Minimal Residual Disease In Hematologic Malignancies (Acta Haematologica 2004)

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Minimal Residual Disease in Hematologic Malignancies

Guest Editors

Pia Raanani, Tel Hashomer/Tel Aviv Andreas Hochhaus, Mannheim

16 ﬁgures, 5 in color, and 8 tables, 2004

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Drug Dosage The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant ﬂow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.

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Vol. 112, No. 1–2, 2004

Contents

Editorial

55 Pathogenesis, Diagnosis and Monitoring of Residual

5 Minimal Residual Disease in Hematological

Malignancies Raanani, P. (Tel Hashomer/Tel Aviv); Hochhaus, A. (Mannheim)

Disease in Acute Promyelocytic Leukaemia Reiter, A.; Lengfelder, E. (Mannheim); Grimwade, D. (London) 68 FLT3 Length Mutations as Marker for Follow-Up

Studies in Acute Myeloid Leukaemia Schnittger, S.; Schoch, C.; Kern, W.; Hiddemann, W.; Haferlach, T. (Munich)

Reviews 8 Minimal Residual Disease Studies by Flow

Cytometry in Acute Leukemia Campana, D.; Coustan-Smith, E. (Memphis, Tenn.) 16 Strategies and Clinical Implications of Chimerism

Diagnostics after Allogeneic Hematopoietic Stem Cell Transplantation Thiede, C.; Bornhäuser, M.; Ehninger, G. (Dresden) 24 The Multiparametric Scanning System for

Evaluation of Minimal Residual Disease in Hematological Malignancies Trakhtenbrot, L.; Rechavi, G.; Amariglio, N. (Tel Hashomer/ Tel Aviv) 30 Standardization of Preanalytical Factors for Minimal

Residual Disease Analysis in Chronic Myelogenous Leukemia Müller, M.C.; Hördt, T.; Paschka, P.; Merx, K.; La Rosée, P.; Hehlmann, R.; Hochhaus, A. (Mannheim) 34 Minimal Residual Disease in Childhood Acute

Lymphoblastic Leukemia: Current Status and Challenges

79 WT1 as a Universal Marker for Minimal Residual

Disease Detection and Quantification in Myeloid Leukemias and in Myelodysplastic Syndrome Cilloni, D.; Saglio, G. (Turin) 85 Molecular Surveillance of Chronic Myeloid Leukemia

Patients in the Imatinib Era – Evaluation of Response and Resistance Paschka, P.; Merx, K.; Hochhaus, A. (Mannheim) 93 Clinical Implications of Minimal Residual Disease

Monitoring for Stem Cell Transplantation after Reduced Intensity and Nonmyeloablative Conditioning Shimoni, A.; Nagler, A. (Tel Hashomer) 105 Molecular and Clinical Follow-Up after Treatment of

Multiple Myeloma Rasmussen, T.; Knudsen, L.M.; Huynh, T.K.; Johnsen, H.E. (Herlev) 111 Significance of Minimal Residual Disease in

Lymphoid Malignancies Brüggemann, M.; Pott, C.; Ritgen, M.; Kneba, M. (Kiel)

Izraeli, S.; Waldman, D. (Tel Hashomer/Tel Aviv) 40 Detection of Minimal Residual Disease in Acute

Myelogenous Leukemia Raanani, P.; Ben-Bassat, I. (Tel Hashomer/Tel Aviv)

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120 Author Index Vol. 112, No. 1–2, 2004 120 Subject Index Vol. 112, No. 1–2, 2004

Editorial Acta Haematol 2004;112:5–7 DOI: 10.1159/000077553

Minimal Residual Disease in Hematological Malignancies Pia Raanani, Tel Hashomer/Tel Aviv

Andreas Hochhaus, Mannheim

Do you prefer quality to quantity? This question has been asked for many years. It is well known that mathematics is the science of pure quantity but what about medicine? For many years, counting cells and identifying them by microscopic inspection have determined the number of normal or abnormal cells in hematological and nonhematological malignancies. During the last decade, several studies have shown that detection and quantification of residual tumor cells significantly correlate with clinical outcome in several types of hematological malignancies. In particular the quantitative measurement of the decrease of the leukemic cell load during the first phases of treatment has a high prognostic value [1]. Detection of minimal residual disease (MRD) is now becoming routinely implemented in treatment protocols and is increasingly used for guiding therapy and for evaluation of new treatment modalities [2]. Methods to detect MRD include technologies designed to detect residual malignant cells beyond the sensitivity of conventional approaches like for example morphology and banding cytogenetics in leukemia. A wide variety of techniques have been developed. The choice of the best method for the particular clinical situation certainly depends on the biology of the individual malignancy, i.e. on the determination of specific markers, which are useful to differentiate between leukemic cells and normal hematopoiesis in leukemic patients. These markers include leukocyte differentiation antigens, fusion transcripts, transcripts overexpressed by mutated or nonmutated genes,

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rearranged genes, and individual markers, like polymorphic repetitive DNA sequences. In this issue we sought to provide a comprehensive overview on the major technologies for the detection of MRD and their clinical applications. Campana and Coustan-Smith critically discuss advantages and disadvantages of flow cytometry methods in acute leukemias. A special direction of MRD detection is the differentiation between donor and recipient hematopoiesis after allogeneic stem cell transplantation as discussed by Thiede et al. The authors also reviewed the technical issues, advantages and limitations of the methods currently used for chimerism analysis. Adding the morphological analysis of small populations of cells to malignant or recipient-associated markers may improve the accuracy of chimerism and MRD testing, and delineate their clinical significance. Trakhtenbrot et al. introduce the simultaneous analysis of morphology, immunophenotyping and FISH on the same cell, i.e. a multiparametric scanning system. For the comparability of results, an agreement should be reached between various laboratories and different multicenter studies on sample source and volumes obtained (or minimum cell counts) for RT-PCR analyses and regarding the general methods of cell purification, RNA extraction and cDNA synthesis. Rigorous, internationally accepted controls need to be implemented. Müller et al. investigated the impact of preanalytical factors and their standardization. Their review introduces some important considerations for the implementation of RT-PCR-based MRD

endpoints into clinical trials. Specific aspects of childhood acute lymphoblastic leukemia (ALL) are elucidated by Izraeli and Waldman. Fusion transcripts or overexpressed genes, like the Wilms tumor gene WT1, represent a common target for MRD analysis in acute myelogenous leukemia (AML). Current standards and their clinical implications are demonstrated by Raanani and Ben-Bassat as well as the dilemmas and unresolved issues in the interpretation of MRD in AML patients. Recently, the genomic structure of acute promyelocytic leukemia (APL) has been described. Structural factors for the pathogenesis of APL and their implications for MRD monitoring are reviewed by Reiter et al. FLT3 length mutations in AML are considered as prognostic markers as well as markers for MRD studies and are also targets for novel treatment modalities. The current view in this respect is summarized by Schnittger et al. Reduced intensity stem cell transplantation techniques are based on individualized additional immunotherapy depending on exact and rapid MRD monitoring. Shimoni and Nagler outline clinical and methodological aspects of MRD in nonmyeloablative transplantations. MRD monitoring in multiple myeloma is part of purging studies of stem cell aphaeresis and evaluation of novel therapies for this disease. The clinical considerations and molecular data are summarized by Rasmussen et al. The clinical need for molecular endpoints has become even more apparent with the introduction of imatinib in the therapy of chronic myelogenous leukemia (CML) and BCR-ABL-positive ALL. A systematic quantitative PCR monitoring emphasized the prognostic impact of residual BCR-ABL transcripts in CML patients [3, 4]. Paschka et al. discuss the clinical applications and standardized approaches for treatment surveillance of CML patients in the imatinib era. The majority of malignant lymphoproliferative disorders display clonal rearrangement of the antigen receptor genes suitable for detection by PCR. According to Brüggemann et al. MRD-based intensification, modification of treatment and guidance of maintenance treatments should be the goal of current clinical studies to determine the prognostic significance of MRD in chronic lymphocytic leukemia and non-Hodgkin’s lymphomas. The WT1 gene is considered a new universal marker for MRD detection and quantification in myeloid malignancies and myelodysplastic syndromes. Cilloni and Saglio review molecular studies using WT1 for the follow-up of these patients. Currently, PCR-based methods represent the most widely accepted technologies for MRD detection. Over the past 15 years, PCR techniques and implications have been optimized and improved. In view of the limited val-

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Acta Haematol 2004;112:5–7

ue of qualitative PCR for treatment surveillance, quantitative PCR assays were developed. Competitive RT-PCR was employed to monitor CML patients after stem cell transplantation [5, 6] or treatment with interferon alpha [7], and patients with acute leukemias harboring specific fusion transcripts (CBFß-MYH11 [8]; AML1-ETO [9, 10]). Despite considerable specificity and sensitivity, these methods were cumbersome and time-consuming and were therefore only employed in specialized laboratories. With the introduction of real-time PCR methodologies and machines quantification of residual disease has been simplified. Results of real-time PCR were readily comparable with competitive PCR data [11, 12]. Thus, quantification of residual disease has been developed as a major diagnostic tool in most studies focusing on treatment optimization in leukemias characterized by fusion transcripts. A variety of real-time PCR instruments are available and different approaches can be applied. To reach reproducible, sensitive and standardized quantitative PCR data, important prerequisites should be considered: (1) The sensitivity that can be obtained in RT-PCR analyses depends on the number of cells and the total amount of RNA analyzed and on the use of a single or nested PCR approach. (2) Unstabilized anticoagulated blood can be processed even in multicenter trials, when processing is guaranteed within 36 h. As an alternative, bedside RNA stabilization could be made available for multicenter studies with central analysis [13]. (3) The level of target gene transcripts per volume cDNA should be related to the expression of a standard gene. However, the heterogeneity of the preanalytical environment of PCR machines and methods, and of the methods to calculate the final results causes confusion among patients, treating physicians and laboratories. The universal acceptance of real-time PCR urgently demands standardization of nomenclature and technologies. At present, at least seven different systems are commercially available using three different methods of fluorescence labeling [2]. However, adjustment of the protocols, standardization of preanalytical considerations and of the methods to calculate the final transcript ratio should result in comparable data. In an attempt to reach an agreement on the minimal requirements for standardization of real-time PCR analyses in leukemias, new international studies have recently been launched. A significant step forward in this direction has been achieved by the recent creation of the European LeukemiaNet. A particular work package within this network has been assigned to MRD analysis in leukemias.

Raanani/Hochhaus

Standardized PCR protocols will serve as a methodological reference [14, 15] and the progress in the development of PCR machines, software and biochemical assays will certainly lead to further improvement. Methodological aspects and clinical applications of MRD monitoring and standardization strategies are the

major topics of this issue – ‘from bench to bedside’. We tried to give a comprehensive overview on the significance of MRD in the evaluation, treatment and follow-up of hematological malignancies. We are most grateful to our international colleagues for their major contributions to the subject.

References 1 van Dongen JJM, MacIntyre EA, Gabert JA, Delabesse E, Rossi V, Saglio G, Gotardi E, Rambaldi A, Dotti G, Griesinger F, Parreira A, Gameiro P, Gonzalez Diaz M, Malec M, Langerak AW, San Miguel JF, Biondi A: Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease. Leukemia 1999;13:1901–1928. 2 van der Velden VHJ, Hochhaus A, Cazzaniga G, Szczepanski T, Gabert J, van Dongen JJM: Detection of minimal residual disease in hematologic malignancies by real-time quantitative PCR: Principles, approaches, and laboratory aspects. Leukemia 2003;17:1013–1034. 3 Hughes TP, Kaeda J, Branford S, Rudzki Z, Hochhaus A, Hensley ML, Gathmann I, Bolton AE, van Hoomissen IC, Goldman JM, Radich JP, for the International Randomized Study of Interferon versus STI571 (IRIS) Study Group: Frequency of major molecular responses to imatinib or interferon alfa plus cytarabine in newly diagnosed chronic myeloid leukemia. N Engl J Med 2003;349:1423–1432. 4 Müller MC, Gattermann N, Lahaye T, Deininger MWN, Berndt A, Fruehauf S, Neubauer A, Fischer T, Hossfeld DK, Schneller F, Krause SW, Nerl C, Sayer HG, Ottmann OG, Waller C, Aulitzky W, le Coutre P, Freund M, Merx K, Paschka P, König H, Kreil S, Berger U, Gschaidmeier H, Hehlmann R, Hochhaus A: Dynamics of BCR-ABL mRNA expression in first line therapy of chronic myelogenous leukemia patients with imatinib or interferon ·/araC. Leukemia 2003;17:2392–2400. 5 Cross NCP, Feng L, Chase A, Bungey J, Hughes TP, Goldman JM: Competitive polymerase chain reaction to estimate the number of BCR-ABL transcripts in chronic myeloid leukemia patients after bone marrow transplantation. Blood 1993;82:1929–1936.

Minimal Residual Disease in Hematological Malignancies

6 Lion T, Henn T, Gaiger A, Kalhs P, Gadner H: Early detection of relapse after bone marrow transplantation in patients with chronic myelogenous leukaemia. Lancet 1993;341:275– 276. 7 Hochhaus A, Reiter A, Saussele S, Reichert A, Emig M, Kaeda J, Schultheis B, Berger U, Shepherd PCA, Allan NC, Hehlmann R, Goldman JM, Cross NCP, for the German CML Study Group and the UK MRC CML Study Group: Molecular heterogeneity in complete cytogenetic responders after interferon-alpha therapy for chronic myelogenous leukemia: Low levels of minimal residual disease are associated with continuing remission. Blood 2000; 95:62–66. 8 Seale JR, Varma S, Swirsky DM, Pandolfi PP, Goldman JM, Cross NC: Quantification of PML-RAR alpha transcripts in acute promyelocytic leukaemia: Explanation for the lack of sensitivity of RT-PCR for the detection of minimal residual disease and induction of the leukaemia-specific mRNA by alpha interferon. Br J Haematol 1996;95:95–101. 9 Tobal K, Newton J, Macheta M, Chang J, Morgenstern G, Evans PA, Morgan G, Lucas GS, Liu Yin JA: Molecular quantitation of minimal residual disease in acute myeloid leukemia with t(8;21) can identify patients in durable remission and predict clinical relapse. Blood 2000;95:815–819. 10 Wattjes MP, Krauter J, Nagel S, Heidenreich O, Ganser A, Heil G: Comparison of nested competitive RT-PCR and real-time RT-PCR for the detection and quantification of AML1/ MTG8 fusion transcripts in t(8;21) positive acute myelogenous leukemia. Leukemia 2000; 14:329–335.

11 Emig M, Saussele S, Wittor H, Weisser A, Reiter A, Willer A, Berger U, Hehlmann R, Cross NCP, Hochhaus A: Accurate and rapid analysis of residual disease in patients with CML using specific fluorescent hybridization probes for real time quantitative RT-PCR. Leukemia 1999;13:1825–1832. 12 Guo JQ, Lin H, Kantarjian H, Talpaz M, Champlin R, Andreef M, Glassman A, Arlinghaus RB: Comparison of competitive-nested and real-time PCR in detecting BCR-ABL fusion transcripts in chronic myeloid leukemia patients. Leukemia 2002;16:2447–2453. 13 Müller MC, Merx K, Weisser A, Kreil S, Lahaye T, Hehlmann R, Hochhaus A: Improvement of molecular monitoring of residual disease in leukemias by bedside RNA stabilization. Leukemia 2002;16:2395–2399. 14 Gabert J, Beillard E, van der Velden VHJ, Bi W, Grimwade D, Pallisgaard N, Barbany G, Cazzaniga G, Cayuela JM, Cavé H, Pane F, Aerts JLE, de Micheli D, Thirion X, Pradel V, Gonza´lez M, Viehmann S, Malec M, Saglio G, van Dongen JJM: Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction (RQ-PCR) of fusion gene transcripts for residual disease detection in leukemia – A Europe Against Cancer Program. Leukemia 2003;17: 2318–2357. 15 Beillard E, Pallisgaard N, van der Velden VHJ, Bi W, Dee R, van der Scoot E, Delabesse E, Macintyre E, Gottardi E, Saglio G, Watzinger F, Lion T, van Dongen JJM, Hokland P, Gabert J: Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using ‘real-time’ quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR) – A Europe Against Cancer Program. Leukemia 2003;17:2474–2486.

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Review Acta Haematol 2004;112:8–15 DOI: 10.1159/000077554

Minimal Residual Disease Studies by Flow Cytometry in Acute Leukemia Dario Campana a, b Elaine Coustan-Smith a a Departments b Department

of Hematology-Oncology and Pathology, St. Jude Children’s Research Hospital, and of Pediatrics, University of Tennessee College of Medicine, Memphis, Tenn., USA

Key Words Acute lymphoblastic leukemia W Acute myeloid leukemia W Flow cytometry W Minimal residual disease

Abstract Minimal residual disease (MRD) assays are increasingly important in the clinical management of patients with acute leukemia. Among the methods available for monitoring MRD, flow cytometry holds great promise for clinical application because of its simplicity and wide availability. Several studies have demonstrated strong correlations between MRD levels by flow cytometry during clinical remission and treatment outcome, lending support to the reliability of this approach. Flow-cytometric detection of MRD is based on the identification of immunophenotypic combinations expressed on leukemic cells but not on normal hematopoietic cells. Its sensitivity depends on the specificity of the immunophenotypes used to track leukemic cells and on the number of cells available for study. Immunophenotypes that allow detection of 1 leukemic cell in 10,000 normal cells can be identified in at least 90% of patients with acute lymphoblastic leukemia; immunophenotypes that allow detection of 1 leukemic cell in 1,000–10,000 normal cells can be identified in at least 85% of patients with acute

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myeloid leukemia. Identification of new markers of leukemia by gene array technology should lead to the design of simple and reliable antibody panels for universal monitoring of MRD. Here we review the relative advantages and disadvantages of flow cytometry for MRD studies, as well as results obtained in correlative studies with treatment outcome. Copyright © 2004 S. Karger AG, Basel

Introduction

Studies of minimal residual disease (MRD) are becoming central to the clinical management of patients with acute leukemia. The most established methods for detecting MRD are polymerase chain reaction (PCR) amplification of antigen receptor genes and of fusion transcripts, and flow-cytometric detection of ectopic or aberrant immunophenotypes [1–6]. The discovery that leukemic cells expressed immunophenotypes not expressed by normal bone marrow and peripheral blood cells provided one of the first opportunities to study MRD [7–9]. Over the years, MRD assays based on immunophenotyping have been improved by consistent advances in the quality and variety of antibodies, by the refinement of flow cytometers, and by the

Dario Campana, MD, PhD Department of Hematology-Oncology St. Jude Children’s Research Hospital, 332 North Lauderdale Memphis, TN 38105 (USA) Tel. +1 901 495 2528, Fax +1 901 495 3749, E-Mail [email protected]

enormous progress in informatics that has occurred during the last decade. The striking correlations of MRD results obtained by flow cytometry with clinical features and treatment outcome provided much credibility to this approach [10–16]. Because of its wide availability and conceptual straightforwardness, flow cytometry is the most accessible method for MRD detection. In this article, we summarize technical issues that we deem important for the productive detection of MRD by flow cytometry, and the relation between results obtained with this method and treatment outcome in patients with acute leukemia.

Advantages and Disadvantages of Flow Cytometry

One specific advantage of flow cytometry over PCRbased assays is that it allows direct quantitation of MRD, rather than extrapolating it from amounts of PCR product. This feature makes quantitation easier and, typically, more accurate [17]. In addition, flow cytometry allows the identification of dying cells and cellular debris. Therefore, leukemic cells irreversibly damaged by chemotherapy and unable to further expand (but capable of producing positive PCR signals) can be excluded from the counts. Obviously, this feature is useful only when analyzing samples freshly collected (e.g., 4 h or less), in which spontaneous apoptosis due to deprivation of survival factors has not yet affected a substantial number of cells. Flow cytometry also has some specific limitations. Extreme sensitivity, such as detection of 1 leukemic cell among 105 or more normal cells, is difficult to achieve consistently by flow cytometry but well within the range of PCR. Such high sensitivity may be desirable, for example, in studies seeking MRD in patients who have a patchy distribution of the disease, or in cell harvests for autografting. Another limitation is that the immunophenotype of leukemic cells may change during the progression of the disease. If these changes affect markers used for monitoring MRD, a false-negative finding may result [10, 18, 19]. The potential adverse effect of this phenomenon is inversely related to the number of marker combinations that can be applied to each patient. That is, if cells express more than one suitable phenotype, the effects of losing one phenotypic pattern may be offset by the persistence of other aberrant patterns. Finally, a general limitation of flow-cytometric assays is that results may not appear as ‘black and white’ as those of PCR. This is because the distinguishing immunophenotypic features of leukemia are

Minimal Residual Disease Studies in Leukemia

often, although not always, the result of quantitative differences in antigen expression between leukemic and normal cells. Nevertheless, objective MRD estimates are possible if one determines the limits of normal antigenic expression using a variety of normal samples, and avoids the use of immunophenotypes that partially overlap that of normal cells.

Markers of Leukemia

Immunophenotypes for MRD Studies To be useful for MRD studies, immunophenotypes must be expressed on leukemic cells and not expressed on normal bone marrow and peripheral blood cells. Proteins that are produced or dysregulated by gene fusions, such as BCR-ABL, AML1-ETO, or PBX-1 in E2A-PBX1 should contribute to such immunophenotypes, but antibodies that allow reliable detection of these proteins by flow cytometry are scarce [20]. The identification of immunophenotypes for effective MRD studies is complicated by variations in the cellular composition and immunophenotype of normal bone marrow that occur with age and exposure to drugs. For example, proportions of early lymphoid progenitors (or ‘hematogones’) are low in the bone marrow of healthy adults and especially low in patients receiving corticosteroids or chemotherapy [21]. By contrast, proportions are high in the bone marrow of young children [22–24], or of patients with malignancies after transplantation or cessation of chemotherapy [25–28]. These conditions may uncover normal cells expressing phenotypes that are undetectable in samples obtained from healthy individuals. Markers for MRD Studies in Acute Lymphoblastic Leukemia Table 1 summarizes combinations of markers used in our laboratory to study MRD in children with acute lymphoblastic leukemia (ALL) and their applicability in consecutive cases studied from January 1999 to July 2002. The reader should refer to the listed references for markers used by other investigators [11, 29–33]. The normal equivalents of T lineage ALL cells are immature T cells. Since these are confined to the thymus whereas leukemic T lymphoblasts can circulate, MRD studies in patients with T lineage ALL consist of searching for cells with the phenotype of immature T cells in the bone marrow or in the peripheral blood. The most useful immunophenotypes for this task are coexpression of T cell markers such as CD3 and CD5 with TdT or CD34 (ta-

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Table 1. Marker combinations used to

study MRD in childhood ALL

Leukemia cell lineage

Marker combination

Applicability, %a

T lineage ALL (n = 39)

TdT/CD5/CD3/(CD19/CD33/HLA-Dr) CD34/CD5/CD3/(CD19/CD33/HLA-Dr) CD19/CD34/CD10/CD38 CD19/CD34/CD10/CD58 CD19/CD34/CD10/CD45 CD19/CD34/CD10/TdT CD19/CD34/CD10/CD66c CD19/CD34/TdT/IgM CD19/CD34/CD10/CD22 CD19/CD34/CD10/CD13 CD19/CD34/CD10/CD15 CD19/CD34/CD10/CD21 CD19/CD34/CD10/CD33 CD19/CD34/CD10/NG-2 CD19/CD34/CD10/CD65

92 21 52 49 47 43 31 17 11 10 10 6 6 5 4

B lineage ALL (n = 169)

n = Number of cases studied. Percentage of patients within each type of leukemia in whom MRD could be studied with the listed antibody combination. Percentages were calculated by including only cases in which intensity of antigen expression was sufficiently different from that of normal bone marrow cells to afford a sensitivity of detection of 1 in 104. a

ble 1) [34]. Other authors indicated that antibody combinations including CD7 and CD3 with CD2 or CD5 may also be aberrantly expressed in a proportion of T-ALL patients [29, 30]. The normal equivalents of B lineage ALL cells are B cell progenitors which normally reside in the bone marrow, and can also be found in low proportions in the peripheral blood. Therefore, MRD studies in B lineage ALL must distinguish leukemic cells from their normal counterparts. This is possible because several molecules can be expressed at abnormally high or low levels in leukemic cells (fig. 1) [26, 31, 33–36]. For example, myeloidassociated markers CD13, CD15, CD33 and CD65, and the mature B cell-associated marker CD21 can be expressed by CD19+CD34+ B lineage ALL cells, whereas normal CD19+CD34+ B cell progenitors do not express these markers or express them very weakly [34]. Expression of CD19, CD10, TdT and CD34 in B lineage ALL can be significantly different (higher or lower) than that of their normal counterparts [11, 34, 37] and CD38 and CD45 (or CD45RA) are often underexpressed in leukemic cells [33, 34]. In efforts to prevent false-negative MRD findings due to immunphenotypic shifts, we use antibody panels that are somewhat more extensive than the ones used in other laboratories.

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Markers for MRD Studies in Acute Myeloid Leukemia Detection of MRD in acute myeloid leukemia (AML) requires the identification of immunophenotypic features that can distinguish leukemic myeloblasts from normal bone marrow myeloid cells. Phenotypic abnormalities in AML include expression of markers normally not expressed on myeloid cells, coexpression of markers normally expressed at different stages of maturation, as well as overexpression and under expression of myeloid markers [38]. Detection of MRD by flow cytometry in AML presents some specific difficulties. Due to their immunophenotypic heterogeneity [39], AML cells usually spread across many areas of each dot plot instead of forming the tight cluster typical of ALL cells. Therefore, with any given marker combination, only a fraction of cells may appear to be phenotypically abnormal. In addition, AML cells often have light scattering properties similar to that of normal cells with high autofluorescence. These features introduce complexity in the analysis, and may reduce the sensitivity of the assay. Nevertheless, sensitive MRD detection in AML is feasible (fig. 2). In recent studies with four-color flow cytometry, we have identified immunophenotypic combinations that allow measurement of MRD with a sensitivity of 1 leukemic cell among 10,000 or more normal cells in 48% of children, and 1 leukemic

Campana/Coustan-Smith

cell among 1,000 cells in an additional 37% [39]. San Miguel et al. [13] found that 175 of 233 adult patients with AML expressed leukemia-associated immunophenotypes, while Venditti et al. [40] detected them in 65 of 93 patients. A recent study by Sievers et al. [41] indicated that residual disease could be studied by flow cytometry

Fig. 1. Immunophenotypic differences between immature B cells in normal and regenerating bone marrow (BM). Bone marrow mononucleated cells were collected from healthy individuals (a, c) and patients with leukemia recovering from chemotherapy but MRD negative by flow cytometry and PCR (b, d). Shown are expression of CD22 and CD10 on selectively gated CD34+CD19+ lymphoid cells (a, b), and expression of TdT and cytoplasmic Ì heavy chains on selectively gated TdT+CD19+ lymphoid cells (c, d). Each dot plot is the overlay of two samples; all dot plots have a similar number of events, ranging from 1,223 to 1,288. Dot plot overlaying was done with the FCS Express software (DeNovo Software, Thornhill, Canada).

Fig. 2. Detection of MRD in AML by four-color flow cytometry. Bone marrow mononuclear cells from 4 healthy

donors (top) and 1 patient with AML at different stages of treatment (bottom) were labeled with antibodies to CD38, CD13, CD34 and CD33. The same number of mononuclear cells were studied in all samples. CD13 and CD38 expression in CD34+CD33+ cells is shown. In normal samples, these cells also express CD13 and/or CD38. In the AML patient, most leukemic cells at diagnosis were CD34+CD33+ but lacked CD13 and CD38 (dashed square). Cells with this phenotype were detectable at weeks 7 and 14 of therapy, a finding that was followed by cytogenetic relapse first and then by clinical relapse.

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in all patients, although this was an assumption because the immunophenotype at diagnosis was not determined. Therefore, the actual prevalence of leukemia-associated immunophenotypes in this series is unclear. Identification of New Markers The advent of arrays that allow genome-wide analysis of gene expression has opened new possibilities to identify markers for MRD studies [42]. To test the validity of this concept, we compared the gene profile of ALL cells to that of purified normal B cell progenitors [43]. Among the F4,000 genes studied, we found over 250 that were overexpressed in more than one leukemic sample. We selected 9 of these genes for which antibodies were easily available and measured expression of the encoded proteins by flow cytometry. Seven proteins (CD58, creatine kinase B, ninjurin1, Ref1, calpastatin, HDJ-2 and annexin VI) were expressed in B lineage ALL cells at higher levels than in normal CD19+CD10+ B cell progenitors. The results with CD58 were in line with a previous report indicating overexpression of this molecule in leukemic cells [44]. CD58 is now one of the most useful markers for the study of MRD in B lineage ALL (table 1). These results suggest that a comparison of the gene profiles of normal and leukemic cells will identify new, widely applicable markers for MRD studies in ALL and in AML, and should ultimately allow the design of simple antibody panels for practical, reliable, and universal monitoring of MRD. Sensitivity and Measurement of MRD Two main variables influence rare cell detection by flow cytometry: (1) the degree of morphologic and phenotypic difference between target cells and the remaining cells, and (2) the number of cells that can be analyzed. Under ideal conditions, i.e. very distinct target cells and a large number of cells (107 or more) available for analysis, the sensitivity of flow cytometry is similar to that of PCR [45]. During analysis of MRD in clinical samples, however, the number of cells that can be analyzed for each set of markers in children is usually less than 1 ! 106. Because a distinct cluster of at least 10–20 dots is necessary to interpret suspect flow-cytometric events, the maximum sensitivity achievable in these circumstances would be 1 in 105 cells. The phenotype of primary leukemic cells may not be as distinct as that of cell lines used in an experimental setting and, in the case of B lineage ALL, sensitivity of detection may be influenced by the treatment interval at which the sample is taken, because of the variable proportion of normal B cell precursors. Therefore, a con-

12

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sistent sensitivity of 1 in 104 is probably the maximum that can be expected during routine MRD testing. Flow cytometry has the potential for a very accurate quantitation of MRD. In experiments with serial dilutions of KOPN-57bi pro-B ALL cells admixed with normal peripheral blood mononuclear cells at ratios of 3 in 105 and higher, the estimate of the leukemic cell content in each mixture was extremely accurate (r2 = 0.999) [17]. In other experiments, we compared the results of multiple measurements of residual leukemia in the same cell mixture [10]. In 23 tests of mixtures containing 1 leukemic cell in 104 normal cells, results were remarkably similar (coefficient of variation = 15%); in 22 tests of mixtures containing 1 leukemic cell in 103 cells, the coefficient of variation was 10%.

Clinical Applications

Prognostic Value of MRD in ALL We used flow cytometry to prospectively study MRD in 195 children with newly diagnosed ALL enrolled in a single-institution chemotherapy program (TOTAL XIII) [10, 12, 15]. We found that detectable MRD (i.e., 60.01% leukemic mononuclear cells) at each time point (day 19 of remission induction therapy, end of remission induction and weeks 14, 32 and 56 of continuation) was significantly associated with a higher rate of relapse. Patients with high levels of MRD at the end of the remission induction therapy (61%) or at week 14 of continuation therapy (60.1%) had a particularly poor outcome. The incidence of relapse among patients with MRD at the end of induction was 7 B 7% if MRD became undetectable at week 14 of continuation therapy, compared with 68 B 16% (SE) if MRD persisted. Notably, 53 of the 112 patients studied at day 19 of remission induction therapy had achieved MRD negativity (!0.01%) despite the short chemotherapy period [15]. The 3-year cumulative incidence of relapse was 1.9 B 1.9%, as compared to 28.4 B 6.4% for patients who were MRD positive at this time point. At all time points, the prognostic value of MRD was independent from that of other known clinical and biologic prognosticators of outcome. Other investigators have used flow cytometry to study MRD in patients with ALL undergoing chemotherapy and found a good correlation with outcome [11, 14, 30]. MRD detected by flow cytometry in bone marrow samples taken prospectively from 24 patients with ALL undergoing stem cell transplantation before starting the conditioning regimen was a significant predictor of outcome [46].

Campana/Coustan-Smith

Detection of MRD in Peripheral Blood We recently compared MRD measurements in 747 pairs of bone marrow and peripheral blood samples collected from 231 children during treatment for newly diagnosed ALL [6, 16]. MRD was detected in both marrow and blood in 78 pairs and in marrow but not in blood in 67 pairs; it was undetectable in the remaining 602 pairs. Findings in marrow and blood were completely concordant in the 179 paired samples from patients with T lineage ALL: for each of the 41 positive marrow samples, the corresponding blood sample was positive. In B lineage ALL, however, only 37 of the 104 positive marrow samples had a corresponding positive blood sample. Results of a recently reported study by another group of investigators are in agreement with the remarkable concordance of MRD results in marrow and blood of patients with TALL [47]. We also observed that peripheral blood MRD in B lineage ALL patients was associated with a very high risk of relapse [16]. MRD Studies in AML A recent study found immunophenotypic abnormalities in 41 of 252 children with AML who responded to initial therapy, a finding that was associated with a poorer outcome [41]. In studies performed in adult patients with AML, the first bone marrow in morphologic remission obtained after induction treatment was found to be very informative [13]. Of the 126 patients studied, 8 had !0.01% leukemic cells and none had relapsed at the time of the report; 37 had 0.01–0.1% leukemic cells and a 3year cumulative relapse rate of 14%; 64 had 0.1–1% leukemic cells and a relapse rate of 50%; 17 had more than 1% residual cells and a relapse rate of 84%. In another study of 51 patients in whom MRD was examined after consolidation, the most predictive MRD cutoff value determined retrospectively was 0.035%: 17 of 22 patients with that level of MRD or higher levels relapsed compared with 5 of 29 patients with lower MRD levels [40]. We recently reported that detection of MRD in children with AML is prognostically important [39]. In patients with AML receiving autologous bone marrow transplantation, levels of MRD measured by flow cytometry in the autograft correlated with disease recurrence [48].

can be spared unnecessarily intense and toxic treatment. By precisely measuring early treatment response in vivo, MRD studies have great potential in this context. In other forms of leukemia, e.g. ALL in adults and AML, novel treatments are urgently needed if one hopes to improve cure rates substantially. Thus, the highest value of MRD assays in these diseases may possibly lie in the rapid measurement of the effect of novel therapies on the leukemic clone. The discovery of a small set of new leukemia-specific markers that can be detected in most cases would extend the benefits of MRD detection to a larger number of patients. Because flow-cytometric analysis of leukemic cell phenotype is performed in nearly all pediatric and adult cancer centers worldwide, newly developed methods of MRD detection can realistically be accessible to most patients. Internet-based methods of rapid file transfer between flow cytometers at remote centers [49] should allow rapid quality control and distance learning and help to set up this methodology in laboratories with experience in leukemia phenotyping but novice at MRD detection. However, many of these centers cannot support the use of complex, costly antibody panels and a reduced panel of antibodies may not allow optimal MRD monitoring. Gene expression analysis with microarrays hold the potential to identify new markers of leukemia for MRD studies. New markers should allow the simplification of the current immunophenotypic panels and the wide applicability of flow-cytometric studies of MRD.

