvHD or VOD arise. 2.1. Total parenteral nutrition The routine use of TPN during transplantation is based on a study performed twenty years ago that randomly assigned 137 previously well nourished patients undergoing BMT for different malignancies (mainly acute leukaemia) to either prophylactic TPN or intravenous maintenance fluids (dextrose, electrolytes, minerals, trace elements and vitamins). Treatment was started during conditioning, and continued for 4 weeks following transplantation. Compared to the control arm, patients receiving TPN had significantly better OS and DFS, and a longer time to relapse. Notably, 61% of control patients eventually required TPN because of a decline in their nutritional status (1). In contrast other studies have not provided strong support for the routine use of TPN in patients HSCT. Moreover, the care of recipients of HSCT has changed significantly over the last twenty years, e.g. hospital stays are shorter due to the increasing use of PBSCT and the use of recombinant haematopoietic growth factors, and a number of questions remain unanswered regarding nutritional support in current management. For instance, in a prospective randomised trial, 57 patients undergoing BMT received either prophylactic TPN or an enteral feeding program (2). TPN was
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associated with significantly more days of diuretic use, more frequent hyperglycaemia and more CVC related complications. Although the patient cohort was small this study suggests that the role of TPN in patients undergoing HSCT deserves further investigation. In study, which was double-blinded, 258 patients were randomly assigned to receive either TPN or hydration in an outpatient setting. Patients who received TPN had a delay in the resumption of 85% of their caloric requirement (16 vs. 10 days), suggesting that the administration of TPN may suppress normal appetite (3). There were no effects of TPN on hospital readmission, disease relapse, or survival. Although this study did not specifically examine whether prophylactic TPN is indicated for patients who receive HSCT, it suggests that after hospital discharge, TPN may not be necessary for all patients. One further study attempted to define subgroups of patients with haematological malignancies receiving intensive chemotherapy (with or without transplantation), who were likely to require TPN (4). They used the following three criteria to define the need for TPN: severe malnutrition at admission, a prolonged (at least 7–10 days) period of minimal oral intake or clinical weight loss exceeding 10% during treatment. Any one of these criteria was sufficient. According to these definitions TPN was found to be necessary in only 55% of patients undergoing HSCT with a range from 37% of autologous recipients to 92% of those receiving an HLA mismatched allograft. 2.2. Caloric, protein and other necessities It is clear that energy expenditure differs between auto- and allo-HSCT recipients. Also, there is a consensus that energy requirements in such patients may exceed 130–150% of the estimated basal energy expenditure. Protein needs are also elevated. The usual amino acid dose in TPN is 1.5–2 g/kg/day. The use of fat as an energy source is safe especially as fat-free TPN can be complicated by hepatic steatosis and glycogenosis. Thus a balanced caloric intake with both fat and carbohydrate is recommended. Long chain triglycerides (LCT), containing saturated fatty acid moieties of 20–24 carbons or a mixture of long chain and medium chain triglycerides (MCT, 6–12 carbons) should provide 30% of caloric intake. MCT seem to be advantageous in TPN over LCT because they are more water soluble, more rapidly cleared from plasma, have protein sparing effects, do not accumulate in the liver or adipose tissue and also do not serve as precursors of eicosanoids (e.g. prostaglandins and leukotrienes) and so might improve immune function and decrease inflammation. However, clinical studies in patients with respiratory illnesses, hepatic dysfunction, and sepsis with multi-system organ failure have suggested that mixtures of MCT and LCT are advantageous compared to LCT alone (5). Electrolytes, minerals, vitamins and trace elements (chromium, zinc, copper, manganese, selenium) 166
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are added to the TPN according to the recommended daily amount. Glutamine supplementation of TPN has been addressed recently, as glutamine is an important precursor for nucleotide synthesis and thus can be a source for rapidly dividing cells, such as gastrointestinal epithelial cells. Although several studies have evaluated the effect of enteral or parenteral administration of glutamine on gastrointestinal toxicity, none have shown a clear preventative or therapeutic effect on intestinal mucositis. On the other hand, prospective studies have suggested positive effects of glutamine administration after HSCT on nitrogen balance, infectious complications and length of hospital stay (6). Preliminary data support the concept that parenteral glutamine may preserve hepatic function in HSCT recipients by maintaining hepatic glutathione concentrations which protect hepatocytes from the oxidant stress of the conditioning regimen. Glutamine supplementation may also have a role in preventing and possibly treating VOD (7). 2.3. Evaluation of nutritional status and monitoring nutritional support Nitrogen balance is considered the most accurate way of assessing nutritional status in HSCT recipients, as it is the direct expression of the imbalance existing between protein breakdown and synthesis. From studies published of TPN in HSCT some kind of consensus can be derived concerning what parameters should be used to monitor nutritional status/support. Daily monitoring of weight (primarily to judge hydration status) is essential, together with electrolytes, BUN/urea, creatinine, and glucose. Liver function tests should be measured at least twice per week as abnormalities can result from TPN. Finally, weekly serum albumin, transferrin (reflective of amino acid intake for visceral protein synthesis) triglyceride and nitrogen balance are also helpful (Table 1). 2.4. Timing of nutritional support Current practice in the timing of TPN is heterogeneous. In the study of Weisdorf et
Table 1: Monitoring of nutritional support during the in-patient stay Daily
Two times a week
Once a week
- weight (fluid balance) - blood glucose - serum electrolytes - BUN - serum creatinine - calorie and protein intake
- liver function tests - serum calcium - serum magnesium - serum phosphorus
- nitrogen balance - serum transferrin - serum albumin - serum triglyceride - serum zinc
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al. (1) (which became the scientific basis of routine administration of TPN in HSCT patients), parenteral nutrition was started before the conditioning regimen and continued to day 28 following HSCT. However in many centres TPN is started only when severe mucositis develops. Others initiate TPN on day 1 post transplant and continue for 2–3 weeks according to the intensity and duration of mucositis. TPN is usually not administered routinely to recipients of auto-HSCT unless prolonged mucositis occurs (8). Simultaneous use of parenteral feeding and oral intake is also contradictory. According to one theory oral intake should be avoided during TPN to minimise the risk of gut contamination. However most studies consider concomitant oral ingestion useful as even small amounts of enteral intake can reduce infection rates or hepatic complications of TPN by avoiding the enteric stasis that results in high levels of endotoxins in the portal venous blood. 2.5. Complications of TPN Complications essentially can be divided into two types: metabolic complications or those connected with the central venous catheter. 2.5.1. Metabolic complications Abnormal liver function is the major metabolic complication of TPN. The initial manifestation is usually an elevation of transaminases, occurring 1–2 weeks after the start of TPN. Increases in serum bilirubin and alkaline phosphatase generally occur 1 or 2 weeks later. These changes often resolve spontaneously without longterm consequences especially if the period of administration of TPN is not longer than 3 months. However there are many other possible causes of elevated liver enzymes after HSCT and the differential diagnosis can be difficult. Drug toxicity, infections, GvHD, VOD, or recurrence of the original malignancy should also be considered (Table 2). If all these possible causes are excluded various other measures can be tried e.g. shortening the period of infusion of TPN from 24 hours to 12–20 hours, introducing some oral feeding, decreasing the non-protein caloric intake and commencing treatment with ursodeoxycholic acid (Table 3). Table 2: Common causes of elevated liver enzymes in the post transplant period Side effects of drugs (methotrexate, cyclosporin-A) Infections (bacterial, fungal, viral) • Veno-occlusive liver disease • Graft versus host disease • Relapse of malignancy • Parental nutrition • •
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Table 3: Treatment modalities in elevated liver enzymes during TPN Search for causes other than TPN (see Table 2) Shorten TPN cycle to 12-20 from 24 hours/day • Reduce the non-protein caloric intake by 10-15% of the total daily calories • Initiate some oral intake if possible • Treat with ursodeoxycholic acid • Utilise oral metronidazole to decrease enteral endotoxin formation • •
2.5.2. Complications related to the central venous catheter Although CVC are indispensable in HSCT, they also represent a significant source of complications including infections, venous thromboembolism, mechanical obstruction, dislodgment and leakage. The most important of these complications are infections, the incidence of which remains high despite the use of aseptic techniques. In a prospective study of 111 HSCT recipients, representing 143 Hickman catheter placements, 44% of patients had positive blood cultures. Most of these (40/63) were coagulase-negative Staphylococci suggesting a primary line infection rather than a secondary contamination from a blood borne source. 2.6. Prevention of central venous catheter related infections A catheter related blood stream infection is defined as bacteraemia or fungaemia in a patient with an intravascular catheter with at least one positive blood culture obtained from a peripheral vein, clinical manifestation of infections (i.e. fever, chills, and/or hypotension) and no apparent source for the blood stream infection except for the catheter. One of the following should be present: • A positive semi-quantitative (more than 15 CFU/catheter segment - i.e. piece of catheter sent for culture) or quantitative (more than 103CFU/catheter segment) culture where the same organism (species and antibiogram) is isolated from the catheter segment and peripheral blood. • Simultaneous quantitative blood cultures with a ratio higher than 5:1 comparing CFU from the CVC sample vs. a peripheral blood sample. • A differential period of CVC culture vs. peripheral blood culture positivity of greater than 2 hours. There are four main possible mechanisms for developing a CVC related infection: • Migration of skin organisms at the insertion site into the cutaneous catheter tract with colonisation of the catheter tip. • Contamination of the catheter hub leading to intraluminal colonisation. • Occasionally, catheters might be haematogenously seeded from another focus of infection and rarely. • Infusate contamination. HAEMATOPOIETIC STEM CELL TRANSPLANTATION
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The most important recommendations concerning the prevention of CVC related blood stream infections are listed in Table 4 based on the guidelines developed in the USA by a working group led by the Infectious Disease Society of America (9).
Table 4: Recommendations for prevention of CVC related blood stream infections
170
Education
Health-care worker education and training for the insertion and maintenance of CVCs is essential. Moreover, periodic assessment of their knowledge of and adherence to is strongly recommended. Trained personnel for the insertion and maintenance of CVCs should be designated.
Catheters and materials
An important pathogenetic determinant is the material from which the device is made. In vitro studies demonstrate that CVCs made of polyethylene or polyvinyl chloride are less resistant to adhesion of micro-organisms than are CVCs made of teflon, silicone or polyurethane. The number of ports must be kept to the minimum required for the patient's management. Cuffed and tunnelled CVCs should be employed if their use is to be prolonged (e.g. allogeneic transplant).
Site of catheter insertion
In adults, a subclavian site is preferred as lower extremity sites are associated with a higher risk of infection (and deep venous thrombosis). Subclavian sites also reduce the risk of infection compared to jugular sites.
Maximal sterile barrier precautions during insertion
Full aseptic techniques should be used at the time of insertion. 2% aqueous chlorhexidine gluconate (preferably), tincture of iodine, or 70% alcohol can be used to prepare the skin before CVC insertion. Organic solvents (e.g. acetone and ether) should not be applied.
Catheter and catheter site care
One port should be designated exclusively for hyperalimentation if a multilumen catheter is used. The routine use of prophylactic intranasal or systemic antibiotics before insertion or during the use of the CVC and antibiotic lock solutions are not recommended. The catheter site can be covered by sterile gauze or a sterile, transparent semi-permeable dressing and the dressings should be replaced whenever they become damp or loosened. Gauze dressings should be replaced at least every two days and transparent dressings every 7 days. The catheter sites must be monitored visually or by palpation regularly and if patients have tenderness at the insertion site and/or fever without obvious source, the dressing should be removed for thorough examination.
Replacement of catheter
Catheters that are no longer essential should be removed promptly. However routine replacement of CVCs to prevent catheter related infections is not advised. CVCs must not be removed on the basis of fever alone. Clinical judgement is required to assess the appropriateness of catheter removal if infection is evidenced elsewhere or if a non-infectious cause of fever is suspected.
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2.7. Conclusions Nutritional support is an integral part of the supportive care of patients receiving HSCT and the main tool remains TPN. Further studies will be required to determine the exact role of TPN in the course of HSCT. Until then, it seems to be prudent to administer TPN to patients undergoing HSCT if they have severe mucositis or gastrointestinal manifestations of GvHD, when a long period of insufficient oral intake is anticipated. The most common complications of TPN are CVC related.
3. Haematopoietic growth factors 3.1. G-CSF and GM-CSF The application of recombinant human haematopoietic growth factors such as G-CSF and GM-CSF have resulted in clear clinical benefits in the post-transplantation setting, especially in auto-BMT and PBSCT. GM-CSF is no longer readily available in Europe but many of the results from clinical trials of GM-CSF can be extrapolated to G-CSF, hence its mention here. Growth factor therapy is usually started 1–3 days after transplantation and continued until the white blood count reaches 5.0–10 x 109/L. 3.1.1. Autologous transplantation Both G-CSF and GM-CSF have been shown to shorten the time to engraftment in randomised phase III trials. Some studies have also demonstrated a reduction in the number of days of antibiotic therapy and length of hospitalisation. In one study of recipients of auto-BMT, GM-CSF therapy reduced both the time to reach an absolute neutrophil count (ANC) of 0.5 x 109/L and the incidence of bacterial infections (15 vs. 28 days and 36 vs. 63% respectively) compared to placebo. Similarly, in another controlled trial GM-CSF therapy was associated with a shorter median time to an ANC of 0.5 x 109/L (14 vs. 21 days) and a shorter duration of hospitalisation (23 vs. 28 days). However, the positive effects in these trials were limited to neutrophil engraftment and neutropenia-related complications. None of the studies have demonstrated enhanced platelet or red blood cell engraftment and none of them showed a survival advantage. The value of colony stimulating factors following auto-PBSCT is less impressive. The addition of G-CSF may further accelerate neutrophil engraftment, with 3–6 fewer days to achieve an ANC of 0.5 x 109/L. There have been no comparative studies of different colony stimulating factors (10). 3.1.2. Allogeneic transplantation In a randomised, controlled trial, 109 patients who underwent allo-BMT received either GM-CSF or placebo following the transplantation of SC. GM-CSF was associated
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with a significant shortening of the time to engraftment (ANC above 0.5 x 109/L 13 vs. 17 days) and a reduction in infectious complications. No differences could be detected in platelet or erythrocyte recovery (11). Another study randomised 54 patients undergoing allo-PBSCT to receive either G-CSF or placebo. The G-CSF arm was associated with significant acceleration of engraftment (median time to ANC above 0.5 x 109/L was 11 vs. 15 days p = 0.008). No differences were found in time to platelet engraftment, red blood cell transfusion independence, incidence of aGvHD or 100 day mortality (12). However the long term benefit of colony stimulating factor therapy following allo-HSCT remains controversial, as it may be associated with an increased risk of GvHD, especially cGvHD. In a retrospective study of 155 recipients sibling transplants at a single centre, patients treated with G-CSF after the transplant (43%) had a significantly higher incidence of grades II-IV acute GvHD (13). Moreover, a recent retrospective EBMT study of 1789 acute leukaemia patients concluded that G-CSF should probably not be used after HSCT from HLA identical sibling donors because it was associated with increased risks of GvHD and TRM (14). In the haplo-identical setting, post-transplant administration of G-CSF was found to impair functional immune recovery by promoting T-helper (Th)-2 immune responses which, unlike Th-1 responses, do not protect against intracellular pathogens and fungi. 3.2. Erythropoietin (EPO) EPO has been used to accelerate the recovery of red blood cells. This idea was supported by the observation that EPO levels after transplantation were lower than calculated for the degree of anaemia. However studies of its usage in this situation have not shown any major benefits. In allo-HSCT some studies have shown a reduction in the median time to transfusion independence but transfusion requirements were not different. There were however subsets of patients at high risk for transfusion in which EPO reduced transfusion needs. Because of the modest and mixed results of the studies, most centres do not use EPO in the early posttransplant setting. A few randomised trials have addressed the efficacy of EPO following autologous transplantation. None found a significant reduction of transfusion requirements. The lack of benefit is particularly true with PBSCT. The use of thrombopoietin for accelerating platelet recovery is still under investigation. 3.3. Conclusion Evidence exists in favour of the use of G-CSF (and GM-CSF) in some transplantation settings. Besides stem cell mobilisation, these include their use for accelerating engraftment following autologous stem cell transplantation (bone marrow and 172
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peripheral blood as well). The prophylactic use of G-CSF or GM-CSF following allogeneic stem cell transplantation is generally not recommended except for the cases of delayed neutrophil engraftment or secondary neutropenia due to infection or drug toxicities.
4. Oral mucositis, mouth care and pain relief Despite the benefits of TPN, mucositis per se remains an important clinical problem. Mucositis is usually managed by the application of topical and systemic pain medications. Many patients require narcotic pain medication and the length of time of intravenous narcotic need is one of the best indicators of the degree of mucositis. Routine mouth care typically consists of oral rinses performed every four hours with sodium bicarbonate or chlorhexidine. Patients should cease using a toothbrush (even a soft brush) while neutropenic and should not restart until the neutrophil count exceeds 1.0 x 109/L. Dentures are generally removed for the duration of mucositis because of discomfort. A variety of mucosal coating agents have been used with or without analgesics: diphenhydramine, corticosteroids, antacids, and sodium hyaluronate. Unfortunately, the evidence supporting benefit for any of these interventions is weak. Analgesia may necessitate systemic narcotics. In a study of 119 BMT recipients patient controlled analgesia with IV morphine, hydromorphone, or sufentanil resulted in nearly equivalent pain relief but side effects were lowest in the morphine group. Thus, morphine was recommended as the opioid of choice for patient controlled analgesia. Mucositis is associated with an increased risk of systemic infection resulting from bacteraemia associated with the breakdown of mucosal barriers. The severity of mucositis and the associated infection risk is a significant cause of morbidity and mortality, and more effective treatment modalities are therefore needed (15). 4.1. Keratinocyte growth factor (KGF, palifermin, Kepivance®) One new approach is the use of KGF. The efficacy of prophylactic intravenous palifermin was demonstrated in a double-blind multicentre trial of 212 patients undergoing HSCT for haematological malignancies. Significantly fewer patients receiving palifermin had grade 3 or 4 mucositis (63 vs. 98 percent with placebo) and the duration of mucositis was shorter (median 6 vs. 9 days). These benefits were associated with significantly less use of opioid analgesics and less frequent requirement for TPN support. On the basis of this study palifermin was approved for the reduction of mucositis following HSCT. However an unsettled issue is the effect of palifermin on GvHD; in an animal model it seemed to decrease the risk of GvHD but in a human clinical trial KGF did not show any effect on GvHD (16).