Acknowledgment This work was supported by grants CA60419 and CA21765 from the National Cancer Institute, by the Rizzo Memorial Grant from the Leukemia Research Foundation, and by the American Lebanese Syrian Associated Charities (ALSAC).

Future Outlook

In some forms of acute leukemia, e.g. childhood ALL, a central issue is the identification of patients who require more aggressive therapy to avert relapse and of those who

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13 San Miguel JF, Vidriales MB, Lopez-Berges C, Diaz-Mediavilla J, Gutierrez N, Canizo C, Ramos F, Calmuntia MJ, Perez JJ, Gonzalez M, Orfao A: Early immunophenotypical evaluation of minimal residual disease in acute myeloid leukemia identifies different patient risk groups and may contribute to postinduction treatment stratification. Blood 2001;98:1746– 1751. 14 Dworzak MN, Froschl G, Printz D, Mann G, Potschger U, Muhlegger N, Fritsch G, Gadner H: Prognostic significance and modalities of flow cytometric minimal residual disease detection in childhood acute lymphoblastic leukemia. Blood 2002;99:1952–1958. 15 Coustan-Smith E, Sancho J, Behm FG, Hancock ML, Razzouk BI, Ribeiro RC, Rivera GK, Rubnitz JE, Sandlund JT, Pui CH, Campana D: Prognostic importance of measuring early clearance of leukemic cells by flow cytometry in childhood acute lymphoblastic leukemia. Blood 2002;100:52–58. 16 Coustan-Smith E, Sancho J, Hancock ML, Razzouk BI, Ribeiro RC, Rivera GK, Rubnitz JE, Sandlund JT, Pui CH, Campana D: Use of peripheral blood instead of bone marrow to monitor residual disease in children with acute lymphoblastic leukemia. Blood 2002;100: 2399–2402. 17 Neale GA, Coustan-Smith E, Pan Q, Chen X, Gruhn B, Stow P, Behm FG, Pui CH, Campana D: Tandem application of flow cytometry and polymerase chain reaction for comprehensive detection of minimal residual disease in childhood acute lymphoblastic leukemia. Leukemia 1999;13:1221–1226. 18 Oelschlagel U, Nowak R, Schaub A, Koppel C, Herbst R, Mohr B, Loffler C, Range U, Gunther H, Assmann M, Siegert E, Wendt E, Huhn R, Brautigam E, Ehninger G: Shift of aberrant antigen expression at relapse or at treatment failure in acute leukemia. Cytometry 2000;42/ 4:247–253. 19 Baer MR, Stewart CC, Dodge RK, Leget G, Sule N, Mrozek K, Schiffer CA, Powell BL, Kolitz JE, Moore JO, Stone RM, Davey FR, Carroll AJ, Larson RA, Bloomfield CD: High frequency of immunophenotype changes in acute myeloid leukemia at relapse: Implications for residual disease detection (Cancer and Leukemia Group B Study 8361). Blood 2001; 97:3574–3580. 20 Sang BC, Shi L, Dias P, Liu L, Wei J, Wang ZX, Monell CR, Behm F, Gruenwald S: Monoclonal antibodies specific to the acute lymphoblastic leukemia t(1;19)-associated E2A/pbx1 chimeric protein: Characterization and diagnostic utility. Blood 1997;89:2909–2914. 21 Paolucci P, Hayward AR, Rapson NT: Pre-B and B cells in children on leukaemia remission maintenance treatment. Clin Exp Immunol 1979;37/2:259–266. 22 Longacre TA, Foucar K, Crago S, Chen IM, Griffith B, Dressler L, McConnell TS, Duncan M, Gribble J: Hematogones: A multiparameter analysis of bone marrow precursor cells. Blood 1989;73:543–552.

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23 Caldwell CW, Poje E, Helikson MA: B-cell precursors in normal pediatric bone marrow. Am J Clin Pathol 1991;95:816–823. 24 Lucio P, Parreira A, van den Beemd MW, van Lochem EG, Van Wering ER, Baars E, PorwitMacDonald A, Bjorklund E, Gaipa G, Biondi A, Orfao A, Janossy G, van Dongen JJ, San Miguel JF: Flow cytometric analysis of normal B cell differentiation: A frame of reference for the detection of minimal residual disease in precursor-B-ALL. Leukemia 1999;13:419– 427. 25 Asma GE, van den Bergh RL, Vossen JM: Regeneration of TdT+, pre-B, and B cells in bone marrow after allogeneic bone marrow transplantation. Transplantation 1987;43: 865–870. 26 Ciudad J, San Miguel JF, Lopez-Berges MC, Garcia MM, Gonzalez M, Vazquez L, del Canizo MC, Lopez A, van Dongen JJ, Orfao A: Detection of abnormalities in B-cell differentiation pattern is a useful tool to predict relapse in precursor-B-ALL. Br J Haematol 1999;104: 695–705. 27 van Lochem EG, Wiegers YM, van den BR, Hahlen K, van Dongen JJ, Hooijkaas H: Regeneration pattern of precursor-B-cells in bone marrow of acute lymphoblastic leukemia patients depends on the type of preceding chemotherapy. Leukemia 2000;14:688–695. 28 McKenna RW, Washington LT, Aquino DB, Picker LJ, Kroft SH: Immunophenotypic analysis of hematogones (B-lymphocyte precursors) in 662 consecutive bone marrow specimens by 4-color flow cytometry. Blood 2001;98:2498– 2507. 29 Porwit-MacDonald A, Bjorklund E, Lucio P, van Lochem EG, Mazur J, Parreira A, van den Beemd MW, Van Wering ER, Baars E, Gaipa G, Biondi A, Ciudad J, van Dongen JJ, San Miguel JF, Orfao A: BIOMED-1 concerted action report: Flow cytometric characterization of CD7+ cell subsets in normal bone marrow as a basis for the diagnosis and follow-up of T cell acute lymphoblastic leukemia (T-ALL). Leukemia 2000;14:816–825. 30 Ciudad J, San Miguel JF, Lopez-Berges MC, Vidriales B, Valverde B, Ocqueteau M, Mateos G, Caballero MD, Hernandez J, Moro MJ, Mateos MV, Orfao A: Prognostic value of immunophenotypic detection of minimal residual disease in acute lymphoblastic leukemia. J Clin Oncol 1998;16:3774–3781. 31 Lucio P, Gaipa G, van Lochem EG, Van Wering ER, Porwit-MacDonald A, Faria T, Bjorklund E, Biondi A, van den Beemd MW, Baars E, Vidriales B, Parreira A, van Dongen JJ, San Miguel JF, Orfao A: BIOMED-I concerted action report: Flow cytometric immunophenotyping of precursor B-ALL with standardized triple-stainings. BIOMED-1 Concerted Action Investigation of Minimal Residual Disease in Acute Leukemia: International Standardization and Clinical Evaluation. Leukemia 2001; 15:1185–1192.

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32 Weir EG, Cowan K, LeBeau P, Borowitz MJ: A limited antibody panel can distinguish B-precursor acute lymphoblastic leukemia from normal B precursors with four color flow cytometry: Implications for residual disease detection. Leukemia 1999;13:558–567. 33 Dworzak MN, Fritsch G, Fleischer C, Printz D, Froschl G, Buchinger P, Mann G, Gadner H: Comparative phenotype mapping of normal vs. malignant pediatric B-lymphopoiesis unveils leukemia-associated aberrations. Exp Hematol 1998;26:305–313. 34 Campana D, Coustan-Smith E: Advances in the immunological monitoring of childhood acute lymphoblastic leukaemia. Best Pract Res Clin Haematol 2002;15/1:1–19. 35 Hurwitz CA, Loken MR, Graham ML, Karp JE, Borowitz MJ, Pullen DJ, Civin CI: Asynchronous antigen expression in B lineage acute lymphoblastic leukemia. Blood 1988;72/1: 299–307. 36 Wells DA, Hall MC, Shulman HM, Loken MR: Occult B cell malignancies can be detected by three-color flow cytometry in patients with cytopenias. Leukemia 1998;12:2015–2023. 37 Lavabre-Bertrand T, Janossy G, Ivory K, Peters R, Secker-Walker L, Porwit-MacDonald A: Leukemia-associated changes identified by quantitative flow cytometry. I. CD10 expression. Cytometry 1994;18/4:209–217. 38 Terstappen LW, Loken MR: Myeloid cell differentiation in normal bone marrow and acute myeloid leukemia assessed by multi-dimensional flow cytometry. Anal Cell Pathol 1990; 2/4:229–240.

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39 Coustan-Smith E, Ribeiro RC, Rubnitz JE, Razzouk BI, Pui CH, Pounds S, Andreansky M, Behm FG, Raimondi SC, Shurtleff SA, Downing JR, Campana D: Clinical significance of residual disease during treatment in childhood acute myeloid leukemia. Br J Haematol 2003;123:243–252. 40 Venditti A, Buccisano F, Del Poeta G, Maurillo L, Tamburini A, Cox C, Battaglia A, Catalano G, Del Moro B, Cudillo L, Postorino M, Masi M, Amadori S: Level of minimal residual disease after consolidation therapy predicts outcome in acute myeloid leukemia. Blood 2000; 96:3948–3952. 41 Sievers EL, Lange BJ, Alonzo TA, Gerbing RB, Bernstein ID, Smith FO, Arceci RJ, Woods WG, Loken MR: Immunophenotypic evidence of leukemia after induction therapy predicts relapse: Results from a prospective Children’s Cancer Group study of 252 patients with acute myeloid leukemia. Blood 2003;101:3398– 3408. 42 Yeoh EJ, Ross ME, Shurtleff SA, Williams WK, Patel D, Behm FG, Raimondi SC, Relling MV, Patel A, Cheng C, Campana D, Wilkins D, Zhou X, Li J, Pui CH, Evans WE, Wong L, Downing JR: Classification, subtype discovery, and prediction of outcome in pediatric acute lymphoblastic leukemia by gene expression profiling. Cancer Cell 2002;1:133–143. 43 Chen JS, Coustan-Smith E, Suzuki T, Neale GA, Mihara K, Pui CH, Campana D: Identification of novel markers for monitoring minimal residual disease in acute lymphoblastic leukemia. Blood 2001;97:2115–2120.

44 De Waele M, Renmans W, Jochmans K, Schots R, Lacor P, Trullemans F, Otten J, Balduck N, Vander GK, Van Camp B, van Schie RC, Van Riet I: Different expression of adhesion molecules on CD34+ cells in AML and B-lineage ALL and their normal bone marrow counterparts. Eur J Haematol 1999;63/3:192–201. 45 Gross HJ, Verwer B, Houck D, Recktenwald D: Detection of rare cells at a frequency of one per million by flow cytometry. Cytometry 1993;14:519–526. 46 Sanchez J, Serrano J, Gomez P, Martinez F, Martin C, Madero L, Herrera C, Garcia JM, Casano J, Torres A: Clinical value of immunological monitoring of minimal residual disease in acute lymphoblastic leukaemia after allogeneic transplantation. Br J Haematol 2002; 116:686–694. 47 van der Velden V, Jacobs DC, Wijkhuijs AJ, Comans-Bitter WM, Willemse MJ, Hahlen K, Kamps WA, Van Wering ER, van Dongen JJ: Minimal residual disease levels in bone marrow and peripheral blood are comparable in children with T cell acute lymphoblastic leukemia (ALL), but not in precursor-B-ALL. Leukemia 2002;16:1432–1436. 48 Reichle A, Rothe G, Krause S, Zaiss M, Ullrich H, Schmitz G, Andreesen R: Transplant characteristics: Minimal residual disease and impaired megakaryocytic colony growth as sensitive parameters for predicting relapse in acute myeloid leukemia. Leukemia 1999;13:1227– 1234. 49 Lorenzana R, Coustan-Smith E, Antillon F, Ribeiro RC, Campana D: Simple methods for the rapid exchange of flow cytometric data between remote centers. Leukemia 1999;14: 336–337.

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Review Acta Haematol 2004;112:16–23 DOI: 10.1159/000077555

Strategies and Clinical Implications of Chimerism Diagnostics after Allogeneic Hematopoietic Stem Cell Transplantation Christian Thiede Martin Bornhäuser Gerhard Ehninger Medizinische Klinik und Poliklinik I, Universitätsklinikum Carl Gustav Carus der Technischen Universität, Dresden, Germany

Key Words Stem cell transplantation W Chimerism W Minimal residual disease W PCR W Leukemia

Abstract Analysis of donor chimerism has become a routine method for the documentation of engraftment after allogeneic hematopoietic stem cell transplantation (HSCT). In recent years several groups have also focused on the application of this technique for the detection of relapsing disease after allogeneic HSCT. This review addresses technical issues (sensitivity, specificity) and discusses the advantages and limitations of methods currently used for chimerism analysis and their usefulness for the detection of MRD. In addition, the potential impact of novel procedures, e.g. subset chimerism or real-time PCR-based procedures, is discussed. Copyright © 2004 S. Karger AG, Basel

Introduction

After allogeneic blood stem cell transplantation, a coexistence of the host and donor lympho- and hematopoietic system will develop. This period, which is temporary in the majority of successful stem cell transplants, is referred to as mixed chimerism, whereas complete chi-

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merism denotes the situation, when all cell lineages are reconstituted by the donor. The evaluation of chimerism after stem cell transplantation has been of central interest since this treatment has been used clinically. Several techniques have been applied for this purpose during the last 30 years, including cytogenetics, isoenzyme analysis and blood group phenotyping [for a review, see 1]. A major improvement in the clinical applicability was made when differentiation of sex chromosomes using fluorescence in situ hybridization (FISH) became possible [2, 3]. This method allows rapid and quantitative evaluation of engraftment; however, it is obviously restricted to the approximately 50% of patients transplanted from a sex-mismatched donor. The invention of the polymerase chain reaction (PCR) was a key step which laid the fundament for the development of modern molecular diagnostics [4]. In the field of chimerism diagnostics, several PCR-based procedures have been developed for the evaluation of engraftment [5–16]. Most of these assays rely on the amplification of highly polymorphic repetitive DNA sequences, i.e. short tandem repeats (STR) or a variable number of tandem repeat (VNTR) sequences. Besides the documentation of engraftment, detection of reappearing leukemic cells has become a key issue in chimerism diagnostics. Numerous publications deal with this application. This review focuses on the use of chimerism analysis for the detection of minimal residual leuke-

Christian Thiede, MD Medizinische Klinik und Poliklinik I Universitätsklinikum Carl Gustav Carus der Technischen Universität Fetscherstrasse 74, DE–01307 Dresden (Germany) Tel. +49 351 458 4704, Fax +49 351 458 5362, E-Mail [email protected]

Table 1. Comparison of different methods for chimerism analysis

Method

Sensitivity1, %

Advantages

Disadvantages

Ref.

XY-FISH

0.1

Quantitative relatively sensitive, fast, standardized technique

Restricted to sex mismatch

2, 3

Real-time PCR

0.1–0.0001

Quantitative, highly sensitive

False positive results possible in SNP-based procedures

23–25

Fast sensitive

Long latency makes it unsuitable for rapidly proliferating diseases

26

Red cell phenotyping STR or VNTR-PCR

5–1

Informative, low amounts of material, rapid

Moderate sensitivity

5–16

YX-FISH and immunohistochemistry (FICTION)

0.1–0.001

Sensitive, cell line specific

Labour intensive, restricted to sex mismatch

27–30

STR-PCR + cell sorting

0.1–0.0001

Sensitive, cell line specific

Labor intensive, requires specific equipment

31–34

* The lower level of host cells detectable is denoted.

The first of these questions is especially important. The term MRD describes a state of leukemic burden which is below the threshold detection level of conventional measures, i.e. morphology. This standard method for assessment of response towards chemotherapy has, by definition, a limit of detection for leukemic blasts in the bone marrow of 5% [17]. Thus any procedure which can measure cells below this level may be suitable to detect leukemic cells. But what level of sensitivity should we approach? Generally one might wish to know precisely which level of MRD has been reached. Thus, it is much more important to quantitatively assess the level of residual leukemia than to deal with merely qualitative analyses. The level of detection achievable can be illustrated by comparing several techniques which are currently used for the detection of residual leukemia after treatment. For example, FISH detection of chromosomal translocations has a level of sensitivity between 0.1 and 8% depending

on the technique used and the specific translocation investigated [recent review in 18]. Flow-cytometric assays, monitoring aberrant antigen expression, might achieve considerably higher levels of sensitivity, between 0.01 and 1% [recent examples in 19, 20]. PCR, however, represents the most sensitive technique reported so far. The exponential amplification of specific target sequences facilitates a limit of detection between 0.1 and 0.01% cells. This sensitivity can be further increased if two consecutive rounds of PCR are applied, which routinely detects 1/105 to 1/106 cells, and which can be further increased and has been used even to demonstrate the presence of leukemia-associated translocations in healthy individuals [21]. The diagnostic message can be substantially augmented when quantitative PCR methods (realtime PCR) are applied, which gives information on the number of starting molecules [22]. Thus, taken together there is a broad range of methods for the detection of residual leukemia. The methods currently in use for chimerism analysis must be interpreted in this context, if discussing their value in the detection of MRD. Table 1 summarizes different techniques for chimerism analysis, their advantages and disadvantages as well as the reported levels of sensitivity. Among these assays, STR-based methods are the most frequently used. These methods usually have a level of sensitivity between 1 and 5%, a range which allows the accurate and reliable monitoring of engraftment. In addition, there is evidence that at least in slowly proliferating

Chimerism Analysis for MRD Detection

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mia and addresses several questions which may be important in this context: (1) Are the current methodologies for chimerism analysis appropriate for the detection of minimal residual disease (MRD)? (2) If so how can they potentially be further improved? (3) If not, which alternatives can be found to sufficiently solve the problem?

Methods for Chimerism Analysis and Their Sensitivity

17

diseases like CML, this level may be sufficient for the routine follow-up. Several reports have shown that increasing mixed chimerism is a sensitive predictor of relapse in cases with persistent positivity for the bcr-abl fusion transcript [35, 36]. However, these data mainly originate from the era before real-time PCR for the bcr-abl mRNA has become widely available [37]. Today, the quantitative information on the bcr-abl copy numbers is regarded as the most important diagnostic tool in this disease [for a review, see 38]. Nevertheless, chimerism analysis may be an important adjunct method to assess the response towards donor leukocyte infusion (DLI) [30, 32]. In addition, analysis of chimerism in bone marrow CD34+ cell before DLI may give valuable information on the risk of developing aplasia after DLI [39]. Chimerism analysis in transplant settings using T cell depletion to schedule T cell add-back and to monitor the success of this intervention may also be of special importance, especially in transplants with dose-reduced conditioning [40]. Conflicting data have been reported on the question of whether standard STR-based procedures are also useful for the early detection of reappearing disease in acute leukemias. There are several reports indicating that a level of about 1% may be sufficient to detect relapse in acute leukemia. Bader et al. [41] described the use of overall chimerism analysis in 30 transplanted children with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) and MDS. Patients who showed mixed chimerism had a significantly higher probability of relapse compared to those with stable complete chimerism. However, there were also patients within this cohort who had increasing host chimerism without relapse as well as 3 patients who had complete chimerism before relapse. A later analysis by the same group of 55 patients confirmed these data [42]. Similar data, indicating that chimerism analysis from unsorted peripheral blood or bone marrow samples is able to detect relapse, were published by several groups [43, 44]. In contrast, other groups were not able to demonstrate a good prediction of relapse by analyzing chimerism from the peripheral blood. Suttorp et al. [9] showed that an RFLP assay with a 1% sensitivity showed complete chimerism 30–86 days before a clinical relapse was diagnosed. They concluded that the kinetics of reappearing leukemia are too rapid to be diagnosed with a detection limit of 1%. Comparable results were seen by other groups [45–48]. Taking these results together, mixed chimerism may precede subsequent relapse, but the interval between the decrease in donor cells using STR or VNTR-PCR and the clinical diagnosis is much too short to make meaningful clinical decisions in the majority of the patients. The

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background of these discrepancies in the reports on the usefulness of chimerism analysis of peripheral blood is not yet clear. The reasons may be technical (e.g. different levels of sensitivity) or different sampling protocols. They may also be related to different patient populations, since the majority of data showing sufficient sensitivity are from pediatric transplantations. However, an increase in sensitivity is clearly desirable to improve the detection range of residual host cells and to potentially increase the time for clinical decision making.

New Techniques for the Detection of MRD Using Chimerism Analysis

As stated above, STR-based methods have a level of sensitivity which is in the range of 1–5%. One additional problem associated with this technique is the diagnostic uncertainty induced by PCR artifacts (stutter signals) [49], which renders a large number of peak constellations insufficient for analysis, because a residual host signal is located within the stutter peak of the donor, and thus cannot be detected accurately [50]. To overcome this limitation, a selection of STRs with a high proportion of donor/ recipient constellations without interference of host and donor signals is desirable. In a recent analysis of 27 different STRs in a population of 203 HLA-matched related donors and recipients, we have identified a subset of markers with a high likelihood of finding such constellations [50a]. However, even if STR-based assays for chimerism analysis may be further improved and may detect leukemic cells in some patients, according to our experience and most of the published data the detection is too late in most of the patients to facilitate early intervention, e.g. tapering of immunosuppressive therapy. Thus in order to detect recurring disease as early as possible, novel assays with higher sensitivity would be a major improvement. Recently, quantitative chimerism analysis using real-time PCR has been reported for this purpose. Realtime PCR, either by the 5)-nuclease assay (TaqMan procedure) or by fluorescent resonance energy transfer, has the advantage that the product yield of the PCR is measured during each cycle, which allows the calculation of the number of starting molecules [for a review, see 22]. Realtime PCR monitoring has become a standard procedure for the evaluation of the response to therapy in acute and chronic leukemias and has been shown to be a sensitive and predictive tool for the guidance of treatment. It relies on the amplification of a specific target sequence, such as fusion oncogenes (e.g. bcr-abl, PML-RARalpha) or B or

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T cell receptor rearrangements [22]. However, in a large proportion of acute leukemias, such specific molecular targets are missing. In male patients transplanted from a female donor, the Y chromosome represents a unique characteristic of recipient cells. Lo et al. [51] were the first to use real-time PCR for the SRY gene on the Y chromosome to detect microchimerism due to fetomaternal transfer in females who have given birth to male children. Fehse et al. [23] have reported a similar method for the evaluation of chimerism after hematopoietic stem cell transplantation (HSCT). However, clinical data on the use of this procedure are still missing. We used real-time PCR for the SRY gene on the Y chromosome to quantify the level of chimerism in 43 male patients transplanted from a female donor [52]. This method was shown to have a lower limit of detection of one male cell in 100,000 female cells. Our results indicate that residual host cells are detectable at very low levels in almost all patients investigated up to 5 years after transplantation. In addition, a gradual decrease of residual host cells was found over time (median 1 month: 0.3%; median 6 months: 0.01%; median 1 year: 0.0061%; median 4 years: 0.0015%). This persistence of host cells over long periods at low levels might explain why earlier reports using end point PCR did not show any impact of the detection of residual host cells [53]. In contrast, using real-time PCR, patients with relapse showed an increase in the percentage of host cells which preceded the haematological relapse by up to 120 days. Thus, real-time PCR might represent an interesting tool to analyze the level of residual host cells. As discussed earlier, the Y chromosome can be used only in a subset of patients. Several groups have now reported real-time PCR assays using other genetic targets, i.e. single nucleotide polymorphisms (SNPs), for the differential amplification of host cells after transplantation. SNPs are mostly biallelic genetic variances which occur on average every 1,000 bp, thus about 3,000,000 of these polymorphisms are present. Alizadeh et al. [54] recently published a set of 11 biallelic SNPs for real-time PCR analysis after allogeneic HSCT. These SNPs could discriminate 90% of the donor and recipient constellations tested. The assay has a minimum sensitivity of 0.1%, which is not as sensitive as for the SRY gene, but at least 10-fold better than for standard STR or VNTR-based methods. Maas et al. [25] also published data on the use of real-time PCR for chimerism analysis with very similar results. In summary, real-time PCR-based procedures will provide an important improvement of the diagnostic inventory for chimerism analysis. However, as shown by several authors, realtime PCR cannot totally replace STR- or VNTR-based

methods. One substantial drawback of this technique is the lower quantitative accuracy [25, 54]. This problem is inherent in the technique, since a change of only one PCR cycle means a 2-fold quantitative difference. This implies that a coefficient of variation of about 20–30% must be considered as normal. In contrast, STR-based procedures, especially when performed in a multiplex PCR, achieve reproducibility values between 4 and 8% [50, 55]. Thus in states of chimerism exceeding 5% host signal, STR analysis remains the method of choice. This technique is also superior to real time in the analysis of subset populations due to the limitations discussed above. Another possibility to increase the sensitivity of a chimerism analysis is the use of specific subpopulations of the peripheral blood or bone marrow. The enrichment of cellular compartments has been shown to increase the sensitivity of chimerism analysis, since specific populations of cells, like B cells, T cells or early stem cells, are enriched from the background of mature granulocytes. Ginsburg and coworkers [56] were the first who described the investigation of cellular subsets after HSCT; van Leeuwen et al. [57] were the first to use highly purified cellular populations. Meanwhile, several groups have reported the analysis of chimerism in specific subsets to detect reappearing leukemic cells [27, 34, 58–61] or to monitor the effect of treatment [30]. Lamb et al. [58] described the use of subset analysis to differentiate suspicious relapse in two cases. In one patient they did not find evidence for relapse, whereas the second patient showed relapse in the CD34+/CD7+ leukemic cells, which was undetectable in the unsorted bone marrow material. Zetterquist et al. [60] compared chimerism analysis in sorted B cells with overall chimerism and PCR for clone specific T cell or B cell receptor rearrangements. Mixed chimerism in the B cell compartment was found in 5 patients who also showed persistence of the clonality marker in the PCR. Mixed chimerism in the B cells was detected 2.5 months prior to the morphological relapse in 3/4 patients with relapse. No relapse was observed in those seven cases with complete donor chimerism in the B cell compartment. Mattson et al. [34] studied 30 patients with AML and MDS. They used immunomagnetic labeling with antibodies against CD33, CD7 or CD45 to enrich the specific subpopulations from the peripheral blood or the bone marrow, achieving a final sensitivity between 2 and 4 ! 10 –4. Mixed chimerism in these populations 1 month after transplantation was observed in 14 of the 30 patients. A relapse was seen in 10/ 14 patients with mixed chimerism in the subpopulations compared to only 2/16 cases with complete chimerism, a

Chimerism Analysis for MRD Detection

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19

difference which was highly significant. Interestingly, mixed chimerism in the peripheral blood seemed to have a lower sensitivity (67%), but a higher specificity (100%) in the prediction of relapse compared to the analysis of bone marrow samples. We have recently performed a study using CD34+ cells as target for the identification of leukemic cells. The basic idea behind this was that the CD34 antigen is expressed on a very small population of normal hematopoietic progenitor cells, but can be frequently detected on blasts of different leukemias [62, 63]. Evaluation of this method showed a minimum sensitivity of 1/40,000 cells [33]. In a panel of 87 prospectively investigated patients, chimerism analysis in the CD34+ cells was performed for the detection of relapse [61]. After a median follow-up of 295 days (range 28–1,152 days), a total of 22 relapses were observed in the 84 patients showing engraftment. Relapse was associated with a decrease in CD34+ cell donor chimerism by up to 97 days in 20/22 patients. In patients with CML and molecular relapse, recurrent disease was associated with a decrease in donor CD34+ cells. Treatment with DLI or imatinib resulted in an increase in donor CD34+ cells and a clearance of bcr-abl-positive cells. Since this assay can be performed with peripheral blood, the investigations can be done at short intervals. These data inspired us to start a randomized prospective multicenter trial comparing chimerism from the peripheral blood and subset chimerism within the CD34 compartment in patients with AML, ALL or MDS whose leukemia blasts express the CD34 antigen. In patients showing a decrease in the proportion of donor CD34+ cells below 60%, immunosuppression is reduced or DLIs are given. First results of this study are inspiring and we have seen responses in several cases of AML and ALL. Thus taken together, these data clearly show that subset analysis is a very sensitive technique, with a limit of detection comparable to nested PCR. This technique adds important information, since it is able to clarify whether reappearing host cells are of leukemic origin or are T cells or other nonmalignant cells. In this article we have not focused specially on the use of chimerism in the setting of dose-reduced conditioning, since the detection of MRD is not substantially different after this form of transplantation. However, since mixed chimerism, especially in T cells, is much more common after dose-reduced preparative regimens, subset analysis is even more important to differentiate persistent mixed T cell chimerism from reappearing leukemic cells.

20

Acta Haematol 2004;112:16–23

Use of Chimerism for Follow-Up after Transplantation: The Dresden Experience

Recently, several groups have published their procedures for the follow-up investigations after allogeneic HSCT [64–69]. These reports are mainly focused on technical issues. As discussed above these questions are certainly important. However, especially for the detection of MRD, the use of the appropriate methods according to the clinical situation is most important. Based on our own experience and the literature data, our current recommendations for chimerism analyses are as follows: During engraftment and during the entire period of mixed macrochimerism (i.e. chimerism between 3 and 97%), STR analysis, preferentially in a multiplex assay, should be used on whole blood or bone marrow samples to quantify chimerism. We usually perform these analyses twice weekly starting at day +5. If graft failure or relapse is clinically suspected, sorting of T cells, NK cells and myeloid cells, preferentially CD34+ cells, might be helpful. Once the chimerism in the peripheral blood has reached a level of more than 97% donor, real-time PCR assays should be used for further monitoring whenever possible. When real-time PCR indicates that the level of residual host cells further declines follow-up monitoring with real-time PCR is recommended at regular intervals. When this method indicates a level of residual host cells below 0.005%, the periods between the analyses may be increased to 2–3 months, because up to now we have not seen relapses when patients were at this low level. When real-time PCR assays cannot be used, standard STR-PCR should be performed at closer intervals. The length of the intervals should be chosen according to the tendency of relapse of the primary disease and the time after transplantation, with more frequent analyses (weekly to every 2 weeks) performed in patients with high risk disease (like AML or ALL) and early after transplantation. This very tight schedule should be followed for the first 2 years after transplantation, since the majority of relapses occur during this period. If real-time PCR shows that the level of host cells does not decline or increases again, subset analysis (including T cells, CD15+ cells and, if possible, CD34+ cells) should be performed in order to clarify the origin of these cells. Based on this information and the clinical situation (i.e. whether there is graft versus host disease present or not and the individual relapse risk), reduction of the immunosuppressive therapy or infusion of T cells might be performed. Thus in conclusion, we believe that chimerism analysis can be performed for the detection of MRD, but the

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diverse methods currently available differ in their abilities. If there is a genetic marker (translocation, IgVH rearrangement) which can be used for real-time PCR, this is certainly the first choice for MRD detection, since these markers are specific for leukemia. If not, chimerism analysis using the most sensitive and quantitative method available should be used. We believe that a strategy like this will allow for a more accurate and reliable assessment of chimerism and might help to identify patients at risk of

a reappearance of leukemia. However, prospective trials will clearly have to show whether these strategies can be used to achieve a longer leukemia-free survival after transplantation.

Acknowledgments This study was supported in part by the Deutsche Krebshilfe, Bonn (grant No. 70-2755 to CT and MB).

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58 Lamb LS Jr, Robbins NF, Abhyankar S, Joyce M, Stetler-Stevenson M, Henslee-Downey PJ, Gee AP: Flow cytometric cell sorting combined with molecular chimerism analysis to detect minimal recurrent leukemia: Good news and bad news. Bone Marrow Transplant 1997;19: 1157–1161. 59 Winiarski J, Gustafsson A, Wester D, Dalianis T: Follow-up of chimerism, including T- and B-lymphocytes and granulocytes in children more than one year after allogeneic bone marrow transplantation. Pediatr Transplant 2000; 4/2:132–139. 60 Zetterquist H, Mattsson J, Uzunel M, NasmanBjork I, Svenberg P, Tammik L, Bayat G, Winiarski J, Ringden O: Mixed chimerism in the B cell lineage is a rapid and sensitive indicator of minimal residual disease in bone marrow transplant recipients with pre-B cell acute lymphoblastic leukemia. Bone Marrow Transplant 2000;25:843–851. 61 Thiede C, Lutterbeck K, Oelschlägel U, Kiehl M, Steudel C, Platzbecker U, Brendel C, Fauser AA, Neubauer A, Ehninger G, Bornhauser M: Detection of relapse by sequential monitoring of chimerism in circulating CD34+ cells. Ann Hematol 2002;81(suppl 2):S27–S28. 62 Maynadie M, Gerland L, Aho S, Girodon F, Bernier M, Brunet C, Campos L, Daliphard S, Deneys V, Falkenrodt A, Jacob MC, Kuhlein E, LeCalvez G, Moskovtchenko P, Philip P, Carli PM, Faure GC, Bene MC: Clinical value of the quantitative expression of the three epitopes of CD34 in 300 cases of acute myeloid leukemia. Haematologica 2002;87:795–803. 63 Serke S, Huhn D: Expression of class I, II and III epitopes of the CD34 antigen by normal and leukemic hemopoietic cells. Cytometry 1996; 26/2:154–160.