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4.2. Amifostine Amifostine is a phosphorylated aminothiol, which has a protective effect on normal tissues against radiation and alkylating agent toxicity. In a retrospective study of 68 patients receiving high dose melphalan prior to auto-HSCT those who received amifostine (35 pts) prior to conditioning had a significantly lower median number of days of analgesic therapy (0 vs. 6 days) and a lower incidence of severe mucositis (21 vs. 51%). There was no difference in haematological recovery between the two groups. The results of other attempts such as the application of TGF-b, IL-11, or laser therapy are even less mature. 4.3. Conclusion Several approaches have been tried for the prophylaxis or therapy of mucositis. However the problem has not yet been solved. A number of new agents promise clinical benefit, either singly or in combination therapy. Further studies are warranted.
5. Prevention and treatment of chemotherapy induced nausea and vomiting The objective of antiemetic treatment in the transplant setting is the complete prevention of nausea and vomiting caused by the conditioning regimen. For the classification of CT induced vomiting and the emetogenic potential of cytotoxic therapy (see Tables 5 and 6). Table 5: Classification of CT induced emesis Acute emesis
Delayed emesis
Anticipatory emesis
Occurs during the first 24 h following CT
Occurs later than 24 h
Conditioned response of patients who developed significant nausea and vomiting during previous CT
Table 6: Classification of emetogenic potential of CT Emetogenic potential High Moderate Low
Probability of vomiting (a) 60–99% 30–59% 1–29%
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The high dose CT/RT used in standard conditioning regimens (see Chapter 6) has itself high emetic potential, and there are some additional factors (consecutive day administration, prior cytotoxic treatment, and other medication e.g. opiate analgesics, which may cause emesis), which can increase the risk of vomiting. 5.1. Prophylaxis and treatment There are few randomised trials specifically studying the issue of nausea/vomiting in the transplant setting. There is a general consensus that a combination of a serotonin receptor antagonist (ondansetron, granisetron, tropisetron) and dexamethasone (8–20 mg IV) should be the standard prophylaxis on days of the conditioning. In case of failure of the prophylaxis adding of further dexamethasone (max 20 mg/day) and/or a benzodiazepine (e.g. lorazepam max 4 mg IV) may help to counter increased patient anxiety and possible anticipatory emesis. An alternative is to switch to a different serotonin antagonist, since there is an incomplete cross-resistance between agents (Figure 1).
Figure 1: Prevention and treatment of conditioning induced nausea/vomiting
On days of conditioning serotonin antagonist od-bd IV + dexamethasone 12 mg od IV
Does prophylactic regimen control emesis?
Yes
Continue
No
1. Rule out anticipatory emesis, consider benzodiazepine (e.g. lorazepam max 4 mg/d IV) 2. Add dexamethasone max 20 mg/d IV 3. Consider switch to another serotonin antagonist 4. Exclude other aetiologies
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Neurokinin–1 receptor antagonist aprepitant represents a new class of antiemetic drugs. When used in combination with serotonin antagonists and corticosteroids, aprepitant appears to provide better protection against both acute and delayed emesis in very highly emetic CT.
References 1. Weisdorf SA, Lysne J, Wind D, et al. Prophylactic total parenteral nutrition on long-term outcome of bone marrow transplantation. Transplantation 1987; 48: 833-838. 2. Szeluga DJ, Stuart RK, Brookmeyer R, et al. Nutritional support of bone marrow parenteral nutrition to en enteral feeding program. Cancer Res 1987; 47: 3309-3316. 3. Charuhas PM, Fosberg KL, Breummer B, et al. A double blind randomized trial comparing outpatient parenteral nutrition with intravenous hydration: Effect on resumption of oral intake after marrow transplantation. J Parenter Enteral Nutr 1997; 21: 157-161. 4. Iestra JA, Fibbe WE, Zwinderman AH, et al. Parenteral nutrition following intensive cytotoxic therapy: An exploratory study on the need for parenteral nutrition after various treatment approaches for haematological malignancies. Bone Marrow Transplant 1999; 23: 933-939. 5. Ulrich H, Pastores SM, Katz DP, et al. Parenteral use of medium-chain triglycerides: A reappraisal. Nutrition 1996; 12: 231-238. 6. Ziegler TR, Young LS, Benfell K, et al. Clinical and metabolic efficacy of glutamine supplemented parenteral nutrition after bone marrow transplantation. A randomized, double-blind, controlled study. Ann Intern Med 1992; 116: 821-828. 7. Brown SA, Goringe A, Fegan C, et al. Parenteral glutamine protects hepatic function during bone parrow transplnatation. Bone Marrow Transplant 1998; 22: 281-284. 8. Muscaritoli M, Grieco G, Capria S, et al. Nutritional and metabolic support in patients undergoing bone marrow transplantation. Am J Clin Nutr 2002; 75: 183-190. 9. O’Grady NP, Alexander M, Patchen Dellinger E, et al. Guidelines for the prevention of intravascular catheter-related infections. CID 2002; 35: 1281-1307. 10.Dekker A, Bulley S, Beyene J, et al. Meta-analysis of randomized controlled trials of prophylactic granulocyte colony-stimulating factor and granulocyte-macrophage colonystimulating factor after autologous and allogeneic stem cell transplantation. J Clin Oncol 2006; 24: 5207-5215. 11.Nemunaitis J, Rosenfeld CS, Ash R, et al. Phase III randomized, double-bind placebo controlled trial of rhGM-CSF following allogeneic bone marrow transplantation. Bone Marrow Transplant 1995; 15: 949-954. 12.Bishop MR, Tarantolo SR, Geller RB, et al. A randomized double-blind trial of filgastrim (granulocyte colony-stimulating factor) versus placebo following allogeneic blood stem cell transplantation. Blood 2000; 96: 80-85. 13.Remberger M, Naseh N, Aschan J, et al. G-CSF given after haematopoietic stem cell transplantation using HLA identical sibling donors is associated to higher incidence of acute GvHD II-IV. Bone Marrow Transplant 2003; 32: 217-223. 14.Ringden O, Labopin M, Gorin NC, et al. Treatment with granulocyte colony-stimulating 176
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factor after allogeneic bone marrow transplantation for acute leukemia increases the risk of graft-versus-host disease and death: A study from the acute leukemia working party of the European Group for Blood and Marrow Transplantation. J Clin Oncol 2004; 22: 416423. 15.Fanning SR, Rybicki L, Kalaycio M, et al. Severe mucositis is associated with reduced survival after autologous stem cell transplantation for lymphoid malignancies. Br J Haematol 2006; 135: 374-381. 16.Blazar BR, Weisdorf DJ, Defor T, et al. Phase 1/2 randomized, placebo-control trial of palifermin to prevent graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (HSCT). Blood 2006; 108: 3216-3222.
Mutiple Choice Questionnaire To find the correct answer, go to http://www.esh.org/ebmt-handbook2008answers.htm 1. Which of the following is the most accurate for assessing nutritional status in HSCT recipients? a) Serum cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Serum glucose. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Nitrogen balance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Anthropometric measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. What is the most common complication of TPN? a) Elevated liver enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Hypertriglyceridaemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Elevated serum creatinine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Catheter-related complications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Which of the following is not considered a standard indication for the use of G-SF? a) Stem cell mobilisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Accelerate engraftment following allo-HSCT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Accelerate engraftment following auto-HSCT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Treat secondary neutropenia following transplant due to drug toxicity . . .
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4. Which statement is not true regarding the CVC? a) Health-care worker education and training for the insertion and maintenance of the CVC is essential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) In adults, a subclavian site is preferred . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Catheters that are no longer essential should be removed promptly . . . . . . d) Polyvinyl chloride catheters are more resistant to adhesion of micro-organisms than teflon, silicone, or polyurethane catheters . . . . . . . . 5. Which statement is not true concerning oral mucositis? a) Palifermin is especially indicated in allo-HSCT as it decreases the risk of GvHD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Oral mucositis per se is a significant cause of morbidity and mortality of HSCT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Patients should stop using a toothbrush while neutropenic . . . . . . . . . . . . . . . d) Routine mouth care typically consists of oral rinses performed every four hours with chlorhexidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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NOTES
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*
CHAPTER 9
Early complications after HSCT
E. Carreras
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CHAPTER 9 • Early complications after HSCT
1. Introduction The high dose of RT and/or CT included in conditioning regimens (see Chapter 6) affects all organs and tissues of the recipient, producing several early and late secondary effects of variable intensity. The most common early effects like nausea, vomiting, mucositis and pain are treated in Chapter 8, and late effects are covered in Chapter 12. In this Chapter we summarise some early complications that, despite not being very frequent, are an important cause of morbidity and mortality.
2. Haemorrhagic cystitis (HC) (1) Pathogenesis HC is produced by direct toxicity of the conditioning regimen on the urothelium or by viral infections affecting the urinary tract. Usually, HC secondary to conditioning appears early after HSCT (several days after receiving CT agents) and can be produced by Cy (or ifosfamide), Bu (especially if combined with Cy), VP or TBI (both uncommon). Viral HC appears later (usually after day +30) and can be due to human polyomavirus type BK or JC, adenovirus type 11 (less frequent) or CMV (exceptional). Incidence HC secondary to CT: 1 to 25%, depending on the preventive measures adopted. The incidence of viral HC is not well established; 5 to 25%, according to the degree of immunosuppression of the recipient. Prophylaxis Continuous irrigation of the bladder during conditioning is effective but is no longer used. Nowadays, HC prophylaxis is based on hyperhydration and Mesna administration. The recommended daily dose of hydration is 3 L/m2. The usual daily dose of Mesna is 1.0–1.5 x daily dose of Cy (e.g. daily dose of Cy: 4.2 g Æ daily dose of Mesna: 4.2–6.3 g) administered IV as: a) continuous infusion in 1 L of 0.9% saline over 12–24 hrs, beginning 4 hours prior to the 1st dose of Cy and ending 12–24 hrs after the last Cy dose; or b) bolus injections, 20% of daily dose of Cy dose administered as a bolus 1/2–1 h before Cy and the remaining daily dose divided into bolus injections q 2–3 h, maintained up to 12–24 after ending Cy. Despite these classical forms of administration, some pharmacodynamic studies have shown that the best method of administering Mesna is to combine a continuous infusion with intermittent bolus injections during the 6 hrs after Cy dose. The presence of acrolein in the bladder 24 h after Cy makes it advisable to prolong Mesna administration 24 hrs after the last Cy dose. Treatment Treatment should be based on a three step approach: 1. Forced hydration plus intensive platelet support. The use of procoagulant agents
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like aminocaproic acid is contraindicated because they favour clot formation in the bladder. 2. Continuous bladder irrigation with saline solution. Some success has been reported with bladder instillation of alumina 1% (risk of encephalopathy if associated renal failure), prostaglandin E2 or E1, GM-CSF or cidofovir (all with limited experience). Similarly, hyperbaric oxygen or oestrogens (2–4 mg q 8 h p.o., Odermann et al., BMT 2000) have also been reported as effective measures. 3. If the previous measures do not solve HC other salvage approaches can be considered: selective embolisation of bladder arteries (one of the simplest and effective measure in the hands of an expert angioradiologist) (2); suprapubic cystotomy; cystoscopy + installation of formalin (very painful, risk of scars and bladder contraction, requires anaesthesia); catheterisation of both urethers to rest the bladder; hypogastric bond (can produce sexual impotence); and, as a last resort, cystectomy.
3. Early complications of vascular origin Injury of the vascular endothelium seems to be the most important initial event in a variety of complications with imprecise diagnostic criteria and overlapping clinical features, which are observed within the first 30–60 days after HSCT. The best defined syndromes resulting from this endothelial injury are: 1) Veno-occlusive disease of the liver; 2) Capillary leak syndrome; 3) Engraftment syndrome; 4) Diffuse alveolar haemorrhage; 5) Thrombotic microangiopathy; 6) Idiopathic pneumonia syndrome; 7) Multiple-organ dysfunction syndrome. Figure 1 shows their common pathogenesis. 3.1. Hepatic veno-occlusive disease (VOD) Definition VOD is the term used to designate the symptoms and signs that appear early after HSCT as a consequence of the conditioning regimen-related hepatic toxicity. This syndrome is characterised by jaundice, fluid retention and tender hepatomegaly appearing in the first 35–40 days after HSCT (3–8). Pathogenesis (3, 6–8) The hepatic metabolism of certain drugs (e.g. Cy) by the cytochrome P-450 enzymatic system produces several toxic metabolites (e.g. acrolein). These toxic metabolites are converted to stable (non-toxic) metabolites by the glutathione enzymatic system (GSH) and eliminated. When this process occurs in patients with a reduced GSH activity, due either to pre-existing liver disease or to the action of agents as Bu, BCNU or TBI that reduce GSH levels, toxic metabolites are not 182
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Figure 1: Common pathogenesis of early complications of vascular origin after HSCT
VEGF: vascular endothelial growth factor; Tx: thromboxane; PG: prostaglandin; TM: thrombomodulin; PAI-1: plasminogen activator inhibitor type 1
metabolised. Toxic metabolites are predominantly located in area 3 of the acinus (around centrilobular veins) because this area is rich in P-450 and poor in glutathione. Consequently, damage to hepatocytes and sinusoidal endothelium occurs predominantly in this anatomic zone. The remaining factors mentioned in Figure 1 can also contribute to the endothelial injury. Experimental models show that the first events after endothelial injury by toxic metabolites are loss of sinusoidal endothelial cell (SEC) fenestrae, formation of gaps within and between SEC, and rounding up or swelling of SEC. Consequently, red blood cells penetrate into space of Disse and dissect off the sinusoidal lining, which embolises downstream and blocks the sinusoids reducing the hepatic venous outflow and producing post-sinusoidal hypertension. In the light of all these observations some authors have proposed the term of sinusoidal obstruction syndrome (SOS) for this complication. Clinical features (3, 4, 6, 8) Classical VOD. Occurs within days after conditioning (from day -1 to +14) and is characterised by presence of: jaundice (in almost 100% of cases), hepatomegaly and/or
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right upper quadrant pain, and weight gain (not attributable to excessive fluid administration) with oedemas and ascites. Late VOD. The same clinical manifestations as classical VOD but developing late after HSCT (one third of cases occur after patient discharge). Mainly observed after conditioning including several alkylating agents in combination (busulfan, melphalan, thiotepa). One third have a biphasic course with an initial transitory peak followed by a definitive late phase (5) VOD with multiorgan failure. The same clinical manifestations as previously described plus: thrombocytopenia (refractoriness to platelet transfusions); pleural effusion / pulmonary infiltrates; progressive renal, cardiac, pulmonary failure; confusion, encephalopathy and coma. Incidence The incidence of VOD ranges from 3 to 54% in the largest series. This variability is the consequence of the presence or absence of a number of well-known risk factors for this complication (see Table 1). In the only prospective multicentre study published the incidence of VOD was 8% in allo-HSCT and 3% in auto-HSCT (6). Diagnostic criteria As for any syndrome the diagnosis of VOD must be established clinically. All HSCT teams use one of the following sets of clinical criteria (3, 4, 6 ,8): Seattle criteria: In the first 20 days after HSCT presence of two or more of the following: bilirubin > 2 mg/dL; hepatomegaly or pain in the right-upper quadrant; weight gain (>2% basal weight). Baltimore criteria: In first 21 days after HSCT, presence of bilirubin >2 mg/dL plus ≥2 of the following: painful hepatomegaly; ascites; weight gain (>5% basal weight). In both, other possible causes of these clinical features should be excluded before accepting the diagnosis of VOD (see differential diagnosis). Additionally, it is necessary to remember that some VOD cases can appear late after HSCT. Additional investigations Other complementary studies that can aid diagnosis are: Haemodynamic study of the liver carried out through the jugular or femoral veins (9): Despite its usefulness, this is only indicated to confirm the diagnosis of VOD before adopting a therapeutic approach that may be potentially hazardous for the patient. An hepatic venous gradient pressure (HVGP) ≥10 mmHg in a patient without previous liver disease allows a precise differential diagnosis with a high degree of specificity. However, a normal HVGP does not exclude the diagnosis of VOD. Liver biopsy: Thrombocytopenia usually present in this phase of HSCT precludes a transparietal liver biopsy; consequently hepatic tissue can only be obtained by means of a transvenous biopsy in the course of a haemodynamic study. In addition to the 184
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Table 1: Risk factors (3, 4, 6–8) Risk
Lower risk < Higher risk
Transplant type Donor type HLA compatibility Stem cell origin T-cell depletion Diagnosis Status of the disease
Syngeneic or autologous < allogeneic Sibling < another relative < unrelated HLA match < any mismatch Peripheral blood < bone marrow With TCD < without TCD non malignant disease < malignant disease Remission < relapse
Conditioning - Intensity - TBI
- Busulfan - Timing Age / Sex Karnofsky index ASAT/ALAT before HSCT Transplant number Previous hepatic irradiation Previous Mylotarg Status of the liver CMV serological status Fever in conditioning Hepatotoxic drugs Genetic predisposition
Cy alone < Cy + TBI < BVC (a) Fractionated TBI < single dose TBI Less than 12 Gy < more than 12 Gy Low dose rate < high dose rate IV BU < adjusted oral Bu < non adjusted oral Bu Interval Cy – TBI 36 hours < 12 hours Younger < older / men < women 100–90 < lower than 90 Normal < high First < second No < yes No < yes (b) Normal < fibrosis < cirrhosis or infiltration Negative < positive Absent < present Progestogens, ketoconazole, CsA, methotrexate, amphotericin B, vancomycin, acyclovir, IV Ig (c) GSTM1 positive < GSTM1 null genotype (d)
The most important risk factors are indicated in bold type. (a) BVC (BCNU, VP, Cy). (b) VOD incidence up to 64% (Wadleigh et al. Blood 2003). (c) Higher incidence of VOD with high-dose IVIg. (d) Srivastava et al., Blood 2004
classical histological changes of VOD (concentric non-thrombotic narrowing of the lumen of small intrahepatic veins) other less specific abnormalities can be observed in patients with a VOD syndrome, including eccentric narrowing of the venular lumen; phlebosclerosis; sinusoidal fibrosis and hepatocyte necrosis. Due to patchy nature of VOD a normal biopsy does not exclude the diagnosis. Ultrasound: A variety of abnormalities can be observed; gallbladder wall thickening, ascites, hepatomegaly and attenuate or reversed portal flow, but all of them are nonspecific. Biological markers: Although the serum of patients with VOD shows an increase in HAEMATOPOIETIC STEM CELL TRANSPLANTATION
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levels of plasminogen activator inhibitor-1 (PAI-1) (marker with the highest specificity and sensitivity for VOD), aminopropeptides of type III collagen, and hyaluronic acid, all these measurements are of little utility in routine clinical practice. Differential diagnosis To accept the diagnosis of VOD all the following possible causes of similar clinical features should be excluded as far as possible, including: • Infections: Cholangitis lenta (sepsis of liver) / fungal infection / viral hepatitis • Immune dysfunctions: Acute GvHD of the liver • Drug toxicity: CsA, azoles, MTX, progestogens, trimethoprim-sulphamethoxazole, TPN, among others • Reduction of venous outflow / increased volume: Constrictive pericarditis / congestive heart failure / fluid overload / renal failure • Others: Pancreatic ascites / chylous ascites / infiltration of the liver. Prophylaxis of VOD (6, 8) (Table 2) Table 2: Prophylaxis of VOD Avoidance of risk factors • When possible delay HSCT if an acute hepatitis exists; adjust Bu dose or use IV Bu; fractionate TBI; avoid hepatotoxic drugs, etc. • In high risk patients, consider allo-RIC HSCT (lower incidence of VOD) Pharmacological The following drugs have been used to prevent VOD from the beginning of conditioning until day +21–30: • Sodium heparin: 100 U/kg/day by continuous infusion. Two randomised studies showed a beneficial effect but others have suggested that it is ineffective and dangerous • Prostaglandin E1: 0.3 µg/kg/h by continuous infusion. Evaluated in several clinical trials usually combined with heparin. When administered alone no beneficial effect was observed • Ursodeoxycholic acid: 600–900 mg/day p.o. Four randomised trials and 2 historically controlled studies have shown a reduction in incidence of VOD and in TRM • N-acetylcysteine. Very limited experience • Low molecular weight heparin: Enoxaparin 40 mg/day or fraxiparin 5000 U/day subcutaneously seem to be relatively safe and may have some effect but a large randomised study is needed to confirm these results • Pre-emptive ATIII replacement: Ineffective • Defibrotide. Several preliminary reports have shown encouraging results
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Treatment of established VOD (6, 8) (Table 3) Table 3: Treatment of established VOD First line therapy Symptomatic (a)
-
Specific (b)
-
Restriction of salt and water intake ± diuretics Maintain intravascular volume and renal perfusion by means of albumin, plasma expanders and transfusions (haematocrit >30%) Defibrotide 6.25 mg/kg IV in 2 h infusion q 6 h IV during 14 days (1) (c) (d) rt-PA 0.05 mg/kg/h during 4 hours (maximum 10 mg/day) for 2–4 days ± sodium heparin 20 U/kg as a bolus (maximum 1000 U) followed by 150 U/kg/day by continuous infusion for 10 days (e)
Other measures Symptomatic (a)
-
Specific
-
Low dose dopamine (effectiveness not demonstrated) Analgesia Paracentesis / thoracocentesis Haemodialysis / haemofiltration Mechanical ventilation TIPS (transvenous intrahepatic portosystemic shunt) (f) Surgical shunt Liver transplantation
rt-PA: recombinant tissue plasminogen activator. (a) Symptomatic treatment should be established first, reserving specifies measures for most severe cases. (b) Although other agents have been used (antithrombin III, prostaglandin, corticosteroids, glutamine/vitamin E, N-acetylcysteine, etc.) the only ones occasionally effective are those mentioned. (c) Defibrotide permits the resolution of 50–55% of severe VOD with multiorgan dysfunction and a 47–60% of survival at day +100 with no secondary effects in adults and children (8, 10). (d) In a randomised study defibrotide at 25 mg/kg/day has shown similar effectiveness to the classical dose of 40 mg/kg /d (8). (e) rt-PA has been shown to be effective only in patients with a non-advanced VOD. Its use is contraindicated in patients with multi-organ dysfunction syndrome (MODS), haemorrhages or severe hypertension. (f) Despite improvement in portal hypertension and ascites, long term efficacy and survival are extremely poor
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VOD evolution (3, 4, 6 ,7) (Table 4) Table 4: VOD evolution Complete resolution on day +100 w/o treatment Complete resolution on day +100 with treatment Non resolution before death (c) or on day +100 Mortality attributable to VOD by day +100 (e)
Classification (a) Frequency (b) Mild VOD 8–23% Moderate VOD 48–64% Severe VOD (d) 23–28% 1–3% of all HSCT 18–28% of all VOD 75–95% of severe VOD (f)
(a) Classification described by Seattle group for retrospective evaluation of VOD. (b) Values observed in two large series (3, 6); (c) In many cases VOD is not the direct cause of death but contributes to it. (d) The severity of VOD can be predicted by means of a mathematical model (Bearman et al., JCO 1993). (e) Data from pre-defibrotide era. (f) The equivalent predicted mortality in non-severe VOD cases ranges between 10 and 20%
3.2. Capillary leak syndrome (CLS) (11) Pathogenesis The injury to the capillary endothelium produces a loss of intravascular fluids into interstitial spaces and the clinical manifestations. Incidence The absence of well-established clinical criteria for its diagnosis precludes an accurate estimation of its incidence. Additionally, the differential diagnosis with VOD, ES or IPS can be very difficult. Clinical features Development, in the first 15 days after HSCT, of: • Weight gain (>3% in 24 hours), and • Generalised oedemas (ascites, pleural effusion, pericarditis) that characteristically does not respond to frusemide treatment. Other features occasionally observed are: tachycardia, hypotension, renal insufficiency of pre-renal origin and hypoalbuminaemia. Differential diagnosis From engraftment syndrome (ES): Its earlier development, the absence of skin rash and the poor response to corticosteroids. From VOD: The absence of jaundice and painful hepatomegaly, and the poor response to furosemide. From IPS: The presence of generalised oedema. Risk factors The use of G-CSG, GM-CSF or K-CSF; high cumulative dose of CT in the pre-HSCT phase; 188
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unrelated or HLA mismatched donor grafts. Treatment To withdraw growth factors. Despite being systematically used the response to corticosteroids is poor. There is no other specific treatment. Evolution There is a high mortality if it progresses to MODS. 3.3. Engraftment syndrome (ES) (12–14) Pathogenesis Massive release of pro-inflammatory cytokines by tissues injured by intensive conditioning and by recovering neutrophils has been hypothesised to play a role. Incidence Variable depending on the diagnostic criteria used. After auto-HSCT: from 5 to up to 25% in patients with breast cancer or autoimmune diseases. After conventional allo-HSCT: only occasionally described (possibly because of the difficult differential diagnosis from GvHD). After allo-RIC: 10% in a recent series (14). Clinical features Development, within 72 hours of the start of neutrophil recovery, of the following major clinical criteria: • High fever of a non-infectious origin (unresponsive to antibiotics and negative cultures); • Skin rash affecting >25% body surface and not attributable to an allergic reaction; • Lung infiltrates or hypoxia not attributable to fluid overload, lung embolism, or congestive heart failure. Other symptoms occasionally observed are diarrhoea, weight gain and liver, kidney or CNS dysfunction (minor criteria). Diagnosis There are no well-established criteria for its diagnosis. Spitzer (12): 3 major criteria or 2 major and one or more minor criteria; Majolino (13): fever with either skin rash, pulmonary infiltrates or diarrhoea; Gorack (14): ≥2 major criteria plus weight gain. Risk factors Most cases of ES have been described since the introduction of growth factors and use of PBSCT. For this reason, a high number of CD34+ cells, faster engraftment and use of growth factors (especially GM-CSF) are considered to be the main risk factors as well as the underlying disease (breast cancer, multiple sclerosis, POEMS syndrome). Treatment MethylPDN 1 mg/kg q 12 h (3 days) with progressive tapering over one week. An appropriate empiric antibiotic treatment should always be maintained due to the difficulty of excluding an infectious origin of the fever.
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Evolution Complete resolution in 1–5 days in >80% of cases if steroids are introduced early. 3.4. Diffuse alveolar haemorrhage (DAH) (15, 16) Pathogenesis Very similar to VOD pathogenesis but affecting the lungs. Incidence The reported incidence ranges from 1 to 5% in auto-HSCT and from 3 to 7% in alloHSCT. Some authors consider that underlying undetected infections can play a role in DAH pathogenesis and postulate that infection-associated alveolar haemorrhage and DAH should be considered as equivalents. Clinical features Despite some cases of late-onset DAH, it is usually diagnosed within the first 30 days after HSCT. The main manifestations are: • Dyspnoea, non productive cough, tachypnoea • Hypoxaemia that can require oxygen-therapy • Chest X-ray or CT with focal or diffuse interstitial or alveolar infiltrates located in middle and inferior lung fields • Bronchoalveolar lavage (BAL) progressively bloodier, and not attributable to infection (absence of pathogens in BAL), thrombocytopenia, fluid overload or heart failure. Successive aliquot of 20 mL, in at least three segmentary bronchi, become pro-gressively more bloodstained (indicating blood in the alveoli). Risk factors DAH is not related to low platelet counts. Factors that favour this complication are older age, previous thoracic radiation, allogeneic donor, myeloablative conditioning, and severe acute GvHD. Treatment After publication of some small retrospective series high-dose methylPDN (250–500 mg q 6 h, 4–5 days and tapering in 2–4 weeks) was considered the treatment of choice. However, many other authors have not observed that corticosteroids modify the poor outcome associated with DAH. Recombinant FVIIa has been used with success in some cases. The possible role of cytokine antagonists and antiinflammatory agents should be evaluated. Evolution The overall mortality rate at 60 days from the onset of the haemorrhage is around 75% despite in many patients the death is not directly related to the haemorrhage. 3.5. Thrombotic microangiopathy (TMA) (17–20) TMA is the term used to describe haemolytic uraemic syndrome (HUS) and thrombotic 190
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thrombocytopenic purpura (TTP) associated with HSCT. Pathogenesis Conditioning regimen related toxicity, together with other triggering factors that are not clearly understood, produces a generalised endothelial dysfunction with intravascular platelet activation and formation of platelet-rich thrombi within the microcirculation. In contrast to classical TTP, ADAMTS13 activity very rarely falls below 10%. Incidence Less than 4% in auto-HSCT. Up to 15% in allo-HSCT (7% in an EBMT survey). Clinical manifestations Usually develop around day +60 but early (day +4) and late (2 years) episodes have been described. Characterised by: • Microangiopathic haemolytic anaemia (MHA) (anaemia, >2–5% schistocytes, LDH and other markers of haemolysis) • Thrombocytopenia or increase in transfusional requirement • Fever of non-infectious origin • Renal dysfunction and/or neurological abnormalities (cortical blindness, seizures, typical images in CNS CT-scan). Diagnostic criteria for HSCT-associated TMA (Table 5) Table 5: Diagnostic criteria for HSCT-associated TMA Blood & Marrow Transplant Clinical Trials Network consensus (18) 1) RBC fragmentation and 2 schistocytes per high-power field on PB smear 2) Concurrent increased serum LDH 3) Concurrent renal (a) and/or neurologic dysfunction w/o other explanations 4) Negative direct and indirect Coombs test International Working Group (19) 1) Increased percentage (>4%) of schistocytes in the blood 2) De novo, prolonged or progressive thrombocytopenia 3) Sudden and persistent increase in LDH 4) Decrease in Hb concentration or increased RBC transfusion requirement 5) Decrease in serum haptoglobin concentration (a) Doubling of serum creatinine from baseline
Risk factors A higher incidence has been observed in patients receiving TBI, calcineurin inhibitors (CNI), sirolimus, urelated or HLA-mismatched donor grafts, or developing GvHD or CMV/fungal infections.
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Clinical forms Two main forms of TMA can be observed: 1. CNI-associated nephrotoxicity (or neurotoxicity) with MHA: Classically develops early after HSCT, is related to toxic levels of CNI, and is reversible after stopping its administration. Usually has a favourable evolution if it improves quickly after stopping CNI. 2. Not associated with CNI toxicity, with two clinical forms: a. Conditioning associated HUS: TMA primarily affecting the kidney, often causing oliguric or anuric renal failure with hypertension, MHA and thrombocytopenia, and b. Fulminating multifactorial TMA: Early after HSCT, renal failure, CNS disturbances, hypertension, MHA and thrombocytopenia associated with GvHD, viral or fungal infection. Most cases have a fatal evolution and do not respond to CNI suppression, plasma exchange or other treatments (see below). Prevention The only reasonable measure is to have a close control (2–3 times per week) of CNI, LDH and creatinine levels. If any of them increase peripheral blood smear, haptoglobin and CNI metabolites should be tested. Treatment The only effective measure in some cases is to inmediately stop CNI, adding another agent for GvHD prophylaxis/treatment (corticosteroids, mycophenolate, azathioprine). Plasma exchange cannot be currently considered the standard of care despite some success (less than 50% of responses and 70–90% of mortality in published series possibly due to selection bias). Some authors have reported successful results with anti-TNF MoAb (etanercept/infliximab), defibrotide, daclizumab, rituximab, and eicosapentaenoic acid. 3.6. Idiopathic pneumonia syndrome (IPS) (21) Pathogenesis The formerly used term of interstitial pneumonia has been progressively abandoned because it does not correspond to the real pathologic findings. Apparently, this syndrome is the result of a diversity of lung insults, including the toxic effects of conditioning, immunologic cell-mediated injury, inflammatory cytokines and, probably, occult pulmonary infections. Incidence As a consequence of the improvement of diagnostic methods the incidence of IPS has reduced from more than 20% in earlier series of allo-HSCT to less than 10% at present time (8.4 and 2.2% after conventional and allo-RIC, respectively, in a recent series). It is uncommon in auto-HSCT setting. 192
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Clinical features Development around day +21 of: • Fever, non-productive cough • Tachypnoea, hypoxaemia • Diffuse alveolar or interstitial infiltrates on X-ray or scan. Diagnosis The diagnosis is confirmed when the previous clinical manifestations are associated with: • Absence of infectious pathology or DAH in FBS, BAL or lung biopsy, and • Absence of other possible pathogens (lung oedema, lung haemorrhage, fat embolism, leukaemic infiltration, or lung toxicity due to leucoagglutinins). Risk factors Myeloablative conditioning, age older than 40, and grade III–IV acute GvHD. With myeloablative HSCT, the use of TBI, especially in patients older than 40 years is an additional risk factor. Treatment Supportive care combined with prophylaxis and treatment of infections. Some patients improve with methylPDN and some successes have been described with antiTNF MoAb (etanercept/infliximab). Evolution Up to 50–70% of patients will die due to a progressive impairment of respiratory function. This percentage reaches 97% if mechanical ventilation is required. 3.7. Multiple-organ dysfunction syndrome (MODS) (22) Pathogenesis All mechanisms previously mentioned. Incidence Unknown, due to the difficulty in differentiating this from the syndromes already described. Clinical features This diagnosis should be considered when early after HSCT a patient presents two or more of the following: • CNS dysfunction (>4 points on the scale of Folstein) • Lung dysfunction (O2sat < 90% in two occasions separated by more than 2 hr in the same day) • Renal dysfunction (creatinine >1.5 mg/dL [>133 mmol/L]) • Hepatic dysfunction (VOD criteria, see 3.1.). Treatment There is no effective treatment and this syndrome is irreversible.