64 Schraml E, Daxberger H, Watzinger F, Lion T: Quantitative analysis of chimerism after allogeneic stem cell transplantation by PCR amplification of microsatellite markers and capillary electrophoresis with fluorescence detection: The Vienna experience. Leukemia 2003; 17/1:224–227. 65 Chalandon Y, Vischer S, Helg C, Chapuis B, Roosnek E: Quantitative analysis of chimerism after allogeneic stem cell transplantation by PCR amplification of microsatellite markers and capillary electrophoresis with fluorescence detection: The Geneva experience. Leukemia 2003;17/1:228–231. 66 Koehl U, Beck O, Esser R, Seifried E, Klingebiel T, Schwabe D, Seidl C: Quantitative analysis of chimerism after allogeneic stem cell transplantation by PCR amplification of microsatellite markers and capillary electrophoresis with fluorescence detection: The Frankfurt experience. Leukemia 2003;17/1:232–236. 67 Kreyenberg H, Holle W, Mohrle S, Niethammer D, Bader P: Quantitative analysis of chimerism after allogeneic stem cell transplantation by PCR amplification of microsatellite markers and capillary electrophoresis with fluorescence detection: The Tuebingen experience. Leukemia 2003;17/1:237–240. 68 Acquaviva C, Duval M, Mirebeau D, Bertin R, Cave H: Quantitative analysis of chimerism after allogeneic stem cell transplantation by PCR amplification of microsatellite markers and capillary electrophoresis with fluorescence detection: The Paris-Robert Debre experience. Leukemia 2003;17/1:241–246. 69 Hancock JP, Goulden NJ, Oakhill A, Steward CG: Quantitative analysis of chimerism after allogeneic bone marrow transplantation using immunomagnetic selection and fluorescent microsatellite PCR. Leukemia 2003;17/1:247– 251.

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Review Acta Haematol 2004;112:24–29 DOI: 10.1159/000077556

The Multiparametric Scanning System for Evaluation of Minimal Residual Disease in Hematological Malignancies Luba Trakhtenbrot Gideon Rechavi Ninette Amariglio Department of Pediatric Hematology-Oncology, Safra Children’s Hospital and Institute of Hematology, The Chaim Sheba Medical Center, Tel Hashomer, and Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel

Key Words Combined analyses W Fluorescence in situ hybridization W Minimal residual disease

Abstract Combined simultaneous analysis of morphology, immunophenotyping and fluorescence in situ hybridization on the same cell offers advantages that may help to disclose the relevance of minimal residual disease (MRD) detection. Morphological analysis of small populations of cells related either to malignancy or recipient-associated markers may improve the accuracy of chimerism and MRD testing and delineate their clinical significance. Copyright © 2004 S. Karger AG, Basel

The level of minimal residual disease (MRD) is an important prognostic factor in hematological malignancies. The detection of MRD at any stage of therapy may have a powerful implication for and impact on the clinical management. New international therapeutic protocols base treatment decisions on the level of MRD [1, 2]. The past decade has brought new technologies to the study of MRD in malignancies because the currently used methodologies are far from accurate and sensitive. Morpholog-

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ical analysis of bone marrow (BM) can identify residual disease involving more than 5% of BM cells and thus lacks the necessary sensitivity for early detection. In addition, it is often difficult to differentiate a small leukemia blast population from normal hematopoietic blasts. When the malignant disease is associated with a specific chromosomal rearrangement conventional cytogenetic analysis and fluorescence in situ hybridization (FISH) are used to identify the MRD population. Both methods have a low sensitivity of approximately 10 –2 that is not significantly better than the sensitivity of a classical morphological analysis. The quantitative accuracy of FISH depends on the observed frequency and the number of cells scored, and thus is improved by scoring more cells [3]. However, scoring a large number of cells is not practical with routine FISH. Multiparametric fluorescence-activated cell sorter (FACS)- and polymerase chain reaction (PCR)-based techniques have a higher sensitivity of 10 –4 to 10 –6, respectively, for the detection of MRD. However, their false-positive rate is still very high [4]. The combination of FACS and FISH [5], FISH and May-Grünwald-Giemsa (MGG) [6], dual-color FISH and confocal laser scanning microscopy [7] and PCR and blast-colony assays FACS/ BrdU/FISH [8] improved to some extent the detection of lineage restriction specificity. However, methods that are both sensitive and specific are still missing and the suit-

Luba Trakhtenbrot, PhD Molecular Cytogenetics Laboratory Institute of Hematology, The Chaim Sheba Medical Center Tel Hashomer, 52621 (Israel) Tel. +972 3 5302128, Fax +972 3 5302397, E-Mail [email protected]

ability of these assays for routine clinical studies may be limited. Moreover, the detection of MRD with tumorspecific tests may not always be correlated with the risk of relapse. Thus in acute myelocytic leukemia (AML) with translocation t(8;21) the presence of MRD detected by PCR identification of AML1-ETO does not appear to be a prognostic factor [9]. In another example, positive RTPCR tests for BCR/ABL fusion transcripts have been reported in healthy individuals [10]. The relevance of the maturational stage of a given cell and the specific signs it presents (e.g. cell surface marker, cytogenetic aberration or PCR-detected sequence of a molecular change) is still disputable. Partial differentiation to more mature cells can occur and these cells may lack the potential to proliferate. The PCR tests do not preserve cellular morphology, and therefore do not determine which cells correlate with MRD. It can be suggested that a full morphological analysis of the MRD cells carrying the chromosomal rearrangement would reveal that there are subclasses of cells which are not relevant to disease progression. Recently, a new multiparametric cell scanning system (DuetTM, BioView Ltd., Rehovot, Israel) was introduced and several investigations illustrating the power of this system in MRD testing were published. This system is based on two important features: (1) automated brightfield and fluorescence scanning and classification of large numbers of cells, which allows rapid and efficient identification of small residual populations of pathological cells, and (2) combined analysis of morphology and/or cytochemistry and FISH on the same cell, thereby enhancing the specificity of pathological cell detection. The methods for peripheral blood (PB) and BM slide preparation, MGG staining, FISH and immunocytochemistry procedures were adapted from standard protocols. These methods as well as bright-field and fluorescence scanning and the combined analysis of morphology and FISH have previously been described in detail [11]. Briefly, slides are prepared from PB or BM samples by density gradient centrifugation and cytospin of the final cell suspension containing 300,000 cells. The slides are air-dried and stained with MGG (Sigma, St. Louis, Mo., USA). In a series of experiments it was shown that no white-blood-cell subset is lost or reduced by the preparative procedure. The slides are then scanned by the bright-field mode of the system, based on a dual mode, fully automated microscope (Axioplan2, Carl Zeiss, Jena, Germany), an XYmotorized stage with an accuracy of 0.2 Ìm (Märzhäuser Wetzlar, Germany), a 3CCD, progressive scan camera

(DXC9000, Sony, Japan) and a computer for the control and analysis of the data. In the bright-field mode, the system scans approximately 10,000 cells/min at a resolution of 0.4 m/pixel and saves the coordinates and images of all cells found on the slides for future reference. It also classifies the cells into six categories according to their morphology: polymorphonuclears and band cells, lymphocytes, normoblasts, myelocytes, blasts and plasma cells (PC). An experienced morphology technician can observe the images and correct the system errors. The MGG is removed with methanol/acetic acid 3:1 and FISH is performed on the same slide by standard procedures using commercially available DNA-FISH probes. The system enables observation of the fluorescent signals in the specific cells using a !100 objective (a magnification of !1,000) in parallel to the morphology of the same cells, which were scanned previously. Slides with FISH signals are searched either manually or automatically for target cells. When a target cell is identified, the system saves its morphological and FISH images, as well as its coordinates. From now on, whenever the cell is presented, both the morphological/immunostaining and the FISH images are viewed simultaneously (fig. 1). Therefore, the system can locate and then characterize these cells for the determination of their potential as MRD by the combination of all these characteristics. Cells can be observed by any combination of morphology, FISH or immunohistochemistry. For example, a large number of cells can be searched for blast morphology or according to CD34 positivity and then the FISH genotype of the cells can be elucidated. Alternatively, the system can search automatically for a certain chromosomal marker and then the morphology of each cell positive for this marker can be shown in parallel and the distribution of the different cells is defined according to their morphology/immunostaining and FISH. The hybridization signals can be removed by incubation in 2! SCC solution for 20 min at room temperature and a second FISH with another probe can be applied to the same slide. In this way the target cells can be characterized by several cytogenetic markers. It was suggested that using the system MRD can be accurately detected in hematological diseases because scanning of a large number of cells enables rapid and efficient identification of rare residual leukemia cells, thus increasing sensitivity. The multiple parameter analysis provides phenotypic and genotypic information on the suspected cell, thereby enhancing the specificity. The hypothesis underlying preliminary studies on combined analyses of morphology and FISH at the level of the single cell is that if the chromosomal abnormality is found in entirely dif-

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ferentiated cells that do not proliferate, there is a reduced risk of relapse. However, if immature, proliferating cells are found to be positive, there is a high probability for relapse. This assumption was confirmed by the study of Bielorai et al. [12] where the combined analysis was used to detect residual leukemic cells in the follow-up of an 8year-old boy with Ph+ acute lymphoblastic leukemia (ALL). Ph(+) ALL patients represent a subgroup with poor prognosis and are candidates for stem cell transplantation in first remission [13]. Since the patient described had no available donor, he was treated by using a highrisk protocol and was closely monitored with PCR and FISH analysis for MRD. Consecutive PCR tests were positive for 2.5 years and FISH analysis showed 0.2–4.5% m-BCR/ABL+ cells in PB, which is within the accepted background range for this method. The combined analysis showed that all the m-BCR/ABL+ cells were mature lymphocytes. It is therefore possible that a small fraction of long-living lymphocytes originating from the leukemic clone survived despite the cytotoxic treatment. Because mature lymphocytes have a long life span in the circulation, this finding supports the fact that the patient was in remission. Moreover, since mature differentiated cells have a low proliferate capacity, there is a low risk of relapse. It can be suggested that the detection of the leukemic marker in differentiated cells made the probability of relapse very low and had an important clinical application. Several investigations are focused on the use of the multiparametric system for chimerism testing and the detection of MRD after allogeneic BM transplantation [14–19]. Chimerism testing is used for routine documentation of engraftment and posttransplant follow-up of allogeneic transplant recipients. In sex-mismatched transplants when the recipient and donor are of different gen-

Fig. 1. Combined analysis of morphology or cytochemistry and FISH on the same cell. a Chimerism analysis and identifying of the

morphology of recipient cells in a PB sample from a male (XY genotype – one green and one red FISH signals) transplanted with female donor cells (XX genotype – two green signals). b Analysis of a BM sample from the patient with AML characterized by two cytogenetic markers: trisomy 8 (three green FISH signals) and trisomy 21 (three red signals). The blast cells show these trisomies, while the PMN cells are normal (two green and two red signals). c Combined chimerism analysis of immunostaining and FISH of an SCID male patient (XY – one green and one red signal) transplanted from a female donor (XX – two green signals). The T cells (CD3+, pink color) are of donor origin (XX), while the B cell (CD19+, brown color) is of host origin.

The Multiparametric System for Evaluation of MRD

der, the proportion of female and male cells is usually determined by analysis of sex chromosome markers, most often using FISH. The aim of these studies was to evaluate the notion that a full morphological analysis of the residual host cells would contribute to the accuracy of the detection of MRD. For this purpose 49 examinations of 31 patients were studied that were either transplanted from a sex-mismatched donor or had a defined chromosomal rearrangement. Results were retrospectively correlated with outcome. It was shown that in sex-mismatched transplants the system could detect very small populations of recipient cells not detected by routine FISH. Moreover, it was demonstrated that use of the system made it possible to recognize the malignant potential of residual cells when the number of recipient cells is very low (5–10%), not indicative of a pending relapse. Thus the clinical significance of MRD detection was improved by identifying the morphology of recipient cells. In 10 patients, the recipient cells were mostly blasts and 7 of them relapsed 2–8 weeks after the examination (3 died of treatment, before clinical relapse could be documented). Among the patients with mature hematopoietic morphology 1 died early during treatment and none of the others relapsed. It was concluded that identification of residual recipient-type cells as blasts predicts imminent relapse and these patients need additional therapy. The authors applied further prospectively the combined analyses for clinical management: when a minute recipient blast population was detected, immune suppression was rapidly withdrawn. Conversely, when residual recipient cells displayed mature hematopoietic morphology, patients were followed by serial testing and it was not necessary to expose them to the hazards of unnecessary treatment. The authors noted that for detection of MRD tumorspecific markers probes are probably superior and more accurate than sex mismatch. Detection of recipientderived cells does not necessarily correlate with the risk of relapse as recipient cells contributing to mixed chimerism may belong to the malignant clone, may belong to normal hematopoiesis, or may be stromal cells. During the first few weeks after standard allogeneic transplantation, recipient cells can still be detected when sensitive tests are used. These are mostly T cells surviving the conditioning regimen. Host cells may be detected at low levels (!1%) even later in the post-transplant course. Hardan et al. [20, 21] used the combined morphological and FISH analysis for the determination of the cell lineage carrying the del(13q) to overcome the pitfalls of conventional FISH in BM samples of multiple myeloma (MM) patients. Del(13q) is the best-studied chromosomal

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aberration in MM, associated with a lower response rate, progression-free survival and overall survival. The FISH technology can detect cryptic chromosome 13 abnormalities not detected by conventional cytogenetic analysis and therefore it is strongly recommended for use in MM patients [22–24]. FISH determination of the chromosome 13 status in the BM cells of MM patients bears a major limitation, i.e. there is a high false-positive rate for the available probes for 13q14 and consequently a high (10– 15%) cutoff level [23–25]. This cutoff level is specifically problematic in BM samples with a low proportion of PC due to the patchy pattern of PC distribution in the BM, difficulty in the BM aspiration and dilution of the sample by blood. In addition, patients with MM and del(13q) carry a substantial number of PC without this abnormality [26]. Recently various methods were developed for the combination of MGG staining with FISH at the single cell level such as the manual search for PC [27], the combined immunofluorescent PC detection and FISH [23, 24, 26] and the purification of PC with anti-CD138-coated magnetic beads [25, 28]. All these methods are time-consuming, may lead to loss of PC, and have limitations related to immunofluorescent PC detection, allowing only the analysis of BM samples with 130% of PC. Hardan et al. [20] used a combined morphological and FISH analysis and found that in most samples there is a spectrum of early and mature BM cells other than PC that are clearly positive for del(13q). This del(13q)-positive population varies in its size and may explain the high false-positive rate. These data were used as a basis for the determination of chromosome 13 status in the PC population: the percentage of the del(13q)-positive cells in the

PC population was counted, as well as the percentage of the del(13q)-positive cells in the non-PC population. The authors introduced a new index for the characterization of the chromosome 13q status in PC population of the BM: the ratio of del(13q)-positive cells in the PC population to del(13q)-positive cells in the non-PC population was defined as del(13q) index. This del(13q) index is high when most of the deletion-carrying cells are PC and low when most of the cells with deletion are non-PC. It is suggested that a combined analysis can be useful for more precise determination of the chromosome 13 status in patients with del(13q) detected by routine FISH in 10% of the cells as well as for the detection of MRD in MM patients with del(13q) detected at diagnosis. In conclusion, combined simultaneous morphological and cytogenetic multiparametric analysis offers advantages that may help disclose the relevance of MRD detection. Adding the morphological analysis of small populations of cells to malignancy or recipient-associated markers may improve the accuracy of chimerism and MRD testing, and delineate their clinical significance. It seems that the detection of MRD, preceding the appearance of overt leukemia, in such a small subpopulation of cells could only be accomplished by a combined morphological and FISH analysis. This technology merits further study in larger-scale trials.

Acknowledgment We thank Ms. Bella Weismann and Dr. Galina Ishuev for excellent technical assistance. G.R. holds the Gregorio and Dora Shapiro Chair in Hematologic Malignancies, Tel Aviv University.

References 1 Campana D, Pui CH: Detection of minimal residual disease in acute lymphoblastic leukemia: Methodological advances and clinical significance. Blood 1995;85:1416–1434. 2 Faderl S, Kurzrock R, Estron Z: Minimal residual disease in hematological disorders. Arch Pathol Lab Med 1999;123:1030–1034. 3 Cohen N, Novikov I, Hardan I, Esa A, BrokSimoni F, Amariglio N, Rechavi G, Ben-Bassat I, Trakhtenbrot L: Standardization criteria for the detection of BCR/ABL fusion in interphase nuclei of CML patients by FISH technique. Cancer Genet Cytogenet 2000;123:102–108.

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4 Engel HH, Goddacre A, Keyhani H, Jiang S, Van NT, Kimmel M, Sanchez-Williams G, Andreeff M: Detection of minimal residual disease in acute myelogenous leukemia and myelodysplastic syndromes in clinical remission by molecular cytogenetics. Blood 1995;86(suppl 1):1059. 5 Engel H, Goodacre A, Keyhani A, Jiang S, Van NT, Kimmel M, Sanchez-Williams G, Andreeff M: Minimal residual disease in acute myelogenous leukemia and myelodysplastic syndromes: A follow-up of patients in clinical remission. Br J Haematol 1997;99:64–75. 6 Bernell P, Arvidson I, Jacobsson B, Hast R: Fluorescence in situ hybridization in combination with morphology detects minimal residual disease in remission and heralds relapse in acute leukemia. Br J Haematol 1996;95:666– 672.

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7 Van Lom K, Houstmuller AB, van Putten WL, Slater RM, Lowenberg B: Cytogenetic clonality analysis of megakaryocytes in myelodysplastic syndrome by dual-color fluorescence in situ hybridization and confocal laser scanning microscopy. Genes Chromosomes Cancer 1999;25: 332–338. 8 Roberts WM, Estrov Z, Ouspenskaia MV, Johnston DA, McClain KL, Zipf TF, Roberts WM: Measurement of residual leukemia during remission in childhood acute lymphoblastic leukemia. N Engl J Med 1997;336:317–323.

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9 Kusec R, Laczika K, Knobl P, Fiedl J, Greinix H, Kahls P, Linkesch W, Schwarzinger I, Mitterbauer G, Purtscher B: AML1-ETO fusion mRNA can be detected in remission blood samples of all patients with t(8:21) acute myeloid leukemia after chemotherapy or autologous bone marrow transplantation. Leukemia 1994;8:735–739. 10 Bose S, Deininger M, Gora-Tybor J, Goldman JM, Melo JV: The presence of typical and atypical BCR-ABL fusion genes in leukocytes of normal individuals: Biologic significance and implications for the assessment of minimal residual disease. Blood 1998;92:3362–3367. 11 Trakhtenbrot L, Reichart M, Shimoni A: Chimerism testing and detection of minimal residual disease after allogeneic hematopoietic transplantation using BioView (DuetTM) combined morphological and cytological analysis. Leukemia 2002;16:1419–1422. 12 Bielorai B, Golan H, Trakhtenbrot L, Reichart M, Toren A, Daniely M, Zilberstein Y, Amariglio N, Rechavi G, Kaplinsky C: Combined analysis of morphology and FISH in follow-up of minimal residual disease (MRD) in a child with Ph+ acute lymphoblastic leukemia (ALL). Cancer Genet Cytogenet 2002;138:64–68. 13 Arico M, Valsecchi MG, Camitta B, Schrappe M, Chessells J, Baruchel A, Gaynon P, Silverman L, Janka-Schaub G, Kamps W, Pui CH, Masera G: Outcome of treatment in children with Philadelphia chromosome-positive acute lymphoblastic leukemia. N Engl J Med 2000; 342:998–1006. 14 Kaplinsky C, Hardan I, Toren A, Reichart M, Daniely M, Zilberstein Y, Bielorai B, Shimoni A, Avigdor A, Nagler A, Rechavi G, Amariglio N, Trakhtenbrot L: Minimal residual disease and chimerism detection after bone-marrow transplantation using an automatic cell scanning system. 43rd ASH Annual Meeting (abstract 5159). Blood 2001;98/2:348b. 15 Kaplinsky C, Hardan I, Toren A, Reichart A, Daniely M, Zilberstein Y, Shimoni A, Avigdor A, Brok-Simoni F, Rechavi G, Amariglio N, Trakhtenbrot L: Combined analysis of morphology and FISH using an automatic cell scanning system, increases the accuracy of leukemia diagnosis. 43rd ASH Annual Meeting (abstract 4451). Blood 2001;98/2:189b.

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16 Shimoni A, Trakhtenbrot L, Reichart M, Daniely M, Zilberstein Y, Brok-Simoni F, Rechavi G, Amariglio N, Kaplinsky C: Chimerism testing and detection of minimal residual disease in a patient with Ph+ acute lymphoblastic leukemia after bone marrow transplantation using multiparametric cell scanning system (BioView Ltd. Israel) (abstract 5176). 43rd ASH Annual Meeting. Blood 2001;98/2:352b. 17 Shimoni A, Nagler A, Kaplinsky C, Reichart M, Avigdor A, Hardan I, Yeshurun M, Daniely M, Zilberstein Y, Amariglio N, Brok-Simoni F, Rechavi G, Trakhtenbrot L: Chimerism testing and detection of minimal residual disease after allogeneic hematopoietic transplantation using BioView (DuetTM) combined morphological and cytogenetical analysis. Leukemia 2002;16: 1413–1418. 18 Shimoni A, Nagler A, Bielorai B, Reichart M, Rothman R, Toren A, Rechavi G, Amariglio N, Trakhtenbrot L: Detection of host blasts but not mature hematopoietic cells among minute host populations remaining after allogenetic transplantation, using combined morphology and cytogenetic analysis, predicts imminent relapse. 44th ASH Annual Meeting (abstract 2485). Blood 2002;100/1:631a. 19 Shimoni A, Trakhtenbrot L, Bielorai B, Reichart M, Rothman R, Toren A, Rechavi G, Nagler A: Application of immune-therapeutic interventions after allogeneic transplantation based on MRD detection with combined morphological and cytogenetic analysis. Biol Bone Marrow Transplant 2003;9:68. 20 Hardan I, Nagler A, Reichart M, Shimoni A, Rothman R, Daniely M, Kaplan T, BrokSimoni F, Rechavi G, Amariglio N, Trakhtenbrot L: Detection of 13q deletion in the bone marrow cells of patients with multiple myeloma using combined morphology and FISH analysis. 44th ASH Annual Meeting (abstract 1522). Blood 2002;100/1:392a. 21 Hardan I, Trakhtenbrot L, Rothman R, Reichart M, Shimoni A, Brok-Simoni F, Rechavi G, Amariglio N, Nagler A: Monitoring of minimal residual disease in the bone marrow of patients with multiple myeloma after allogeneic stem cell transplantation by combined morphological and cytogenetic analysis. 9th International Workshop of Multiple Myeloma (abstract 210). Hematol J 2003;4:S76.

22 Zojer N, Konigsberg R, Ackermann J, Fritz E, Dallinger S, Kromer E, Kaufmann H, Riedl L, Gisslinger H, Schreiber S, Heinz R, Ludwig H, Huber H, Drach J: Deletion of 13q14 remains an independent adverse prognostic variable in multiple myeloma despite its frequent detection by interphase fluorescence in situ hybridization. Blood 2002;95:1925–1930. 23 Fonseca R, Harrington D, Oken M, Dewald GW, Bailey RJ, Van Wier SA, Henderson KJ, Blood EA, Rajkumar SV, Kay N, Van Ness B, Greipp PR: Biological and prognostic significance of interphase fluorescence in situ hybridization detection of chromosome 13 abnormalities (delta 13) in multiple myeloma: An Eastern Cooperative Oncology Group study. Cancer Res 2002;62:715–720. 24 Facon T, Avet-Loiseau H, Guillerm G, Moreau P, Genevieve F, Zandecki M, Lai JL, Leleu X, Jouet JP, Bauters F, Harousseau JL, Bataille R, Mary JY: Chromosome 13 abnormalities identified by FISH analysis and serum beta2-microglobulin produce a powerful myeloma staging system for patients receiving high-dose therapy. Blood 2001;97:1566–1571. 25 Avet-Loiseau H, Facon T, Grosbois B, Magrangeas F, Rapp MJ, Harousseau JL, Minvielle S, Bataille R: Oncogenesis of multiple myeloma: 14q32 and 13q chromosomal abnormalities are not randomly distributed, but correlate with natural history, immunological features, and clinical presentation. Blood 2002;99:2185– 2191. 26 Nickenig C, Lang NK, Schoch C, Hiddemann W, Haferlach T: New insights into the biology of multiple myeloma using a combination of May-Grunwald-Giemsa staining and fluorescence in situ hybridization techniques at the single cell level. Ann Hematol 2001;80:662– 668. 27 Ng MH, Kan A, Chung YF, Wong IH, Lo KW, Wickham NW, Lei KI, Lee JC: Combined morphological and interphase fluorescence in situ hybridization study in multiple myeloma of Chinese patients. Am J Pathol 1999;154:15– 22. 28 Harrison CJ, Mazzullo H, Cheung KL, Gerrard G, Jalali GR, Mehta A, Osier DG, Orchard KH: Cytogenetics of multiple myeloma: Interpretation of fluorescence in situ hybridization results. Br J Haematol 2003;120:944–952.

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Review Acta Haematol 2004;112:30–33 DOI: 10.1159/000077557

Standardization of Preanalytical Factors for Minimal Residual Disease Analysis in Chronic Myelogenous Leukemia Martin C. Müller Tanja Hördt Peter Paschka Kirsten Merx Paul La Rosée Rüdiger Hehlmann Andreas Hochhaus III. Medizinische Universitätsklinik Mannheim der Universität Heidelberg, Mannheim, Deutschland

Key Words BCR-ABL W Minimal residual disease W Real-time quantitative polymerase chain reaction W RNA stabilization

Abstract Optimal sample quality is a prerequisite to generate valid data in the detection of minimal residual disease (MRD) in leukemias. Thus, the risk of obtaining ‘false’-negative results is increased when both quality and quantity of RNA are suboptimal. Factors which affect the sensitivity and consequently the validity of MRD results are reviewed. RNA degradation in unstabilized peripheral blood (PB) samples does not play a major role in samples being processed on the day of blood collection. However, the simulation of sample shipping at room temperature with a delay of sample processing of up to 3 days causes a dramatic loss of intact RNA. RNA degradation can be prevented by the use of a bedside RNA stabilization system. Additionally, the stabilizing procedure is capable of keeping real-time quantitative polymerase chain reaction (RQ-PCR) results comparable whether the sample is processed immediately or with a delay of up to 3 days. Consistent quantitative data cannot be obtained with unstabilized blood samples. Furthermore, the opti-

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mum volume of PB required for MRD diagnostics in patients with BCR-ABL-positive chronic myelogenous leukemia in complete cytogenetic remission is revisited. Ten milliliters of PB is sufficient for processing on the day of blood collection whereas the use of only 5 ml PB may result in false-negative results. Standardization of preanalytical and analytical factors is necessary to provide a comparability of RQ-PCR results from different laboratories within multicenter studies. The definition of ‘undetectable BCR-ABL’ in an individual patient should take these preanalytical parameters into consideration. Copyright © 2004 S. Karger AG, Basel

Introduction

Qualitative and real-time quantitative polymerase chain reaction (RQ-PCR) assays are well established for the detection of BCR-ABL. However, investigators are using different protocols which is important when it comes to interpretation of minimal residual disease (MRD) data. The drawback of this variety of techniques is the noncomparability of the results caused by nonstandardized procedures concerning the volume of used peripheral blood (PB), transit time from patient to laboratory, temperature during transport, RNA extraction meth-

Dr. Martin C. Müller III. Medizinische Universitätsklinik Fakultät für Klinische Medizin Mannheim der Universität Heidelberg Wiesbadener Strasse 7–11, DE–68305 Mannheim (Germany) Tel. +49 621 383 4232, Fax +49 621 383 4248, E-Mail [email protected]

od, cDNA synthesis, and PCR protocols using a variety of different standards. Besides general recommendations for good laboratory practice in MRD laboratories [1], we review approaches to minimizing preanalytical variability for the detection and quantification of BCR-ABL mRNA in patients with chronic myelogenous leukemia (CML). RQ-PCR has a significant prognostic impact on CML patients after stem cell transplantation [2], interferon-· [3], and imatinib therapy [4–6].

Stabilization of RNA in PB Samples

A crucial point for the standardization of MRD detection is the instability of RNA in vitro. Since most of the PB or bone marrow samples are not processed at the center of blood collection RNA degrades time-dependently during shipment to a specialized laboratory. When shipment of EDTA-anticoagulated PB is simulated by room temperature incubation for 72 h, a loss of intact RNA down to 3–13% compared to immediate processing of PB was shown [7]. The same study demonstrated that the RNA stabilization and extraction method PAXgene (PreAnalytiX, Hombrechtikon, Switzerland) significantly reduces the loss of RNA up to 40–44%. When tested specifically for quantitative detection of BCR-ABL, ABL, and G6PD in Philadelphia-positive CML, this protocol produced stable ratios of BCR-ABL/ABL (r = 0.99, p ! 0.0001) and BCR-ABL/G6PD correlating the measures after 2 with 72 h incubation at room temperature. In contrast, using unstabilized PB leads to a poor correlation between quantitative data generated from immediately versus delayed processed material (r = 0.65, p = 0.03). Delayed processing of unstabilized PB may not only negatively impact quantitative PCR. In this regard, we observed two false-negative PCR results in 2 out of 11 CML patients (18%), whereas PCR after stabilization revealed BCR-ABL-positive results in 11/11 samples (100%). Similar results concerning inhibition of RNA degradation using the PAXgene system were obtained by others with focus on different PCR applications [8]. PB is directly drawn into prefilled tubes containing stabilizing agent (PAXgene Blood RNA Tubes®, PreAnalytiX) using a standard BD Vacutainer® Blood Collection Set (Becton Dickinson, Franklin Lakes, N.J., USA). 2.5 ml of PB can be obtained within each tube. After a recommended incubation time of at least 2 h at room temperature RNA will be extracted using a silica gel matrix-based method (PAXgene Blood RNA Kit ®).

Preanalytical Factors in MRD Analysis

Impact of the Amount of Starting Material on PCR Sensitivity

Melo et al. [9] performed a systematic investigation addressing the adequate amount of starting material in samples with low level transcripts. The use of suboptimal starting template material leads to a random, unpredictable amplification of detectable products, which raises questions about the reliability of single negative PCR tests. On the other hand, the use of 400 ml PB of healthy individuals revealed BCR-ABL in very low concentrations in 12 of 16 and 22 of 73 cases [10, 11]. To determine the volume of PB necessary for a sensitive MRD analysis, we evaluated two RNA extraction methods using 5 or 10 ml of freshly drawn PB, each in patients with a complete cytogenetic response on imatinib therapy (11 male, 4 female, median age 65 years, range 44–67 years). The laborious but very efficient CsCl gradient ultracentrifugation method was compared to the PAXgene extraction method. Ultracentrifugation was performed for 16 h at 36,000 rpm (L60 Ultracentrifuge, Rotor SW55 Ti, Palo Alto, Calif., USA). Reverse transcription and consecutive RQ-PCR was implemented as described [12]. In cases of negative RQ-PCR a more sensitive qualitative 2-step ‘nested’ PCR for BCR-ABL transcripts was performed [13]. The median BCR-ABL/ABL ratios after RNA extraction of both 10 ml PB (CsCl 0.22% vs. PAXgene 0.23%, n = 8, nonsignificant) and 5 ml PB (CsCl 0.47% vs. PAXgene 0.58%, n = 7, nonsignificant, fig. 1) were not different. Comparable ratios were obtained using G6PD as an alternative housekeeping gene: median BCR-ABL/G6PD ratio using 10 ml PB (CsCl 0.032% vs. PAXgene 0.022%, n = 8, nonsignificant) and 5 ml PB (CsCl 0.050% vs. PAXgene 0.031%, n = 7, nonsignificant). The CsCl procedure achieved a significantly higher RNA yield comparing the median ABL transcript numbers using 10 ml PB (CsCl 20,800/2 Ìl cDNA vs. PAXgene 9,200/2 Ìl cDNA, n = 8, p = 0.0011) or 5 ml PB (CsCl 15,000/2 Ìl cDNA vs. PAXgene 3,800/2 Ìl cDNA, n = 7, p = 0.0041, fig. 2). The performance of a sensitive 2-step ‘nested’ PCR because of a negative 1-step RQ-PCR was necessary in 4 of the 7 patients when using 5 ml PB and PAXgene extraction (arrows in fig. 1) compared to 1 of 7 patients using the CsCl extraction. One patient being monitored with 5 ml PB even became ‘false’ BCR-ABLnegative after PAXgene extraction while the CsCl procedure still revealed low levels of BCR-ABL. In contrast, in all of the 8 monitored with 10 ml PB, low levels of BCRABL were consistently detected independently of the RNA extraction method applied [14].

Acta Haematol 2004;112:30–33

31

50000 Total ABL transcripts per 2 µl cDNA

Ratio BCR-ABL/ABL (%)

10

1

0.1

0.01

30000

20000

10000

negative

MEDIAN:

40000

0

10 ml Cs Cl

10 ml PAX

5 ml Cs Cl

5 ml PAX

0.22

0.23

0.47

0.58

n.s.

n.s.

MEDIAN:

10 ml CsCl

10 ml PAX

5 ml CsCl

5 ml PAX

20800

9200

15000

3800

p=0.0011

p=0.0041

Fig. 1. Comparison of 5 vs. 10 ml PB for MRD detection in CML

Fig. 2. Assessment of RNA yield by quantification of the housekeep-

patients in complete cytogenetic response: ratios of BCR-ABL/ABL after PAXgene extraction vs. CsCl gradient ultracentrifugation. Horizontal arrows mark samples which needed the more sensitive 2-step ‘nested’ PCR because of negative 1-step RQ-PCR.

ing gene ABL: comparison of 5 vs. 10 ml PB after PAXgene extraction or CsCl gradient ultracentrifugation.