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References 1. Sencer SF, Haake RJ, Weisdorf DJ. Hemorrhagic cystitis after bone marrow transplantation. Risk factors and complications. Transplantation 1993; 56: 875-879. 2. Gine E, Rovira M, Real I, et al. Successful treatment of severe hemorrhagic cystitis after hemopoietic celltransplantation by selective embolization of the vesical arteries. Bone Marrow Transplant 2003; 31: 923-925. 3. McDonald GB, Hinds MS, Fisher LD et al. Veno-occlusive disease of the liver and multiorgan failure after bone marrow transplantation: A cohort study of 355 patients. Ann Intern Med 1993; 118: 255-267. 4. Carreras E, Bertz H, Arcese W, et al. Incidence and outcome of hepatic veno-occlusive disease (VOD) after blood or marrow transplantation (BMT): A prospective cohort study of the European group for Blood and Marrow Transplantation (EBMT). Blood 1998; 92: 3599-3604. 5. Lee JL, Gooley T, Bensinger W, et al. Veno-occlusive disease of the liver after busulfan, melphalan, and thiotepa conditioning therapy: Incidence, risk factors, and outcome. Biol Blood & Marrow Transplant 1999; 5: 306-315. 6. Carreras E. Veno-occlusive disease of the liver after hematopoietic cell transplantation. Eu J Haematol 2000; 64: 281-291. 7. DeLeve LD, Shulman HM, McDonald GB. Toxic injury to hepatic sinusoids: Sinusoidal obstruction syndrome (veno-occlusive disease). Semin Liver Dis 2002; 22: 27-42. 8. Ho VT, Linden E, Revta C, Richardson PG. Hepatic veno-occlusive disease after hematopoietic stem cell transplantation: Review and update on the use of defibrotide. Semin Thromb Hemost 2007; 33: 373-388. 9. Carreras E, Gra_ena A, Navasa M, et al. Transjugular liver biopsy in BMT. Bone Marrow Transplant 1993; 11: 21-26. 10.Richardson PG, Murakami C, Jin Z et al. Multi-institutional use of defibrotide in 88 patients after stem cell transplantation with severe veno-occlusive disease and multisystem organ failure: response without significant toxicity in a high-risk population and factors predictive of outcome. Blood 2002; 100: 4337-4343. 11.Nürnberger W, Willers R, Burdach S, Göbel U. Risk factors for capillary leak-age syndrome after bone marrow transplantation. Ann Hematol 1997; 74: 221-224. 12.Speizer TR. Engraftment syndrome following hematopoietic stem cell transplantation. Bone Marrow Transplant 2001; 27: 893-898. 13.Maiolino A, Biasoli I, Lima J, et al. Engraftment syndrome following autologous hematopoietic stem cell transplantation: Definition of diagnostic criteria. Bone Marrow Transplant 2003; 31: 393-397. 14.Gorak E, Geller N, Srinivasan R, et al. Engraftment syndrome after nonmyeloablative allogeneic hematopoietic stem cell transplantation: Incidence and effects on survival. Biol Blood Marrow Transplant 2005; 11: 542-550. 15.Afessa B, Tefferi A, Litzow MR et al. Diffuse alveolar hemorrhage in hematopoietic stem cell transplant recipients. Am J Respir Crit Care Med 2001; 166: 641-645. 16.Majhail NS, Parks K, Defor TE, Weisdorf DJ. Diffuse alveolar hemorrhage and infectionassociated alveolar hemorrhage following hematopoietic stem cell transplantation: 194
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Related and high-risk clinical syndromes. Biol Blood Marrow Transplant 2006;12: 10381046. 17.Daly AS, Xenocostas A, Lipton JH. Transplantation-associated thrombotic microangiopathy: Twenty-two years later. Bone Marrow Transplant 2002; 30: 709-715. 18.Ho VT, Cutler C, Carter S, et al. Blood and marrow transplant clinical trials network toxicity committee consensus summary: Thrombotic microangiopathy after hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2005;11: 571-575. 19.Ruutu T, Barosi G, Benjamin RJ, et al. European Group for Blood and Marrow Transplantation; European LeukemiaNet. Diagnostic criteria for hematopoietic stem cell transplantassociated microangiopathy: Results of a consensus process by an International Working Group. Haematologica. 2007; 92: 95-100. 20.Batts ED, Lazarus HM. Diagnosis and treatment of transplantation-associated thrombotic microangiopathy: Real progress or are we still waiting? Bone Marrow Transplant 2007 Jul 2; [Epub ahead of print]. 21.Fukuda T, Hackman RC, Guthrie KA, et al. Risks and outcomes of idiopathic pneumonia syndrome after nonmyeloablative and conventional conditioning regimens for allogeneic hematopoietic stem cell transplantation. Blood 2003; 102: 2777-2785. 22.Gordon B, Lyden E, Lynch J, et al. Central nervous system dysfunction as the first manifestation of multiorgan dysunction syndrome in stem cell transplant patients. Bone Marrow Transplant 2000; 25: 78-83.
Mutiple Choice Questionnaire To find the correct answer, go to http://www.esh.org/ebmt-handbook2008answers.htm 1. Late-onset haemorrhagic cystitis usually is produced by: a) The direct action of cyclophosphamide on the bladder. . . . . . . . . . . . . . . . . . . . . b) The sum of several toxic factors that produce a bladder damage . . . . . . . . . . c) A polyomavirus infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) A bacterial infection of the urinary tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . e) The neutropenia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Which of the following complications could not be attributed to an endothelial dysfunction? a) Engraftment syndrome. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Veno-occlusive disease of the liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Haemorrhagic cystitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Leak capillary syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . e) Thrombotic microangiopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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3. Which of the following is not a clinical manifestation of VOD? a) Weight gain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Painful hepatomegaly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Ascites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Platelet refractoriness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . e) Diarrhoea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. All but one of the following are classical manifestations of engraftment syndrome, which one? a) Skin rash . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Back pain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Fever . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Hypoxaemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . e) Diarrhoea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Which is the main cause of thrombotic microangiopathy after HSCT? a) Bacterial infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Graft allo-reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Immunological phenomena . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Cyclosporin toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . e) Renal failure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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*
CHAPTER 10
Infections after HSCT
C. Cordonnier
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1. Introduction Despite considerable progress in the management of the complications of HSCT, infection remains an important cause of post-transplant morbidity and mortality, mainly after allogeneic HSCT. The major advances in the management of infectious complications have come from better understanding of the mechanisms of the complex depression of immunity observed during the first months after transplant and their role in the predisposition to given infections, and also from well-designed therapeutic trials. Although the proportion of infectious deaths after allogeneic HSCT has decreased over the last two decades (1), much remains to be done to further decrease this risk and implement more efficient preventive and prophylactic strategies adapted to this high-risk population. Even though the risk of infectious deaths is much lower after autologous transplant, the risks of the procedure are greater than those of conventional chemotherapy, and preventive policies should be implemented in any transplant program.
2. The timing of immune reconstitution determines the timing of infections Stem cell transplantation offers a unique model of gradual immune reconstitution, and illustrates perfectly the relationship between the type of immune deficiency and the occurrence of infection due to special pathogens. Immune reconstitution after HSCT is the topic of another Chapter of this book, and will not be detailed here. However, some basic observations deserve to be emphasised. After allogeneic HSCT following conventional (i.e. myeloablative) conditioning regimens, the sequence of infections can be divided into three periods: 1. The first is the aplastic phase following the conditioning regimen until neutrophil recovery from the donated marrow. During this phase, the infectious complications of HSCT patients are not very different from those encountered in other profoundly neutropenic patients such as acute leukaemia patients, except, in most cases, for more severe mucosal damage, especially after total body irradiation. This is also the beginning of the at-risk period for fungal infections, mainly aspergillosis. Viral infections, especially HSV, are also common. Infection-related mortality at this time is mainly due to severe bacterial sepsis, pneumonia, and fungal infections. 2. The second phase corresponds to the period from initial marrow engraftment to at least the third or fourth month, and is characterised by cell-mediated immune deficiency with decreased number and function of specific and non-specific
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cytotoxic cells. For many years CMV infection, which is mainly due to reactivation, was the greatest problem during this phase. However, the routine practice of early diagnosis through PCR or antigenaemia and prophylactic or pre-emptive therapy have both been shown to decrease mortality due to CMV disease. Other viral diseases, less frequent than CMV, have also been described during this phase, especially those due to adenovirus and enteric and respiratory viruses. The occurrence and severity of GvHD is the main factor delaying immune recovery and favouring infections. 3. The third phase, beginning at the fourth month, is considered to be the late posttransplantation period. Here again, immune reconstitution is mainly influenced by the presence and severity of chronic GvHD. Most patients have immunoglobulin deficiency, particularly of IgG2, which is responsible for a decrease in the response to polysaccharide antigens. Allogeneic HSCT recipients are particularly vulnerable to encapsulated bacteria (e.g. S. pneumoniae and H. influenzae). In the absence of chronic GvHD, this deficiency will often be transient and will resolve over time. In other cases, it may persist indefinitely. However, even in these cases, active immunisation may be beneficial, especially with conjugate vaccines. Although most infectious mortality is observed during the first 6 months after transplant, late infections may be life-threatening (particularly S. pneumoniae) and should not be neglected, especially in patients with chronic GvHD. The development of non-myeloablative approaches has modified the timing and features of the main infectious complications after transplant, although their overall incidence is not greatly changed. Neutropenia almost disappears or is significantly reduced, as is mucositis, and therefore the risk for early bacterial or fungal infections decreases (2). However, patients who undergo reduced intensity conditioning (RIC) regimens are usually older than those who receive conventional conditionings. As the main risk factor for severe infections is GvHD, and as, until now, the risk of GvHD is roughly comparable whatever the conditioning regimen, patients who receive reduced intensity conditioning remain at risk of infections, especially viral and fungal infections. Actually, although we may agree that RIC regimens decrease the overall transplant-related mortality, there is no clear evidence from the literature that RIC decreases infection-related mortality (3, 4). After autologous HSCT, morbidity and mortality related to infection is much lower. Since more than 95% of autologous HSCT are performed with peripheral blood stem cells, neutropenia generally lasts no more than 14 days, and the risk of bacterial sepsis is close to that observed in similar patients after chemotherapy. Fungal risk is extremely low and one may consider that these patients are not at risk for 200
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Aspergillus, and in standard situations they do not need rooms with filtered air. However, in addition to the risk of the autologous HSCT procedure itself, these patients may also be at risk due to previous immunosuppressive factors, such as prolonged steroid therapy, or hypogammaglobulinemia, which may favour other, non neutropenia-related, infections. Late infections may also occur after autologous transplant, but are exceptional without total body irradiation.
3. Clinical management of fever and infectious complications Because allogeneic transplant patients are those at higher risk for opportunistic infections and severe outcome, most of the recommendations given below mainly apply to these patients, but autologous transplant recipients should benefit from similar approaches, at least for the first 2 months after transplant. Two special clinical settings are taken as examples of the management of these patients when infection is probable, or even just possible, because they require special attention with essential diagnostic and therapeutic procedures. 3.1. Fever Febrile neutropenia is a special situation where bacterial infection must be the main target of empirical anti-infective therapy. There are not many differences between febrile neutropenia in patients at the neutropenic phase of stem cell transplant, and febrile neutropenia following chemotherapy, except that patients conditioned with total body irradiation are more likely to suffer from mucositis which is one of the main factors for streptococcal bacteraemia. Only 30% of febrile neutropenic episodes are microbiologically documented, with 20% due to Gram-positive cocci - mainly coagulase negative staphylococci - and 10% due to Gram-negative bacteria. As in any febrile neutropenia, broad-spectrum antibiotics should be administered promptly after blood cultures and sampling of any clinical site of infection whenever possible. Guidelines from the recent ECIL 1 (European Conference on Infections in Leukaemia) have clarified the need for aminoglycosides (5) and glycopeptides (6) in febrile neutropenia: the group considers that in standard situations betalactam monotherapy is as efficacious as betalactam plus aminoglycoside combination, both as initial empirical therapy and in case of persistent fever. Therefore, considering their potential toxicity, the use of aminoglycosides is not recommended, except in case of severe sepsis or septic shock where aminoglycosides are an initial option for first line antibiotic therapy, but where they should be quickly reconsidered according to the clinical outcome (5). Similarly, the use of glycopeptides as initial antibacterial therapy - combined with a betalactam - should be restricted to patients with hypotension or shock, skin or soft tissue infections (including high suspicion of central catheter infection) or in patients
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with known colonisation with methicillin - resistant S. aureus (6). If fever persists after 3 to 5 days of empirical antibacterials, fungal and viral infection must be considered, and an empirical antifungal treatment must be administered (7). Several antifungals may be used in this indication, but liposomal amphotericin B and caspofungin are recommended (8). Routine screening for fungal infection by galactomannan antigenaemia and lung CT-scan offers new hope of moving from the classical empirical strategy to a more risk-targeted, so-called “pre-emptive”, strategy (9, 10), but this strategy is, until now, not standardised for allogeneic HSCT recipients. When a transplant patient is febrile but not neutropenic, any infection should be considered and possible diagnoses should be made according to the clinical presentation and timing of fever after transplant. Although fever can be a feature of GvHD, most febrile episodes in transplant patients are due to infection, and diagnostic methods should be used to identify the responsible pathogen. The main difference between neutropenic and non-neutropenic patients is that in nonneutropenic patients, it is usually possible to investigate the patient properly before starting anti-infective drugs. For example, when an episode of pneumonia occurs within the first 6 months of transplant, and has developed over several days or weeks, it is usually possible to wait for fibre-optic bronchoscopy and BAL, whenever possible within 24h, before giving antibiotics. However, it is not always advisable to wait even in non-neutropenic patients, e.g. those with hypoxemic pneumonia, or severe sepsis. 3.2. Pneumonia Pneumonia complicates the course of half of allogeneic HSCT recipients treated with conventional conditioning regimens. There are many possible causes of pulmonary infiltrates observed after transplant. However, two thirds are due to infection. Pulmonary oedema, pulmonary embolism, alveolar haemorrhage, and alveolar proteinosis, may also occur, especially within the first 3 months after transplant. Fibre-optic bronchoscopy with BAL is the main diagnostic tool. It must be performed early in the course of pneumonia, before administration of antibacterials whenever possible, and a protected sample should be obtained for quantitative microbiological assessment, by either brushing or aspiration. BAL has a limited diagnostic yield for fungal infections. However, despite this limitation, it is the procedure with the highest risk/benefit ratio for first-line investigation. Since the use of prophylactic or preemptive treatment for CMV infection, which makes the occurrence of CMV pneumonia very rare, special attention must be paid to the possibility of respiratory virus pneumonia. Transbronchial lung biopsy is associated with a greater risk of pneumothorax and bleeding, and no greater diagnostic benefit for most causes of pneumonia in the transplant setting, except for mycobacterial and fungal infections. Open lung biopsy should be considered in sub-acute pneumonia when BAL does not 202
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provide a clear diagnosis. Although there is not much available data about the value of indirect non-invasive procedures in such patients (i.e., legionella, pneumococcal, cryptococcal antigens in blood and/or urine), such procedures deserve to be used since they may easily provide indirect evidence of the cause of pneumonia.
4. Bacterial infections Two important points should be remembered: - Timing and immune reconstitution are key points in making a possible diagnosis at the bedside of a transplant patient suspected of bacterial infection. - Additionally, HSCT patients are among the most immunodepressed patients in the hospital. Therefore, they are at risk for nosocomial infections, depending on the environmental measures and the epidemiology of the centre. Bacterial infections occurring during the neutropenic phase are usually the same as those observed during any chemotherapy-induced neutropenia, i.e. streptococci and Gram-negative bacteria. Neutrophil recovery usually marks the end of the bacterial risk for most autologous HSCT patients, but not for allogeneic HSCT recipients who remain at high risk for nosocomial infection for many weeks after neutrophil recovery, especially when they stay in the hospital for the treatment of severe GvHD with multiple complications, and keep their central IV line. After 2 months, the risk for encapsulated bacteria, mainly for S. pneumoniae and H. influenzae, appears and is strongly - but not exclusively - linked to chronic GvHD. Specific deficiencies in anti-S. pneumoniae and anti-PRP antibodies - the main H. influenzae type B capsular polysaccharide - have been noted, which may predispose the patient to these infections. S. pneumoniae may cause bacteraemia and/or pneumonia or sinusitis. Fulminant fatal outcomes may be observed, even years after transplant. S. pneumoniae infections are mainly observed in patients with GvHD and in those who have received TBI (11). Although it rarely causes fulminant disease, H. influenzae may be responsible for upper and lower respiratory tract infection, and bacteraemia. Other rare bacterial infections have been reported after transplant, i.e., mycobacterial or legionella infection. However, due to the rarity of these cases, the timing and risk factors are not well identified. 4.1. Prevention of bacterial infections During the early phase of transplant, three preventive measures must be considered: a) Because of the hospital environment and its resistant bacteria, the physical environment of transplant patients should aim to decrease the risk of nosocomial infection, as in any immunocompromised patient. Different measures can be implemented. The easiest is simple protection including mask, gloves, and
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gowns, which is usually enough for autologous transplant patients. Although there is no clear demonstration in the literature that such measures are superior to hand washing alone in preventing cross-transmission, this kind of isolation is usually easier to control enforce than hand washing alone, and such visible protection usually discourages the staff from making unnecessary approaches to the patient during the neutropenic phase. The control of room air quality through filtration (HEPA) is the main measure that can be taken to decrease the risk of aspergillosis but there is no demonstration that such air treatment has any impact on the incidence of bacterial pneumonia. Water control is also necessary, in any units caring for high-risk patients, to avoid contamination of the patients with Legionella sp. or P. aeruginosa. b) The second measure is the prevention of bacterial translocation by oral antibacterials - absorbable such as quinolones, or non-absorbable such as nonabsorbable gut decontamination - and a diet low in microbes. The gut is the main reservoir for Gram-negative bacteria, and a source of subsequent entry into the body. A diet low in microbes is generally recommended for these patients, but the measures vary from one centre to another. The minimal measures are to avoid fresh vegetables and fruits, and any food suspected to content high quantities of bacteria. This is a logical recommendation, although its benefit alone has never been clearly shown. Gut decontamination is differently approached in different centres and countries. Quinolones have been shown, in comparative trials, to decrease the risk for Gram-negative bacteraemia and the number of days of fever in patients with prolonged neutropenia. They are widely used for allogeneic HSCT patients in Europe, especially ciprofloxacin. In acute leukaemia patients and autologous HSCT recipients, a large trial comparing levofloxacin to placebo has shown that the use of levofloxacin during the neutropenic phase significantly reduces the risk of fever, bacterial infection and bacteraemia, and especially of Gram-negative infections, reducing the cost of intravenous antibacterials for febrile neutropenia (12). So far, there are no data on levofloxacin in allogeneic HSCT patients. Once started, quinolones should be given until the first febrile episode, or until the recovery of neutropenia in the absence of fever. However, quinolones may select Gram-negative quinolone-resistant bacteria. Therefore, their use is not recommended in units with a high level of quinolone resistance among Gramnegative bacteria, and should be associated with periodic monitoring of the local epidemiology (13). The other option, widely used in some European countries, is the use of non-absorbable antibiotics (i.e. colimycine and aminoglycosides). They similarly reduce the risk for Gram-negative bacteraemia, but historical trials failed to show any survival benefit. Non-absorbable gut decontamination 204
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has constraints, especially the need to be combined with sterile food and with frequent microbiological controls of the stools to look for the possible selection of resistant strains. Whatever the option (quinolones, or non-absorbable gut decontamination), an antifungal prophylaxis must also be given to decrease the risk of overgrowth of yeasts in the gut. c) The third measure is the management of central IV lines. Catheters may be the source of bacteraemia, with significant morbidity and potential mortality. During neutropenia, it is controversial whether a catheter may be left in situ when blood cultures have documented a pathogen, except in the case of methicillin resistant S. aureus, Candida sp., Bacillus sp. and Corynebacterium JK, and any hospitalacquired resistant pathogen, such as P. aeruginosa or Acinetobacter sp, for which there are clear recommendations to take the catheter out (2). Central catheter management should be formalised in any transplant unit in written procedures. From the third month after transplant, transplant patients may be profoundly hypogammaglobulinaemic (immune globulin levels below 3 g/L). These patients should receive IV immunoglobulin replacement to maintain immunoglobulin serum levels over 3 g/L, especially when they have chronic GvHD and are receiving immunosuppressive therapy. The most logical and cost-effective approach to prevent S. pneumoniae and H. influenzae infections is active immunisation. For S. pneumoniae, the historical trials with polysaccharide vaccines have been disappointing since they show that patients with GvHD, or receiving steroids, those with the higher risk of infection, have a poor antibody response, especially before 6 months post-transplant. Therefore, long-term antibiotic prophylaxis with penicillin is still the practice in most centres for allogeneic transplant recipients, but the development of penicillin resistance makes this policy unsatisfactory. The recent availability of a conjugate pneumococcal vaccine offers new hopes of improving the protection of HSCT patients since the conjugation of the pneumococcal polysaccharides to proteins elicits a T-cell dependent immune response. A first study shows that 3 doses of the heptavalent pneumococcal conjugate vaccine, given at 3, 6 and 12 months after allogeneic HSCT induces protective IgG concentrations in 60% of the patients regardless of donor immunisation (14). Another study in children showed that 3 doses of the heptavalent vaccine given from 6 months allows protective antibody levels in more than two thirds of the patients (15). A prospective EBMT trial recently completed shows that the administration can be given as soon as 3 months post-transplant. Similarly, the H. influenzae type-B tetanus conjugate vaccine has been shown to be highly effective, giving a satisfactory response in 85% of immunised HSCT
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patients, and should be administered from 4 months. Probably because of the high immunogenicity of such conjugate vaccines, the response does not seem to be influenced by the presence of GvHD. Therefore, these vaccines are recommended in all patients, and even more in those with GvHD who are at higher risk of infection. The main recommendations of the IDWP for active immunisation in transplant patients are summarised in Table 1 (16).
5. Fungal infections Aspergillus is the most serious fungal infection after allogeneic stem cell transplant (17), and is the main cause of infectious death (18). Reported incidences vary from 0 to 20% of transplants. The most common site is the lung and GvHD is the main risk factor. A first peak of incidence occurs during the neutropenic period, particularly in leukaemic patients who may have been previously colonised. A second wave of aspergillosis occurs during the 2nd and 3rd months after transplant and is strongly linked to GvHD; this is now more important than the first peak and late cases are more and more frequent. In patients previously infected by Aspergillus during induction or consolidation treatment phases, fungal recurrence may occur in roughly one third of the patients (19) and the use of a secondary antifungal Aspergillus prophylaxis is widely admitted, although the optimal choice of the drug is not fixed. Despite major improvements in the treatment of fungal infections (20), the mortality of Aspergillus in allogeneic HSCT patients remains over 50% in recent series. PCR and galactomannan antigenaemia may help in the early detection of Aspergillus infections. Candida infection is more rare and has no special clinical presentation in transplant patients when compared to other haematology patients. More and more nonAspergillus, non-Candida infections are reported in HSCT patients. Pneumonias due to endemic fungi, such as histoplasmosis or coccidioidomycosis, particularly in North America, must be considered in these patients, as well as the emerging fungi, including Trichosporon, Alternaria, Fusarium, and the Mucorales. Any of these fungi can cause pneumonia in transplant patients, as in similarly immunocompromised hosts, and their management needs a close collaboration between the mycologist and the clinician. 5.1. Prevention of fungal infections The most efficient protection from fungal air-born infections during the neutropenic phase of allogeneic transplant is the use of air filtration with positive pressure. This kind of isolation has been shown to decrease the early risk of Aspergillus in 206
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Table 1: EBMT recommendations for immunisations of long-term survivors after allogeneic and autologous stem cell transplantation (16) Available forms
Available data in HSCT patients
Recommended after HSCT
Strength No of of recommen- doses dation+
Time Improved after HSCT by donor (months) vaccination
BACTERIAL VACCINES S. pneumoniae
PS Conjugate PS
Yes Yes
Yes AII Yes (subgroups) CII
1 3
12 Unclear
No Yes
H. influenzae type B
Conjugate PS
Yes
Yes
BII
3
6
Yes
N. meningitidis types A and C N. meningitidis type C
PS
Yes
Individual assessment
CII
1
6-12
Unknown
Conjugate PS
No
1
6
Unknown
BCG
Live
No
Contra-indicated EII
NA
NA
Unknown
Tetanus
Toxoid
Yes
Yes
BII
3
6-12
Yes
Diphtheria
Toxoid
Yes
Yes
BII
3
6-12
Likely
B. pertussis#
Acellular, toxoid +/- other antigens
Yes
See text
CIII
3
6-12
Unknown
Inactivated
Yes
Yes, yearly
AII
1
4-6
Unknown
Inactivated polio Inactivated
Yes
Yes
BII
3
6-12
Unknown
VIRAL VACCINES Influenza
Hepatitis B
Inactivated plasma Yes or recombinant DNA derived
See text
BII
3
6-12
Yes
Hepatitis A virus
Inactivated
No
In endemic areas CIII and in travellers
3
6-12
Unknown
Measles§
Live
Yes
Individual assessment
BII
1
24*
Unknown
Rubella§
Live
Yes
Individual assessment
BIII
1
24*
Unknown
Mumps§
Live
Yes
Individual assessment
CIII
1
24*
Unknown
Varicella
Live
Limited
Individual assessment
CIII
Unclear Before HSCT Unknown or at 24*
Yellow fever
Live
Limited
Individual asses- CIII sment, travellers
1
24*
Unknown
+The recommendations are graded according to the CDC system; *Not in patients with chronic GvHD or ongoing immunosuppression; #Combination vaccines including also tetanus, diphtheria, and pertussis with or without HIB and poliovirus components are available; §Usually combined in a MMR vaccine; BCG: Bacille Calmette-Guérin; HIB: Haemophilus influenzae type b; PS: Polysaccharide
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allogeneic HSCT. Good management of air filters, and effective collaboration with the infection control unit of the hospital are of paramount importance for allogeneic transplant units, and for haematology departments in general. Epidemics of mould infections have been reported in stem cell transplant units and have led to interruption of transplant programs until the infections can be controlled (21). However, air filtration, of course, does not provide any protection from yeast infections. In two randomised trials, fluconazole prophylaxis at the dose of 400 mg/day has been shown to reduce the risk for invasive fungal infections, especially Candida infections, and is associated with a long-term survival benefit. However, because non-albicans Candida are more and more frequent in haematology patients, the benefit of fluconazole prophylaxis is probably lower than ten years ago, and anyway unsatisfactory considering the main concern of the transplant community is mould infections. Recently, Ullmann et al. have shown that posaconazole (600 mg/d), when given from the onset of GvHD, significantly decreases the incidence of proven and probable invasive fungal infection, especially Aspergillus (22). In autologous HSCT, except in centres with a high incidence of candidaemia, the risk of invasive Candida infection is low, and therefore the use of fluconazole in this setting is optional (23). 5.2. Treatment of fungal infections Voriconazole, a triazole antifungal agent, is fungicidal active against Aspergillus and Candida species, including non-albicans Candida, and other rare fungal infections such as Fusarium or Scedosporium species which occur in stem cell transplant patients. In the first-line treatment of aspergillosis, it has been shown to be more effective and better tolerated than amphotericin B (20). Despite a substantial number of side effects (including visual disturbances, confusion, skin reactions, and liverfunction abnormalities), and potential drug interactions, especially with cyclosporin, voriconazole is widely used after stem cell transplant. A recent, large prospective study comparing 3 vs. 10 mg/kg/d of liposomal amphotericin B in first line treatment of Aspergillus infections did not find any advantage to the 10 mg dose, but showed comparable results of the 3 mg/kg/d arm with the voriconazole study (24). Echinocandins have been studied in refractory aspergillosis with encouraging results but no study of first-line therapy has been published so far. Echinocandins are excellent candidates for combinations with either azoles, or polyenes. However, there are, until now, no prospective data published on first line therapy of fungal infections with echinocandin combinations. These combinations will be extremely expensive, and there is no evidence as yet that they will provide better results than 208
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single drug regimens. Prospective, comparative trials need to be done before any conclusion can be drawn. The ECIL 1 has recently published treatment guidelines for Candida and Aspergillus infections (25).
6. Viral infections Viral infections are frequent after HSCT. They may be life threatening, especially when affecting lung, liver, or central nervous system in allogeneic HSCT recipients. The availability of new antiviral agents in the last 2–3 years, together with data from comparative trials, have allowed a better control of herpes virus infections. However, due to the subsequent decrease in CMV infection and CMV disease, new viral infections have emerged, especially due to respiratory viruses and adenovirus. Herpes simplex virus (HSV) infections are extremely common, due to reactivation in seropositive patients. The main early manifestation is mucous lesions, difficult to distinguish from chemotherapy-induced mucositis in the absence of viral documentation. These lesions are painful and maybe the portal of entry of bacteria from the gut. In seropositive patients, prophylaxis with acyclovir or valaciclovir is recommended to decrease the risk of reactivation during the early phase of transplant. In case of HSV infection, IV acyclovir is usually effective. Acyclovir resistance is rare in HSCT patients, but this possibility must be considered in case of HSV disease documented during prophylaxis. Due to common mechanisms at the cellular level (thymidine-kinase dependent), resistance to acyclovir is associated with resistance to ganciclovir and famciclovir. The best choice in case of acyclovir resistance of HSV is foscavir. Cytomegalovirus (CMV) disease has historically been a main cause of death in allogeneic transplant patients, except where both donor and recipient are seronegative. Since the demonstration that CMV infection usually precedes CMV disease, and considering the poor prognosis of CMV disease even when treated, two strategies - prophylactic and pre-emptive - have been developed in order to reduce the risk of CMV disease. First, prophylactic trials comparing IV ganciclovir versus placebo showed that ganciclovir prevents the risk of CMV infection and disease, but does not improve survival and additionally favours the delay of specific immune reconstitution, and consequently, the occurrence of late CMV infection. This prophylactic strategy was finally not more effective than a pre-emptive strategy. Therefore, prophylactic strategies are usually reserved for patients with high-risk of CMV disease, such as mismatched transplant recipients. For other patients, a preemptive strategy is more cost-effective. Controlled trials are needed to know
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whether the oral pro-drug of ganciclovir, valganciclovir, will be as effective and no more toxic than the IV ganciclovir. First experiences are encouraging but suggest caution is needed because of the different pharmacokinetics of the two drugs (26). The clinical impact of theses differences should be explored further. Although most first line pre-emptive strategies have used ganciclovir, foscavir has been shown, in an EBMT comparative trial, as effective and no more toxic than ganciclovir (27). Both can be used as first-line treatment of CMV infection, for an initial duration of 2 weeks. If CMV is still detected after 2 weeks of therapy, an additional course of 2 weeks should be given. Cidofovir has been studied only in uncontrolled trials and because of its toxicity profile; its use should be reserved for second line preemptive therapy. These strategies must be based on sensitive diagnostic methods of CMV infection. Allogeneic HSCT recipients must be monitored for CMV in peripheral blood at least weekly with a sensitive method, until day 100 after transplant, and longer in case of prolonged GvHD or previous CMV reactivation. Antigenaemia testing detects CMV antigen pp65 in leukocytes by immune staining with monoclonal antibodies. This test is semiquantitative and rapid. PCR is also widely used. The quantification of viral load seems to be important since higher levels of CMV DNA are indicators for a higher risk of CMV disease. The availability of Real Time or Light Cycler technologies has improved the quantitative evaluation of the viral load. Detection of mRNA by nucleic acid sequence-based amplification (NASBA) is also available and has been shown to be similarly effective as pp65 antigenaemia or detection of DNA by PCR for the early detection of CMV infection. Because the risk of CMV disease is extremely low after autologous transplant, systematic CMV screening is not recommended in this population, except in high-risk patients, like those receiving CD34-selected grafts, or patients who have recently received fludarabine, 2-CDA, or alemtuzumab. In additionally to antiviral strategies, CMV prophylaxis must include transfusion policies to avoid acquisition of CMV through blood products, especially for CMV seronegative recipients of seronegative donors. These patients must receive blood products either from CMV seronegative donors exclusively, or leukocyte-depleted products. Although less established by prospective trials, the same policy is logical for CMV seronegative recipients of autologous transplants. Varicella-zoster virus (VZV) infections occur both after allogeneic and autologous transplants. Primary varicella may be severe in both populations. IV acyclovir is the therapy of choice. Valaciclovir can also be used, especially in late infections, and in patients who do not receive high-dose immunosuppressive drugs. These patients, when given valaciclovir, must be correctly managed in case of rash extension or 210
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systemic manifestations, and informed of the possible need for switching to the IV form of the antiviral. Human herpes virus 6 (HHV6) infection after allogeneic HSCT has been associated with pneumonia, delayed marrow engraftment, and particularly with prolonged thrombocytopenia and encephalitis. HHV6-DNA is frequently detected in blood during the first months after transplant in asymptomatic patients, so that its implication in clinical symptoms is usually difficult to establish, except in encephalitis when HHV6-DNA is detected in CSF. Epstein-Barr virus associated lymphoproliferative disease (EBV-LPD) is a lifethreatening complication occurring after allogeneic HSCT. Monitoring of the EBV viral load by quantitative PCR permits the early detection of EBV reactivation that may lead to EBV-LPD. Recipients of a T-cell-depleted HSCT, or patients conditioned with antithymocyte globulins are at higher risk of EBV-LPD. In these patients, preemptive therapy of EBV reactivation with rituximab has been shown to improve outcome. Infusion of EBV-specific cytotoxic T-cells has also been studied in highrisk patients with elevated EBV-DNA levels. However, the exact indications for preemptive therapy based on EBV-viral load for preventing EBV-LPD are not yet clear and should highly depend on the transplant population. Prospective trials are needed. 6.1. Respiratory virus infections Respiratory viruses, including respiratory syncytial virus (RSV), parainfluenza virus, rhinovirus, and influenza virus, appear to be now more frequent than CMV pneumonia. A prospective study from the EBMT showed an incidence of respiratory virus pneumonia of 2.1% in allogeneic, and 0.2% in autologous transplant patients (28). Most cases in this series were due to RSV or influenza A. The mortality of these infections also varies among series, and with the time after transplant and the degree of immunosuppression, but it may be as high as 80% in RSV pneumonia. Few data are available in the literature on the efficacy of antivirals in RSV pneumonia. Due to the rarity of the disease, and the poor prognosis of the lower tract infections, prospective trials are extremely difficult to perform. In the European experience, there was no clear advantage or disadvantage of adding IV ribavirin to either aerosolised ribavirin or to IV immune globulins. The best therapeutic option for RSV pneumonia is not established. Due to the risk of spread in the transplant unit, it is important to diagnose these patients very early, and to prevent transmission in the ward. Oseltamivir has been used in an open study to control an influenza A outbreak and appears to be safe in allogeneic transplant recipients (29).