Considering that this trial was performed using immediately processed PB after only 2 h of incubation at room temperature, the problem of RNA degradation could be neglected. Thus, one might conclude that at least 10 ml of PB are needed for MRD analysis provided that early processing of the material is guaranteed. Higher volumes of PB should be used for longer transfer times without stabilization.

patients with particularly low tumor burden might be caused by inadequate volumes of starting material. At least 10 ml of PB seem to be sufficient in CML patients with an excellent response to therapy (complete cytogenetic response) when processed on the same day. Furthermore the amount of usable RNA can be significantly increased by the use of the PAXgene stabilization method in case the arrival at the laboratory is not guaranteed on the day of blood collection. The PAXgene system represents an easy to use procedure which immediately stabilizes PB after phlebotomy and makes possible a standardized RNA extraction. Investigators of future clinical studies will need to agree on common preanalytical and analytical procedures such as cDNA synthesis and PCR protocols to achieve comparable MRD results. Our conclusions are derived from the experience with CML patients. However, the proven prognostic significance of PCR monitoring in other leukemic disorders calls for the use of optimized and standardized protocols regardless of the molecular target.

cDNA Synthesis

Different cDNA synthesis procedures should be individually tested for each application. To our knowledge, a comparative evaluation of cDNA synthesis protocols prior to BCR-ABL RT-PCR has not been reported as yet. However, best experiences have been made by using random hexamer priming.

Summary and Future Directions

Optimal sample quality is indispensable for molecular monitoring of leukemic fusion transcripts after therapy. However, the amount of usable RNA after arrival in the specialized laboratory depends on a multitude of factors. The problem of insufficient sensitivity in samples of

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Acta Haematol 2004;112:30–33

Acknowledgement The work was supported by the competence network ‘Acute and chronic leukemias’, sponsored by the German Bundesministerium für Bildung und Forschung (BMBF), Projektträger Gesundheitsforschung e.V. – DLR, 01 GI9980/6.

Müller/Hördt/Paschka/Merx/La Rosée/ Hehlmann/Hochhaus

References 1 Neumaier M, Braun A, Wagener C: Fundamentals of quality assessment of molecular amplification methods in clinical diagnostics. International Federation of Clinical Chemistry Scientific Division Committee on Molecular Biology Techniques. Clin Chem 1998;44:12– 26. 2 Cross NC, Feng L, Chase A, Bungey J, Hughes TP, Goldman JM: Competitive polymerase chain reaction to estimate the number of BCRABL transcripts in chronic myeloid leukemia patients after bone marrow transplantation. Blood 1993;82:1929–1936. 3 Hochhaus A, Reiter A, Saussele S, Reichert A, Emig M, Kaeda J, Schultheis B, Berger U, Shepherd PC, Allan NC, Hehlmann R, Goldman JM, Cross NC: Molecular heterogeneity in complete cytogenetic responders after interferon-alpha therapy for chronic myelogenous leukemia: Low levels of minimal residual disease are associated with continuing remission. German CML Study Group and the UK MRC CML Study Group. Blood 2000;95:62–66. 4 Merx K, Müller MC, Kreil S, Lahaye T, Paschka P, Schoch C, Weisser A, Kuhn C, Berger U, Gschaidmeier H, Hehlmann R, Hochhaus A: Early reduction of BCR-ABL mRNA transcript levels predicts cytogenetic response in chronic phase CML patients treated with imatinib after failure of interferon alpha. Leukemia 2002;16: 1579–1583.

Preanalytical Factors in MRD Analysis

5 Paschka P, Müller MC, Merx K, Kreil S, Schoch C, Lahaye T, Weisser A, Petzold A, Konig H, Berger U, Gschaidmeier H, Hehlmann R, Hochhaus A: Molecular monitoring of response to imatinib (Glivec®) in CML patients pretreated with interferon alpha. Low levels of residual disease are associated with continuous remission. Leukemia 2003;17: 1687–1694. 6 Müller MC, Gattermann N, Lahaye T, Deininger MW, Berndt A, Fruehauf S, Neubauer A, Fischer T, Hossfeld DK, Schneller F, Krause SW, Nerl C, Sayer HG, Ottmann OG, Waller C, Aulitzky W, le Coutre P, Freund M, Merx K, Paschka P, König H, Kreil S, Berger U, Gschaidmeier H, Hehlmann R, Hochhaus A: Dynamics of BCR-ABL mRNA expression in first line therapy of chronic myelogenous leukemia patients with imatinib or interferon t/araC. Leukemia 2003;17:2392–2400. 7 Müller MC, Merx K, Weisser A, Kreil S, Lahaye T, Hehlmann R, Hochhaus A: Improvement of molecular monitoring of residual disease in leukemias by bedside RNA stabilization. Leukemia 2002;16:2395–2399. 8 Rainen L, Oelmueller U, Jurgensen S, Wyrich R, Ballas C, Schram J, Herdman C, BankaitisDavis D, Nicholls N, Trollinger D, Tryon V: Stabilization of mRNA expression in whole blood samples. Clin Chem 2002;48:1883– 1890.

9 Melo JV, Yan XH, Diamond J, Lin F, Cross NC, Goldman JM: Reverse transcription/polymerase chain reaction (RT/PCR) amplification of very small numbers of transcripts: The risk in misinterpreting negative results. Leukemia 1996;10:1217–1221. 10 Biernaux C, Loos M, Sels A, Huez G, Stryckmans P: Detection of major bcr-abl gene expression at a very low level in blood cells of some healthy individuals. Blood 1995;86: 3118–3122. 11 Bose S, Deininger M, Gora-Tybor J, Goldman JM, Melo JV: The presence of typical and atypical BCR-ABL fusion genes in leukocytes of normal individuals: Biologic significance and implications for the assessment of minimal residual disease. Blood 1998;92:3362–3367. 12 Emig M, Saussele S, Wittor H, Weisser A, Reiter A, Willer A, Berger U, Hehlmann R, Cross NC, Hochhaus A: Accurate and rapid analysis of residual disease in patients with CML using specific fluorescent hybridization probes for real time quantitative RT-PCR. Leukemia 1999;13:1825–1832. 13 Cross NC, Feng L, Bungey J, Goldman JM: Minimal residual disease after bone marrow transplant for chronic myeloid leukaemia detected by the polymerase chain reaction. Leuk Lymphoma 1993;11(suppl 1):39–43. 14 Müller MC, Hördt T, Paschka P, Weisser A, Petzold A, Merx K, König H, Hehlmann R, Hochhaus A: The impact of preanalytical and analytical standardization for the detection of minimal residual disease in CML patients after therapy (abstract). Blood 2002;100:548a.

Acta Haematol 2004;112:30–33

33

Review Acta Haematol 2004;112:34–39 DOI: 10.1159/000077558

Minimal Residual Disease in Childhood Acute Lymphoblastic Leukemia: Current Status and Challenges Shai Izraeli Dalia Waldman Department of Pediatric Hemato-Oncology and the Cancer Research Center, Safra Children Hospital, Sheba Medical Center, Tel Hashomer, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

Key Words Acute lymphoblastic leukemia W Minimal residual disease W Childhood W FACS W PCR

Abstract The pace of disappearance of leukemic blasts in response to therapy has long been recognized as the most important prognostic factor in childhood acute lymphoblastic leukemia (ALL). Recent technological advancements enable detection of submicroscopic leukemic cells. The extent of reduction in the level of minimal residual disease (MRD) during the first phase of therapy can be exploited for improved risk classification of children with ALL. Current prospective studies test the hypothesis that tailoring treatment to the level of MRD will improve patients’ outcome. Copyright © 2004 S. Karger AG, Basel

Introduction

Modern treatment protocols lead to morphological complete remission in 95–98% of children with acute lymphoblastic leukemia (ALL) [1–3]. If treatment is discontinued at this stage all children will eventually relapse.

ABC

© 2004 S. Karger AG, Basel 0001–5792/04/1122–0034$21.00/0

Fax + 41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/aha

Indeed, all prospective clinical studies have shown that ALL should be treated for at least 2 years. These facts indicate that at the end of remission induction chemotherapy not all clonogenic malignant lymphoblasts are killed although most of the patients are in clinical and morphological remission. Moreover, despite intensive therapy throughout the period of clinical remission, 25– 30% of the patients will eventually relapse. Thus a low level of clonogenic malignant cells remains in a substantial proportion of the patients even after completion of chemotherapy. Since leukemic cells have to constitute at least 1–5% of the nucleated cells in the bone marrow in order to be detected by microscopic examination, morphological examination is clearly inadequate for evaluation of the quality of remission in patients with ALL. Therefore more sensitive techniques for the detection of rare leukemic cells are required. This is the rationale behind the recent incorporation of modern techniques of detection of minimal residual disease (MRD) into treatment protocols of childhood ALL. Our purpose here is to highlight the achievements and current challenges of MRD detection in childhood ALL. For a more detailed information about methodologies and clinical trials the interested readers are referred to several excellent recent reviews and to other papers in this special edition of Acta Haematologica [4–7].

Shai Izraeli, MD Pediatric Hemato-Oncology, Sheba Medical Center Tel Hashomer, IL–52621 Ramat Gan (Israel) Tel. +972 3 5303037, Fax +972 3 5303031 E-Mail [email protected]

A clinically useful technique for detection of MRD needs to fulfill the following minimal criteria: (1) It should reliably discriminate between malignant and normal cells. This is not trivial since leukemia is characterized by arrest in the normal developmental program. Thus, leukemic blasts are similar in many respects to normal lymphoid precursors. (2) It should be significantly more sensitive than morphological microscopic examination. Therefore, the threshold for detection should be at least one leukemic cell among 1,000 normal cells (i.e. at least !1:10 –3, and preferably 1:10 –4 to 10 –6). (3) The method should allow clear and precise quantification of MRD. (4) The leukemic-specific markers utilized for MRD detection should be stably expressed by the leukemic cell throughout the course of the disease. Since malignant cells are naturally genetically unstable, some potential markers present at the time of diagnosis may be lost later in the disease. A search for such an unstable marker later in the disease may lead, therefore, to false-negative result. (5) The technique should be easily reproducible within the same laboratory and between different laboratories. This is essential for conduction of large multicenter studies. Ideally, the techniques should be simple and reproducible enough to be eventually incorporated into the routine clinical laboratory, similarly to cytogenetic and immunophenotyping techniques that are currently routinely used in most large hematological clinical centers. (6) The results should be available fast enough to allow for timely clinical therapeutic decisions. (7) Cost-effectiveness is extremely important for global incorporation into the routine treatment of childhood ALL. The currently available approaches can be divided into two groups: methodologies that are based on the identification of abnormal phenotypes and techniques that follow aberrant genotypes of the leukemic cell. The specific phenotype of a leukemic cell is manifested by its morphology and by the aberrant expression of surface and intracellular proteins. Multiparametric flow cytometry (FACS-MRD) has been used for the last decade for the sensitive detection and quantification of MRD [4, 8]. (An updated review of this technology by Campana and Coustan-Smith [9] who pioneered this approach for MRD detection can be found in this issue). The advantages of this technology are as follows: (1) Adequate sensitivity (one leukemic cell can be detected within 104 normal cells). (2) The analysis is done with equipment that is already being used routinely for immunophenotyping of

leukemia. Thus potentially this technique can become routine and be less dependent on centrally placed sophisticated laboratories. (3) It is possible to distinguish living from dead or apoptotic leukemia cells. (4) The method is quick and relatively cheap. There are, however, a few disadvantages that currently delay the implementation of FACS-MRD into the clinical routine: (1) The analysis is quite complex and depends on the expertise of the operator. This problem may be overcome by modern communication technology. In the near future it may be possible to analyze centrally results obtained in the local centers. (2) It is difficult to distinguish between normal regenerating bone marrow progenitors and residual blasts of B cell precursor leukemias (BCP-ALL), which is the most common type of childhood leukemia. Thus the sensitivity of FACS-MRD is better for T-ALL than BCP-ALL. (3) The analysis can be reliably done only on fresh cells. Thus expert operators have to continuously be available locally for the analysis, which is a major limitation for multicenter studies. This problem may be solved by the approach delineated in (1). (4) Instability of antigenic expression on leukemic cells (lineage switch, loss of antigens) during or after the treatment course. This problem can be solved by choosing several leukemia-specific antigens for follow-up of MRD. Detection of MRD by quantification of leukemia-specific genetic lesions is done by either DNA- or RNA-based methodologies. There are a few technical issues with these two nucleic acids that may be of clinical importance and thus deserve mentioning here. Genomic DNA is relatively stable and easy to obtain. It may be shipped at room temperature, a big advantage for multicenter studies. The amount of DNA is equal in all somatic cells. Therefore the actual quantity of leukemia-specific DNA correlates with the number of leukemic cells. Because of its stability, however, it is impossible to distinguish between DNA that originates in living and dying leukemic cells. Unlike DNA, RNA is an extremely unstable molecule. To protect themselves from RNA viruses mammalian cells express large amounts of stable enzymes that degrade RNA (RNAases). These enzymes are ubiquitous and stable. Thus RNA has to be prepared either from fresh cells or from cells that were snap-frozen and kept at a temperature lower than –70 ° C. These limitations also hold for shipment of RNA. These are major limitations for routine clinical applications. In the near future, however, thanks to significant efforts made by the biotechnology industry, it may be possible to keep and send RNA at room temperature. Another potentially important issue is the fact that

MRD in Childhood ALL

Acta Haematol 2004;112:34–39

Methodologies – What Does the Clinician Need to Know?

35

the amount of RNA of a specific gene varies between cells and within a single cell at different time points. Thus, theoretically the same quantity of a leukemia-specific RNA may originate in few cells possessing a lot of this RNA species or in many more cells each producing a small quantity of this transcript. So, for example, a large number dormant leukemic cells that produce only small amounts of the fusion gene bcr/abl may not be detected by an RNA PCR approach (RT-PCR) [10]. Whether this problem is of a theoretical or practical value needs to be determined by clinical trials where DNA (or FACS) MRD will be compared with RT-PCR MRD in the same patients. Such studies are in progress [11]. The most widely studied DNA-based MRD methodology is based on the identification of clonospecific rearrangements of immunoglobulins or T cell receptors (IgTcRPCR) [6]. This approach exploits the physiologic process of somatic rearrangement of Ig and TcR gene loci that occur during early differentiation of any lymphocyte. Thus, any single lymphocyte carries a unique rearrangement that is not shared by any other lymphoid cell. This process ensures the level of diversity of the immune response against an unlimited number of antigens. Since leukemia is clonal, i.e. it originates in one lymphoid cell, all the leukemic cells of a particular person carry the same Ig and/or TcR rearrangements. Because leukemic cells are genetically unstable they often (190% of the cases) carry multiple rearrangements, a fact that facilitates the usefulness of using these rearrangements as a clonal marker for MRD detection. The details of the techniques are described elsewhere [6, 12]. Its big advantages are the exquisite sensitivity (at least 10 –5), reliability, reproducibility and its applicability to more than 90% of children with ALL. Its biggest disadvantages are the costs and complexity. Fluorescence in situ hybridization (FISH) allows the detection of clonal structural and numerical chromosomal abnormalities in the vast majority (at least 70%) of childhood ALL. Currently the technique is well standardized. Unlike conventional cytogenetics it does not require dividing cells and can be performed on smears of bone marrow or peripheral blood. Its major disadvantage is the lack of suitable sensitivity (1–5%) for MRD detection. New promising automated methodologies that combine morphological and FISH examinations are being developed [13–15]. Detection and quantification of leukemia-specific mRNA are rapidly gaining popularity as a reliable, sensitive method at relatively low costs. Such RNA transcripts can be found in up to 70% of leukemic samples. They con-

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Acta Haematol 2004;112:34–39

sist of either fusion mRNA resulting from chromosomal translocations (e.g. BCR/ABL, TEL/AML1, E2A/PBX1) or other transcripts that are specifically elevated in ALL [WT1 in B lineage ALL and HOX11L2 and SIL-SCL (TAL1) in T ALL]. The recent development of RT-PCR techniques of automated precise quantification (RQ-PCR such as TaqManTM and LightcyclerTM technologies) will facilitate standardization and the applicability of these techniques [16, 17]. Which MRD methodology is better for MRD monitoring in childhood ALL? This issue has not been resolved yet. So far most of the experience has been with FACSMRD and IgTcR-PCR. Direct comparisons between these methodologies in a single institution [18] have shown them to be largely equivocal. However, FACSMRD has not been compared directly with PCR in a multicenter trial. Another problem with FACS-MRD is a lack of international standardization. For example, the St. Jude group defines MRD by FACS as the percentage of blasts among normal mononuclear cells while the Austrian group defines MRD as the absolute number of blasts per microliter [8, 19]. Until comparative multicenter standardized studies are published, IgTcR-PCR remains the benchmark, to which any method should be compared. Interestingly, independent studies using either FACS or IgTcR-PCR have reached similar conclusions (see below). It is quite possible, therefore, that because of their lower costs FACS and fusion mRNA detection will replace IgTcR-PCR in the future as the standard methodology for MRD detection.

Clinical Significance of MRD Monitoring

Several large retrospective analyses of samples collected during prospective clinical trials yielded strikingly similar results (table 1) [7, 8, 19–23]. It appears that the pace of reduction in leukemic burden during the first few weeks of therapy is the strongest prognostic indicator that overrides any other conventional prognostic factors. Thus in several independent protocols MRD levels higher that 1% at the end of induction or more than 0.1% before consolidation were found to identify patients with an extremely bad prognosis who may benefit from an intensification of therapy. These results strengthen and refine previous observations made by the international BFM group that the extent of early response to therapy is the most important prognostic factor. Poor response to 1 week of prednisone at the beginning of therapy, defined as more than 1,000 blasts on day 8, characterizes a small group of

Izraeli/Waldman

Table 1. Selected major studies of MRD in childhood ALL

Study (group)

Patients

MRD methodology

Major conclusions

EORTC [26]

178

IgTcR-PCR (Genescan)

MRD 61% at end of induction or MRD 60.1% at later time point high risk for relapse

iBFM 90 [20]

240

IgTcR-PCR and TAL1 deletion

MRD at day 33 (tp1) and 78 (tp2) defining risk: high risk MRD 61% at tp1 or MRD 60. 1% at tp2 3 years EFS = 16%; low risk MRD ! 0.01% at tp1, 2 3 years EFS = 98%

St Jude [19]

195

FACS-MRD

High risk: MRD 61% at end of induction or MRD 60.1% at week 14 or persistence till week 33

Austrian BFM 95 [8]

108

FACS-MRD

High risk 1 1 blast/Ìl at day 14, 33 and week 12 100% relapse compared to 6% relapse in all others

NOPHO [21]

104

IgTcR-PCR

MRD ! 0.01% at day 29 identifies patients with extremely good prognosis

Australia [23]

85

IgTcR-PCR

Detectable levels of MRD at 12 and 24 months predictive of relapse

Austrian BFM [24]

68

IgTcR-PCR

In 20% of patients MRD is already below 0.01% at day 15; identifies a group with excellent prognosis

226

FACS-MRD

Level of MRD are identical in peripheral blood and BM for T lineage ALL; peripheral blood MRD is lower than in the BM of B-lineage ALL; detectable levels of MRD in peripheral blood of B-lineage ALL at the end of induction are associated with 80% chance of relapse

59

IgTcR-PCR

MRD levels prior BMT predict relapse

St Jude [22]

iBFM [27]

tp = Time point; EFS = event-free survival; BM = bone marrow; BMT = bone marrow transplant.

poor prognosis patients. Still most of the relapses occur in the majority of patients that are good prednisone responders. Monitoring of molecular MRD has refined this group. It seems to identify 15% of patients with ALL, in whom most of the relapses will occur. Conversely, in about 40% of the patients the frequency of leukemic cells at the end of first months of therapy is less than 0.01% [20]. Amazingly MRD is already undetectable in about 20% of the patients at day 15 of therapy in the BFM protocol [24]! These patients have close to 100% chance of a long-term disease-free survival. Thus, for the first time it is possible to prospectively identify those patients who may benefit from reduction in therapy. This assumption requires some caution, however. The excellent prognosis of the rapid responders may actually indicate that the type of therapy given is effective and suitable. Therefore, a reduction in treatment intensity may result in an increased rate of relapse. These hypotheses are currently being tested in the cooperative AIEOP-BFM ALL2000 clinical protocol for childhood ALL, which is mainly based on MRD assessment at day +33 and day +78 by two Ig/TcR targets with a sensitivity of at least 10 –4. Patients receive the same

induction therapy and, according to MRD assessment at two time points and some clinical features [presence of t(4;11) or t(9;22), response to prednisone in the first week, clinical remission after induction], patients are stratified into three different risk groups with tailored therapy. Intensification of treatment of the high risk group and reduction of treatment of the low risk group is tested in a randomized fashion. The long-term results of this study and comparable studies are likely to show whether this more sensitive and specific evaluation of remission and early response to treatment could speed up further improvement in the cure rates of children with ALL. The value of MRD monitoring during maintenance therapy and off therapy is less clear [20, 23, 25]. Most patients become MRD negative. The absence of residual disease after remission induction is associated with a good prognosis. If multiple bone marrow samples are analyzed during follow-up, a steady decrease of MRD levels to becoming undetectable is observed in childhood ALL patients. The persistence of residual blasts beyond 4–6 months or the reemergence of residual disease, even at the level of 1 ! 10 –4, predicts clinical relapse [23, 26]. However, most of the relapses occur in MRD-negative patients

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Acta Haematol 2004;112:34–39

37

and therefore late determination of MRD is of limited clinical value. Finally, the value of MRD monitoring has also been shown in the follow-up of relapsed patients. Pretransplant levels highly correlate with the outcome of stem cell transplantation [27].

Questions and Challenges

Over the last decade sensitive methodologies for MRD detection have been developed and the prognostic power of MRD monitoring during early phases of therapy has clearly been proven. The availability of these technologies provides now the opportunity to answer few critical questions: 1 Would tailoring treatment by risk classification based on MRD improve outcome of children with ALL? In particular is it appropriate to reduce toxic treatment to the large group of patients, in which MRD levels drop below 0.01% after 4 weeks of therapy? 2 What sensitivity should we aim for? Is 10 –4 enough or is it too much? Clearly, if a reduction in therapy proves futile, MRD methodologies do not need to be so sensitive. In addition, since leukemia may be a patchy dis-

ease, an extremely sensitive determination of MRD in one bone marrow sample may not reflect the total body burden of leukemia. Indeed a real challenge is developing new methodologies that will make it possible to estimate the whole body burden and distribution of leukemia during remission. 3 What is the ideal technology for MRD detection? How can MRD quantification be made more cost-effective? 4 Should MRD be followed off therapy? Should the definition of relapse be changed [28]? Is it useful to diagnose molecular relapse based on rising levels of MRD? 5 Could some of the MRD positivity off therapy represent premalignant nonclonogenic clones [29]? If yes, what is their significance? The answers to these questions will determine whether the amazing technological achievements will also translate into improved outcome of children with ALL.

Acknowledgments We wish to thank Drs. Gianni Cazzaniga and Andrea Biondi for sharing an unpublished manuscript with us. Supported in part by the Israel Cancer Association.

References 1 Schrappe M, Reiter A, Zimmermann M, Harbott J, Ludwig WD, Henze G, Gadner H, Odenwald E, Riehm H: Long-term results of four consecutive trials in childhood ALL performed by the ALL-BFM study group from 1981 to 1995. Berlin-Frankfurt-Munster. Leukemia 2000;14:2205–2222. 2 Schrappe M, Camitta B, Pui CH, Eden T, Gaynon P, Gustafsson G, Janka-Schaub GE, Kamps W, Masera G, Sallan S, Tsuchida M, Vilmer E: Long-term results of large prospective trials in childhood acute lymphoblastic leukemia. Leukemia 2000;14:2193–2194. 3 Pui CH, Relling MV, Campana D, Evans WE: Childhood acute lymphoblastic leukemia. Rev Clin Exp Hematol 2002;6:161–180, 200–202. 4 Campana D: Determination of minimal residual disease in leukaemia patients. Br J Haematol 2003;121:823–838. 5 Cazzaniga G, d’Aniello E, Corral L, Biondi A: Results of minimal residual disease (MRD) evaluation and MRD-based treatment stratification in childhood ALL. Best Pract Res Clin Haematol 2002;15:623–638. 6 Szczepanski T, Orfao A, van der Velden VH, San Miguel JF, van Dongen JJ: Minimal residual disease in leukaemia patients. Lancet Oncol 2001;2:409–417.

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7 Biondi A, Valsecchi MG, Seriu T, D’Aniello E, Willemse MJ, Fasching K, Pannunzio A, Gadner H, Schrappe M, Kamps WA, Bartram CR, van Dongen JJ, Panzer-Grumayer ER: Molecular detection of minimal residual disease is a strong predictive factor of relapse in childhood B-lineage acute lymphoblastic leukemia with medium risk features. A case control study of the International BFM study group. Leukemia 2000;14:1939–1943. 8 Dworzak MN, Froschl G, Printz D, Mann G, Potschger U, Muhlegger N, Fritsch G, Gadner H: Prognostic significance and modalities of flow cytometric minimal residual disease detection in childhood acute lymphoblastic leukemia. Blood 2002;99:1952–1958. 9 Campana D, Coustan-Smith E: Minimal residual disease studies by flow cytometry in acute leukemia. Acta Haematol 2004;112:8–15. 10 Izraeli S, Janssen JW, Haas OA, Harbott J, Brok-Simoni F, Walther JU, Kovar H, Henn T, Ludwig WD, Reiter A, Gadner H: Detection and clinical relevance of genetic abnormalities in pediatric acute lymphoblastic leukemia: A comparison between cytogenetic and polymerase chain reaction analyses. Leukemia 1993;7:671–678.

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11 Drunat S, Olivi M, Brunie G, Grandchamp B, Vilmer E, Bieche I, Cave H: Quantification of TEL-AML1 transcript for minimal residual disease assessment in childhood acute lymphoblastic leukaemia. Br J Haematol 2001;114: 281–289. 12 Brüggemann M, Pott C, Ritgen M, Kneba M: Significance of minimal residual disease in lymphoid malignancies. Acta Haematol 2004; 112:111–119. 13 Shimoni A, Nagler A, Kaplinsky C, Reichart M, Avigdor A, Hardan I, Yeshurun M, Daniely M, Zilberstein Y, Amariglio N, Brok-Simoni F, Rechavi G, Trakhtenbrot L: Chimerism testing and detection of minimal residual disease after allogeneic hematopoietic transplantation using the bioView (Duet) combined morphological and cytogenetical analysis. Leukemia 2002;16: 1413–1422. 14 Kaplinsky C, Trakhtenbrot L, Hardan I, Reichart M, Daniely M, Toren A, Amariglio N, Rechavi G, Izraeli S: Tetraploid myeloid cells in donors of peripheral blood stem cells treated with rhG-CSF. Bone Marrow Transplant 2003; 32:31–34. 15 Trakhtenbrot L, Rechavi G, Amariglio N: The multiparametric scanning system for evaluation of minimal residual disease in hematological malignancies. Acta Haematol 2004;112:24– 29.

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16 Cazzaniga G, Rossi V, Biondi A: Monitoring minimal residual disease using chromosomal translocations in childhood ALL. Best Pract Res Clin Haematol 2002;15:21–35. 17 Cazzaniga G, Lanciotti M, Rossi V, Di Martino D, Arico M, Valsecchi MG, Basso G, Masera G, Micalizzi C, Biondi A: Prospective molecular monitoring of BCR/ABL transcript in children with Ph+ acute lymphoblastic leukaemia unravels differences in treatment response. Br J Haematol 2002;119:445–453. 18 Neale GA, Coustan-Smith E, Pan Q, Chen X, Gruhn B, Stow P, Behm FG, Pui CH, Campana D: Tandem application of flow cytometry and polymerase chain reaction for comprehensive detection of minimal residual disease in childhood acute lymphoblastic leukemia. Leukemia 1999;13:1221–1226. 19 Campana D, Neale GA, Coustan-Smith E, Pui CH: Detection of minimal residual disease in acute lymphoblastic leukemia: The St Jude experience. Leukemia 2001;15:278–279. 20 van Dongen JJ, Seriu T, Panzer-Grumayer ER, Biondi A, Pongers-Willemse MJ, Corral L, Stolz F, Schrappe M, Masera G, Kamps WA, Gadner H, van Wering ER, Ludwig WD, Basso G, de Bruijn MA, Cazzaniga G, Hettinger K, van der Does-van den Berg A, Hop WC, Riehm H, Bartram CR: Prognostic value of minimal residual disease in acute lymphoblastic leukaemia in childhood. Lancet 1998;352:1731– 1738.

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21 Nyvold C, Madsen HO, Ryder LP, Seyfarth J, Svejgaard A, Clausen N, Wesenberg F, Jonsson OG, Forestier E, Schmiegelow K: Precise quantification of minimal residual disease at day 29 allows identification of children with acute lymphoblastic leukemia and an excellent outcome. Blood 2002;99:1253–1258. 22 Coustan-Smith E, Sancho J, Hancock ML, Razzouk BI, Ribeiro RC, Rivera GK, Rubnitz JE, Sandlund JT, Pui CH, Campana D: Use of peripheral blood instead of bone marrow to monitor residual disease in children with acute lymphoblastic leukemia. Blood 2002;100: 2399–2402. 23 Marshall GM, Haber M, Kwan E, Zhu L, Ferrara D, Xue C, Brisco MJ, Sykes PJ, Morley A, Webster B, Dalla Pozza L, Waters K, Norris MD: Importance of minimal residual disease testing during the second year of therapy for children with acute lymphoblastic leukemia. J Clin Oncol 2003;21:704–709. 24 Panzer-Grumayer ER, Schneider M, Panzer S, Fasching K, Gadner H: Rapid molecular response during early induction chemotherapy predicts a good outcome in childhood acute lymphoblastic leukemia. Blood 2000;95:790– 794.

25 Campana D, Neale GA, Coustan-Smith E, Pui CH: Detection of minimal residual disease in acute lymphoblastic leukemia: The St Jude experience. Leukemia 2001;15:278–279. 26 Cave H, van der Werff ten Bosch J, Suciu S, Guidal C, Waterkeyn C, Otten J, Bakkus M, Thielemans K, Grandchamp B, Vilmer E: Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia. European Organization for Research and Treatment of Cancer – Childhood Leukemia Cooperative Group. N Engl J Med 1998;339:591–598. 27 Bader P, Hancock J, Kreyenberg H, Goulden NJ, Niethammer D, Oakhill A, Steward CG, Handgretinger R, Beck JF, Klingebiel T: Minimal residual disease (MRD) status prior to allogeneic stem cell transplantation is a powerful predictor for post-transplant outcome in children with ALL. Leukemia 2002;16:1668– 1672. 28 Lion T, Izraeli S, Henn T, Gaiger A, Mor W, Gadner H: Monitoring of residual disease in chronic myelogenous leukemia by quantitative polymerase chain reaction. Leukemia 1992;6: 495–499. 29 Greaves M: Silence of the leukemic clone. N Engl J Med 1997;336:367–369.

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Review Acta Haematol 2004;112:40–54 DOI: 10.1159/000077559

Detection of Minimal Residual Disease in Acute Myelogenous Leukemia P. Raanani I. Ben-Bassat Institute of Hematology, Chaim Sheba Medical Center, Tel Hashomer and Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel

Key Words AML W MRD W FISH W Cytogenetics W RT-PCR

Abstract Acute myelogenous leukemia (AML) is considered to be in complete remission when fewer than 5% of the cells in bone marrow are blasts. Nevertheless, approximately two thirds of patients relapse due to persisting leukemic blasts. The persistence of these cells, below the threshold of morphological detection, is termed minimal residual disease (MRD) and various methods are used for its detection. These methods include classical cytogenetics, fluorescence in situ hybridization, qualitative and quantitative RT-PCR and multiparametric flow cytometry. Currently, less than half of the AML patients have a specific marker detectable by RT-PCR techniques. The major specific molecular markers are involvement of the MLL gene with up to 50 different partners and partial tandem duplications, the core binding factor leukemias with AML1/ ETO and CBFß/MYH11 rearrangements, PML/RAR· in acute promyelocytic leukemia, internal tandem duplications and mutations of FLT3 and some other rare translocations. In addition, several other genes show abnormal expression levels in AML, including the Wilms tumor gene, the PRAME gene and Ig/TCR rearrangements.

ABC

© 2004 S. Karger AG, Basel 0001–5792/04/1122–0040$21.00/0

Fax + 41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/aha

Most of these genetic abnormalities can be detected by qualitative but more importantly by quantitative RT-PCR. The kinetics of disappearance of molecular markers in AML differs between the various types of leukemias, although at least a 2 log reduction of transcript after induction chemotherapy is necessary for long-term remission in all types. Conversely, the change of PCR from negativity to positivity is highly predictive of relapse. Whereas in acute lymphoblastic leukemia, multiparametric flow cytometry is an established method for MRD detection, this is less so in AML. The reason is the absence of well-characterized leukemia-specific antigens and the existence of phenotypic changes at relapse. On the other hand, this method is convenient due to its simplicity and universal applicability. In conclusion, several methods can be used for MRD detection in AML patients; each has its pros and cons. Several issues still remain to be settled including the choice of the best method and the timing for MRD monitoring and above all the practical clinical implications of MRD in the various types of AML. Copyright © 2004 S. Karger AG, Basel

Dr. Pia Raanani Institute of Hematology Chaim Sheba Medical Center, Tel Hashomer IL–52621 Tel Hashomer (Israel) Tel. +972 35302564, Fax +972 35340167, E-Mail [email protected]

Introduction

Measurement is the cornerstone of all branches of science including medicine in general and hematology in particular. For many years, counting cells and identifying them by microscopic inspection have determined the number of normal or abnormal cells in the bone marrow (BM). These traditional methods have very limited sensitivity but recently developed, more sensitive techniques are providing new information of significant value [1]. At diagnosis patients with acute leukemia may have a total of approximately 1012 malignant cells. The disease is considered to be in complete remission (CR) when fewer than 5% of the cells in BM samples are blasts. In quantitative terms: a 12 log leukemic cell kill is induced leaving a total leukemic burden of 1010 neoplastic cells. From that point until overt clinical relapse the level of leukemic cells in the body is largely unknown, resulting in clinical management strategies that do not discriminate among patients by their levels of residual disease. Thus, patients with 1010 leukemic cells are treated with the same regimen as those with much lower levels or, perhaps, with no leukemia at all [2, 3]. CR rates as high as 70–80% have been reported in adult patients with acute myelogenous leukemia (AML) [4–10], but approximately 60–70% patients will eventually have a relapse due to the persistence of residual leukemic cells surviving after chemotherapy. The persistence of residual malignant cells below the threshold of conventional morphological findings is termed minimal residual disease (MRD) and may identify patients at a higher risk of relapse [4]. The final goal of detecting low numbers of residual leukemic cells is to obtain a more precise evaluation of the effectiveness of treatment in order to give information on disease prognosis, design patient-adapted post-remission therapies, assess information on the response to chemotherapy, predict impending relapses prior to clinical manifestations, make a better assessment of the quality of the stem cells harvested for autologous transplant and the efficacy of purging methods and facilitate early therapeutic interventions (i.e. donor lymphocyte infusions) following transplantation [11, 12]. The study of MRD uses modern technical methods to identify disease far below the detection threshold of routine pathology.