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6.2. Adenovirus infections Adenovirus infections can be a cause of severe disseminated infections in allogeneic HSCT recipients. Patients receiving mismatched or unrelated donor transplants, patients with severe acute GvHD, patients showing isolation from multiple sites or from blood are at high risk of developing organ involvement due to adenovirus. There are currently no established strategies for prophylaxis or treatment of adenovirus disease. Spontaneous recovery may be observed. However, in high-risk patients such as children receiving mismatched transplants, adenovirus infection usually develops into adenovirus disease, which has a high mortality rate, and pre-emptive treatment should be logical but has not been studied extensively. A recent retrospective study by the IDWP of the EBMT has showed that cidofovir was effective in 10/16 patients with invasive adenovirus disease (30). Ribavirin has been used in case reports with varying outcome. Both drugs - cidofovir and ribavirin - may be effective in preemptive therapy. Prospective controlled studies are needed to define the best strategy.
7. Other infections Toxoplasmosis occurring after HSCT has been mainly investigated in Europe, due to a higher seroprevalence of the disease when compared to US. Patients at risk are those who are seropositive for toxoplasmosis before transplant, irrespectively of the serology of the donor. Blood PCR allows early detection of toxoplasma reactivation. A prospective study from the IDWP on 106 allogeneic toxoplasma seropositive recipients, screened weekly by blood PCR, showed an incidence of PCR-documented infection of 16%, and an incidence of toxoplasma disease of 6% up to 6 months post-transplant. All patients developing disease were previously or simultaneously PCR-positive in blood (31). Most of these reactivations occur in patients with GvHD, while trimethoprim-sulfamethoxazole has been stopped for side effects, and replaced by aerosolised pentamidine for P. jiroveci prophylaxis. Whether asymptomatic toxoplasma infection documented by blood PCR should be treated is not yet clear. However, there are reports in the literature where high-risk patients, with severe GvHD, developed toxoplasma infection which evolved, in the lack of specific therapy, to toxoplasma disease with a high mortality rate. P. jiroveci pneumonia must be prevented in allogeneic stem cell transplant recipients from engraftment to at least 6 months, even longer in case of prolonged immunosuppression. The best option is trimethoprim-sulfamethoxazole. In case of intolerance, alternatives are dapsone or aerosolised pentamidine, but the latter does not provide optimal protection (32). 212
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8. Other preventive strategies Many issues regarding infectious complications in HSCT patients remain to be explored, especially in allogeneic HSCT recipients. Active immunisation against tetanus, poliovirus, and diphtheria is highly recommended in all transplant populations, due to the usual loss of specific immunity both after autologous and allogeneic stem cell transplantation. This is the only way to allow leukaemic patients to have similar levels of immunity to these pathogens - even rarely encountered - as the normal population. Immunisation with live vaccines is classically prohibited in immunocompromised patients. However, transplant patients may require active immunisation with vaccines which only exist in a live form, i.e. measles, mumps, or yellow fever. Ljungman et al. have showed that live vaccine to measles, mumps and rubella may be safely administered to transplant children, as far as they are at more than 2 years after transplant, have no GvHD, and are not receiving immunosuppressive drugs. Immune globulins have been administered for years to allogeneic HSCT recipients to try to prevent the occurrence of GvHD and to reduce infectious mortality. A French double-blind study comparing 3 doses of IVIg to placebo in recipients of HLA-identical sibling donors shows that IVIg administration did not affect the incidence of infections over the first 6 months after transplant, the occurrence of GvHD, or survival. On the other hand, severe veno-occlusive disease was significantly more frequent in patients receiving high doses of IVIG (250 mg/kg and 500 mg/kg/weekly) (18). Similar findings have been reported in another randomised trial in unrelated transplant patients. Consequently, considering the availability of other strategies now available for infection prophylaxis, and considering the high cost of IVIg, prophylactic administration of IVIg is not recommended in allogeneic transplant recipients.
References 1. Gratwohl A, Brand R, Frassoni F, et al. Cause of death after allogeneic haematopoietic stem cell transplantation in early leukaemias: An EBMT analysis of lethal infectious complications and changes over calendar time. Bone Marrow Transplant 2005; 36: 757769. 2. Junghanss C, Marr KA, Carter RA, et al. Incidence and outcome of bacterial and fungal infections following nonmyeloablative compared with myeloablative allogeneic hematopoietic stem cell transplantation: A matched control study. Biol Blood Marrow Transplant 2002; 8: 512-520. 3. Diaconescu R, Flowers C, Storer B, et al. Morbidity and mortality with nonmyeloablative compared to myeloablative conditioning before hematopoeitic cell transplantation from HLA matched related donors. Blood 2004; 104: 1550-1558. 4. Scott BL, Sandmaier BM, Storer B, et al. Myeloablative vs nonmyeloablative allogeneic
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transplantation for patients with myelodysplastic syndrome or acute myelogenous leukemia with multilineage dysplasia: A retrospective analysis. Leukemia 2006; 20: 128-135. 5. Drgona L, Paul M, Bucaneve G, et al. The need for aminoglycosides in combination with betalactams for high-risk, febrile neutropenic patients with leukaemia. Eur J Cancer 2007; Suppl. 5: 13-22. 6. Cometta O, Marchetti O, Calandra T. Empirical use of anti-Gram positive antibiotics in febrile neutropenic cancer patients with acute leukaemia. Eur J Cancer 2007; Suppl. 5: 23-31. 7. Hughes WT, Armstrong D, Bodey GP, et al. 2002 guidelines for the use of antimicrobial agents in neutropenic patients with cancer. Clin Infect Dis 2002; 34: 730-751. 8. Marchetti O, Cordonnier C, Calandra T. Empirical antifungal therapy in neutropenic cancer patients with persistent fever. Eur J Cancer 2007; Suppl. 5: 32-42. 9. Maertens J, Theunissen K, Verhoef G, et al. Galactomannan and computed tomographybased pre-emptive antifungal therapy in neutropenic patients at high risk for invasive fungal infection: A prospective feasibility study. Clin Infect Dis 2005; 41: 1242-1250. 10.Cordonnier C, Pautas C, Maury S, et al. Empirical versus pre-emptive antifungal strategy in hig-risk febrile neutropenic patients: A prospective randomized study. 48 th ASH Annual meeting, Orlando, Florida, 9-12 December 2006. 11.Engelhard D, Cordonnier C, Shaw PJ, et al. Early and late invasive pneumococcal infection following bone marrow and stem cell transplantation. Br J Haematol 2002; 117: 444-450. 12.Bucaneve G, Micozzi A, Menicheti F, et al. Levofloxacin to prevent bacterial infection in patients with cancer and neutropenia. N Engl J Med 2005; 353: 977-987. 13.Bucaneve G, Castagnola E, Viscoli C, et al. Quinolone prophylaxis for bacterial infections in afebrile high risk neutropenic patients. Eur J Cancer 2007; Suppl. 5: 5-12. 14.Molrine DC, Antin JH, Guinan EC, et al. Donor immunization with pneumococcal conjugate vaccine and early protective antibody responses following allogeneic hematopoietic cell transplantation. Blood 2003; 101: 831-836. 15.Meisel R, Kuypers L, Dirksen U, et al. Pneumococcal conjugate vaccine provides early protective antibody responses in children after related and unrelated allogeneic hematopoietic stem cell transplantation. Blood 2007; 109: 2322-2326. 16.Ljungman P, Engelhard D, Locasciulli A, et al. Vaccination of stem cell transplant recipients. Recommendations of the Infectious Diseases Working Party of the EBMT. Bone Marrow Transplant 2005; 35:737-746. 17.Marr KA. Epidemiology and outcome of mold infections in hematopoietic stem cell transplant recipients. Clin Infect Dis 2002; 34: 909-917. 18.Cordonnier C, Chevret S, Legrand M, et al. Should immunoglobulin therapy be used in allogeneic transplantation? A randomized, double-blind, dose-effect, placebo-controlled, multicenter trial. Ann Intern Med 2003; 139: 8-18. 19.Offner F, Cordonnier C, Ljungman P, et al. Impact of previous aspergillosis on the outcome of bone marrow transplantation. Clin Infect Dis 1998; 26: 1098-1103. 20.Herbrecht R, Denning DW, Patterson TF, et al. Voriconazole versus Amphotericin B for primary therapy of invasive aspergillosis. New Engl J Med 2002; 347: 408-415. 21.Gratwohl A, McCann S, Byrne JL, et al. Outbreaks of infectious disease in stem cell transplant units: A silent cause of death for patients and transplant programmes. Bone Marrow 214
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Transplant 2002; 29: S 53. 22.Ullmann A, Lipton J, Vesole DH, et al. Posaconazole or Fluconazole for prophylaxis in severe graft-versus-host disease. N Engl J Med 2007; 356: 335-347 23.Maertens J, Frere P, Lass-Flörl C, et al. Primary antifungal prophylaxis in leukaemia patients. Eur J Cancer 2007; Suppl. 5: 43-48. 24.Cornely OA, Maertens J, Bresnik M, et al. Liposomal amphotericin B as initial therapy for invasive mold infection: A randomized trial comparing a high-loading dose regimen with standard dosing. Clin Infect Dis 2007; 44: 1298-1306. 25.Herbrecht R, Flückiger U, Gachot B, et al. Treatment of invasive Candida and Aspergillus infections in adult haematological patients. Eur J Cancer 2007; Suppl. 5: 49-59. 26.Einsele H, Reusser P, Bornhaüser, et al. Oral valganciclovir leads to higher exposure to ganciclovir than intravenous ganciclovir in patients following allogeneic stem cell transplantation. Blood 2006; 107: 3002-3008. 27.Reusser P, Einsele H, Lee J, et al. Randomized multicenter trial of foscarnet versus ganciclovir for preemptive therapy of cytomegalovirus infection after allogeneic stem cell transplantation. Blood 2002; 99: 1159-1164. 28.Ljungman P, Ward KN, Crooks BNA, et al. Respiratory virus infections after stem cell transplantation. A prospective study from the Infectious Diseases Working Party of the European Group for Blood and Marrow Transplantation. Bone Marrow Transplant 2001; 28: 479-484. 29.Vu D, Peck AJ, Nichols WG, et al. Safety and tolerability of oseltamivir prophylaxis in hematopoietic stem cell transplant recipients: A retrospective case-control study. Clin Infect Dis 2007; 45: 187-193. 30.Ljungman P, Ribaud P, Eyrich M, et al. Cidofovir for adenovirus infection after allogeneic stem cell transplantion (HSCT). A retrospective survey of the Infectious Diseases Working Party of the European Group for Blood and Marrow Transplantation. Bone Marrow Transplant 2003; 31: 481-486. 31.Martino R, Bretagne S, Einsele H, et al. Early Detection of Toxoplasma Infection by Molecular Monitoring of Toxoplasma gondii in Peripheral Blood after Allogeneic Stem Cell Transplantation. Clin Infect Dis 2005; 40: 67-78. 32.Center for Disease Control. Guidelines for preventing opportunistic infections among hematopoeitic stem cell transplant recipients. Recommendations of CDC, the Infectious Diseases Society of America, and the American Society of Blood and Marrow Transplantation. October 20, 2000 / 49 (RR10); 1-128. (www.cdc.gov).
Mutiple Choice Questionnaire To find the correct answer, go to http://www.esh.org/ebmt-handbook2008answers.htm 1. Among these statements comparing RIC regimens and conventional regimens, one is true. Which one? a) RIC decreases the incidence of encapsulated bacterial infections . . . . . . . . . HAEMATOPOIETIC STEM CELL TRANSPLANTATION
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b) RIC increases the incidence of early bacterial infections . . . . . . . . . . . . . . . . . . . c) RIC decreases the incidence of fungal infections . . . . . . . . . . . . . . . . . . . . . . . . . . . d) RIC delays the occurrence of CMV infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Among these statements about S. pneumoniae vaccination, one is true. Which one? a) The conjugate pneumococcal vaccine is a live vaccine . . . . . . . . . . . . . . . . . . . . . b) The conjugate pneumococcal vaccine includes more antigens than the olysaccharide vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) The polysaccharide vaccine is recommended in allogeneic SCT recipients from 3 months after transplant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) The conjugate pneumococcal vaccine is more immunogenic because it induces a T-cell response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Among these statements about the use of antibacterials in febrile neutropenic patients, one is true. Which one? a) The use of aminoglycosides is not recommended in febrile neutropenic patients without severe sepsis or septic shock. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) The use of glycopeptides in combination with a betalactam is recommended as first line treatment of febrile neutropenic patients in standard situations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) In neutropenic patients, the risk of S. aureus bacteraemia is increased in the presence of severe mucositis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) In neutropenic patients, the risk of streptococcal bacteraemia is increased in the presence of catheter infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Among these statements about antifungal strategies in allo-HSCT, one is true. Which one? a) Posaconazole prophylaxis decreases the incidence of invasive fungal infection when given from transplant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Posaconazole prophylaxis decreases the incidence of invasive fungal infection when given from the onset of GvHD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Increasing the dose of liposomal Amphotericin B from 3 to 10 mg/kg in first line treatment of aspergillosis improves the clinical response . . . . d) It has been shown in a prospective study that the combination of 216
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caspofungin with voriconazole is more efficient than voriconazole alone in first line treatment of aspergillosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Among these statements about toxoplasmosis after allo-HSCT, one is true. Which one? a) Toxoplasmosis after allo-HSCT occurs almost exclusively in patients who were seropositive for toxoplasma before transplant . . . . . . . . . . . . . . . . . . . . . . . . b) Reactivation documented by blood PCR is observed in approximately 30–40% of seropositive allogeneic transplant recipients. . . . . . . . . . . . . . . . . . . c) Asymptomatic toxoplasma infection never develops into toxoplasma disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Aerosolised pentamidine prophylaxis is effective against both P. jiroveci and toxoplasma infection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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*
CHAPTER 11
Graft versus host disease
A. Devergie
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CHAPTER 11 • Graft vs. host disease
Graft versus host disease (GvHD) is the most frequent complication after allogeneic haematopoietic stem cell transplantation (HSCT). GvHD can occur despite aggressive immunosuppressive prophylaxis even when the donor is a “perfectly” matched (HLA-identical) sibling. It is a consequence of interactions between antigen (Ag)presenting cells of the recipient and mature T-cells of the donor.
1. History When the first allografts were performed, it was shown that patients given marrow from donors other than monozygotic twins were likely to develop what was then called “secondary disease”. Clinical manifestations involved the skin, intestinal tract and liver. They were similar to those observed in mice neonatally transplanted with allogeneic spleen cells (runt disease) and in some immunodeficient children who had received blood transfusions. Patients given allo-HSCT fulfilled the three conditions necessary for the development of GvHD, as defined by Billingham in 1966 (1): • Administration of immunocompetent cells • Histo-incompatibility between donor and recipient • Inability of the recipient to destroy or inactivate the transfused or transplanted cells.
2. Pathophysiology A 3-step process reflects the current view of the development of acute GvHD (Table 1) (2). Phase 1: Effect of conditioning: Underlying malignancy, effects of previous therapies and conditioning-related tissue damage lead to generation of large amounts of inflammatory cytokines such as TNF-a and IL-1 that enhance early activation of host Ag-presenting cells (APCs). Translocation of lipopolysaccharide (LPS) across damaged intestinal mucosa activates the innate immune system and promotes the inflammatory cytokine cascade.
Table 1: Immunobiology of aGvHD as a 3-step process Phase
Cells
Cytokines, chemokines
1
Effect of conditioning
Host APCs (dendritic cells) Epithelial cell damage
TNF-a, IL-1 Adhesion molecules, LPS
2
T-cell activation
Donor T-cells (mainly CD4+) Host APCs
IL-2 IFN-g
3
Cellular and inflammatory effector phase
CTLs NK cells
“Cytokine storm” TNF-a / IL-1 / nitric oxide
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Phase 2: T-cell activation: In this milieu, transplanted naïve donor T lymphocytes (and other cellular compartments) interact with host APCs, are activated and express cytokines such as IFN-g, IL-2 and TNF-a (among others), leading to T-cell expansion. Phase 3: Cellular and inflammatory effector phase: This phase is a complex cascade of multiple effectors including cytotoxic effector cells (CTLs), using Fas- and perforin-mediated mechanisms, natural killer (NK) cells and large granular lymphocytes (LGLs), followed by the generation of inflammatory cytokines, such as TNF-a, IL-1 and macrophage-derived nitric oxide. Interactions of innate (LGL) and adaptive (alloreactive T-cells) immune responses lead to organ damage. Additional complexity has been added by the recent description of regulatory cell populations including regulatory T-cells (Treg), regulatory APC populations and perhaps mesenchymal stem cells (3).
3. Classification of GvHD The traditional definition of acute (aGvHD) or chronic GvHD (cGvHD) was based on the time of onset after transplantation (less or more than 100 days after HSCT). This distinction is no longer tenable. For instance, aGvHD may present beyond 3 months in patients who have received reduced-intensity conditioning (RIC), and symptoms characteristic of cGvHD may occur before D 100. Furthermore, some signs and symptoms are common to both cGvHD and aGvHD. The recent National Institutes of Health (NIH) Consensus Conference proposed a definition of aGvHD or cGvHD, each with 2 subcategories, based on the specificity of signs and symptoms rather than the criterion of time of onset (Table 2) (4).