Methodologies for Detecting MRD in AML

If the original leukemic cell carries a molecular or antigenic marker that distinguishes it from nonleukemic cells, then all cells of the leukemic clone will exhibit the same marker. This property allows the application of sensitive new techniques that use either the polymerase chain reaction (PCR) or antibody to detect or quantify leukemic cells [1]. Sensitive methods to detect residual disease include ‘classic’ metaphase cytogenetics, cell cytometry studies and molecular genetic studies such as PCR and fluorescence in situ hybridization (FISH) detection of specific genetic targets. Each method has relative advantages, disadvantages and sensitivity for detecting MRD [13]. The morphological analysis of marrow smears, while suitable for all patients, has a low sensitivity of 5%. Conversely, the other methods including conventional cytogenetics, FISH, multiparametric flow cytometry and PCR are much more specific and sensitive [14–17]. Conventional Cytogenetics Karyotype analysis at diagnosis is essential and allows the detection of clonal abnormalities including structural and numerical changes specific to the leukemic clone. Conventional cytogenetics is applicable for 60–70% of the patients, uses a patient-specific marker but has a sensitivity of only 1–5% and is laborious. Cytogenetics remains one of the most important prognostic factors in AML and can be applied during remission to detect residual leukemia. An additional benefit of this approach is its ability to detect the acquisition of new clonal aberrations with disease progression [18]. Fluorescence in situ Hybridization FISH can increase sensitivity by screening nondividing interphase cells using chromosome-specific or locus-specific probes. The sensitivity of FISH is between 10 –1 and 10 –3 and the detection of numerical losses or gains of chromosomes is generally more sensitive than the detection of translocations [13]. FISH also allows the identification of cryptic translocations that may be clinically relevant. However, this method is applicable to only about 40–60% of the cases and its sensitivity is limited by the presence of false-positive cells due to colocalization of two signals [15–18]. Nucleic Acid Amplification Techniques The most sensitive approach for detecting MRD involves nucleic acid amplification using the PCR. The PCR must have a specific genetic lesion as the ‘finger-

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print’ of the malignancy in order for PCR-driven reactions to have the desired sensitivity and specificity. Most commonly, the genetic target providing the ‘fingerprint’ is from the unique juxtaposition of genes occurring from chromosomal translocations [13]. Detection of MRD by PCR has become an essential tool for molecular monitoring of AML. Currently, specific translocation markers are available for 40–50% of AML patients. Expression markers may widen this spectrum to 70–90%. PCR is highly sensitive (up to 1 ! 10 –6) but technically demanding, susceptible to false-positives due to contamination and applicable only to those with specific chromosomal aberrations (about 30–40% of cases). The common finding for all molecular subtypes in AML is that the transcript levels are at least 1 log higher in BM than in peripheral blood (PB) [14–17, 19]. Initial studies of MRD in AML relied on the reversetranscriptase polymerase chain reaction (RT-PCR) assay being either positive or negative. The application of highly sensitive RT-PCR methods to detect MRD in leukemias has revealed the persistence of low levels of leukemic cells in many patients in long-term remission and therefore considered ‘cured’ of their disease. While qualitative RT-PCR methods have been clinically useful in monitoring MRD in specific leukemias, in certain types of leukemia the qualitative detection of the respective fusion gene transcripts did not distinguish patients in durable remission from those at high risk of relapse. For this reason quantifying target genes as a measure of residual leukemia provides a better means for monitoring MRD [18]. Quantification of the level of transcripts of a target gene can be carried out either by the end point (competitive) RT-PCR or the cycle-cycle (real-time) techniques. Competitive RT-PCR assays are labor intensive and use manual protocols that may require up to 48 h to produce results. They require exponential amplification for precise quantifications and the final amount of the PCR product is very sensitive to slight variations in the reaction components that need to be rigorously controlled. To overcome these shortcomings, real-time RT-PCR techniques for quantification of target sequences have been developed. The principle behind this technique is to estimate the level of PCR products as they accumulate rather than estimating the level of the final products. At present three main types of real-time RT-PCR techniques are available. In the SYBR Green I technique, fluorescence is increased upon binding to double-stranded DNA. During the extension phase, more and more SYBR Green I binds to the PCR product, resulting in an increased fluorescence. Consequently, during each subsequent PCR cycle

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more fluorescence signal will be detected. In the hydrolysis probe technique, the hydrolysis probe is conjugated with a quencher fluorochrome, which absorbs the fluorescence of the reporter fluorochrome as long as the probe is intact. As Taq polymerase has also the activity of endonuclease upon amplification of the target sequence, the hydrolysis probe is displaced and subsequently hydrolyzed by the Taq polymerase. This displaces the reporter from the quencher fluorochrome and thus the fluorescence of the reporter fluorochrome becomes detectable. Consequently, during each subsequent PCR cycle more fluorescence signal will be detected. Reactions are characterized by the number of cycles after which the fluorescent signal is first detected (due to the accumulation of enough reporter molecules released) – CT. In the third method, the hybridization probes technique, one probe is labeled with a donor fluorochrome at the 3) end and a second probe is labeled with an acceptor fluorochrome. When the two fluorochromes are in close vicinity, the emitted light of the donor fluorochrome will excite the acceptor fluorochrome. This results in the emission of fluorescence, which subsequently can be detected. Consequently, during each subsequent PCR cycle more fluorescence signal will be detected [19, 20]. Although a recent comparison of real-time RT-PCR with competitive RT-PCR using samples from t(8;21) AML patients showed that these two methodologies are comparable for sensitivity, linearity and reproducibility, the former method of analysis appears to offer technical advantages by providing absolute quantification of the target sequence, expanding the dynamic range of quantification to over six orders of magnitude, eliminating the post-PCR processing, and reducing labor and carry-over contamination [21]. Real-time RT-PCR is now as sensitive as conventional two-step PCR and could improve as well as facilitate clinical decision making. This method has been applied to a variety of molecular markers. For most markers, a lack of decline of transcript levels by less than 2 logs after chemotherapy has been established as a poor prognostic sign. Moreover, increases in transcript levels are almost invariably associated with relapse [14–17, 19]. Multiparametric Flow Cytometry Normal cells display a variety of cell surface antigens in a strictly defined sequence according to cell type and differentiation status. The malignant process often disturbs this programmed expression and the aberrant expression of cell surface antigens can thus be used to distinguish malignant cells from normal cells. Cells labeled with a panel of fluorescent antibodies can be detected and

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quantified by a florescence-activated cell sorter (FACS). The patterns of aberrant expression can ‘fingerprint’ a malignant clone with a sensitivity of 10 –1 to 10 –4. Flow cytometry is potentially quite rapid. However, to fully optimize its sensitivity for detecting the leukemic clones, combinations of several antibodies are needed to define the aberrant antigen expression, requiring considerable technical expertise [13]. In 2- or 3-color immunofluorescence analysis the leukemic cells demonstrate multiple differences from their presumed physiological counterparts due to different light scattering properties and an aberrant or asynchronous antigen expression. Using this method it is possible to define an aberrant leukemic immunophenotype in nearly 70–80% of all cases of AML. The most frequent abnormal immunophenotype found is an asynchronous myeloid antigen expression, and the most frequent lymphoid markers coexpressed with myeloid antigens are T cell-associated antigens, especially CD7 [12, 14–17, 22, 23].

Detection of MRD in AML with Genetic Abnormalities by Nucleic Acid Amplification Techniques

The clinical prognostic value of genetic markers in AML has qualitative and quantitative aspects: predictive value at diagnosis by definition of cytogenetic/molecular risk groups and predictive value of remission/relapse linked to the kinetics of molecular markers during the course of the disease. Currently, 40–50% of AML patients have a specific marker detectable by PCR techniques. The major specific molecular markers with an expected frequency of 11% in de novo unselected AML patients are involvement of the MLL gene on chromosome band 11q23 with about 50 partners (10–20%) [19, 24, 25] followed by the core binding factor (CBF) leukemias with AML1/ETO (7–14%) and CBFß/MYH11 (3–10%) rearrangements [19, 26]. PML/RAR· and variant rearrangements (PLZF) reviewed elsewhere in this issue are confined to acute promyelocytic leukemia and occur in 5–10% of de novo AML [19, 27, 28]. Internal tandem duplications (ITD) and mutations of FLT3 also reviewed elsewhere in this issue are present in 10–30% of de novo AML [19, 29–31]. Partial tandem duplications (PTD) of MLL were detected in 3.4% unselected patients [19, 32]. In addition, a number of other PCR-amplifiable translocations with an occurrence of less than 1% such as BCR/ABL, DEK/CAN, or TLS/ERG, MOZ/CBF and translocations involving EVI 1 have been recognized [17, 19, 24, 25].

Detection of Minimal Residual Disease in Acute Myelogenous Leukemia

Abnormal expressions of several other genes not involved in translocations include an increased transcript number of the Wilms tumor gene (WT1) reviewed elsewhere in this issue, the PRAME gene and Ig/TCR rearrangements [19, 33–35]. The kinetics of disappearance of molecular markers in AML are influenced by the various therapeutic regimens but are mainly different between the various types; for example, while molecular remission is achieved within the first 6 months in patients with acute promyelocytic leukemia, PCR markers may persist for several years in apparently cured patients with CBF leukemias [19, 26, 36, 37]. CBF Leukemias AML with translocation t(8;21)(q22;q22) and pericentric inversion of chromosome 16, inv(16)(p13q22) share some unique phenotypic and clinical properties. Moreover, both leukemia types are usually included in the cytogenetic group of adult AML with a favorable prognosis with more than 50% of patients obtaining a long-term CR. Both types of disease are referred to collectively as CBF leukemias resulting in the rearrangement, respectively, of alpha (CBF· or AML1) or beta (CBFß) subunits of the same transcription factor CBF that plays a key role in hematopoiesis. In particular, genomic rearrangements in t(8;21) and inv(16) leukemia fuse CBF· with the ETO gene (or MTG) encoding a transcription factor and CBFß with the smooth muscle myosin heavy chain gene MYH11, respectively [38, 39]. In normal hematopoietic cells, CBF·/ß regulate the transcription of a number of genes important for hematopoiesis, including IL-1, IL-3, GM-CSF, the CSF1 receptor, myeloperoxidase, BCL2, the immunoglobulin heavychain and T cell receptor genes, and the multidrug resistance gene, MDR 1 that encodes P-glycoprotein. The AML-associated t(8;21), inv(16) and related t(16;16) and the complex t(3;21)(21q22) associated with AML and myelodysplastic syndrome disrupt the transcriptional activation function of CBF·/ß and block the induction of the expression of critical target genes [40]. The resulting hybrid mRNA can be detected by the RT-PCR assay and exploited for both diagnostic and follow-up purposes. In t(8;21)(q22q22) or inv(16)(p13q22), failure to detect submicroscopic (cryptic) rearrangements of the involvedgenes leads to false-negative results. Sensitive molecular methodologies such as RT-PCR have been successfully used to detect cryptic CBF abnormalities in diagnostic samples of AML patients with karyotypes that are otherwise negative for t(8;21)(q22;q22) or inv(16)(13q22) [41, 42].

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t(8;21)(q22;q22) The t(8;21)(q22;q22) translocation was first described by Rowley [43] in 1973. The fusion gene AML1/ETO is transcribed from the derivative chromosome 8 and results in a chimeric AML1/ETO protein. The AML1/ETO fusion gene most likely is involved in the transformation of hematopoietic cells by blocking the AML1-dependent transcription of genes critical to normal hematopoiesis. The t(8;21)(q22;q22) occurs in approximately 10% of adult AML patients. These patients are generally of a younger age (mostly !60 years). In most cases, the t(8;21) is accompanied by an FAB M2 morphology or less often an M1 subtype. The immunophenotype of patients with t(8;21) is characterized by an overexpression of the CD34 antigen, in some cases with an aberrant coexpression of the CD19 antigen. Also the CD56 antigen can be expressed by t(8;21)-positive AML blasts. AML with t(8;21) is associated with a favorable response to chemotherapy with both a high remission rate and long-term disease-free survival [44, 45]. However, despite the relatively good prognosis relapse remains a major problem, especially in the first 2 years of remission. Standard cytogenetic analysis is the most commonly used method to detect t(8;21). The t(8;21) can also be identified by FISH or by RT-PCR. Since the breakpoints for the t(8;21) cluster within one single intron of the AML1 gene, an identical AML1/ETO fusion transcript can be detected in all patients at initial diagnosis when specific oligonucleotide primers for AML1 upstream and ETO downstream of the breakpoint are used for PCR amplification. Several studies found AML1/ETO transcripts in patients with no cytogenetic evidence of this aberration. These findings indicate that the sensitivity for the detection of a t(8;21)can be increased by molecular screening for all AML patients [36]. Surprisingly, detection of the AML1/ETO fusion transcript by nested RT-PCR has been reported in patients in long-term CR treated with either consolidation chemotherapy or autologous stem cell transplantation. Similarly, these findings suggest that the complete eradication of AML1/ETO and RT-PCR negativity are not invariably needed to achieve cure from AML with t(8;21). Studies by qualitative RT-PCR for detecting MRD in t(8;21)-positive AML have yielded discrepant results. Using sensitive RT-PCR assays (detecting 1 leukemia cell in 105 to 106 normal cells), several groups have shown that following chemotherapy, AML1/ETO transcripts can be detected in most patients in long-term remission [46–49]. Moreover, persistence of residual disease has also been

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described after autologous BM transplantation. Persistence of the hybrid gene has also been detected in BM or PB samples from patients undergoing allogeneic stem cell transplantation, despite a variety of conditioning regimens and occurrence of acute and/or chronic graft-versushost disease [50–55]. The biological significance of this finding is uncertain, although it suggests that the t(8;21) is only one step in the multistep pathogenesis of AML. However, there have also been reports of the absence of AML1/ETO transcripts in a significant number of long-term remitters. In prospectively studied patients, negative PCR results correlated with an absence of relapse. Furthermore, early negative results with one-step RT-PCR technique, i.e. before consolidation chemotherapy seemed to portend a good prognosis, suggest that RT-PCR analysis at this time point could be clinically useful. For patients who test consistently positive during remission, qualitative RT-PCR assays for AML1/ETO transcripts are of limited value in monitoring MRD. Consequently, quantitative RT-PCR methods have been developed and applied successfully to monitor MRD in remission patients. Tobal et al. [56] and Tobal and Yin [57], using a sensitive competitive RT-PCR method to quantify AML1/ETO transcripts, reported the results of sequential monitoring of MRD in BM and PB samples of patients with t(8;21)-positive AML. In the 21 patients studied, they showed that in general a successful course of induction chemotherapy produced a reduction of 2–3 logs in the level of AML1/ETO transcripts followed by a further 2–3 log after consolidation intensification treatment. Their data showed that serial quantification of AML1/ETO transcripts could identify patients in durable remission and also allowed the establishment of critical MRD thresholds that were predictive of imminent hematological relapse. They found that patients in durable remission had levels of AML1/ETO transcripts !1 ! 103 molecules/mg RNA in BM and !1 ! 102 molecules/mg RNA in PB. They identified threshold levels, termed relapse-increased risk threshold of 1103 to !0.71 ! 105 molecules/mg of RNA in BM and of 1102 to !2.27 ! 103 molecules/mg of RNA in PB. Levels above the upper limit of these thresholds were indicative of hematological relapse within 3–6 months. Data published to date from real-time RT-PCR quantification of MRD in t(8;21)-positive AML are limited to small numbers of patients and are generally consistent with the findings obtained with competitive assays [18, 31, 58]. The decrease of transcripts after induction therapy varies between 2 and 4 logs [25, 28, 36, 50, 59]. There are indications that a slow decrease (!2 logs) in the BM

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may be associated with relapse [60]. A 1–3 log difference between BM and PB was observed in complete hematological remission [19, 28]. Viehmann et al. [61] have recently analyzed 15 AML1/ETO-positive children using real-time RT-PCR enrolled in the multicenter therapy trial AML-BFM 98. A linear decrease of 2–4 logs could be seen from the start of therapy until the beginning of consolidation in most of the children. Most cases remained positive at a low level during the consolidation treatments. Three of 4 children who relapsed showed an increase of the AML1/ETO fusion transcript 6, 9, and 11 weeks before relapse. Recently, Schnittger et al. [62] by using the real-time RT-PCR method evaluated the prognostic significance of quantitative fusion transcripts of PML/RAR·, AML1/ ETO and CBFß/MYH11 in 142 patients. They established a new prognostic score based on the combination of transcript levels at two checkpoints: the 75 percentile of the expression ratio at diagnosis and the median expression ratio after consolidation therapy. This score differentiated between a group with 100% event-free survival and a significantly worse group in each of the three AML categories. Patients at high risk of treatment failure had high levels of fusion gene expression at diagnosis and/or less than 3 logs of tumor reduction during the first 3–4 months of therapy. The demonstration that patients often continue to have MRD for years while in morphological remission suggests that the way in which patients are ‘cured’ is not by elimination of all disease, but rather by debulking to a critical level. The coexistence of MRD in a ‘cured’ patient might be due to a preleukemic clone that possesses the molecular signature of the disease, but lacks other genetic ‘hits’ needed for progression to frank leukemia. Alternatively, epigenetic factors, such as cytokine exposure released from accessory cells, may promote or inhibit the outgrowth of dormant leukemia clones. Lastly, perhaps the immune system controls the outgrowth of the malignancy. Temporary immunosuppression might allow the escape and growth of the malignant clone to a level of a ‘critical mass’ that is unmanageable by the immune system [49, 63]. Another explanation is that the residual cells expressing the hybrid transcript are no longer clonogenic, i.e. they have lost the capacity to reexpand the leukemic clone [64]. Varella-Garcia et al. [65] characterized the cells that express AML1/ETO transcripts in remission marrow using cell sorting and FISH with a set of probes from each side of both translocation breakpoints. Remission samples from 29 patients were studied. An analysis of un-

sorted marrow samples revealed that residual t(8;21) cells could be detected at levels of 0.05–0.19% in approximately 60% of remission samples, and that levels of 0.6% and greater were associated with relapse. In a patient who had serial remission samples available for study, the combination of cell sorting and FISH demonstrated that the t(8;21) was present in CD34+ cells in early remission, but in CD34– cells in later remission. This observation suggests that t(8;21) cells in patients in long-term remission undergo differentiation, in contrast to t(8;21) cells in leukemic and early remission samples, which are CD34+. The findings of Varella-Garcia et al. are consistent with those of Miyamoto et al. [66, 67], who demonstrated that AML1/ETO transcripts were present in a fraction of stem cells, monocytes, and B cells in remission marrow, and in a fraction of B cells in leukemic marrow, but not in T cells. AML1/ETO transcripts were also demonstrated in a fraction of colony-forming cells of erythroid, granulocyte-macrophage, and/or megakaryocytic lineages in both leukemic and remission marrow. These data strongly suggest that the acquisition of the t(8;21) occurs at the level of stem cells capable of differentiating into B cells as well as all myeloid lineages, and that a fraction of the AML1/ ETO-expressing stem cells undergo additional oncogenic events that ultimately lead to transformation into AML [66, 67]. In conclusion, the persistence of AML1/ETO transcripts in many studies suggests that at least in patients with t(8;21) AML, a clinical cure of the leukemia may not require the eradication of all leukemic cells. In this context, it is possible that the development of overt leukemia requires additional mutations to express the transformed phenotype in t(8;21) AML.

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inv(16)(p13;q22) and t(16;16)(p13;q22) AML-M4Eo according to the FAB classification is often associated with rearrangements of chromosome 16, mostly involving 16p13 and 16q22, leading either to pericentric inversion, inv(16)(p13q22) or, less commonly, to a translocation between the homologous chromosomes, t(16;16)(p13q22). The pericentric inversion inv(16;16) (p13;q22) is observed in approximately 10% of de novo AML, mostly classified as FAB M4Eo subtype [68, 69]. Several studies have suggested that the presence of inv(16) is an important prognostic indicator of AML patients, as it is associated with a relatively favorable outcome: 90% of patients obtain CR, with nearly 50% being disease-free survivors at 5 years. Despite the favorable response to chemotherapy in this group, many patients relapse [69].

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The inv(16) may be difficult to detect with conventional cytogenetics, particularly when poor quality metaphases are obtained from leukemic cells. Moreover, conventional cytogenetics cannot detect cryptic deletion sequences centromeric to the p-arm breakpoint which has been described in a subset of inv(16) AML patients, accounting for 18–33% of cases. The RT-PCR is able to detect CBFß/MYH11 fusion transcripts in the majority of inv(16)-positive cases at diagnosis. However, some cases are RT-PCR negative. This finding might be caused by the diversity in the amount of inv(16) transcripts together with the presence of alternatively spliced transcripts or poor quality RNA [69]. Several reports suggest that the CBFß/MYH11 fusion mRNA can often be detected in patients in long-term remission after chemotherapy [70]. Qualitative RT-PCR for the detection of CBFß/MYH11 transcripts during remission has yielded conflicting results and the monitoring of MRD with such endpoint assays in this subset of AML is of limited value. Results of qualitative RT-PCR confirm that virtually every patient is PCR positive in early post-remission BM samples. Nevertheless, a gradual decline of MRD occurs between 4 and 12 months after CR achievement with PCR negativity achieved with slow kinetics after 6–18 months in patients remaining in long-term CR, while persisting positivity indicates relapse. This indicates that even without further chemotherapy, a progressive clearing of residual leukemic cells occurs. This supports the possibility that biological mechanisms might contribute to the eradication of residual leukemic cells, even in a ‘nontransplant’ setting. Long-term survivors in complete cytogenetic remission usually ultimately tested PCR negative, suggesting that ‘cure’ of inv(16) leukemia is associated with PCR negativity. This observation is apparently in sharp contrast with the findings reported in t(8;21) leukemia, where long-term complete CR is usually associated with persistence of MRD [19, 38, 68]. Preliminary data suggest that quantitative RT-PCR may be more useful. A limited number of studies have so far addressed the quantitation of CBFß/MYH11 transcripts in inv(16) leukemia at diagnosis and during followup. Both competitive and real-time amplification technologies were employed in small patient series analyzed retrospectively. Guerrasio et al. [38], in their study of 16 patients, observed a dramatic decrease (2–3 logs) of the leukemic transcript level after induction/consolidation treatment with further reduction after autologous or allogeneic transplant. Most patients (14/16) achieved a reduction in CBFß/MYH11 fusion transcripts to less than 10

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copies/104 ABL copies by using the real-time method by the end of the planned treatment in at least one BM sample. A threshold level of CBFß/MYH11 transcripts could be established by distinguishing between patients who relapsed and those who remained in CR. Patients who never relapsed fell below the level of one copy of leukemic transcripts/104 ABL copies (i.e. they became PCR negative). While the fusion gene transcript level at diagnosis was not predictive of the subsequent outcome, the level at later time points identified patients at high and low risk of relapse (threshold values: 100 copies/104 ABL copies after induction and 10 copies/104 ABL after consolidation therapy). Furthermore, negative PCR status, using the qualitative assay, was strongly associated with prolonged CR in keeping with the results obtained by real-time amplification. Buonamici et al. [71] showed that remission samples with CBFß/MYH11:ABL ratios above 0.25% corresponded with a high risk of relapse whereas ratios below 0.12% were associated with durable remission. These findings are in agreement with other studies where the degree of reduction of leukemic transcripts was predictive of clinical outcome [38]. According to quantitative RT-PCR studies, a more than 2 log decline after induction therapy was found in patients achieving long-term CR [38, 72, 73]. Patients with a less than 2 log early reduction are at high risk of relapse [73]. However, relapses also occur in patients with sufficient initial molecular responses and relapses from PCR negativity have also been observed [17, 21, 38]. Nevertheless, there is strong evidence that high copy numbers after the end of consolidation treatment are associated with hematological relapse. Increasing transcript levels predict relapse 2 months before hematological relapse in the BM [19, 21, 73, 74]. In conclusion, data suggests that RT-PCR positivity in rearrangements of chromosome 16 involving 16p13 and 16q22 gradually decreases over time, but most patients remain positive until their 1st year in CR, at least in their BM. PCR positivity at this stage may be compatible with long-term remission or cure. Therefore, qualitative RTPCR results seem to have a limited predictive value. However, the degree of reduction of leukemic transcripts over time is predictive of the clinical outcome. This represents an important achievement from the clinical point of view as it may allow treatment modification during CR according to risk group. t(15;17)(q22;q21) This has been reviewed elsewhere in this issue.

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11q23 and Partners Structural lesions involving chromosome 11 band q23 are among the most common cytogenetic abnormalities associated with hematopoietic malignancies [75]. The gene on chromosome 11 that is targeted by 11q23 rearrangements is referred to as MLL for ‘mixed lineage leukemia’. The MLL gene is also known as ALL1, HTRX1 or HRX [76, 77]. Translocations involving the MLL gene are not associated with a specific lineage or subtype of leukemia and have been described in AML, acute lymphoblastic leukemia (ALL), biphenotypic or mixed-lineage leukemia and even in cases of lymphomas. The diversity of phenotypes might be caused by the great variety of chromosomal fusion partners [78]. Translocations involving 11q23 with up to 50 different partners resulting in MLL fusion genes have been described in ALL, as well as in 5–10% of AML [75, 79, 80]. The most common translocations are t(4;11)(q21;q23), t(9;11)(q22;q23) and t(11;19)(q23;p13) [75, 79, 80]. Regardless of their association with other high-risk factors at presentation, 11q23 rearrangements are strongly predictive of a poor clinical outcome. Previous studies have shown that the outcome of patients with AML with t(9;11) involving the MLL/ AF9 fusion gene is more favorable than that of patients with other 11q23 abnormalities. In contrast, t(6;11) (MLL/AF6 chimeric gene) translocations have a low molecular remission and high relapse rate [80]. Few studies have been performed on MRD in AML patients with MLL rearrangements probably due to the high number of possible translocations. Recently, Scholl et al. [81] analyzed samples from 8 patients with t(9;11)-positive AML by a real-time RTPCR assay that they established for the identification of MRD by quantification of the most frequent fusion transcripts resulting from t(9;11)(p22;q23). The sensitivity of the assay was comparable to that of qualitative singleround RT-PCR. Normalized copy numbers were positive at diagnosis and relapse. Samples from only 2 of 7 patients collected at the time of CR became negative. The 5 cases in CR still had positive copy numbers. In addition to rearrangements involving various partner genes, MLL rearrangements can be found within MLL as the result from PTD usually spanning exons 2 through 6 or exons 2 through 8 [82, 83]. Translocations of the MLL gene and PTD seem to be mutually exclusive. The clinical outcome in this AML subgroup is also unfavorable [32, 79, 84, 85]. While MLL rearrangement assessment by molecular methods is difficult due to the high number of partner genes, flow cytometry can provide additional data on

MLL status, establishing the abnormal immunophenotype that will be used in the MRD study [75].

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Rare Translocations Some rare genotypic targets in AML can be used for the detection of residual disease. Data on MRD monitoring by RT-PCR methods in these rare cases are scanty and mostly confined to case reports or small numbers of patients [86, 87]. Of particular interest is AML with t(6;9) that is associated with basophilia and carries a poor prognosis. The t(6;9) gives rise to the DEK/CAN fusion transcript, which has also been used to monitor residual disease [87, 88]. Other rare genotypic targets are inv3(q21;q23), t(3;3)(q21;q26), t(3;21)(q26;q22), t(9;22) (q24;q11), t(8;16)(p11;p13) and t(16;21)(p11;q22) with the molecular targets EVI 1, AML 1/EVI 1, BCR/ABL, MOZ/CBP and TLS/FUS-EG, respectively [18]. FLT-3 Mutations and ITD These have been reviewed elsewhere in this issue. Expression Markers An abnormal expression of several genes not involved in translocations has been noticed in AML. PRAME PRAME (preferentially expressed antigen of melanoma) has been identified to code for a tumor antigen consisting of 509 amino acids recognized by cytotoxic T cells that was originally established from a melanoma patient [89]. It has been reported that this gene is expressed in a variety of cancer cells including leukemic cells using semiquantitative RT-PCR [90–93]. Matsushita et al. [34] screened 98 Japanese patients with leukemia and lymphoma for the expression of the PRAME gene using semiquantitative RT-PCR. Forty-two percent showed high levels of PRAME expression. Eight of these patients were then monitored using real-time RTPCR for a period of 10–37 months. Significant reductions in the PRAME expression were observed in all patients after chemotherapy. An increased expression was detected in the 2 patients who relapsed, 1 of whom before cytological diagnosis. Quantitative monitoring of the PRAME gene using the real-time RT-PCR method may be useful for detecting MRD and to predict subsequent relapse, especially in patients without known genetic markers. Wilms Tumor Gene (WT1) This has been reviewed elsewhere in this issue.

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New Expression Markers Since the development of the microarray methodology it has become possible to study the gene expression pattern of a large amount of genes relevant to tumor development. Larramendy et al. [94] analyzed the gene expression profile of 15 patients with AML at diagnosis using the cDNA microarray analysis to discover the pattern of under- and overexpressed genes. Their results revealed 50 differentially expressed genes in at least 3 out of 15 patients. Twenty-two genes were upregulated and 28 genes were downregulated. Almost all genes tested by realtime RT-PCR showed the same expression profiles. Among the overexpressed genes there were: FGFR1, MYC, NPM1, DEK, BCL2, HOXA4 and CSF1R – some of them previously associated with chromosomal translocations. Schoch et al. [95] performed microarray analyses on a cohort of leukemia samples from 37 AML patients with the three distinct subtypes of AML: t(8;21), inv(16) and t(15;17). They demonstrated an unequivocal association between disease-specific translocations and distinct gene expression profile. For each of the three subtypes of AML patterns of gene expression were identified that were homogenous within all samples of the respective subgroup and clearly differed between the three subgroups. By using two different strategies for microarray data analyses, this study revealed a unique correlation between AML-specific cytogenetic aberrations and gene expression profiles. Levy-Nissenbaum et al. [96] have recently shown by using a gene expression array that PYST2, a member of the PYST subfamily of dual-specificity phosphatases that play an important role in various signaling pathways, is highly expressed in leukocytes derived from AML patients. The gene expression array ClonTechs was used to identify genes that were differentially expressed in ‘leukemic-phase leukocytes’ derived from untreated AML patients and in ‘remission-phase leukocytes’ obtained from the same patients following induction of remission. Focusing on genes that were more highly expressed by the leukemic- than by the remission-phase leukocytes in 3 AML patients, they showed that the expression of PYST2 was 37-fold higher in the leukemic than in the remission phase. These results were verified in another 12 patients and in 8 leukemic cell lines [97, 98]. In conclusion, by using gene expression profiling, overexpression of various genes could be identified in AML patients. These genes probably have a role in the development and progression of AML. Moreover, as already shown for WT1 and PYST2, the assessment of levels of

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expression of these genes may be used for MRD evaluation in AML patients during the course of treatment.

Detection of MRD in AML by Multiparametric Flow Cytometry

Campana and Coustan-Smith [99] also discuss this method for MRD detection in AML in this issue. Immunophenotypic analysis of leukemic cells using multiparametric flow cytometry is a convenient method for the investigation of MRD due to its speed and relative simplicity [2, 12, 96, 100–106]. Immunophenotyping has proved to be an attractive approach for MRD investigation particularly in ALL. Although reported data in the literature are still scanty for AML patients, several authors have shown that this technique could be used for prognostic evaluation. The advantages of this method are its rather high sensitivity, 10 –3 to 10 –5, depending on the type of antigen used, and its universal applicability since AML cells have usually leukemia-specific immunophenotypes. In order to use immunophenotypic investigation for MRD, leukemic cells must display aberrant antigenic profiles that allow their distinction from normal hematopoietic cells even when present in very low frequencies and such antigenic characteristics should remain stable during the course of the disease. The majority of AML patients (about 80%) display leukemia-associated phenotypes that can be used for follow-up. These phenotypes result from cross-lineage antigen expression, antigen overexpression, ectopic antigen coexpression, abnormal light scatter pattern and asynchronous antigen expression. Flow cytometry immunophenotypic detection of MRD has two major pitfalls: the absence of well-characterized leukemia-specific antigens and the existence of phenotypic changes at relapse that could induce false-negative results. As a result, this method is less accurate in AML than in ALL as regards both false-positive and falsenegative results [22, 107]. There are conflicting data regarding the exact time point at which MRD levels can differentiate between high- and low-risk patients, i.e. at which time point during treatment immunophenotypic MRD evaluation has prognostic significance. San Miguel et al. [108] using multiparametric flow cytometry evaluated the level of MRD in the first CR BM. In 126 patients with AML who displayed aberrant phenotypes at diagnosis, four different risk categories were identified: very low risk with fewer than 10 –4 cells, low risk with 10 –4 to 10 –3 cells, intermediate risk with fewer than

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10 –3 to 10 –2 cells, and high-risk group with more than 10 –2 residual aberrant cells. The MRD level influenced relapse-free survival and overall survival and was the most powerful independent prognostic factor followed by cytogenetics and number of cycles to achieve CR. Venditti et al. [109] have used flow cytometry to quantify MRD in 63 patients with AML. No significant correlation was found between the level of MRD after induction and disease outcome. After consolidation, a threshold of 3.5 ! 10 –4 residual leukemic cells divided the 57 evaluable patients into two distinct groups above and below this threshold with a relapse rate of 81 and 27%, respectively. Munoz et al. [75] used the identification of abnormal immunophenotypes for the detection of MRD in AML patients with MLL rearrangements including translocations and PTD involving the MLL gene. The patients with MLL involvement usually expressed myeloid and monocytic antigens including CD33, CD13, CD117, CD64, HLA-DR and CD34. All patients showed at least one aberrant phenotype at diagnosis. The high incidence of aberrant phenotypes in these patients could be applied for MRD study. The most frequently encountered immunologic aberration that made it possible to detect residual leukemic cells was the coexpression of CD15 and CD117 followed by the coexpression of immature antigens such as CD34 or CD117 with mature monocytic and granulocytic markers (CD64 or CD11b). MRD studies were carried out in BM samples from patients in CR after the induction, intensification and stem cell transplants. All patients who eventually relapsed were MRD positive according to an immunophenotype analysis. Conversely, only 1 patient who was MRD positive remained in CR. According to Munoz et al. [75], flow cytometry could be applied to MRD detection in most MLL+ patients given the high incidence of aberrant phenotypes. This technique is especially suitable for patients with PTD, in whom RT-PCR methods cannot be easily employed. Another antigen amenable for flow cytometry estimation uses c-kit monoclonal antibodies that identify an extracellular epitope of the protooncogene and class III receptor tyrosine kinases c-kit, which has been shown to be the receptor for the stem cell factor or Steel factor and clustered as CD117. This antigen is expressed by between 1 and 4% of normal human BM mononuclear cells, including multipotent, erythroid and myeloid-committed progenitor cells. Preliminary reports have shown that the CD117 antigen is coexpressed in 40–60% of CD34+ BM cells and in 50–85% of the CD34+/CD38+ cells. Scolnik

et al. [110] have shown that CD117 is expressed at a much higher level in myeloblasts than in normal myeloid precursors. The reported incidence of CD117+ AML ranges between 23 and 87%. Therefore, a quantitative estimation of CD117 together with CD34 might be a useful combination to detect MRD in AML, because both markers are positive and highly expressed in a substantial proportion of AML cases while they are only present in a minority of BM normal progenitor cells at a much lower density [111]. The combination of CD117 and other myeloid-associated antigens such as CD11b and CD15 may also be used for monitoring MRD in AML patients because it defines a subpopulation of myeloid cells that are either absent or present in very small numbers in normal human BM [111–113].

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Conclusion

Studies of MRD using molecular as well as flow cytometry approaches have shown that monitoring of residual disease may not only help to assess the efficacy of treatment but also to enable the identification of risk groups (see table 1). At diagnosis karyotype analysis is essential but it is not always sufficient. Since molecular screening at diagnosis for the most common fusion transcripts increases detection rate, AML patients should ideally be screened at diagnosis for the relatively common transcripts. AML patients with molecular target genes should be followed by RT-PCR. Since there are conflicting data regarding the persistence of AML/ETO and CBFß/ MYH11 transcripts in long-term remitters of t(8;21)- and inv(16)-positive AML patients, respectively, serial quantification of MRD in these patients, preferably with realtime RT-PCR, is recommended as it can provide prognostic information on the response to treatment, identify patients who will achieve durable remission and predict clinical relapse. For patients without a specific molecular target, multiparametric flow cytometry analysis may prove to be useful. Although phenotypic switch at relapse is common, the use of a large number of multiple antibody panels might overcome this problem. Should we introduce the MRD data into daily clinical practice and decide on treatment options based on the MRD measurements in AML? The answer is, cautiously, yes. It is now quite obvious that a less than 2 log reduction of transcript after induction and particularly the change of PCR from negativity to positivity are highly predictive of

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Table 1. MRD detection in AML

Leukemia type Core binding factor AML

t(8;21)(q22;q22)

inv(16)(p13;q22)/ t(16;16)(p13;q22)

APL

Sensitivity

Increased relapse risk

RT-PCR

1 step: 10 –2–10 –3 nested: 10 –5–10 –6

PCR negativity not necessary for durable remission

Q-RT-PCR

10 –4–10 –5

Less than 3 log reduction in transcript during early months of therapy; slow decrease; critical levels of transcript in durable remission

RT-PCR

1 step: 10 –2–10 –3 nested: 10 –5–10 –6

PCR negativity not necessarily achieved until 1 year in CR

Q-RT-PCR

10 –4–10 –5

Less than 2 log reduction after induction

Flow cytometry

10 –1–10 –4

Q-RT-PCR

10 –4–10 –5

t(15;17)(q22;q21)

AML

11q23 and partners

AML

FLT-3 mutations and ITD

AML

MRD method

PRAME

see Reiter et al. [114] Residual leukemic cells detected by flow cytometry after induction and/or intensification

see Schnittger et al. [115]

WT-1

Increasing levels

see Cilloni and Saglio [116]

New expression markers

Microarray analysis

–

Increased expression during and after treatment

Q-RT-PCR = Quantitative reverse transcription polymerase chain reaction.

relapse. The existing data are most convincing for acute promyelocytic leukemia and for the CBF leukemias. Many other issues regarding MRD in AML still remain to be resolved. These include the kinetics of transcript reduction during the course of therapy and, further, the best source of the samples for MRD (i.e. PB vs. BM), the

optimal frequency and timing of MRD monitoring and most importantly what is the clinical role of MRD information, i.e. could it provide the biological basis for therapeutic decisions that would improve the outcome of AML patients.

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Review Acta Haematol 2004;112:55–67 DOI: 10.1159/000077560

Pathogenesis, Diagnosis and Monitoring of Residual Disease in Acute Promyelocytic Leukaemia Andreas Reiter a Eva Lengfelder a David Grimwade b, c a III.

Medizinische Universitätsklinik, Klinikum Mannheim, Fakultät für Klinische Medizin Mannheim der Universität Heidelberg, Mannheim, Germany; b Department of Medical and Molecular Genetics, Guy’s, King’s and St. Thomas’ School of Medicine and c Department of Haematology, University College London Hospitals, London, UK

Key Words Acute promyelocytic leukaemia W Promyelocytic leukaemia W Retinoic acid receptor alpha

Abstract The clinical course of acute promyelocytic leukaemia (APL) has changed over the last 25 years from one that was fatal for the majority of patients to representing one of the most curable subtypes of acute myeloid leukaemia. Besides improved supportive care this has mainly been achieved through the introduction of novel targeted therapies in the form of all-trans retinoic acid (ATRA) and arsenic trioxide that specifically address the underlying molecular lesion. APL is characterized by chromosomal rearrangements of 17q21 leading to the formation of fusion proteins involving retinoic acid receptor alpha (RARA). To date five different fusion partners of RARA have been identified, but the vast majority of cases are characterized by the presence of the t(15;17)(q22;q12–21), which involves the promyelocytic leukaemia (PML) gene. The identification of different breakpoint microclusters within RARA intron 2 suggests that sequence-associated or structural factors play a role in the formation of the t(15;17). In addition, the compari-

ABC

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son of forward and reverse genomic junctions has revealed microhomologies, deletions and/or duplications of either gene consistent with the hypothesis that the t(15;17) occurs by non-homologous recombination of DNA after processing of the double strand breaks by a dysfunctional DNA damage repair mechanism. The detection of the PML-RARA fusion gene by reverse-transcription polymerase chain reaction (RT-PCR) is routinely used for diagnosis and monitoring of minimal residual disease (MRD). In PML-RARA-positive APL about 70% of patients are expected to be cured with a combination of ATRA and anthracycline-based chemotherapy. However, relapse remains a major problem. The identification of patients at high risk of relapse and the development of risk-adapted treatment schedules are therefore clearly the most challenging tasks in the treatment of APL. Recent studies have shown that pre-emptive chemotherapy at the time of molecular relapse improves survival compared to treatment at the point of haematological relapse. Quantitative RT-PCR technology is expected to further improve the predictive value of MRD monitoring and therefore to guide therapy in order to reduce the rate of relapses and to increase rates of cure in high-risk patients. Copyright © 2004 S. Karger AG, Basel

Dr. Andreas Reiter, III. Medizinische Universitätsklinik Klinikum Mannheim gGmbH, Fakultät für Klinische Medizin der Universität Heidelberg Wiesbadener Strasse 7–11, DE–68305 Mannheim (Germany) Tel. +49 621 383 4115, Fax +49 621 383 4201 E-Mail [email protected]

Clinical Course

Acute promyelocytic leukaemia (APL; FAB M3) accounts for 10–15% of de novo cases of acute myeloid leukaemia (AML) and is defined by a unique genetic feature, the aberrant transcriptional property of the disrupted retinoic acid receptor alpha (RARA) protein leading to the particular morphological and clinical characteristics [1]. The process of leukaemic transformation is accompanied by a block in differentiation such that the marrow becomes replaced by abnormal promyelocytes [2, 3]. The disease was formerly associated with a high induction death rate resulting from severe coagulopathy, frequently inducing fatal cerebral haemorrhage. In addition, a high relapse rate was common. However, the clinical course of this leukaemia has changed dramatically from one that led to the demise of the majority of patients to one of the most favourable subsets of AML. This phenomenon reflects improvements in supportive care as well as the introduction of agents such as all-trans retinoic acid (ATRA) or more recently arsenic trioxide (ATO) that directly target the underlying molecular lesion [4, 5]. APL is uniquely sensitive to ATRA and clinical studies have shown that this agent not only ameliorates the life-threatening coagulopathy, but also in combination with chemotherapy confers a significant improvement in outcome such that approximately 70% of patients are now cured of their disease [6–17]. The best results have been achieved in trials in which ATRA is given concurrently with anthracycline-based chemotherapy during induction [9– 13, 18]. In addition, it has become evident that ATRA may also have a positive effect during maintenance therapy after successful consolidation therapy [11, 13]. However, relapse remains a serious problem and therapy for patients who relapse or are refractory to ATRA-based regimens is not standardized. One potential treatment approach involves the use of arsenic-based compounds which have recently been shown to induce impressive complete remission (CR) rates of more than 80% in relapsed patients; results regarding their efficacy as a component of first-line therapy are awaited from several ongoing trials [19–24]. In addition, monoclonal antibodies have been shown to have activity in this disease [25–27], whilst histone deacetylase (HDAC) inhibitors, demethylating agents and FLT3 inhibitors may provide novel dimensions to APL therapy in the future [28, 29]. In view of the favourable outcome following ATRA and anthracycline-based chemotherapy, autologous or allogeneic stem cell transplantation (SCT) are no longer routinely undertaken in first CR; however, such approaches should still

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be considered as the consolidation treatment of choice for the relatively small group of patients who relapse after first-line therapy [30, 31].

Molecular Basis of APL

Molecular Characterization of APL-Associated Translocations The vast majority of cases are characterized by the presence of the t(15;17)(q22;q12–21) [32, 33], which leads to a fusion of the promyelocytic leukaemia (PML) gene on chromosome band 15q22 and the retinoic acid receptor alpha (RARA) gene at 17q12–21 [34–39]. Approximately 10% of cases with morphologically typical APL have other cytogenetic abnormalities or a normal karyotype; nevertheless, in the vast majority of such cases a cryptic rearrangement of PML and RARA can be identified resulting from insertion events or more complex rearrangements [40–46]. The presence of the PML-RARA fusion protein has a dramatic effect on nuclear architecture, leading to disruption of multiprotein nuclear body structures (known as PML nuclear bodies), through interaction with wild-type PML, which is a critical structural component of this organelle. This phenomenon forms the basis of the PML immunostaining method for rapid diagnosis of PML-RARA-associated APL. In addition to the PML-RARA rearrangement, which accounts for over 95% of APL cases, four alternative fusion partners for RARA have been identified, namely PLZF, NPM, NuMA and STAT5b as a result of t(11;17) (q23;q12–21), t(5;17)(q35;q12–21), t(11;17)(q13;q21) and der(17), respectively [47–51]. The biological properties conferred by the fusion partner are not yet known in full detail but have a critical influence upon clinical and biological characteristics, particularly the sensitivity to ATRA or ATO [52, 53]. APL cases involving NPM or NuMA are sensitive to retinoids; APL with the PLZFRARA rearrangement, on the other hand, is worth noting because it is resistant to ATRA as well as ATO and is associated with a relatively poor prognosis [29, 54–56]. An important challenge has been to understand the apparent paradox as to how disruption of the steroid hormone transcription factor RARA through APL-associated translocations mediates a block in myeloid differentiation, whilst exposure to pharmacological levels of its ligand (retinoic acid, RA) in the form of ATRA mediates terminal differentiation of the leukaemic clone in the majority of cases. The mechanisms accounting for these phenomena are considered in more detail below. How-

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Fig. 1. Functional domains of the RARA

protein and structure of the X-RARA fusion proteins generated by all known reciprocal translocations involving the RARA gene at 17q12–21.

Fig. 2. Location of the PML and RARA genomic breakpoint cluster regions (bcr). Numbers indicate exons. The dark grey box shows a cluster of repetitive elements within RARA intron 2.

ever, over the last few years it has become apparent that retinoid pathways, particularly RARA, play an important role in modulating myeloid differentiation. RARA has a number of functional regions, which mediate DNA binding, interaction with co-activators and co-repressors, ligand and retinoid X receptors (see fig. 1), with the latter interaction being essential for high affinity binding to DNA response elements. Interestingly breakpoints in all molecularly defined subsets of APL occur within the same breakpoint cluster region, thereby preserving these functional domains serving to highlight the importance of deregulation of RARA in the pathogenesis of the disease and accounting for the characteristic block in differentiation that typifies this subgroup of AML [1]. Moreover, the identification of mutations in the ligand-binding domain of the PML-RARA oncoprotein in cases of ATRA-resistant APL has provided important in vivo evidence that

the clinical response to RA is mediated through the fusion protein itself [57]. The genomic breakpoints in the t(15;17) are known to occur within three different PML breakpoint cluster regions (bcr) on chromosome 15 and within RARA intron 2 on chromosome 17 (fig. 2) [1]. Rarely, breakpoints have been detected outside these regions [58, 59]. Bcr1 and bcr3 correspond to PML introns 6 and 3, respectively, whereas bcr2 is located within PML exon 6 or very occasionally within PML exon 5 [1]. Bcr1 breakpoints occur in approximately 55% of patients and result in an mRNA fusion of PML exon 6 to RARA exon 3, conventionally referred to as the L (long) isoform [reviewed in 1]. Bcr3 breakpoints occur in approximately 40% of cases and result in an mRNA fusion of PML exon 3 to RARA exon 3, referred to as the S (short) isoform [1]. Bcr2 breakpoints occur in approximately 5% of patients and usually result

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Fig. 3. Positions of the PML-RARA genomic breakpoints within RARA intron 2. A number of breakpoint microclusters were observed for patients with particular PML breakpoints [65].

in a tripartite mRNA fusion, in which part of PML exon 6 is fused to an insert sequence and then to RARA exon 3, where the insert is usually between 3 and 127 bp long and is derived from RARA intron 2 [44, 60–63]. Some cases have no inserted nucleotides and instead utilize a cryptic splice site within PML exon 6 to splice part of this exon in frame to RARA exon 3. Collectively these transcripts are referred to as the V (variable) isoform [reviewed in 64, 65]. RARA intron 2 is 16.9 kb in length and contains large stretches of Alu elements at its 5) end. Although the positions of the breakpoints in RARA intron 2 are variable between patients, three significant breakpoint microclusters have been identified within this region (fig. 3), suggesting that sequence-associated or structural factors play a role in the formation of the t(15;17) [65]. The finding of significant microclusters suggests the possibility that some features of these regions make them prone to breakage. There is no evidence that the location of a breakpoint in PML has any relationship to the location of the corresponding breakpoint in RARA. Although some sequence motifs previously implicated in illegitimate recombination were found in the microcluster regions, these associa-

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tions were not significant. The comparison of forward and reverse genomic junctions revealed microhomologies, deletions and/or duplications of either gene in all but 1 of 29 cases examined, in which a complex rearrangement with an inversion of the PML-derived sequence was found [65]. These findings are consistent with the hypothesis that the t(15;17) occurs by non-homologous recombination of DNA after processing of the double strand breaks by a dysfunctional DNA damage repair mechanism [65]. As described above, the majority of bcr2 cases are characterized by an insertion of a variable number of nucleotides between part of PML exon 5 or 6 and RARA exon 3 in the mature mRNA. The inserted nucleotides are derived from distinct regions of the RARA intron 2, with the beginning of the mRNA insert sequence corresponding precisely to the genomic translocation breakpoint. Since in bcr2 cases disruption of PML occurs within an exon, the position of the other breakpoint within the RARA gene on chromosome 17 must be restricted to regions that provide an in-frame sequence followed by a functional cryptic splice site. The finding of a significant clustering of bcr2 cases in RARA intron 2 might, therefore, indicate the presence of a particularly good cryptic

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splice site as well as, or instead of, a region that is prone to breakage [65]. Molecular Pathogenesis of APL Common features of all APL-associated fusion proteins are the retained DNA- and ligand-binding domains of RARA, fused to structurally unrelated N-terminal moieties of the fusion partners (X), which contribute additional dimerization motifs. Normal X-proteins can exhibit growth suppressor and pro-apoptotic activity; at physiological levels of RA, on the other hand, RARA binds co-activators in preference to co-repressor complexes, leading to transcriptional activation at retinoid response elements, which is implicated in normal myeloid differentiation. In contrast to wild-type RARA, at physiological levels of retinoid the X-RARA fusion proteins function as transcriptional repressors at retinoid response elements of downstream target genes through recruitment of nuclear co-repressor/HDAC complexes and DNA methyltransferases leading to the characteristic differentiation block [reviewed in 52, 53, 66–68]. APL may, therefore, reflect the combined effect of deregulation of Xdependent (disrupted growth suppressor/pro-apoptotic pathways) and RARA-dependent pathways (differentiation block). Conversely, at pharmacological levels of ATRA, ligand binding to the fusion protein leads to displacement of co-repressor complexes in favour of co-activators and transcriptional activation of downstream target genes associated with induction of differentiation. ATRA also leads to degradation of the PML-RARA oncoprotein, which may contribute to the response, and leads to a restoration of nuclear architecture with reformation of PML nuclear bodies. The differentiation process culminates in the upregulation of TRAIL and the induction of apoptosis [69]. The reversal of the differentiation block by ATRA is not seen in PLZF-RARA-positive APL, essentially because co-repressors are additionally bound to the PLZF moiety; they are not displaced in the presence of ATRA. The key role of co-repressor/HDAC complexes in the pathogenesis of APL may suggest that HDAC inhibitors could play a therapeutic role in the future. However, from experiments in transgenic mice there is growing evidence that the aberrant transcriptional properties of XRARA fusion proteins may be insufficient to mediate APL and additional oncogenic events may be required [70–73], although the lack of penetrance and long latency period could be a reflection of targeting inappropriate progenitors. Recent work has highlighted the importance of abnormalities of signal transduction pathways as cooperating events in the development of AML, e.g. activat-

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ing mutations of the FLT3 tyrosine kinase receptor, which cause a rapid-onset APL if co-expressed with the PMLRARA fusion in transgenic mice [72, 73]. The mechanisms by which ATO induces clinical remission in APL are incompletely understood; however it appears to induce a partial differentiation of the APL blasts combined with an induction of apoptosis [reviewed in 74]. The partial differentiation may be accounted for by the displacement of the HDAC/co-repressor complex from PML-RARA, together with an induction of the degradation of the fusion protein. Pro-apoptotic effects appear to be largely mediated at the level of the mitochondrion through direct action on the transition pore leading to a collapse of transmembrane potentials and triggering of the caspase cascade; however, a number of other pathways have been implicated in ATO responses [reviewed in 74]. Intriguingly, APL cases with the PLZF-RARA fusion are resistant to ATO, although the reasons for this remain obscure.

Diagnostic Tools

In addition to conventional morphology, several laboratory techniques are available for the confirmation of suspected APL including cytogenetic analysis, fluorescence in situ hybridization (FISH), PML immunostaining, Southern blot analysis and reverse-transcriptase polymerase chain reaction (RT-PCR). The most rapid test for PML-RARA-positive APL is provided by PML immunofluorescence using polyclonal or monoclonal PML antisera through detection of a characteristic microparticulate staining pattern (130 nuclear dots). In contrast, only a wild-type staining pattern (typically 10–20 nuclear dots) is seen in cases where alternative fusion partners are fused to RARA or in other subsets of AML [46, 75]. This test is particularly valuable in cases where the diagnosis of APL is uncertain, enabling prompt initiation of targeted therapy in the form of ATRA and ensuring careful monitoring of the coagulation profile in patients confirmed to harbour the PML-RARA fusion. The specific translocation t(15;17) is not identified by conventional cytogenetics in nearly 10% of cases of suspected APL [76]; however, the majority of such cases are PML-RARA positive as determined by simultaneously performed FISH or RT-PCR [12]. These apparently falsenegative results may be accounted for in some instances by technical failure, whilst in others, as mentioned above, they are due to insertion events or complex aberrations [45, 46]. However, conventional cytogenetics, which

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should be considered a mandatory component of the diagnostic work-up for all cases of AML, is a pivotal tool for the identification of alternative translocations involving chromosome band 17q12–21 and the presence of secondary chromosomal aberrations, even though the significance of the latter has yet to be determined. A cytogenetic analysis may also be helpful in revealing cases incorrectly diagnosed as APL that are unlikely to benefit from ATRA or ATO [12]. The identification of the rarer subset of APL with the t(11;17)(q23;q21)/PLZF-RARA fusion (approximately 1% of cases) is usually feasible on the basis of distinct morphological features and CD56 positivity [56]. This is clinically important in the light of the resistance of this group of patients to ATRA or ATO as single-agent therapies. For patients with PML-RARA-negative APL, lacking abnormalities of 17q12–21 by chromosomal analysis, screening for underlying RARA rearrangements can be undertaken most conveniently by FISH, since Southern blot analysis is quite cumbersome and time-consuming. For those cases confirmed to have RARA rearrangements, further characterization may be undertaken to identify the fusion partner. RT-PCR for the PML-RARA fusion should be considered a mandatory part of the routine laboratory work-up for all patients with suspected APL. This approach has the potential to provide a rapid confirmatory test; indeed, documentation of an underlying PML-RARA fusion is essential for patients being treated according to less intensive ATRA and anthracycline-based regimens designed by the GIMEMA and PETHEMA groups, since patients lacking this rearrangement are likely to be undertreated using such protocols. RT-PCR is also of value for defining PML breakpoint locations which are associated with specific disease characteristics and possibly prognostic information, as well as identifying targets for subsequent minimal residual disease (MRD) assessment [77–81]. RTPCR analyses have established that reciprocal RARAPML transcipts are expressed in 70–80% of cases. Detection of RARA-PML is of interest, since it facilitates PML breakpoint assignment and provides an additional and potentially more sensitive target for MRD monitoring [12, 82]. When performing RT-PCR assays it is imperative that strict precautions are undertaken to avoid falsepositive results due to contamination and suitable checks take into account the possibility of false-negative results due to poor RNA/cDNA quality.

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Minimal Residual Disease

The vast majority of patients are in complete haematological remission after the induction courses with ATRA and intensive chemotherapy. More than 70% of these patients are expected to be cured by subsequent consolidation and maintenance therapy, but this number also reflects the fact that about 30% of patients will ultimately relapse after the end of consolidation. Of these only about two thirds achieve a second remission, which is associated with a 2-year survival rate of about 40% [83]. The identification of different risk groups and the development of risk-adapted treatment schedules are, therefore, clearly the most challenging tasks in the treatment of APL. Lowrisk patients might also be cured with less intensive consolidation, particularly in view of the potential risks of chemotherapy-related toxicity. In contrast, possible treatment modifications for high-risk patients could include more intensive induction or consolidation, the use of autologous or allogeneic SCT in first remission or alternative treatment approaches like ATO, HDAC inhibitors or antibodies targeting CD33. Some pretreatment patient characteristics have been identified for the prediction of the individual risk of relapse, e.g. presenting leukocyte and platelet counts [12, 84, 85], CD56 status [86, 87], presence of FLT3 mutations [88] and PML breakpoint pattern [1], but all lack the precision to ensure optimal individual treatment modifications. This has provided the rationale for the monitoring of MRD by detection of PML-RARA fusion transcripts with conventional qualitative RT-PCR assays during and after first-line or relapse treatment schedules including autologous and allogeneic transplant procedures [10, 12, 14, 15, 81, 82, 89–94]. However, it has become evident that the interpretation of qualitative RT-PCR results is rather complex. A minority of patients (!10%) have been found to test PML-RARApositive after the end of consolidation, which is highly predictive of subsequent overt haematological relapse [10, 12, 14, 15]; this can, however, be averted by additional therapy such as allogeneic BMT [95]. Hence, achievement of PCR negativity as determined by conventional nested RT-PCR assays (sensitivity threshold: 1 in 104) has become a major therapeutic goal in APL. Nevertheless, achievement of this goal cannot be equated with cure, since 20–30% patients testing PCR negative in the marrow at the end of consolidation ultimately relapse [10, 12, 82, 93, 96]. In an attempt to improve the predictive value of MRD assessment, the GIMEMA group undertook 3monthly bone marrow analyses post-consolidation. This revealed that about 70% of relapses could be successfully

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predicted on the basis of prior molecular relapse, with over 80% of molecular conversions occurring within the first 6 months after completion of consolidation. Median time from molecular conversion to frank relapse was 3 months (range: 1–14 months). As a consequence, patients were monitored prospectively after first-line therapy. Those who converted to PCR-positive in two consecutive specimens taken from the marrow were given anticipated salvage therapy including standard doses of ATRA for 30 days, followed by consolidation chemotherapy and autologous SCT. Preliminary data suggest that initiation of second-line therapy may be more effective and less toxic at the time of molecular relapse than at the point of haematological relapse resulting in improved long-term survival [97]. These data clearly demonstrate that PML-RARA positivity is providing important prognostic information for directing necessary additional treatment intensification for the small subgroup of patients who are at high risk of relapse and affords the opportunity to limit treatment and toxicity in patients who are at low risk of relapse or even cured. The observation that frank relapse of APL occurs in some patients despite documentation of prior PCR negativity has highlighted several shortcomings of the conventional assays. Based on current evidence, a bone marrow specimen should be preferred because analyses from the peripheral blood are less sensitive than those based upon RNA derived from the marrow. In comparison to assays for the detection of BCR-ABL and AML1-ETO transcripts, conventional nested RT-PCR for the PML-RARA fusion is relatively insensitive reaching a level of 10 –3 to 10 –4. This has been ascribed to instability of PML-RARA transcripts, inefficiency of the RT step [92] and the GCrich nature of the fusion gene template [98]. However, development of quantitative ‘real-time’ RT-PCR methodologies (RQ-PCR) has revealed that the relative level of leukaemia-associated fusion gene expression is the key determinant of assay sensitivity, with PML-RARA being less highly expressed in primary leukaemic cells as compared to BCR-ABL or AML1-ETO [99]. Technical improvements have been evaluated including the use of gene-specific primers in the RT step, prolongation of the RT reaction and ‘hot start’ PCR resulting in higher sensitivity. However, as far as qualitative assays are concerned, enhancing the sensitivity was not found to be particularly advantageous. In particular, the predictive value of the assay was reduced due to the detection of PML-RARA transcripts in patients in long-term remission who were likely to be cured of APL [92, 100]. Recently developed bedside RNA stabilization kits may be helpful for better

standardization and reliability of MRD detection within multicentre and international trials [101]. Several strategies have been developed to improve the predictive value of MRD monitoring in APL. It was suggested that the kinetics of clearance of residual leukaemic cells might be a prognostic marker, but clinical studies provided conflicting data. Whereas the GIMEMA group found no correlation between PCR status after induction and subsequent risk of relapse, the MRC study found that the detection of transcripts at any stage following induction or during consolidation was associated with an increased risk of relapse, with PCR status following the third course of chemotherapy being most predictive [12]. Another strategy is the use of modified or alternative assays with increased sensitivity for the detection of the PML-RARA fusion gene or the reciprocal RARA-PML fusion gene which is an interesting additional target for monitoring residual disease because it can be detected with a higher sensitivity of about 1 log. However, this increase in sensitivity did not improve the predictive value of MRD assessment at the end of consolidation because RARA-PML-negative patients relapsed and RARA-PML-positive ones did not [12]. However, for patients that express RARA-PML, serial monitoring of this transcript in parallel with the more conventional PML-RARA assay could be of value, providing an earlier warning of impending relapse and hence permitting a more rapid initiation of pre-emptive therapy. As discussed above, modified assays with an improved sensitivity of up to 10 –6 detect residual PML-RARA fusion transcripts in the majority of patients in long-term remission and make it difficult to distinguish between patients prone to relapse and those who are still likely to be cured [98]. MRD assessment is particularly useful in planning treatment approach in patients with ‘high-risk’ APL, which may be defined as persistent PCR positivity following completion of therapy, or cases in molecular or frank relapse. One potential treatment option is ATO which can achieve molecular remission in the context of frank or molecular relapse. However, subsequent sequential MRD monitoring is important, since the persistence or reappearance of PML-RARA transcripts demands additional therapy to prevent relapse. Patients with ‘high-risk’ disease are candidates for transplantation procedures. Although the number of patients studied to date is relatively small due to the favourable prognosis of APL following ATRA and anthracycline-based chemotherapy, MRD assessment appears to be useful in planning the timing and type of transplant, as well as determining the need for

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additional therapy in the post-transplant setting. Patients with detectable PML-RARA transcripts have a high rate of relapse if subjected to autologous transplant procedures, particularly if the graft also tests PCR positive [1, 102, 103]. Such patients would be more ideally treated with allogeneic transplantation if this is an option, since this approach may be curative in this situation [95]. If no donor is available, additional therapy should be given to aim to achieve PCR negativity before any autologous transplant is contemplated. Molecular surveillance in the post-transplant period has been shown to be valuable in directing the need for treatment modification to avert relapse. Similar to other acute and chronic leukaemias patients in long-term remission have been found to be PML-RARA negative after autologous or allogeneic SCT [104] and evidence to date suggests that patients with PML-RARA transcripts still detectable 3 months posttransplant are destined to relapse in the absence of intervention [95, 102, 104, 105]. There are a variety of treatment approaches that could be employed in this setting [106]. One option is ATRA, which induced prolonged remission associated with the achievement of PCR negativity in two children with significant levels of MRD following ABMT undertaken in second CR [107]. ATO has also been found to be effective, when used alone or in combination with ATRA in patients with residual disease or relapse after allogeneic BMT, leading to molecular remission associated with full donor chimaerism [108, 109]. A more recent study has provided the first evidence suggesting a graft versus APL effect, with reversal of PCR positivity after allogeneic BMT following cyclosporin A withdrawal [95].

Quantitative RT-PCR

Because conventional qualitative RT-PCR fails to detect significant residual disease in a proportion of patients who ultimately go on to relapse [12, 93] and more sensitive assays detect residual disease in the majority of patients but do not distinguish between patients who are in long-term remission and those at high risk of relapse [98], sensitive quantitative approaches have been developed for the evaluation of the kinetics of residual disease. These are either based on assays using a competitor molecule [100] as internal standard or real-time quantification using hybridization or hydrolysis technology [110–113]. The expression of PML-RARA transcripts is usually normalized using a housekeeping gene, e.g. as the quotient of PML-RARA mRNA copies and GAPDH or ABL mRNA

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copies. The sensitivity level is at 10 –4 to 10 –5 and is marginally more sensitive than conventional RT-PCR [99]. In several relatively small studies it was demonstrated that a sensitive and accurate quantification of PMLRARA fusion transcripts may provide a superior approach for monitoring MRD compared to qualitative RTPCR [100, 110–112, 114], with the majority of investigated patients showing significantly higher levels of PMLRARA transcript levels before the onset of clinical relapse. In addition to providing more reliable information regarding the kinetics of molecular response to therapy or evidence of rising transcript levels prior to relapse, a major advantage of RQ-PCR is the capacity for the parallel quantification of endogenous control genes allowing the identification of poor quality samples, thus making possible the exclusion of false-negative samples with transcript numbers below a certain threshold. The largest series so far evaluating RQ-PCR for MRD monitoring in APL patients was a retrospective analysis undertaken by the US Intergroup evaluating 123 patients treated on protocol 0129 [113], which included randomizations for ATRA as a component of induction and/or maintenance therapy. PML-RARA transcript levels were normalized to the expression of GAPDH as the endogenous control gene. This study showed that patients with relatively high PMLRARA transcript levels at the end of consolidation (PMLRARA/GAPDH copy ratio 110 –5) had a significantly increased risk of relapse in comparison to patients with lower or undetectable PML-RARA transcripts (disease free survival 33 vs. 65% at 3 years). Interestingly, half of the patients who ultimately relapsed had levels of MRD that were below the detection threshold of the PMLRARA RQ-PCR assay, suggesting that treatment decisions cannot be solely based on PCR status at the end of consolidation therapy. This underlines the importance of molecular surveillance after completion of therapy to identify additional subsets patients at high risk of relapse. Intriguingly the Intergroup study revealed intermittent PCR positivity in patients in remission who appear to be cured of their disease. This suggests that immunological mechanisms may play a role in maintaining remission in patients with APL and could also lend support to the hypothesis that PML-RARA alone is insufficient to mediate the disease. The observation of this phenomenon further underlines the importance of serial monitoring in patients, such that trends in the transcript level may be appreciated, enabling treatment decisions to be based upon a rising level of PML-RARA transcripts rather than a single PCR-positive assay. Ongoing studies are seeking to address the optimal frequency of follow-up MRD

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assessment and whether peripheral blood could be used as an alternative to bone marrow in this context. A key issue is whether peripheral blood affords sufficient sensitivity during monitoring after the completion of therapy when early recognition of impending relapse is clinically important due to the potential for improved survival with early intervention with pre-emptive therapy. It is, therefore, expected that the advent of quantitative RT-PCR assays will yield further improvements in patient care, by enabling the development of risk-adapted treatment strategies on the basis of PCR profiles. This is particularly important for patients who would also be good candidates for allogeneic stem cell transplantation because the potential for cure is counterbalanced by a high rate of early mortality and long-term morbidity. Conversely, a variety of alternative and less toxic treatment options exist including conventional chemotherapy,

ATO, antibodies targeting CD33 and autologous SCT. In contrast, patients who are cured of APL are still at an inherent risk of treatment complications including secondary myelodysplasia and leukaemia. The automation will, therefore, facilitate appropriate quality control and standardization but the merit of interventions based on the detection of residual leukaemia still remains to be further substantiated in prospective studies.

Acknowledgment This work was supported by the Forschungsfonds der Fakultät für Klinische Medizin Mannheim der Universität Heidelberg and by the German Bundesminister für Bildung und Forschung (BMBF) through the Kompetenznetz ‘Akute und chronische Leukämien’. DG was supported by the Leukaemia Research Fund of Great Britain.