4. Incidence and risk factors The median incidence of clinically significant (grade II-IV) aGvHD is about 40% but
Table 2: Definition of acute and chronic GvHD Category
Time of manifestation
aGvHD features
cGvHD features
Classic
£ 100 days
Yes
No
Persistent, recurrent, late onset
> 100 days
Yes
No
Classic
No time limit
No
Yes
Overlap syndrome
No time limit
Yes
Yes
aGvHD
cGvHD
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ranges from 10 to 80% according to a number of risk factors (Table 3). The risk of aGvHD increases with the use of unrelated donors, multiparous female donor, older age of the recipient, graft type (cord blood has a lower rate and peripheral blood stem cells have a higher rate than marrow), and certain conditioning regimens. Although the central role of HLA matching in HSCT is well established, it has become apparent that other genetic systems affect the development of GvHD. Minor histocompatibility antigens (mHA) are peptides from polymorphic cellular proteins, encoded by regions outside the major histocompatibility complex (MHC). Interestingly, some mHA are expressed mainly by malignant cells and represent attractive target for the induction of Graft-versus-Leukaemia (GvL) effect without promoting GvHD. Genes controlling inflammatory processes, such as cytokines, chemokines and their receptors, can modulate GvHD. Gene polymorphisms affecting IL-1, IL-6, IL-10, TNF, TGF-b, and IFN-g have all been implicated in the incidence and severity of GvHD, both in experimental models and immunogenetic analysis of retrospective clinical data (5). These findings suggest that, in the future, the genotyping of patients and/or donors with respect to a panel of cytokines, chemokines, pharmacogenes, etc. will possibly complement histocompatibility typing and increase our ability to predict the risk of transplant-related toxicity (6).
Table 3: Risk factors for the development of GvHD Donor
Recipient
HLA compatibility (related/unrelated) Sex mismatched (FÆM) Alloimmunisation (parity, transfusions) Source of SC (PBSC > BM > CB)
Age Conditioning regimen Prevention of GvHD
5. Histopathology Apoptosis of cells in the tissue layer responsible for proliferation and regeneration is a typical histologic feature of GvHD and is the final event of the allogeneic reaction (Phase 3: Cellular and inflammatory effector phase). The targets are epithelial cells, including basal and suprabasal cells of the epidermis, the intestinal epithelium and the biliary duct epithelium. In the three target organs, the characteristic lesion of GvHD is the same, with infiltrating immune cells in the vicinity of the apoptotic cell giving a feature classically termed “satellite cell necrosis”. 5.1. Skin biopsies Skin biopsies are widely used in the diagnosis of a GvHD. The dermoepidermal junction
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is most severely affected: There is epidermal and basal cell vacuolar degeneration, disorganisation of epidermal cell maturation, eosinophilic body formation and melanocyte incontinence. In the first 3 weeks after transplant, the microscopic lesions induced by the conditioning regimen, i.e. lymphocytic infiltrates and epithelial damage, can be very similar to early lesions of GvHD. When the histological lesions are mild, it can be useful to look for satellite cell necrosis by electron microscopy, or to use an anti-TNF antibody to stain activated intraepithelial lymphocytes. In very severe forms of aGvHD with epidermal necrolysis (Lyell syndrome) clinical diagnosis is obvious. Skin biopsy would show satellite cell necrosis of all the keratinocytes of the basal layer leading to the dissociation of the upper part of the epidermis from the basal layer. 5.2. Gut biopsies Gut biopsies are performed as a diagnostic procedure to explore gastro-intestinal (GI) tract symptoms, mainly nausea or diarrhoea. Rectal or colon biopsies have been generally replaced by gastro-duodenal biopsies. The target cells for GvHD are basal epithelial cells localised in the epithelium of the crypts, which undergo apoptosis in the close vicinity of intraepithelial lymphocytes. In the acute phase of GvHD, it is important to distinguish lesions of GvHD from those induced by virus, particularly CMV. Classically, epithelial cells infected by CMV have voluminous clear inclusions and become necrotic. However, in GI lesions of recent onset, viral inclusions can be difficult to see and, in some cases, CMV and GvHD can be associated. Therefore, the possibility of CMV infection should be excluded using antibodies directed against CMV early proteins. 5.3. Liver biopsies Liver biopsies are performed only to rule out viral infection or drug toxicity when isolated hepatic GvHD is suspected. The target is the epithelium of the biliary canal and the lymphocytic infiltrates are mainly localised in portal tracts. Foci of necrotic hepatocytes can be observed, but there is no sclerosis in the acute phase of GvHD. Hepatic small bile ducts show segmental disruption, injury to the periductular epithelium, bile duct atypia and cellular degeneration. Cholestasis may be present.
6. Acute GvHD 6.1. Clinical manifestations and grading 6.1.1. Skin A maculo-papular rash, often involving the palms and soles usually marks the onset 222
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of aGvHD. Lesions may be pruritic and/or painful. The rash then spreads and can involve the entire body surface. In more severe cases, bullae can form and surface areas can desquamate, leading to extremely painful denudation associated with protein loss. 6.1.2. Liver Liver involvement results in cholestatic hepatopathy, with or without jaundice, in which the cholestatic enzymes are substantially raised whilst the transaminases show only non-specific changes. The clinical diagnosis of aGvHD of the liver is difficult since distinguishing liver impairment due to therapy associated hepatotoxicity, infection, veno-occlusive disease or GvHD is not always possible. 6.1.3. Gastro-intestinal Involvement of the GI tract primarily manifests as nausea and green watery diarrhoea. The enteral fluid loss is used as a measure of gut involvement. Severe abdominal pain, bloody diarrhoea and massive enteral fluid losses accompany advanced disease. A variant of mild enteric GvHD involving only upper GI tract has been described. Symptoms include anorexia and nausea without diarrhoea and this usually responds well to immunosuppressive therapy. 6.1.4. Concomitant signs of acute GvHD These include fever, decrease in performance status and weight loss. Other tissues such as lymphoid organs, mucous membranes, conjunctivae, exocrine glands and the bronchi may also be involved, but these are not included in the clinical staging and grading established by Glucksberg and more recently by the International Bone Marrow Transplant Registry (IBMTR). 6.1.5. Grading In 1974, Glucksberg published the first aGvHD classification (7). Each organ is staged from 0 to 4 (Table 4). These stages are combined to calculate an overall grade including objective assessment of organ function and subjective assessment of performance status (Table 5). It is routine practice to dichotomise GvHD severity into clinically insignificant grades 0–I and clinically significant grades II–IV. In recognition of the system’s limitations, a Consensus Workshop was held in 1995 and a modified grading was system proposed (Table 6) (8). The latter system retained the objective organ staging criteria of the Glucksberg system but excluded the subjective criteria of clinical performance. Thus, a revised system was developed by the IBMTR (Table 7) (9). To compare prospectively the Glucksberg and IBMTR classifications, a prospective
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multicentre study was conducted in 607 patients receiving T-cell replete allografts in 18 transplantation centres (10). Patients were scored weekly for aGvHD. The 2 classifications performed similarly in explaining variability in survival by aGvHD score, although the Glucksberg classification predicted early survival better. There was less physician bias or error in assigning grades with the IBMTR scoring system. With either system, only the maximum observed grade had prognostic significance for survival.
Table 4: Grading system: Stage for each organ Stage
Skin / Maculo-papular rash
Liver / Bilirubin
GI / Diarrhoea
+
<25% of body surface
34-50 mmol/L
> 500 mL
++
25-50% of body surface
51-102 mmol/L
> 1000 mL
+++
Generalised erythroderma
103-255 mmol/L
> 1500 mL
++++
Generalised erythroderma with bullae formation and desquamation
> 255 mmol/L
Severe abdominal pain with or without ileus
Table 5: Overall grading system (Glucksberg) Grade of aGvHD
Degree of organ involvement
I
Skin: + to ++
II
Skin: + to +++ Gut and/or liver: + Mild decrease in clinical performance
III
Skin: ++ to +++ Gut and/or liver: ++ to +++ Marked decrease in clinical performance
IV
Skin: ++ to ++++ Gut and/or liver: ++ to ++++ Extreme decrease in clinical performance
Table 6: Consensus Conference on aGvHD grading
224
Grade
Skin
Liver
Gut
I
Stage 1–2
0
0
II
Stage 3 or
Stage 1 or
Stage 1
III
–
Stage 2–3 or
Stage 2–4
IV
Stage 4 or
Stage 4
–
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Table 7: IBMTR Severity Index for aGvHD Skin
Liver
GI
INDEX
Stage (max)
Extent of rash
Stage (max)
Bilirubin (µmol/L)
Stage (max)
Diarrhoea (mL/d)
A
1
<25%
0
<34
0
<500
B
2
25–50% or
1–2
34–102
1–2
550–1500
C
3
>50% or
3
103–255 or
3
>1500
D
4
Bullae or
4
>255 or
4
Pain, ileus
Thus, the authors failed to demonstrate a clear advantage of one system for grading aGvHD. 6.2. Prevention and treatment Acute GvHD is the major cause of early TRM. Mortality is due not only to GvHD itself but also to treatment related complications, both being responsible for a profound immune deficiency with frequent opportunistic infections. A major goal is prevention of GvHD. However, to date, it has not been possible to separate the deleterious effect of GvHD from the beneficial GvL effect. This explains why it is difficult to define the best strategy to prevent GvHD. Once GvHD occurs, the most important predictor of long-term survival is the primary response to therapy, as results with secondary treatments have been disappointing. 6.2.1. Prevention of GvHD a) Post transplant immunosuppressive treatment of the recipient (Table 8) Currently, most centres use a combination of a calcineurin inhibitor (cyclosporin (CsA) or tacrolimus) with “short course” methotrexate (MTX). Although other regimens are being explored, this standard regimen, described twenty years ago (11), has been shown repeatedly to result in a reasonable balance of GvHD and GvL in matched sibling transplants after ablative conditioning regimen. For higher risk groups or groups receiving non-conventional grafts (such as mismatched donors, older patients, reduced intensity regimen etc.), the best prophylaxis is less clearly established and other immunosuppressive drugs have been used: Sirolimus has been used in combination with tacrolimus and low dose (5 mg/m2) short MTX or with tacrolimus alone with prompt engraftment and minimal TRM (12). The efficacy of mycophenolate mofetil (MMF) associated with CsA has been studied mainly after RIC regimens (13). MMF may substitute MTX in the standard CsA combination because of less mucositis and overall good tolerance.
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Table 8: GvHD prophylaxis agents and mechanisms of action Mechanism of action
Dose
Cyclosporin (CsA)
Calcineurin inhibitor: blockade of T-cell activation
3 mg/kg IV
Tacrolimus
Calcineurin inhibitor: blockade of T-cell activation
0,02 mg/kg IV
Methotrexate (MTX)
Antimetabolite, folic acid analogue
15 mg/m2 D+1, 10 mg/m2 D+3, +6 and +11
Methylprednisolone (MP)
Receptor-mediated lympholysis + additional mechanisms
0,5–1 mg/kg
Mycophenolate mofetil (MMF)
Inhibition of DNA synthesis: lymphocyte apoptosis
1.5 to 3 g/d
Sirolimus (Rapamycine)
Macrolide antibiotic: blockade of T- and B-cell activation
12 mg D–3 then 4 mg/d
Antithymocyte globulin (ATG)
Rabbit or equine antibodies against human T-cells
2,5 mg/kg/d x 4
Alemtuzumab (Campath-1H)
Humanised monoclonal antibody to CD52
MethylPDN is the first line treatment for aGvHD, but its role in the prevention of GvHD is controversial. Several combination using prednisone in combination with MTX, CsA or both have been reported without substantial benefit. b) In vitro or in vivo T-cell depletion (TCD) of the transplant For patients receiving mismatched grafts, more intensive immunosuppression is usually needed. Methods of ex vivo TCD as well as pharmacologic in vivo TCD (anti-thymocyte globulin, alemtuzumab) have been used. In general, these methods reduce aGvHD but increase the incidence of infection (due to delayed reconstitution of the immune system) and the incidence of relapse (due to a decreased GvL effect). Several randomised or comparative studies have been performed comparing in vitro TCD to CsA + MTX, but so far, it has not been conclusively established whether TCD can improve LFS (14). 6.2.2. Treatment of aGvHD a) Primary treatment The most important predictor of long-term survival is the primary response of GvHD to therapy. Methyl-prednisolone (MP) at a dose of 2 mg/kg/d is the best initial therapy for aGvHD. This treatment, associated with a calcineurin inhibitor, is given for 7 to 14 days, and then tapered slowly if there is a complete response to therapy. 226
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Complete responses occur in 25 to 40% of patients with grade II to IV aGvHD. However, the likelihood of response decreases with increasing severity of the disease and the dynamic of responses may differ between target organs and between patients. Failure of therapy is usually defined as: • Progression after 3 days, or • No change after 7 days, or • Incomplete response after 14 days. b) Secondary treatment Patients in whom initial therapy has failed will receive a secondary treatment. Table 9 provides a summary of available agents. Numerous strategies have been used but only very few controlled studies have been conducted and there are no criteria to identify patients who are likely to respond to any second line treatment. Overall, results have been disappointing. The rate of partial and complete response to second line therapy varies from 35 to 70%, but the 6–12 months survival is low, in the order of 30%, in most large trials, because of high incidence of infectious complications or recurrence of GvHD. Treatment of steroid-refractory aGvHD has not made major advances in the last decade, as shown in several reviews recently published (15–17). In patients with predominant GvHD of the skin, which is refractory to steroids, the use of phototherapy may be an attractive and non-toxic strategy. In case of visceral
Table 9: Second line treatment of steroid-refractory aGvHD Methylprednisolone (2–5 mg/kg) Immunosuppressive drugs: - Tacrolimus, MMF, sirolimus (if not used for prophylaxis) Oral non-absorbable steroids (in case of GI involvement) Anti-thymocyte globulin Monoclonal antibodies: - Anti-IL-2 receptor (CD25) antibody: Inolinomab, basiliximab, daclizumab, denileukin difitox - Anti-TNFa antibody: Infliximab, etanercept - Anti-CD52 antibody (broad specificity T-cell antibody): Alemtuzumab (Campath 1H) - Anti-CD147 antibody (anti activated T- and B-cells): ABX/CBL - Anti-CD3 (broad specificity T-cell antibody): Visilizumab, OKT3 Pentostatin: - Inhibitor of adenosine-deaminase Extracorporeal photopheresis: - Suppression of T-cell reactivity and cytokine release; induction of regulatory T-cells Mesenchymal stem cells: - Immunomodulatory and tissue repairing effect
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GvHD, despite concerns about toxicity, second line treatment can be either polyclonal antibodies (i.e. ATG) or monoclonal antibodies. Other approaches, such as the use of mesenchymal stem cells, need to be explored in prospective randomised trials. There is agreement that efforts must be aimed at reducing steroid exposure and protecting the patient against infectious complications. c) Supportive care Supportive measures are very important. They include GI rest, parenteral hyperalimentation, replacement therapy of enteral losses of fluids, pain control, and infectious prophylaxis. Nevertheless, despite all preventive measures, viral, bacterial and mycotic infections are common and are the most frequent causes of death in patients with severe aGvHD. Ultimately, the outcome of aGvHD depends on two main factors: the overall grade of aGvHD and the response to treatment.