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94 Gameiro P, Vieira S, Carrara P, Silva AL, Diamond J, Botelho S de, Mehta AB, Prentice HG, Guimaraes JE, Hoffbrand AV, Foroni L, Parreira A: The PML-RAR alpha transcript in long-term follow-up of acute promyelocytic leukemia patients. Haematologica 2001;86: 577–585. 95 Lo Coco F, Romano A, Mencarelli A, Diverio D, Iori AP, Moleti ML, de Santis S, Cerretti R, Mandelli F, Arcese W: Allogeneic stem cell transplantation for advanced APL: Results in patients treated in second molecular remission or with molecularly persistent disease. Leukemia 2003;17:1930–1933. 96 Devaraj PE, Foroni L, Prentice HG, Hoffbrand VA, Secker-Walker LM: Relapse of acute promyelocytic leukemia follows serial negative RT-PCR assays: A cautionary tale. Leuk Res 1996;20:733–737. 97 Lo Coco F, De Santis S, Esposito A, Divona M, Diverio D: Molecular monitoring of hematologic malignancies: Current and future issues. Semin Hematol 2002;39(2 suppl 1):14–17. 98 Tobal K, Liu Yin JA: RT-PCR method with increased sensitivity shows persistence of PML-RARA fusion transcripts in patients in long-term remission of APL. Leukemia 1998; 12:1349–1354. 99 Gabert J, Beillard E, van der Velden VHJ, Bi W, Grimwade D, Pallisgaard N, Barbany G, Cazzaniga G, Cayuela JM, Cave H, Pane F, Aerts JL, De Micheli D, Thirion X, Pradel V, Gonzalez M, Viehmann S, Malec M, Saglio G, van Dongen JJ: Standardization and quality control studies of ‘real time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia – a Europe Against Cancer program. Leukemia 2003;17:2318– 2357. 100 Tobal K, Moore H, Macheta M, Yin JA: Monitoring minimal residual disease and predicting relapse in APL by quantitating PMLRARalpha transcripts with a sensitive competitive RT-PCR method. Leukemia 2001; 15:1060–1065. 101 Müller MC, Merx K, Weisser A, Kreil S, Lahaye T, Hehlmann R, Hochhaus A: Improvement of molecular monitoring of residual disease in leukemias by bedside RNA stabilization. Leukemia 2002;16:2395–2399. 102 Meloni G, Diverio D, Vignetti M, Avvisati G, Capria S, Petti MC, Mandelli F, Lo Coco F: Autologous bone marrow transplantation for acute promyelocytic leukemia in second remission: Prognostic relevance of pretransplant minimal residual disease assessment by reverse-transcription polymerase chain reaction of the PML/RAR alpha fusion gene. Blood 1997;90:1321–1325.

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103 Thomas X, Dombret H, Cordonnier C, Pigneux A, Gardin C, Guerci A, Vekhoff A, Sadoun A, Stamatoullas A, Fegueux N, Maloisel F, Cahn JY, Reman O, Gratecos N, Berthou C, Huguet F, Kotoucek P, Travade P, Buzyn A, de Revel T, Vilque JP, Naccache P, Chomienne C, Degos L, Fenaux P: Treatment of relapsing acute promyelocytic leukemia by all-trans retinoic acid therapy followed by timed sequential chemotherapy and stem cell transplantation. APL Study Group. Acute promyelocytic leukemia. Leukemia 2000;14: 1006–1013. 104 Roman J, Martin C, Torres A, Jimenez MA, Andres P, Flores R, de la Torre MJ, Sanchez J, Serrano J, Falcon M: Absence of detectable PML-RAR alpha fusion transcripts in longterm remission patients after BMT for acute promyelocytic leukemia. Bone Marrow Transplant 1997;19:679–683. 105 Takatsuki H, Umemura T, Sadamura S, Yamashita S, Goto T, Abe Y, Yufu Y, Inaba S, Nishimura J, Nawata H: Detection of minimal residual disease by reverse transcriptase polymerase chain reaction for the PML/RAR alpha fusion mRNA: A study in patients with acute promyelocytic leukemia following peripheral stem cell transplantation. Leukemia 1995;9:889–892.

PML-RARA in APL

106 Lengfelder E, Gnad U, Büchner T, Hehlmann R: Treatment of relapsed acute promyelocytic leukemia. Onkologie 2003;26:373–379. 107 Grimwade D, Jamal R, Goulden N, Kempski H, Mastrangelo S, Veys P: Salvage of patients with acute promyelocytic leukaemia with residual disease following ABMT performed in second CR using all-trans retinoic acid. Br J Haematol 1998;103:559–562. 108 Tedeschi A, Cairoli R, Marenco P, Nosari A, Tresoldi E, Di Bona E, Montillo M, Morra E: Molecular remission and reconstitution of a full chimera with arsenic trioxide in a patient with acute promyelocytic leukemia relapsed after allogeneic bone marrow transplantation. Leukemia 2002;16:2455–2456. 109 Grigg A, Kimber R, Szer J: Prolonged molecular remission after arsenic trioxide and alltrans retinoic acid for acute promyelocytic leukemia relapsed after allogeneic stem cell transplantation. Leukemia 2003;17:1916– 1917. 110 Cassinat B, Zassadowski F, Balitrand N, Barbey C, Rain JD, Fenaux P, Degos L, Vidaud M, Chomienne C: Quantitation of minimal residual disease in acute promyelocytic leukemia patients with t(15;17) translocation using real-time RT-PCR. Leukemia 2000;14:324– 328.

111 Visani G, Buonamici S, Malagola M, Isidori A, Piccaluga PP, Martinelli G, Ottaviani E, Grafone T, Baccarani M, Tura S: Pulsed ATRA as single therapy restores long-term remission in PML-RARalpha-positive acute promyelocytic leukemia patients: Real time quantification of minimal residual disease. A pilot study. Leukemia 2001;15:1696–1700. 112 Slack JL, Bi W, Livak KJ, Beaubier N, Yu M, Clark M, Kim SH, Gallagher RE, Willman CL: Pre-clinical validation of a novel, highly sensitive assay to detect PML-RARalpha mRNA using real-time reverse-transcription polymerase chain reaction. J Mol Diagn 2001;3/4:141–149. 113 Gallagher RE, Yeap BY, Bi W, Livak KJ, Beaubier N, Rao S, Bloomfield CD, Appelbaum FR, Tallman MS, Slack JL, Willman CL: Quantitative real-time RT-PCR analysis of PML-RAR alpha mRNA in acute promyelocytic leukemia: Assessment of prognostic significance in adult patients from intergroup protocol 0129. Blood 2003;101:2521–2528. 114 Gu BW, Hu J, Xu L, Yan H, Jin WR, Zhu YM, Zhao WL, Niu C, Cao Q, Su XY, Gu J, Ying HY, Chen Y, Xiong SM, Shen ZX, Chen Z, Chen SJ: Feasibility and clinical significance of real-time quantitative RT-PCR assay of PML-RARalpha fusion transcript in patients with acute promyelocytic leukemia. Hematol J 2001;2:330–340.

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Review Acta Haematol 2004;112:68–78 DOI: 10.1159/000077561

FLT3 Length Mutations as Marker for Follow-Up Studies in Acute Myeloid Leukaemia Susanne Schnittger Claudia Schoch Wolfgang Kern Wolfgang Hiddemann Torsten Haferlach Laboratory for Leukaemia Diagnostics, Department of Internal Medicine III, Ludwig Maximilians University of Munich, University Hospital Grosshadern, Munich, Germany

Key Words FLT3-LM W Minimal residual disease W AML W FLT3-ITD W Follow-up studies

Abstract Length mutations within the FLT3 gene (FLT3-LM) can be found in 23% of acute myeloid leukaemia (AML) and thus are the most frequent mutations in AML. FLT3-LM are highly correlated with AML with normal karyotype and other cytogenetic aberrations of the prognostically intermediate group. This group is supposed to be a mixed group of AML with differences in the underlying pathogenesis. For more individualized treatment options it would be helpful to better characterize this large AML group not only by molecular mutations but also use these markers for the definition of minimal residual disease (MRD). However, so far the cytogenetically intermediate AML has been lacking suitable markers for PCRbased MRD detection like the fusion genes in the prognostically favourable subgroups. The suitability of the FLT3-LM as a target for PCR-based MRD was discussed controversially as it seemed to be a rather unstable

ABC

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marker. Thus, we aimed at the evaluation of FLT3-LM as a marker for residual disease in a large cohort of AML. Paired samples of 97 patients with AML at diagnosis and at relapse were analyzed. It could be shown that in only four cases a loss of the length mutation was detected. This is in the range of other well-characterized AML relapsing with a different geno- and/or phenotype. In contrast, a change in the ratio of the mutated allele in comparison to the wild-type allele was frequently observed. In detail, the FLT3-LM showed a tendency to accumulate during disease progression and was found more frequently at relapse than at diagnosis. In addition, 45 patients were analyzed at different time points during and after therapy. Using conventional PCR it clearly could be shown that for most of the patients positive at presentation FLT3-LM is a reliable PCR marker for monitoring treatment response. Even an early detection of relapse was possible in some cases. Copyright © 2004 S. Karger AG, Basel

Dr. rer. nat. Susanne Schnittger, Laboratory for Leukaemia Diagnostics Department of Internal Medicine III, University Hospital Grosshadern Ludwig Maximilians University, Marchioninistrasse 15 DE–81377 München (Germany), Tel. +49 89 7095 4970, Fax +49 89 7095 4971 E-Mail [email protected]

Introduction

tional control of therapy response by cytomorphology, assessment of MRD by flow cytometry is of growing importance [23]. However, polymerase chain reaction (PCR)-based methods are still the most sensitive ones for the detection of residual leukaemic cells. The application of PCR to quantify MRD so far was based on the presence of fusion genes or their respective fusion transcripts, which occur exclusively in the respective subentities of AML but not in normal bone marrow. In patients with AML the detection of MRD by quantitative PCR has been shown to be feasible and prognostically relevant [24– 32]. However, in AML with normal karyotype or other prognostically intermediate aberrations like trisomies 8 or 11 and del(9q) PCR was not applicable due to the lack of a target suitable for PCR. The portion of cases assessable may now be extended by the inclusion of cases with FLT3-LM. To analyze whether FLT3-LM is a valuable marker for follow-up controls two different analyses were performed: (1) the stability was assessed by paired analysis of samples from diagnosis and relapse and (2) FLT3 status from samples during and after therapy was compared to the status of remission, fluorescence in situ hybridization (FISH) and cytogenetics.

FLT3 is a member of fms-like receptor tyrosine kinases and is expressed in early haematopoietic stem cells [1, 2]. The gene coding for this receptor is targeted by mutations in the juxtamembrane domain in 20–23% of all unselected acute myeloid leukaemias (AML) [3–8]. In addition, point mutations in the activation domain have been described in an additional 6–7% of AML [9–11]. Thus, FLT3 is the gene most commonly targeted by mutations in AML. The mutations in exon 14 and/or exon 15, the part of the gene that is coding for the juxtamembrane domain, have been described to be internal tandem duplications (ITD) resulting in the term FLT3-ITD. However, as we have found that not all of these mutations are simple ITDs but about 30% have insertions of extranucleotides between the duplicated stretches and some cases are even more complicated, we will refer to this mutations as FLT3 length mutations (FLT3-LM) [8]. The elongation of the juxtamembrane domain results in a conformation change that leads to autoactivation of the receptor through a constitutive phosphorylation [12] and can induce IL-3-independent growth in model systems [13, 14]. The FLT3-LM has been shown to be strongly correlated with AML and myelodysplastic syndromes of the RAEB and RAEBt subtypes [8, 15]. In many different study groups the FLT3-LM has been shown to be strongly correlated with the prognostically intermediate karyotype group. This large group of AML is supposed to be a pool of insufficiently characterized AML with different molecular mutations. Now it is becoming clear that the FLT3LM defines a prognostically worse subset within this group [3, 5–8, 16, 17]. Today the karyotype of the leukemic cells is considered the most important parameter indicating the prognosis in patients with AML [18, 19]. Although further pretherapeutically defined prognostic parameters have been identified such as age of the patient and AML occurring as a secondary disease the prognosis of patients within the respective subgroups defined by these parameters is still heterogeneous. As a consequence, the implementation of therapy-dependent parameters into stratification systems has been approached. Along this line, the degree of reduction of the leukemic cell mass following the first course of induction therapy as well as the time to achieve complete remission have been demonstrated to independently impact the prognosis [20–22]. These studies thus have proved the concept of prognostication based on therapy-dependent factors. The most prominent therapy-related factor is the level of minimal residual disease (MRD). Beside the conven-

Sample Preparation Mononucleated bone marrow cells were obtained by Ficoll-Hypaque density gradient centrifugation. Total RNA was extracted from 107 cells with RNeasy (Qiagen, Hilden, Germany) (1997–2000) or mRNA with the MagnaPureLC mRNA Kit I (Roche Diagnostics, Mannheim, Germany) (since January 2001). The cDNA synthesis of 1–2 Ìg total RNA or mRNA from an equivalent of 107 cells was performed using 300 U Superscript II (GibcoBRL/Invitrogen, Karlsruhe, Germany) and random hexamer primers (Pharmacia, Freiburg,

FLT3-LM as MRD Marker

Acta Haematol 2004;112:68–78

Patients, Materials and Methods Patient Samples Bone marrow or peripheral blood samples were sent by overnight service to our lab. All were diagnosed as having AML according to standard French-American-British (FAB) criteria [33–38] and were referred between July 1997 and December 2002 for cytomorphological, cytogenetic, molecular genetic and multiparameter flow cytometry analysis. Cytogenetic and FISH Analysis Cytogenetic G-banding analysis was performed with standard methods [39]. The definition of a cytogenetic clone and descriptions of karyotypes followed the International System for Human Cytogenetic Nomenclature [40]. For FISH the commercially available probes CEP#8SG, LSID7S486/CEP7, LSIPMLRARA, LSICBFB, and LSIAML1/ETO were used according to the manufacturer’s instructions (Vysis, Bergisch Gladbach, Germany).

69

Germany). DNA was extracted using a simple salting-out procedure [41]. PCR at Diagnosis PCR and semiquantitative estimation of the FLT3-LM status was performed as has been described [8]. Strict precautions were taken to prevent contamination. Water instead of cDNA was included as a blank sample in each experiment. Amplification products were analyzed on 2% agarose gels stained with ethidium bromide. Sequencing For direct sequencing of the length mutations the amplified fragments were cut from the agarose gels and isolated with Quiaex II (Qiagen) following the manufacturer’s instructions. Approximately 100 ng of purified PCR products were directly sequenced with 3.3 pmol of primers as described above with the Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Weiterstadt, Germany). After initial denaturation at 95 ° C for 5 min, 25 cycles at 94 ° C for 15 s and 60 ° C for 4 min were performed. Cases with a relatively small length of the mutations or those with status 1 or 5 mutations were subcloned using the TOPO II cloning system (Invitrogen, Karlsruhe, Germany) prior to sequence analysis. Sequence detection was performed on an ABI 310 sequence detection system (Applied Biosystems). Efficiencies, Reproducibility and Sensitivities of the PCR Assays The sensitivity of the PCR assays was assessed by performing limited dilution series of DNA and cDNA of diagnostic patient samples with a different status of FLT3-LM into cDNA of samples without FLT3-LM.

Results

Frequency of FLT3-LM at Presentation and in Relapse In total 2,338 samples were analyzed, 2,135 were from newly diagnosed AML and 203 from relapsed AML. FLT3-LM were found in 461 (21.6%) of the diagnostic samples at first diagnosis and in 62 (30.5%) of the relapses. Thus the frequency is higher in relapsed AML (table 1) (p = 0.005). Semiquantitative Analysis of the Mutations Analysis of the amplification fragments on agarose gels revealed that the band representing the mutation was not always of the same intensity as the wild-type allele. Thus, we divided the FLT3-LM into five categories: (1) mutant fragment less intense than wild-type band (status 1), (2) mutant fragment with the same intensity as wild-type band (status 2), (3) mutant fragment more intense than wild-type band (status 3), (4) only mutant fragment and loss of wild-type band (status 4), and (5) presence of more than one mutant fragment (status 5) (fig. 1). Type 1 has to be interpreted as subclone in an otherwise FLT3-LM-neg-

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Table 1. FLT3-LM status according to a semiquantitative estima-

tion in a cohort of 2,135 AML patients at diagnosis and 203 at relapse Mutation status

Diagnoses (461/2,135; 21.6%), %

Relapse (62/203; 30.5%), %

1 2 3 4 5

10 60 11 6 13

– 16 32 52 –

ative population, type 2 as an equal amount of wild-type and mutated allele, type 3 as loss of the wild type in a subclone, and type 4 as complete loss of the wild type. In type 5 there were two different FLT3-LM in different clones. At diagnosis 70% of the patients with FLT3-LM have status 1 and 2 mutations, whereas a loss or partial loss of the wild-type allele was found in only 17%. In contrast, in relapses status 1 and 5 was never detected and status 2 in only 16%. Most of the relapsed FLT3-LM-positive cases had a partial (32%) or complete (52%) loss of the WTFLT3. Use of DNA and cDNA for PCR Diagnostics The PCR screening for FLT3-LM was done at the genomic and cDNA level in parallel for most of the cases. For some cases with limited material the analysis was restricted to the cDNA level. No major differences concerning the intensities of the mutated allele in comparison to the wild-type allele were observed, suggesting that there were no major expression differences between wild-type and mutated alleles. At diagnosis it was possible to detect an FLT3 expression in every sample. In contrast, in follow-up samples of patients in complete clinical remission, it was not always possible to obtain an amplification product of FLT3 by PCR. This is in accordance with previous studies that have shown FLT3 expression on early haematological and leukaemic cells but not on differentiated haematological cells [2]. Thus for follow-up controls DNA is highly recommended as diagnostic material. Paired Samples from Diagnosis and Relapse In total the FLT3 status was assessed in 97 paired samples at diagnosis and at relapse (table 2). Fifty-one patients were negative at both time points. Thirty-eight

Schnittger/Schoch/Kern/Hiddemann/ Haferlach

Fig. 1. Assessment of the FLT3-LM status according to mutation/wild-type relation.

Table 2. FLT3-LM in paired presentation

Patients

and relapse samples: present study and review of the literature Nakano et al. [48] Kottaridis et al. [16] Hovland et al. [42] Shih et al. [44] Present study

28 44 2 108 97

Total

278

FLT3 status at presentation/relapse –/–

–/+

+/–

+/+

16 20 – 83 51

6 4 1 8 4

1 5 – 1 4

5* 15* 1 16* 38

170 (61.2%) 23 (8.3%) 11 (4%)

75 (27%)

cases were positive for an FLT3-LM at both time points. All 3 cases with a second relapse retained the FLT3-LM at that time point. A gain of an FLT3-LM was found in 4 cases. A loss of the mutation was detected in only 4 cases (IDs 2, 41, 42, 43), three of whom had only a status 1 mutation at presentation (table 3), meaning that at diagnosis only a subclone of all leukemic cells had the mutation. Of the 4 cases with a gain of the FLT3 mutation, in two (IDs 44 and 47) also a change of the FAB from M4 to M2 and M6 to M1, a change of the immunophenotype and in case 47 also a different karyotype were observed raising the question of whether these two might be secondary AML rather than relapsed AML. In addition, in

case 46 the same FAB but a complete change of the karyotype was observed. Focussing on the FLT3 status (relation of the mutated to the WT-FLT3) in more detail (table 4 and 5) we found stability of the status in 12 cases (24.5%); however, a progression from lower to higher status was detected in 30 cases (63%) and a regression to lower status or loss of the FLT3-LM in only 6 cases (12.2%). Thus the overall maintenance of an FLT3-LM in relapse was relatively stable with (45/49; 91.8%). However the mutation had a tendency to accumulate during progression of leukaemia.

FLT3-LM as MRD Marker

Acta Haematol 2004;112:68–78

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Table 3. Paired samples of patients with FLT3-LM at diagnosis and/or relapse (n = 47) ID

FAB

FLT3-LM Karyotype status

ID

FAB

M0 M0 M1 M1 M1 M1 M1 M1

3 3 2 – 2 3 2 3

27 PD 27 Rel 28 PD 28 Rel

M0 2 (Chlorom) M0 3 M2 2 M2 3

5 PD 5 Rel 6 PD 6 Rel 7 PD 7 Rel 8 PD 8 Rel 9 PD 9 Rel 10 PD 10 Rel 11 PD 11 Rel 12 PD 12 Rel 13 PD 13 1. Rel

M1 M1 M1 M1 M1/M2 ? M2/M4 M2 M2 M2 M2 M2 M2 M2. M2 M2 M2 M2

4 2 4 4 2 4 1 3 4 4 1 4 2 3 2 4 2 2

29 PD 29 Rel 30 PD

M4 M4 tM4

2 3 5

30 Rel 31 PD

M1 M5a

2 1

31 Rel

M5a

2

32 PD 32 Rel

M5b M5b

2 2

33 PD 33 Rel 34 PD 34 Rel 35 PD 35 Rel

M1 M1 M1 M1 M2 M2

1 3 1 4 2 4

13 2. Rel

M2

2

14 PD 14 Rel 15 PD 15 Rel 16 PD 16 Rel 17 PD 17 Rel 18 PD 18 Rel

M4 ? M4 M4 M4 M4 M4 M4 M4/M5 M4/M5

5 5 2 3 3 3 2 3 2 2

36 PD 36 Rel 37 PD 37 Rel 38 PD

M1 M2 M5b M5b M3v

2 4 2 2 1

19 PD 19 Rel 20 PD 20 1. Rel 20 2. Rel 21 PD 21 Rel 22 PD 22 Rel 23 PD 23 1. Rel 23 2. Rel 24 PD 24 Rel

M4 M4Rez M5b M5b M5b M5b M5bRez M5b M1 AML AML AML M2 M2

3 4 3 4 4 3 4 1 4 2 2 2 2 2

38 Rel 39 PD 39 Rel 40 PD 40 Rel 41 PD 41 Rel 42 PD 42 Rel 43 PD

M3v M5a M5a M1 M1 M2 M2 sAML sAML M4eo

3 1 4 1 4 1 0 1 0 1

43 Rel

M4eo

0

25 PD 25 Rel 26 PD 26 Rel

M0/M1 M0 M0 M0

2 3 2 2

44 PD 44 Rel 45 PD 45 Rel 46 PD 46 Rel 47 PD 47 Rel

M4 M2 M1 M1 M1 M1 M6 M1

0 3 0 1 0 2 0 4

1 PD 1 Rel 2 PD 2 Rel 3 PD 3 Rel 4 PD 4 Rel

72

46,XX [24] 46,XX [5] 46,XX [25] 46,XX 46,XX [25] 46,XX [8] 46,XX [25] 46,X,del(X)(q11),t(7;18)(q22;p11.2)[8]/ 46,XX [7] 48,XY,+8,+10 [10] 46,XY [25] 46,XY [20] n.d. 46,XY [6] n.d. 46,XX [12] n.d. 46,XX [25] 46,XX [6] 46,XY [25] 46,XY [21] 46,XY [22] 46,XY [25] 46,XY [25] 49,XY,+8,+13,+19 [20] 46,XX [24] 46,XX,t(1;19;3)(p32;q13?;p21)[25]/ 46,XX [2] 46,XX,t(1;19;3)(p32;q13?;p21)[14]/ 46,XX [1] 46,XX [25] 46,XX [25] 46,XX [25] 46,XX [25] 46,XX [25] 46,XX [25] 46,XX [25] 46,XX [15] 46,XY [25] 48,XY,t(2;3)(q31;p21),+8,+8,t(10;17) (q22;q22), del(13)(q14q31)[13]/46,XY [12] 46,XX [25] 46,XX [25] 46,XX [20] 46,XX [20] 46,XX [20] 46,XX [25] 46,XX [25] 46,XX [25] 46,XX [25] 46,XY [15] 46,XY,t(10;12)(q22;p13)[6]/46,XY [6] 46,XY,t(10;12)(q22;p13)[14]/46,XY [1] 46,XY,t(8;21)(q22;q22) [25] 46,XY,t(1;5)(q21;q33),t(8;21)(q22;q22)[6]/ 46,XY,t(1;5)(q21;q33),t(8;21)(q22;q22), del(11)(p13) [4] 46,XY,der(5)t(5;11)(q22;?)[9]/46,XY [11] 46,XY [20] 46,XY,t(11;19)(q13;p13)[17]/46,XY [3] 46,XY,t(11;19)(q13;p13)[9]/46,XY [1]

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FLT3-LM Karyotype status 48,XX,+8,+22[4]/46,XX [20] 50,XX,+X,+8,+10,+13 [3] 47,XX,del(5)(q15q33)+11 [20] 47,XX,del(5)(q15q33)+11[9]/ 47,idem,del(17)(q23)[9]/ 47,idem,add(7)(q3?4)[2] 46,XX,inv(3)(q21q26)[17]/46,XX [8] 46,XX,inv(3)(q21q26)[7]/46,XX [13] 46,X,i(X)(q10)[4] 46,XX [19] 46,XX [20] 46,XY,del(11)(q13q21) or del(11)(q21q23)[6]/47,idem,+8[13]/ 46,XY [3] 46,XY,del(11)(q13q21) or del(11)(q21q23)[1]/47,idem,+8 [19] 47,XY,+8[16]/46,XY [4] 47,XY,+8 [15] 47,XY,t(1;18)(p22;q23),+8,del(11) (p11.2p15) [5] 46,XX [25] 46,XX [15] 46,XX [22] 46,XX [25] 46,XY,der(7)t(1;7)(q31;q32)[8]/46,XY [8] 46,XY,t(3;6)(q21;p12),der(7)t(1;7) (q31;q32),der(10)t(8;10)(q22;q26),t(12;15) (q13;q22)[13]/46,XY [2] 46,XX [21] 46,XX [20] 46,XX [25] 46,XX [25] 46,XY,t(15;17)(q22;q12)[3]46,XY, der(15)t(15;17)(q22;q12),ider(17)(q10) t(15;17) (q22;q12) [17] 46,XY,t(15;17)(q22;q21)[18]/46,XY [2] 46,XX [25] 46,XX [25] 46,XY [21] 46,XY [18] 46,XX,t(8;21)(q22;q22)[19]/46,XX [1] 46,XX,t(8;21)(q22;q22)[11]/46,XX [4] 47,XY,+8 [15] 47,XY,+8[4]/46,XY [16] 46,XY,inv(16)(p13q22)[2]/ 47,XY,+8,inv(16)(p13q22)[1]49,XY,del(1) (q21),+8,+13,inv(16)(p13q22),+21 [4] 46,XY,inv(16)(p13q22) [1]\n47,XY,+8,inv(16)(p13q22) 46,XX [25] 46,XX [20] 46,XX [26] 46,XX [20] 46,XY,del(7)(q21)[5]/46,XY [13] 47,XY,+13[2]/47,XY,+15[2]/46,XY [7] 47,XY,+8[12]/46,XY [8] 46,XY,t(1;17)(q21;q23),inv(6)(p21q27), t(9;13)(q21;q14)[5]/46,XY [16]

PD = Primary diagnosis; Rel = relapse; n.d. = not done.

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Table 4. FLT3-LM status in paired samples at presentation and relapse (n = 49)

FLT3 status at diagnosis 1 2 3 4

5

0

0

0

0

1

1

1

2

2

3

1

2

4

5

FLT3 status at relapse 1 2 3

4

5

1

2

3

4

2

3

4

3

4

4

0

0

2

2

Patients –

3

1

1

1

1

1

1

3

5

9

5

3

3

1

1

1

6

2

Total patients constant: n = 12

progression: n = 30

regression: n = 6

Table 5. Karyotype in paired samples at presentation and at relapses in patients with FLT3-LM

Diagnosis

Relapse

Total (n = 44)

Constant (n = 26)

normal karyotype aberrant karyotype

normal karyotype same aberrant karyotype

23 4

Progression (n = 9)

normal karyotype aberrant karyotype

aberrant karyotype same aberrant karyotype with additional aberrations

5 4

Regression (n = 6)

aberrant karyotype aberrant karyotype

normal karyotype less aberrant karyotype

3 3

Change (n = 3)

aberrant karyotyp

regression + progression

3

Comparison of FLT3 Status and Karyotype Karyotype at diagnosis and relapse was available in 44 paired samples. Most of the cases (n = 23) had a normal karyotype at both time points and 4 cases had equal aberrant karyotypes of the prognostically intermediate karyotype group. Thus 27/44 cases (61.4%) had stable karyotypes. However, of these 27 cases with stable karyotype 18 had a progression of the FLT3-LM. Five cases with normal karyotype had a chromosomal aberration at relapse. In addition, 5 cases with aberrations had additional aberrations at relapse. All these aberrations were of the prognostically intermediate group as is characteristic for cases with FLT3-LM; thus there was no change with regard to the cytogenetically defined risk group but only gains of a so-called additional chromosomal aberration in the sense of progression. Five of the 10 cases with karyotype progression in parallel showed progression of the FLT3 status. In 6 cases a karyotype regression was detected, 3 of these had a regression and a progression in combination. One (ID 43) also revealed regression of FLT3-LM; however, 5 of these cases revealed a progression of the FLT3LM. Thus cytogenetic regression can be replaced by a

molecular progression. Four cases (ID 5, 30, 46, 47) had both a cytogenetic as well as a molecular regression.

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Acta Haematol 2004;112:68–78

Evaluation of FLT3-LM as Follow-Up Marker Using Conventional PCR The sensitivity as estimated by the limited dilution series of FLT3-LM-positive patients’ RNA or DNA from the time of diagnosis in samples negative for the mutation was dependent on the strength of the initial mutation status and was between 1:100 and 1:1,000 (fig. 2). The course of the disease could be followed according to the wildtype/mutation ratio of the amplification product. In total 174 bone marrow samples of 45 patients were analyzed during or after therapy. The median sample number per patient was 4 and the median follow-up time of sampling was 12 months. In 6 cases it was possible to detect FLT3-LM positivity after previous PCR negativity 1–3 months before cytomorphological relapse. In additional 4 cases a pending relapse was detectable due to persistent PCR positivity of the FLT3-LM (examples are depicted in fig. 3).

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FISH and PCR In some cases it was possible to perform FISH analysis or PCR using a second marker in parallel to FLT3-LM PCR. For 11 patients with chromosome aberrations at diagnosis a FISH marker was available for follow-up. Four patients with reciprocal translocations could be analyzed with respective colocalization probes [AML1-ETO (n = 2), PML-RARA and CBFB-MYH11]. Four cases had a trisomy 8 that was followed by a centromere 8 probe. One had a 5q- and 2 a 7q- that were analyzed with probes from the respective deletion region. In the 4 cases with AML1-ETO, PML-RARA, and CBFB-MYH11 fusion

Fig. 2. Limited dilution assays of FLT-LM-positive DNA of patients

with different status at diagnosis into FLT3-LM-negative DNA showing the detection limits of FLT3-LM by conventional PCR.

Fig. 3. Follow-up analyses in some exemplary patients showing the possibility of controlling therapy response and of detecting relapses early. Rel = Relapse; CR = complete remission; BMT = bone marrow transplantation.

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Schnittger/Schoch/Kern/Hiddemann/ Haferlach

FLT3-LM is the most frequent genetic marker in AML. It is most commonly found in the intermediate risk group, a subgroup of AML where PCR markers for followup controls have been missing so far. Therefore, it was suggested that an FLT3-LM should be used as a follow-up marker [8]. However, many studies reported on the instability of this marker during the course of the disease [42– 45]. Here we performed a comprehensive analysis regarding the applicability of the FLT3-LM as a follow-up marker. We and others have previously shown that the amount of FLT3-LM in comparison to the wild-type allele is heterogeneous [7, 8]. In the present study in 17% of the patients at diagnosis and in 84% at relapse partial or complete loss of the wild-type allele was found. Although the clinical significance of FLT3-LM per se is still discussed controversely the loss of the wild-type allele was reported to be clearly associated with a worse outcome [7, 46]. These patients have a higher white blood cell count, a higher percentage of bone marrow blasts, and a shorter overall and disease-free survival [7, 16, 46]. The loss of WT-FLT3 is of interest with respect to functional aspects. As a mutant FLT3 in a mutant/wild-type heterodimer can trans-phosphorylate the wild-type chain [47]; it implies that a mutant homodimer has some gain of function more than simply activating the kinase. Alternatively, formation of the homodimer may reflect an underlying mechanism of genetic instability that has other unknown genomic consequences that may, in turn, influence clinical outcome.

Different incidences of the patients with loss of the wild type were described in different studies. Whereas Whitman et al. [46] found in 8/23 (35%) of their patients a level of mutant greater than the wild-type allele (WT), Fröhling et al. [17] found it in only 1/71 (1.4%). To some extent, this may reflect differences in the cut-off value for loss of wt allele and stresses the need for a truly quantitative assessment of the FLT3 mutation status even at diagnosis. We found a higher FLT3-LM ratio in comparison to the wt in 11% at diagnosis, in 32% at relapse, and complete loss of the WT-FLT3 in 6% at diagnosis and 52% at relapse. Thus a loss of the WT-FLT3 is not only associated with a worse prognosis but, in addition, accumulates during the course of the disease and thus seems to be a marker for more progressed disease. Several studies have been published investigating FLT3 mutations in paired presentation and relapse samples [8, 42–44, 48]. In some of these studies 7–15% of all AML revealed a gain of the FLT3-LM at relapse [43, 44]. Like in the study presented many of the patients lost their wt at relapse. This accumulation of the FLT3-LM implies a role of FLT3-LM in leukaemia progression and onset of relapse. Taking the results of these previous studies together with those of the study presented only 88% of the analyzed patients maintained the same FLT3 status (FLT3LM positive or negative) at both time points. Consequently, as was suggested previously [45] the FLT3-LM should be regarded with caution with respect to its usefulness as a follow-up marker. Patients that were positive for an FLT3-LM at presentation often showed an increased mutant level at relapse, usually with evidence of the loss of wild-type alleles. Where more than one mutation had been detected at presentation, usually only one was dominant at relapse [8, 43, 44]. Thus accumulation in the sense of progression seems to be the most common direction of the instability of the FLT3-LM and does not interfere with the applicability for MRD diagnostics. However, in some studies a significant proportion of patients either gained (8.3%) or lost (4%) an FLT3-LM at relapse, and some patients have been reported with a loss of the presentation LM and a gain of a completely new one at relapse [42–44]. These data imply that FLT3-LM are secondary events, arising in an already transformed clone, which induce the outgrowth of a subclone as a result of an additional proliferative advantage conferred by the FLT3-LM. Further support for the secondary etiology of the FLT3-LM is given by those cases that carry the FLT3LM in only a subset of their leukaemic cells (status 1 mutation in the study presented). In our study all cases

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Acta Haematol 2004;112:68–78

gene-specific PCR and FISH were done in parallel to FLT3-PCR. In each of these cases fusion gene-specific PCR was the most sensitive method. In 6 cases (1 ! AML1-ETO, 1 ! PML-RARA, 2 ! +8, 1 ! 7q-, 1 ! 5q-) FISH and FLT3 revealed comparable results during follow-up. One case with AML1-ETO and the one with CBFB-MYH11 and low FLT3-LM at diagnosis lost their FLT3-LM at relapse. Two of the cases with trisomy 8, 1 with 7q- and the 1 with 5q-, lost these chromosomal aberrations at relapse but retained the FLT3-LM or even revealed a progression of the FLT3-LM. Thus, for patients with fusion genes, fusion gene-specific PCR or even FISH clearly are superior to FLT3-PCR. However, in cases with other aberrations like trisomy 8, 5q- or 7qFLT3-LM PCR seems to be the more reliable follow-up marker.