7. Chronic GvHD 7.1. Clinical manifestations and grading Chronic GvHD is the primary cause of late morbidity and non-relapse mortality in transplant survivors. Chronic GvHD has features resembling autoimmune and other immunologic disorders such as scleroderma, Sjögren syndrome, primary biliary cirrhosis, wasting syndrome, bronchiolitis obliterans (BO), immune cytopenias and chronic immunodeficiency. Symptoms usually present within 3 years after allogeneic HCT and are often preceded by a history of aGvHD. Manifestations of cGvHD may be restricted to a single organ or tissue or may be widespread. Chronic GvHD can lead to debilitating consequences, e.g. joint contractures, loss of sight, end-stage lung disease and profound chronic immunosuppression. Risk factors for cGvHD have been identified, including prior aGvHD, older patient age, the use of female donors for male recipients, use of donor lymphocyte infusion, use of unrelated or HLA-mismatched donors, and, more recently, the use of PBSC as a source of stem cells. 7.1. Diagnosis and staging The list of signs and symptoms of cGvHD, as established by the NIH working group report on diagnosis and staging (4), is shown in Table 10, with a distinction between diagnostic signs (that establish the diagnosis of cGvHD without the need for further testing or symptom), distinctive signs (not found in aGvHD but not sufficient to establish the diagnosis of cGvHD), other features of cGvHD (non-specific) 228
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Table 10: Signs and symptoms of cGvHD Organ/site
“Diagnostic”
Skin
Poikiloderma Depigmentation Lichen planuslike features
Nails
Other
Both acute and chronic Erythema, maculopapular rash, pruritus
Dystrophy
Scalp Mouth
“Distinctive”
Alopecia Lichen planus
Eyes
Xerostomia
Mucositis
Keratoconjunctivitis Photophobia, sicca blepharitis
Genitalia
Lichen planus
GI tract
Strictures of oesophagus
Exocrine pancreatic insufficiency
Liver
Anorexia, weight loss Bilirubin or alkaline phosphatase > 2 x upper limit of normal
Lung
Bronchiolitis obliterans
Muscles, fascia, joints
Fasciitis, joint contractures
Myositis
Cramps, arthralgias
Haematopoietic and immune
Thrombocytopenia eosinophilia, lymphopenia
Other
Ascites, pericardial or pleural effusions
and common signs or symptoms (found in both aGvHD and cGvHD). The diagnosis of cGvHD requires at least one diagnostic manifestation of cGvHD or at least one distinctive manifestation, with the diagnosis confirmed by pertinent biopsy or laboratory test. Historically, cGvHD was classified as limited or extensive on the basis of a small retrospective study (18). The NIH working group proposed a new global scoring system, which includes both the number of organs or sites involved and the severity within each affected organ: score 0 = no symptoms, score 1 = mild symptoms, score 2 = moderate symptoms and score 3 = severe symptoms. Mild cGvHD involves only 1 or 2 organs (except lung) with a maximum score of 1. Moderate cGvHD involves at least 1 organ with score 2, or 3 or more organs with score 1 (or lung score 1). Severe cGvHD indicates a score of 3 in any organ (or score 2 in the lung) (Table 11). HAEMATOPOIETIC STEM CELL TRANSPLANTATION
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Table 11: Global scoring of cGvHD, according to the number of sites and the severity for each organ or site Number of organs
Mild cGvHD
Moderate cGvHD
Severe cGvHD
1 organ or site
Score 1
Score 2
Score 3
2 organs or sites
Score 1
Score 2
Score 3
3 or more organs
Score 1
Score 3
Lung involvement
Score 1
Score 2
7.3. Prevention and treatment Despite the fact that significant aGvHD is one of the strongest predictors for the development of cGvHD, successful efforts to reduce aGvHD with combinations of immunosuppressive agents, such as tacrolimus or MMF, have not resulted in a reduced incidence of cGvHD (19). Other methods, such as in vivo or in vitro TCD have been studied but the benefits due to a decreased incidence of acute and cGvHD need to be balanced against the consequences of delayed immune reconstitution. The role of prolonged administration of CsA for cGvHD prophylaxis has been studied and has shown only a non-significant reduction in the risk of extensive cGvHD with no difference in overall survival or disease free survival (20). 7.3.1. Primary treatment A combination of CsA and prednisolone has been the standard frontline therapy for cGvHD for almost 20 years (21). The initial dose of steroid ranges from 1 to 1.5 mg/kg/day for at least 2 weeks then the dose is slowly tapered, according to response. Duration of therapy is also determined by response, but is prolonged, usually continuing for close to 12 months, even in patients achieving complete resolution. The morbidity associated with steroid therapy is significant, including avascular necrosis, glucose intolerance requiring administration of insulin, infections, hypertension, changes in body habitus, cutaneous atrophy, cataracts, osteoporosis, emotional lability, interference with sleep, and growth retardation in children. So far, the addition of other immunosuppressive drug to standard upfront therapy, such as thalidomide, has not led to any significant difference in cGvHD response rate or survival (22). There is a need for randomised prospective trials to investigate the addition of other immunosuppressive drugs to upfront therapy in an effort to improve outcomes and ameliorate steroid-related side-effects. Such a trial, sponsored by the EBMT, has been activated in 2007 (double-blind placebo-controlled trial comparing CsA plus steroids with or without myfortic (the biologically active component of MMF) as primary treatment for extensive cGvHD). 230
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CHAPTER 11 • Graft vs. host disease
7.3.2. Salvage therapy There is no standard second-line therapy for patients who have failed primary treatment. Various approaches have been developed, including low dose total lymphoid irradiation, PUVA therapy, extracorporeal photochemotherapy, MMF, tacrolimus, and thalidomide. All these treatments have been reported to improve clinical manifestations (23). Recent publications have described encouraging responses following therapy with rituximab (anti-CD20 monoclonal antibody) (24). The efficacy of sirolimus (25) or of pentostatin (26) has also been investigated and some objective responses were described. 7.3.3. Antimicrobial prophylaxis and supportive care Infection is the leading cause of death among patients with cGvHD. Antimicrobial prophylaxis is a very important aspect of the treatment of these patients. Infections with Streptococcus pneumoniae and Haemophilus influenzae are frequent and must be prevented with prophylactic antibiotics and/or vaccination. Pneumocystis pneumonia occurring late after HSCT is strongly associated with cGvHD and prophylaxis must be given to patients receiving long-term immunosuppressive treatment. Additional treatments include ursodeoxycholic acid in patients with hepatic GvHD, anti-osteoporosis agents, and artificial tears in patients with eye involvement. A multidisciplinary approach to the management of patients with cGvHD is essential, including dermatologists, ophthalmologists, stomatologists, gynaecologists and others. In retrospective series of patients with extensive cGvHD, only 10 to 30% became long-term survivors. Late mortality is related not only to opportunistic infections but also to an increased incidence of secondary cancers.
8. Conclusion In conclusion, early institution of effective immunosuppressive therapy has changed the outcome for patients with GvHD, but larger studies demonstrating safety and efficacy of second line treatments for patients non-responsive to standard therapy with steroids are mandatory.
References 1. Billingham RE. The biology of graft versus host reactions. Harvey lectures 1966-1967; 62: 71-78. 2. Reddy P, Ferrara JLM. Immunobiology of acute graft-versus-host disease. Blood Reviews 2003; 17: 187-194. HAEMATOPOIETIC STEM CELL TRANSPLANTATION
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3. Tse WT, Pendleton JD, Beyer Wm, et al. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: Implication in transplantation. Transplantation 2003; 75: 389-397. 4. Filipovich AH, Weisdorf D, Pavletic S, et al. National Institutes of Health consensus development project on criteria for clinical trials in chronic Graft-versus-host Disease: I. Diagosis and Staging Working group report. Biol Blood Marrow Transplant 2005; 11: 945956. 5. Dickinson AM, Charron D. Non-HLA immunogenetics in hematopoietic stem cell transplantation. Curr opin Immunol 2005; 17: 517-525. 6. Baron C, Somogyi R, Greller LD, et al. Prediction of graft-versus-host disease in humans by donor gene-expression profiling. PLoS Med 2007; 4: e23. 7. Glucksberg H, Storb R, Fefer A, et al. Clinical manifestations of graft versus host disease in human recipients of marrow from HLA-matched sibling donors. Transplantation 1974; 18: 295-304. 8. Przepiorka D, Weisdorf D, Martin P, et al. Consensus conference on acute GvHD grading. Bone Marrow Transplant 1995; 15: 825-828. 9. Rowlings PA, Przepiorka D, Klein JP, et al. IBMTR severity index for grading acute GvHD: retrospective comparison with Glucksberg grade. Br J Haematol 1997; 97: 855-864. 10.Cahn JY, Klein JP, Lee SJ, et al. Prospective evaluation of 2 acute GvHD grading system: A joint Société française de Greffe de Moelle et Thérapie Cellulaire (SFGM-TC), Dana Farber Cancer institute (DFCI), and International Bone Marrow Transplant Registry (IBMTR) prospective study. Blood 2005; 106: 1495-1500. 11.Storb R, Deeg HJ, Whitehead J, et al. Methotrexate and cyclosporine compared with cyclosporine alone for prophylaxis of acute graft versus host disease after marrow transplantation for leukaemia. N Engl J Med 1986; 314: 729-735. 12.Cutler C. Li S, Ho VT, et al. Extended follow-up of methotrexate-free immunosuppression using sirolimus and tacrolimus in related and unrelated donor peripheral blood stem cell transplantation. Blood 2007; 109: 3108-3114. 13.Niederwieser D, Maris M, Shizuru JA, et al. Low-dose total body irradiation and fludarabine followed by hematopoietic cell transplantation from HLA-matched or mismatched unrelated donors and post grafting immunosuppression with cyclosporine and mycophenolate mofetil can induce durable complete chimerism and sustained remission in patients with haematological diseases. Blood 2003; 101: 1620-1629. 14.Wagner JE, Thompson JS, Carter S, et al. Effect of GvHD prophylaxis on 3-year DFS in recipients of unrelated donor bone marrow (T-cell Depletion Trial): A multi-centre, randomised phase II-III trial Lancet 2005; 366: 733-741. 15.Kim SS. Treatment options in steroid-refractory acute GvHD following hematopoietic stem cell transplantation. The Annals of Pharmacotherapy 2007; 41:1436-1444. 16.Bacigalupo A. Management of acute GVHD. Br J Haematol 2007; 137: 87-98. 17.Deeg JH. How I treat refractory acute GVHD. Blood 2007; 109: 4119-4126. 18.Shulmann H, Sullivan KM, Weiden PL, et al. Chronic graft-versus-host disease in man: A clinic pathologic study of 20 long term Seattle patients. Am J Med 1980; 69: 204-217. 19.Fraser CJ, Baker KS. The management and outcome of chronic graft-versus-host disease. Br J Haematol 2007; 138: 131-145. 232
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20.Kansu E, Gooley T, Flowers ME, et al. Administration of cyclosporine for 24 months compared with 6 months for prevention of chronic graft-versus-host disease: A prospective randomized clinical trial. Blood 2001; 98: 3868-3870. 21.Sullivan KM, Witherspoon RP, Storb R, et al. Alternating day cyclosporine and prednisone for treatment of high-risk chronic graft-versus-host disease. Blood 1988; 72: 555-561. 22.Arora M, Wagner JE, Davies SM, et al. Randomized clinical trial of thalidomide, cyclosporine and prednisone versus cyclosporine and prednisone as initial therapy for chronic GvHD. Biol Blood Marrow Transplant 2001; 7: 265-273. 23.Vogelsang GB. How I treat chronic graft-versus-host disease. Blood 2001; 97: 1196-1201. 24.Cutler C, Miklos D, Kim HT, et al. Rituximab for steroid-refractory chronic graft-versushost disease. Blood 2006; 108: 756-762. 25.Jurado M, Vallejo C, Perez-Simon JA, et al. Sirolimus as part of immunosuppressive therapy for refractory chronic graft-versus-host disease. Biol Blood Marrow Transplant 2007; 13: 701-706. 26.Jacobsohn DA, Chen AR, Zahurak M, et al. Phase II study of pentostatin in patients with corticosteroid-refractory chronic graft-versus-host disease. J Clin Oncol 2007; 25: 42554261.
Mutiple Choice Questionnaire To find the correct answer, go to http://www.esh.org/ebmt-handbook2008answers.htm 1. The prognosis of aGvHD is mainly related to: a) The diagnosis of the initial disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) The age of the donor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) The % of body surface involved . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) The response of GvHD to initial therapy with 2 mg/kg methyl prednisolone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. One factor is not significantly associated with an increased risk of cGvHD: a) The use of a male donor for a female recipient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) The previous occurrence of an aGvHD grade ≥ I . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) The use of PBSC as a source of stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) The age of the recipient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. T-cell depletion of the transplant is: a) Associated with a better long term survival in all patients when compared with a non T-cell depleted transplant . . . . . . . . . . . . . . . . . . . . . . . . . . . . HAEMATOPOIETIC STEM CELL TRANSPLANTATION
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b) Associated with an increased risk of cGvHD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) An effective prophylaxis of aGvHD but is associated with an increased risk of graft failure/relapse/opportunistic infections . . . . . . . . . . . . . . . . . . . . . . . d) Associated with an increased risk of secondary solid tumour . . . . . . . . . . . . . . 4. The “classical” prevention of GvHD is: a) A combination of methylprednisolone 1 mg/kg/d and a calcineurin inhibitor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) A combination of a calcineurin inhibitor and short course methotrexate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Mycophenolate mofetil alone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) A course of ATG during 10 days . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Chronic GvHD is: a) Associated with an increased risk of relapse of the leukaemia . . . . . . . . . . . . b) Associated with an increased risk of secondary cancer . . . . . . . . . . . . . . . . . . . . . c) Associated with an increased risk of veno-occlusive disease . . . . . . . . . . . . . . d) Observed only after a grade II to IV aGvHD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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NOTES
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*
CHAPTER 12
Long term survivorship, general health status, quality of life and late complications after HSCT
A. Tichelli, C-P. Schwarze, G. Socié
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CHAPTER 12 • Late effects
1. Introduction Large numbers of patients now survive long term following haematopoietic stem cell transplantation (HSCT). The aim of HSCT is not only to cure the patients from their primary disease, but also to allow the long-term survivor to obtain a normal health status, to return to work or to school and to have a normal social life. Immediate survival is therefore no longer the sole concern. Still, HSCT remains associated with considerable morbidity and mortality. Long-term health status and the development of late events related to HSCT have therefore gained increasing interest. Secondary malignant diseases are of particular clinical concern as more patients survive the early phase after transplantation and remain free of their original disease. Nonmalignant late effects are heterogeneous, and although often non-life threatening they significantly impair the quality of life of long-term survivors. This Chapter presents an overview of these malignant and non-malignant late complications, describes the consequences of these late events on the general health status, social integration and quality of life in long-term survivors, and provides recommendations regarding their prevention and early treatment (1, 2). Readers will find extensive literature summary and references in recently published reviews (3–5).
2. Non malignant late complications (Table 1) (2) 2.1. Late ocular effects 2.1.1. Ocular complications of the posterior segment These can be divided into microvascular retinopathy, optic disk oedema, haemorrhagic complications and infectious retinitis. Fungal infections typically occur within 120 days of HSCT, while Herpes zoster, CMV and Toxoplasma retinitis occur later. Ischaemic retinopathy with cotton-wool spots and optic-disk oedema has been described in 10% of patients following HSCT. Microvascular retinopathy occurs mainly after TBI conditioned allogeneic HSCT in patients receiving cyclosporin as GvHD prophylaxis. In most cases, the retinal lesions resolve with withdrawal or reduction of immunosuppressive therapy. 2.1.2. Ocular complications of the anterior segment The two most common late complications affecting the anterior segment are cataract formation and kerato-conjunctivitis sicca syndrome. Posterior subcapsular cataracts have long been recognised in recipients of HSCT as one of most frequent late complications of TBI. After single dose TBI almost all patients develop cataracts within 3 to 4 years and most if not all require surgical repair. Although the probability of developing cataracts after fractionated TBI is
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around 30% at 3 years the incidence may be more than 80% 6 to 10 years post HSCT (6, 7). Use of TBI in general and, single fractionations in particular and the use of
Table 1: Non-malignant late tissue and organ toxicity in long-term survivors Organ
Clinical manifestation
Risk factors
Monitoring
Intervention
Eye
Cataracts
Radiation Steroids
Split lamp examination
Fractionation of TBI Surgical repair
Keratoconjunctivitis Radiation GvHD
Schirmer test
Treatment of GvHD Topical lubricants Topical steroids
Restrictive or dilated Anthracyclines cardiomyopathy Mediastinal Arrhythmia radiotherapy Autonomic neuropathy
LVEF 24-hour ECG
Treatment of cardiac insufficiency Pace maker
Infection GvHD Smoking
Pulmonary function testing Chest radiographic testing if clinically indicated
Treatment of infection Immune globulin replacement Treatment of GvHD
Restrictive lung disease
Radiation Chemotherapy Infection
Pulmonary function testing Chest radiographic testing if clinically indicated
Fractionation of radiation Lung shielding Treatment of infection Steroids
Chronic hepatitis C
HVC infection Iron overload
Liver tests Hepatitis serologies Viral load if positive
Treatment of hepatitis C Treatment of iron overload (phlebotomy or chelation therapy)
Liver cirrhosis
HCV infection Iron overload
Ferritin
Heart
Respira- Chronic obstructive tory tract lung disease Bronchiolitis obliterans
Liver
Chronic GvHD of the liver Kidney
Nephropathy
TBI Chemotherapy (platinum) Cyclosporin
Liver biopsy if indicated
Treatment of GvHD
Renal function tests
Control of hypertension
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Organ
Clinical manifestation
Skeletal
Monitoring
Intervention
Avascular necrosis Steroids of the bone Radiation
Radiographic testing
Avoidance of long term treatment with steroids Symptomatic relief of pain Orthopoedic measures Surgical repair
Osteoporosis
Dual photon densitometry
Sex hormone replacement Treatment of osteoporosis (bisphosphonates, calcium, vitamin D)
Chronic stomatitis Chronic GvHD Radiation
Oral inspection
Oral hygiene Treatment of GvHD
Dental late effects
Radiation Chronic GvHD
Dental inspection
Prophylaxis: oral hygiene, instruction for dental care, brushing teeth, application of fluoride Treatment of caries
Thyroid gland
Hypothyroidism
Radiation
TSH, T4 annually
Thyroid hormone replacement
Gonadal function
Gonadal failure
Radiation Chemotherapy
FSH, LH, testosterone (males), oestradiol (females)
Hormone replacement Sperm banking in males
Fertility
Infertility
Radiation Chemotherapy
FSH, LH, testosterone (males), oestradiol (females) Sperm analysis
Cryopreservation of sperm fluid before HSCT
Nervous system
Leukoencephalopathy
Cranial radiation Intrathecal chemotherapy
Evaluation according to symptoms
Peripheral neuropathy
Chemotherapy GvHD
Atherosclerosis Cerebrovascular events Cardiovascular events Peripheral vascular events
Established Cardiovascular risk cardiovascular risk factors factors GvHD (?) Radiation (?)
Oral
Vascular compartment
Risk factors
Steroids, cyclosporin, tacrolimus Hypogonadism, TBI, chemotherapy Immobility
Correction of cardiovascular risk factors
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steroid treatment for longer than 3 months are associated with a significant risk of cataract development. The development of cataracts is also more likely if the TBI is delivered at a high dose rate. Finally, in prospective studies of the incidence of cataracts according to various risk factors, patients who receive cyclophosphamide and TBI (Cy/TBI) have a higher incidence of cataract formation than those treated with busulfan and cyclophosphamide (BuCy). The only treatment for cataract is to surgically remove the opacified lens from the eye to restore transparency of the visual axis. Today, cataract surgery is a low risk procedure and improves visual acuity in 95% of eyes that have no other pathology. Keratoconjunctivitis sicca syndrome is usually part of a more