Discussion

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with a loss of the FLT3-LM at relapse revealed only a low FLT3-LM/wt ratio at diagnosis. One of the cases had a t(8;21) and one an inv(16) which are subtypes with a rare incidence of FLT3-LM. Also in these cases there seems to be only an FLT3-LM-positive subclone at diagnosis which got lost at relapse. Also these cases had cytogenetically normal clones at diagnosis and not at relapse. Thus in cases with only a low mutation ratio at diagnosis the results of the follow-up analysis should be regarded with special care. Detailed cytomorphological, cytogenetic, and immunophenotypic analysis of our cases with a loss of the FLT3-LM at relapse revealed a shift of the FAB type and immunophenotype in 2 cases and a complete change of the karyotype in another 2 cases, suggesting that these AML might be secondary AML instead of relapses. Taking together our data and data published in the literature (table 2), only 4% have a loss of the FLT3-LM. This would be the subgroup that would escape early detection of relapse. However, from our experience this is in the same range as other leukaemias (for example, those followed up with AML1-ETO or CBFB-MYH11) ‘relapse’ with a different AML. In all of these cases an impending relapse cannot be detected with molecular methods. In addition, therapy response in the early course of the disease can also be applied in these 4% of cases. For individual patients it was possible to apply different methods of follow-up controls in parallel. It was shown that in patients with fusion genes, fusion gene-specific PCR was clearly superior to FLT3-PCR because it is quantitative instead of semiquantitative and more sensitive. Even the fusion gene-specific FISH analysis was more sensitive in these cases, because all these cases had a very low FLT3-LM status at diagnosis. In contrast, in cases with other aberrations like trisomy 8, 5q- or 7qFLT3-LM was more reliable than FISH specific for the

respective chromosome aberrations, because all these chromosome aberrations were unstable at relapse and FLT3-PCR although being not highly sensitive with a sensitivity of 1:1,000 is more sensitive than FISH. To make the FLT3-LM assessment truly semiquantitative, we suggest that GeneScan analysis should be used at diagnosis for better prognostication [7]. During follow-up this method would also improve the estimation of the reduction of the leukemic cells in comparison to standard PCR and gel electrophoresis. It could be shown that realtime quantification with patient-specific primers for individual FLT3-LM is applicable and highly specific and sensitive. Thus in the future a highly sensitive and quantitative PCR may still improve the use of FLT3-LM as a follow-up marker. However, this approach is time consuming and expensive and for prospective assessment of the FLT3-LM in clinical studies it does not seem to be feasible for most of the diagnostic labs. As FLT3-LM characterizes an unfavourable subset of the intermediate group with an increased risk for relapse it is of high importance to monitor especially this group. With the present study it could be shown that FLT3-LM indeed is a reliable marker to assess therapy response and it was even possible to detect relapse early up to 3 months before a clinical relapse. In 94% of cases with FLT3-LM at diagnosis it is also present at relapse. However, the amount of the mutation in relation to the wild-type allele is increasing in 63% of the cases. It seems to be a common signature of the FLT-LM that the status is increasing in the course of unfavourable disease. Although the applicability of FLT3-LM as marker for follow-up controls was regarded quite critically [43, 45] the results of the presented study are encouraging. This study using conventional qualitative PCR shows that FLT3 is a reliable marker for follow-up controls in most of the patients carrying an FLT3-LM at diagnosis.

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42 Hovland R, Gjertsen BT, Bruserud O: Acute myelogenous leukemia with internal tandem duplication of the Flt3 gene appearing or altering at the time of relapse: A report of two cases. Leuk Lymphoma 2002;43:2027–2029. 43 Kottaridis PD, Gale RE, Langabeer SE, Frew ME, Bowen DT, Linch DC: Studies of FLT3 mutations in paired presentation and relapse samples from patients with acute myeloid leukemia: Implications for the role of FLT3 mutations in leukemogenesis, minimal residual disease detection, and possible therapy with FLT3 inhibitors. Blood 2002;100:2393–2398. 44 Shih LY, Huang CF, Wu JH, Lin TL, Dunn P, Wang PN, Kuo MC, Lai CL, Hsu HC: Internal tandem duplication of FLT3 in relapsed acute myeloid leukemia: A comparative analysis of bone marrow samples from 108 adult patients at diagnosis and relapse. Blood 2002;100: 2387–2392. 45 Gilliland DG: Murky waters for MRD detection in AML: Flighty FLT3/ITDs. Blood 2002; 100:2277B. 46 Whitman SP, Archer KJ, Feng L, Baldus C, Becknell B, Carlson BD, Carroll AJ, Mrozek K, Vardiman JW, George SL, Kolitz JE, Larson RA, Bloomfield CD, Caligiuri MA: Absence of the wild-type allele predicts poor prognosis in adult de novo acute myeloid leukemia with normal cytogenetics and the internal tandem duplication of FLT3:A cancer and leukemia group B study. Cancer Res 2001;61:7233– 7239.

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47 Kottaridis PD, Gale RE, Linch DC: Flt3 mutations and leukaemia. Br J Haematol 2003;122: 523–538. 48 Nakano Y, Kiyoi H, Miyawaki S, Asou N, Ohno R, Saito H, Naoe T: Molecular evolution of acute myeloid leukaemia in relapse: Unstable N-ras and FLT3 genes compared with p53 gene. Br J Haematol 1999;104:659–664. 49 Schnittger S, Schoch C, Kern W, Haferlach T, Hiddemann W: FLT3-LM and MLL-PTD as markers for PCR-based detection of minimal residual disease (MRD) in AML with normal karyotype. Blood 2001;98:581A. 50 Hovland R, Gjertsen BT, Bruserud O: Acute myelogenous leukemia with internal tandem duplication of the Flt3 gene appearing or altering at the time of relapse: a report of two cases. Leuk Lymphoma 2002;43:2027–2029. 51 Stirewalt DL, Willman CL, Radich JP: Quantitative, realt-ime polymerase chain reactions for FLT3 internal tandem duplications are highly sensitive and specific. Leuk Res 2001;25:10851088.

Schnittger/Schoch/Kern/Hiddemann/ Haferlach

Review Acta Haematol 2004;112:79–84 DOI: 10.1159/000077562

WT1 as a Universal Marker for Minimal Residual Disease Detection and Quantification in Myeloid Leukemias and in Myelodysplastic Syndrome Daniela Cilloni Giuseppe Saglio Division of Hematology and Internal Medicine, Department of Clinical and Biological Sciences, University of Turin, Turin, Italy

Key Words Acute leukemia W Minimal residual disease W WT1

presence of MRD in all the patients affected by acute and chronic leukemias as well as myelodysplastic syndromes. Copyright © 2004 S. Karger AG, Basel

Abstract Monitoring of acute leukemia patients during and after treatment for the presence of remaining leukemic cells (minimal residual disease, MRD) has been shown to give major insight into the effectiveness of treatment. However, so far the applicability of this strategy has been limited to those leukemia subsets characterized by genetic markers amenable to sensitive detection by PCR. Although PCR for immunoglobulin and T cell receptor gene rearrangement represents the gold standard for MRD detection in most cases of ALL without any fusion gene transcripts as molecular markers available, the situation in AML is more complicated because, at present, more than 50% of them lack any sort of clonality markers suitable for MRD monitoring. Thus, a number of studies have been performed in an attempt to identify cytogenetic and molecular abnormalities associated with leukemic transformation. In this paper we describe the effectiveness of the quantitative assessment of the Wilms tumor gene (WT1) transcript as a molecular marker for the detection of the leukemic clone useful for monitoring the

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The Wilms Tumor Gene

Over the last 20 years attention was focused on the WT1 gene because of its involvement in the pathogenesis of a particular childhood kidney tumor [1–3]. The WT1 gene, cloned in 1990 by Call et al. [4], encodes for a protein with the characteristics of a zinc finger transcription factor. So far, few genes have been found which are physiologically regulated by WT1, among them the epidermal growth factor receptor, syndecan 1, bcl-2, amphiregulin and E-cadherin [5–9]. Unlike the ubiquitous expression of other tumor suppressor genes such as RB1 and p53, the expression of WT1 is restricted to a small number of tissues [10]. WT1 mRNA was detected in baboon and in mouse kidney and spleen [11]. Therefore, in situ, mRNA hybridization experiments have shown that the expression of WT1 also occurs in the testis, ovaries, myometrium, stromal cells of the uterus, mesothelial cell linings of body cavities and

Giuseppe Saglio, MD Department of Clinical and Biological Sciences of the University of Turin San Luigi Hospital, Gonzole 10 IT–10043 Orbassano-Torino (Italy) Tel. +39 011 9026610, Fax +39 11 9038636, E-Mail [email protected]

visceral organs such as the heart, lung, intestine, liver and in the supportive stroma and splenic capsule of the spleen [12]. In contrast, several other tissues and cell lines were negative for WT1 expression [11]. Although the role of the WT1 gene in the development of malignancies in the kidney appears quite well defined, currently its potential function in human hematopoiesis still needs to be clarified. WT1 may contribute to the development of blood cells as suggested by its expression in early hematopoietic precursors and its rapid downregulation following differentiation in primary blood cells and leukemia-derived cell lines [13–16]. The role of WT1 in the leukemogenesis process appears controversial. The majority of human acute myeloid (AML) and lymphoblastic (ALL) leukemias express high levels of wild-type WT1 [17, 18] suggesting that this tumor suppressor might have paradoxical oncogenic activity in the hematopoietic cells. Attempts to define the consequences of WT1 expression in leukemia-derived cell lines have also produced conflicting results, reporting enhanced cellular proliferation in some systems but induction of growth arrest in others [19–23]. Svedberg et al. [23] and Deuel et al. [20] demonstrated that cell lines permanently transfected with WT1 constructs showed defects in the response to differentiating agents and that this inhibiting effect may contribute to the genesis of leukemia [19, 20, 23]. Recent studies by Ellisen et al. [24] have demonstrated that the effect of WT1 on hematopoietic precursor subsets appears to be stage specific, being an inducer of cellular differentiation in lineage-committed precursors while it enhances cellular quiescence in more primitive cells. Although the potential usefulness of WT1 expression as a panleukemic marker was envisaged by Inoue et al. [21] several years ago, its introduction into clinical practice was limited principally by the background expression level normally detected in bone marrow (BM) samples from healthy volunteers that derived from a restricted subset of normal hematopoietic precursors. Consequently, WT1 was designated as a characteristic marker of cell immaturity [14]. More recent data obtained using quantitative RT-PCR methods established that sorted populations of normal progenitors express WT1 at very low levels, sometimes even undetectable by very sensitive methods of nested RT-PCR [25–28]. The recent introduction of the quantitative PCR as a routine technique available in an increasing number of laboratories provides the WT1 with a new significance in terms of a universal marker for MRD detection.

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WT1 Expression in Acute Leukemias and in Myelodysplastic Syndromes

In the last few years an increasing number of papers have been published on the overexpression of WT1 in different types of hematological malignancies focusing on acute and chronic leukemias [17, 18, 29, 30]. Moreover, basing the assessment of WT1 transcript on a precise method of quantitative PCR, all the groups agree in finding a significant difference between the expression levels in normal controls and in leukemic samples, in this way overcoming the obstacle represented by the low amount of WT1 transcript present in normal hematopoiesis. Using a real-time quantitative PCR, we were able to demonstrate the presence of high levels of WT1 in the majority of cases of AML and ALL (fig. 1) at the onset of disease as well as in the different phases of chronic myeloid leukemias [21]. In addition the patients affected by myelodysplastic syndromes (MDS) also express an increased amount of WT1 transcript with a variable level according to the subtypes of MDS [31]. By analyzing a large number of normal BM and peripheral blood (PB) samples (32 BM and 45 PB) we also established that the majority of the PB samples scored negative and the median number of WT1 copies/104 ABL copies detected in the positive samples was very low (4 with a range of 1–22). By contrast, all the normal BM samples scored positive, but, even if higher than in PB samples, the median number of WT1 copies remained low (median value 78, range 3–180). To gain further insight into the theory which attributes the abnormal levels of WT1 expression to the presence of cells with a high degree of immaturity, we tested WT1 expression in regenerating BM samples obtained from AML patients in complete remission during recovery from chemotherapy-induced aplasia. The median value of WT1 was similar (74 WT1 copies/104 ABL) to that detected in normal BM. Similar results (median value 50; range 40–61) were obtained by analyzing several samples of enriched CD34-positive cells obtained from normal PB stem cell donors. These data confirm that the CD34-positive cell compartment is not responsible for the increased WT1 expression detected in leukemias. These results lead to the conclusion that the WT1 overexpression found in leukemic samples is not due to the degree of immaturity of the cell population, but intrinsically related to the presence of leukemic cells. Conversely, the quantitative assessment carried out in 71 AML BM and 14 PB samples showed a median WT1 copy number of 27,669 in BM samples (ranges: 1,081–

Cilloni/Saglio

Fig. 1. WT1 copy number evaluated by RQ-PCR in BM samples from healthy volunteers and in different subtypes of

AML and ALL. The differences in WT1 expression levels in the different subgroups of AML are significant from a statistical point of view only in acute promyelocytic leukemia cases. In ALL cases, the BM samples characterized by the translocation t(4;11) and t(9;22) express significantly higher levels of WT1 than in all other cases whereas the t(1;19) translocation defines a subgroup which presents a low WT1 expression.

121,806) and 10,244 in PB samples (range 758– 86,140). No statistical difference between cytogenetic groups and WT1 expression levels was found except for the t(15;17) APL cases (10 cases analyzed) that expressed a significantly higher amount of WT1 with respect to all other groups considered together. In line with the results obtained in our study, Kreuzer et al. [32] analyzing 43 BM samples from AML patients were also able to demonstrate a significant difference between the normal controls and the leukemic samples and similar data have also been observed in the series of childhood AML analyzed by Trka et al. [30]. WT1 overexpression has also been found in all the ALL cases tested by us (48 BM and 16 PB samples), but the median copy number is generally lower than in AML with a median of 1,388 copies (range 212–34,708) in PB and of 13,807 (range 318–94,682) in BM. The t(4;11) and

the t(9;22) cases represented the two cytogenetically defined groups expressing levels of WT1 which were significantly higher than in all the other cases, whereas in contrast the t(1;19) translocation seems to define a subgroup which presents a low WT1 expression. Concordant data have also been reported in the series of 12 ALL cases analyzed by Kreuzer et al. [32]. Finally, we tested the WT1 expression in a large series of MDS patients (also including a number of secondary leukemias). The data we obtained show that in most MDS, including approximately two thirds of refractory anemia (RA), WT1 expressed is above the range observed in normal controls in both BM and PB and that its expression is directly correlated to the type of MDS, being higher in a statistically significant manner in RA with respect to RAEB and to secondary AML. In addition, even within each subgroup, there is a very good association between

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the level of WT1 expression and the blast percentage and the presence of cytogenetic alterations. This finally results in a very strict correlation, highly significant from the statistical point of view, between the amount of WT1 transcripts in the BM and IPSS, the most widely accepted risk scoring system for MDS. The capacity of WT1 levels to distinguish between different prognostic IPSS subgroups in MDS appears particularly high for patients of the intermediate-low risk compared to patients of the intermediate-high risk group, where a level of 103 copies of WT1 every 104 ABL copies seems to represent a discriminating threshold. As already suggested by Tamaki et al. [33], our data show that a longitudinal monitoring of the WT1 levels may represent a good marker to establish disease progression in MDS and, in addition, it may also help to distinguish patients prone to progress from those who probably will not. If there is enough agreement on the pattern of WT1 expression in leukemia cases [25, 30, 32], major discrepancies still exists about the possible prognostic significance of the amount of WT1 present at the onset of the disease. Whereas Schmid et al. [34] and Gaiger et al. [26] reported that WT1 levels detected at diagnosis in both AML or ALL have no prognostic significance on patients’ outcome, other studies have shown that patients with high levels of WT1 transcripts at diagnosis tended to have a worse outcome compared to those with low levels. In agreement with these data, Trka et al. [30] recently reported in a pediatric group of AML a significant difference in terms of event-free survival and leukemia-free survival for patients with high versus low WT1 levels at presentation of leukemia. To better clarify this point, larger series of patients need to be analyzed with a well-standardized method of quantitative assessment of the amount of WT1.

WT1 as a Marker of MRD

Since the RQ-PCR methods clearly distinguish between the level of WT1 transcripts in normal and leukemic cells, WT1 expression can represent a molecular marker extremely useful in the clinical setting of MRD assessment. In particular in cases lacking cytogenetic lesions, it may help to establish the response to therapy and to monitor the behavior of the leukemia clone during follow-up. To validate the role of WT1 as a marker of MRD, we studied a number of patients bearing a fusion gene transcript suitable for the quantitative assessment of the

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amount of MRD by RQ-PCR and performed a simultaneous analysis of the amount of WT1 at sequential time intervals during the follow-up [25]. The WT1 levels were shown to be strictly parallel to the behavior of the other molecular markers (fusion gene transcripts) used for the MRD monitoring. Furthermore, increased WT1 expression above the range found in normal BM and/or in normal PB samples during follow-up of AML patients was always found to be predictive of an impending hematological relapse even in AML patients lacking additional molecular markers. The increase of the WT1 levels could also precede the occurrence of the overt hematological relapse by some months, although the kinetics of the relapse appears highly variable. In contrast, normal WT1 values have always been found to be associated with persisting remissions. Therefore, evaluation of the WT1 expression could represent a sort of universal marker that can allow a rather sensitive evaluation of the MRD in all AML patients with a degree of sensitivity that can be estimated to reach in most cases between 10 –3 and 10 –4 [25, 30, 32]. Furthermore, the findings of extremely low and often undetectable WT1 levels in the PB of normal individuals and in leukemia patients in continuous complete remission suggest that PB could be even more sensitive than BM in revealing impending relapses. Although this point still needs to be demonstrated, replacement of BM with PB sampling could greatly improve patients’ compliance and considerably simplify molecular monitoring. The clinical application of the WT1 gene as a molecular marker for MRD detection was already suggested by Inoue et al. [35] in 1996 and confirmed later on by different studies. Recently Trka et al. [30] reported data obtained in a pediatric group of AML patients in whom the presence of MRD was assessed by flow-cytometric analysis for the blast cell count. They found a strict correlation between the levels of WT1 transcript and the percentage of blast cells detected by flow-cytometric analysis. Their results confirm the finding that clinical remission is constantly associated with low levels of WT1 while increasing values are associated with relapse. Finally, similarly to what was demonstrated for AML [25, 30, 32], WT1 levels also seem to represent a good marker for MRD detection in MDS patients treated with intensive therapies aimed at disease eradication [31].

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WT1 as a Marker of MRD after BM Transplantation

The usefulness of the WT1 quantitative assessment by RQ-PCR as a marker for MRD detection after allogeneic BM transplantation has recently been investigated by Ogawa et al. [36] in a series of 72 patients affected by different types of leukemias, AMLs, ALLs and CMLs. According to their data, the amount of WT1 transcripts after BM transplantation is lower than in BM samples of normal controls, and therefore, in this particular setting the determination of the amount of WT1 by RQ-PCR acquires a highly significant value in terms of the possibility of being predictive of imminent relapses. Unpublished data from our group are concordant with the conclusion that even in the transplant setting, as already demonstrated for acute leukemia patients treated with intensive chemotherapy, the determination of the amount of WT1 can represent a useful marker to monitor the persistence or the reappearance of leukemic cells and that the finding

of increasing amounts of WT1 transcript during follow-up is predictive of relapse. However, we were not able to find differences in the amount of WT1 transcript expressed by normal controls with respect to transplanted patients in persisting remission soon after transplant or later on. This discrepancy is more probably due to differences in the procedures used. For this reason, although new quantitative real-time procedures promise to simplify the protocols that are currently in use, standardization and the introduction of rigorous, internationally accepted controls are required to enable RQ-PCR for WT1 transcript quantitative assessment to become a robust and routine basis for therapeutic decisions.

Acknowledgments This work was supported by grants from AIRC (Associazione Italiana per la Ricerca sul Cancro) and AIL (Associazione Italiana contro le Leucemie).

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8 Hosono S, Gross I, English MA, Haira KM, Fearon ER, Licht JD: E-cadherin is a WT1 target gene. J Biol Chem 2000;275:10943–10953. 9 Lee SB, Huang K, Palmer R, Troung VB, Herzlinger D, Kolquist KA, Wong J, Paulding C, Yoon SK, Gerald W, Oliner JD, Haber DA: The Wilms tumor suppressor WT1 encodes a transcriptional activator of amphiregulin. Cell 1999;98:663–673. 10 Park S, Schalling M, Bernard A, Maheswaran S, Shipley GC, Roberts D, Fletcher J, Schpman R, Rheinwald J, Demetri G: The Wilms tumour gene WT1 is expressed in murine mesoderm-derived tissues and mutated in a human mesothelioma. Nat Genet 1993;4:415–420. 11 Little M, Holmes G, Walsh P: WT1:What has the last decade told us? Bioessay 1999;21:191– 202. 12 Pritchard-Jones K, Fleming S: Cell types expressing the Wilms’ tumour gene (WT1) in Wilms’ tumour: Implications for tumour histogenesis. Oncogene 1991;6:2211–2220. 13 Baird PM, Simmons PJ: Expression of the Wilms tumor gene (WT1) in normal hemopoiesis. Exp Hematol 1997;25:312–320. 14 Maurer U, Brieger J, Weidmann E, Mitrou PS, Hoelzer D, Bergmann L: The Wilms’ tumor gene is expressed in a subset of CD34+ progenitors and downregulated early in the course of differentiation in vitro. Exp Hematol 1997;25: 945–950.

15 Menssen HD, Renkl HJ, Entezami M, Thiel E: Wilms’ tumor gene expression in human CD34+ hematopoietic progenitors during fetal development and early clonogenic growth. Blood 1997;89:3486–3487. 16 Sekiya M, Adachi M, Hinoda Y, Imai K, Yachi A: Downregulation of the Wilms’ tumor gene (wt1) during myelomonocytic differentiation in HL60 cells. Blood 1994;83:1876–1882. 17 Menssen HD, Renkl HJ, Rodeck U, Maurer J, Nutter M, Schwartz S, Reinhardt R, Thiel E: Presence of Wilms’ tumor gene (WT1) transcripts and WT1 nuclear protein in the majority of human acute leukemias. Leukemia 1995; 9:1060–1067. 18 Miwa H, Beran M, Saunders GF: Expression of the Wilms’ tumor gene (WT1) in human leukemias. Leukemia 1992;6:405–409. 19 Algar EM, Khromykh T, Smith SI, Blackburn DM, Bryson GJ, Smith PJ: A WT1 antisense oligonucleotide inhibits proliferation and induces apoptosis in myeloid leukemia cell lines. Oncogene 1996;12:1005–1014. 20 Deuel TF, Guan LS, Wang ZY: Wilms’ tumor gene product WT1 arrests macrophage differentiation of HL60 cells through its zinc-finger domain. Biochem Biophys Res Commun 1999; 254:192–196. 21 Inoue K, Ogawa H, Sonoda Y, Kimura T, Sakabe H, Oka Y, Miyake S, Tamaki H, Oji Y, Yamagami T, Tatekawa T, Soma T, Kishimoto T, Sugiyama H: Aberrant overexpression of the Wilms tumor gene (WT1) in human leukemia. Blood 1997;89:1405–1412.

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22 Smith SI, Weil D, Johnson GR, Boyd AW, Li CL: Expression of the Wilms’ tumor suppressor gene, WT1 is upregulated by leukemia inhibitory factor and induces monocytic differentiation in M1 leukemic cells. Blood 1998;91:764– 773. 23 Svedberg H, Chylicki K, Baldetorp B, Rauscher FL 3rd, Gullberg U: Constitutive expression of the Wilms’ tumor gene (WT1) in the leukemic cell line U937 blocks part of the differentiation program. Oncogene 1998;16:925– 932. 24 Ellisen LW, Carlesso N, Cheng T, Scadden DT, Haber DA: The Wilms tumor suppressor WT1 directs stage-specific quiescence and differentiation of human hematopoietic progenitors cells. EMBO J 2001;20:1897–1909. 25 Cilloni D, Gottardi E, De Micheli D, Serra A, Volpe G, Messa F, Rege-Cambrin G, Guerrasio A, Divona M, Lo Coco F, Saglio G: Quantitative assessment of WT1 expression by real time quantitative PCR may be a useful tool for monitoring minimal residual disease in acute leukemia patients. Leukemia 2002;16:2115– 2121. 26 Gaiger A, Schmid D, Heinze G, Linnerth B, Greinix H, Halhs P, Tisljar K, Prigljnger S, Mitterbauer M, Novak M, Mitterbauer G, Mannhalter C, Haas OA, Lechner K, Jäger U: Detection of the WT1 transcript by RT-PCR in complete remission has no prognostic relevance in de novo acute myeloid leukemia. Leukemia 1998;12:1886–1894.

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27 Inoue K, Tamaki H, Ogawa H, Oka Y, Soma T, Tatekawa T, Oji Y, Tsuboi A, Kim EH, Kawakami M, Akiyama T, Kischimoto T, Sugiyama H: Wilms’ tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells. Blood 1998;91:2969–2976. 28 Maurer U, Weidmann E, Karakas T, Hoelzer D, Bergmann L: Wilms tumor gene (wt1) mRNA is equally expressed in blast cells from acute myeloid leukemia and normal CD34+ progenitors. Blood 1997;90:4230–4232. 29 Menssen HD, Siehl JM, Thiel E: Wilms tumor gene (WT1) expression as a panleukemic marker. Int J Hematol 2002;76:103–109. 30 Trka J, Kalinova M, Hrusak O, Zuna J, Krejci O, Madzo J, Sedlacek P, Vavra V, Michalova K, Jarosova M, Stary J: Real time quantitative PCR detection of WT1 gene expression in children with AML: Prognostic significance, correlation with disease status and residual disease detection by flow cytometry. Leukemia 2002; 16:1381–1389. 31 Cilloni D, Messa F, Gottardi E, Fava M, Scaravaglio P, Bertini M, Girotto M, Marinone C, Ferrero D, Gallamini A, Levis A, Saglio G: Very significant correlation between WT1 expression level and the IPSS score in patients with myelodysplastic syndromes. J Clin Oncol 2003;21:1988–1995. 32 Kreuzer KA, Saborowski A, Lupberger J, Appelt C, Na IK, Le Coutre P, Schmidt CA: Fluorescent 5)-exonuclease assay for the absolute quantification of Wilms’ tumour gene (WT1) mRNA: Implications for monitoring human leukemias. Br J Haematol 2001;114:313–318.

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33 Tamaki H, Ogawa H, Ohyashiki K, Ohyashiki JH, Iwama H, Inoue K, Soma T, Oka Y, Tatekawa T, Oji Y, Tsuboi A, Kim EH, Kawakami M, Fuchigami K, Tomonaga M, Toyama K, Aozasa K, Kishimoto T, Sugiyama H: The Wilms’ tumor gene WT1 is a good marker for diagnosis of disease progression of myelodysplastic syndromes. Leukemia 1999;13:393– 399. 34 Schmid D, Heinze G, Linnerth B: Prognostic significance of WT1 gene expression at diagnosis in adult de novo acute myeloid leukemia. Leukemia 1997;11:639–643. 35 Inoue K, Ogawa H, Yamagami T, Soma T, Tani Y, Tatekawa T, Oji Y, Tamaki H, Kyo T, Dohy H, Hiraoka A, Masaoka T, Koshimoto T, Sugiyama H: Log-term follow-up of minimal residual disease in leukemia patients by monitoring WT1 (Wilms tumor gene) expression levels. Blood 1996;88:2267–2278. 36 Ogawa H, Tamaki H, Ikegame K Soma T, Kawakami M, Tsuboi A, Kim EH, Hosen N, Murakami H, Fujioka T, Masuda T, Taniguchi Y, Nischida S, Oji Y, Oka J, Sugiyama H: The usefulness of monitoring WT1 gene transcripts for the prediction and management of relapse following allogeneic stem cell transplantation in acute type leukemia. Blood 2003;101:1698– 1704.

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Review Acta Haematol 2004;112:85–92 DOI: 10.1159/000077563

Molecular Surveillance of Chronic Myeloid Leukemia Patients in the Imatinib Era – Evaluation of Response and Resistance Peter Paschka Kirsten Merx Andreas Hochhaus III. Medizinische Klinik, Fakultät für Klinische Medizin Mannheim der Universität Heidelberg, Mannheim, Germany

Key Words Chronic myeloid leukemia W Fluorescence in situ hybridization W Imatinib W Minimal residual disease W Molecular cytogenetics W Quantitative RT-PCR

Abstract Residual disease in chronic myeloid leukemia patients may be assessed by various molecular methods. After imatinib treatment a significant proportion of patients achieve complete cytogenetic remission (CCR) and a sensitive method is necessary to monitor treatment response and to detect early signs of relapse. Reversetranscriptase polymerase chain reaction (RT-PCR) is by far the most sensitive approach to assess residual disease in this group of patients. Qualitative PCR methods give only limited information about the residual leukemic mass. Quantitative RT-PCR (Q-PCR) assays enable to monitor the kinetics of residual BCR-ABL transcripts over time in patients with a good response to imatinib. Early Q-PCR results on imatinib treatment can help to identify individuals who are likely to have a good response. In chronic phase patients after CCR, Q-PCR may identify patients who are likely to continue with their CCR or to relapse and may help to optimize treatment for this group of patients. The definition of molecular surrogate endpoints beyond CCR for studies which are currently planned demands standardization of the nomenclature and of technologies to measure these targets. Copyright © 2004 S. Karger AG, Basel

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Introduction

Nonquantitative polymerase chain reaction (PCR) methods have been developed to detect various types of BCR-ABL transcripts, which arise as the consequence of the reciprocal translocation t(9;22)(q34;q11) forming the Philadelphia (Ph) chromosome [1–3]. Despite its high sensitivity nested reverse-transcriptase (RT)-PCR is limited by the qualitative nature of the assay and the considerable risk of contamination. Quantitative RT-PCR assays including competitive or real-time RT-PCR procedures (Q-PCR) were developed to monitor the evolution of residual leukemic load over time to better assess response to treatment in patients with chronic myeloid leukemia (CML) [4–12]. Recently, molecular endpoints and minimal residual disease (MRD) analysis have started to be implemented in prospective treatment protocols. Molecular data are increasingly being used for the evaluation of the efficacy of imatinib-based treatment protocols [13–17].

Assessing MRD

Minimal Residual Disease A major objective of residual disease analysis in CML is an improved response assessment to therapy in individual patients or evaluating new treatment modalities, e.g. imatinib, in a cohort of patients. Phase II data demonstrate complete cytogenetic remission (CCR) in 41% [18]

Dr. med. Peter Paschka III. Medizinische Universitätsklinik, Fakultät für Klinische Medizin Mannheim der Universität Heidelberg, Wiesbadener Strasse 7–11, DE–68305 Mannheim (Germany) Tel. +49 621 383 4232, Fax +49 621 383 3833 E-Mail [email protected]

of chronic phase (CP) patients after a failure of interferon-· (IFN) therapy, in 17% [19] of accelerated phase patients, and even 7% of patients in myeloid blast crisis [20]. In newly diagnosed patients, the proportion of CCR after 18 months of therapy amounts to 76% [21]. Conventional cytogenetic analysis using banding techniques is generally employed in clinical practice to detect the Ph chromosome and additional chromosomal aberrations and to monitor the response to treatment. Considering that patients at diagnosis or at relapse usually harbor a tumor load of about 1012 cells [22], patients with negative cytogenetic analysis, which has a routine sensitivity of about 1–10%, may still harbor up to 1010 leukemic cells [23]. The presence of malignant cells in CCR patients are referred to as ‘minimal residual disease’ (MRD). In CML patients treated with IFN the degree of tumor load reduction, i.e. the residual amount of BCR-ABL+ cells, has been shown to be an important prognostic factor for the stability of achieved CCR [24]. After CCR, cytogenetic analysis does not provide any information about the amount of residual leukemic mass. Moreover, conventional cytogenetic analysis requires dividing cells and cannot be applied in Ph-negative, but BCR-ABL+ CML patients. An advantage of the cytogenetic analysis is the detection of other chromosomal aberrations which may indicate acceleration of the disease or clonal proliferation of Ph-negative hematopoiesis [25–28]. The optimal method for MRD analysis should be sensitive, easy to perform, should preferably be using peripheral blood (PB), and frequent testing should be possible in order to assess response to treatment and to recognize early signs of relapse. Quantitative MRD data could help to stratify patients according to the probability of response to imatinib or to risk of relapse, which is of particular importance in developing risk-orientated treatment protocols [13, 15–17]. Fluorescence in situ Hybridization Analysis The use of fluorescence in situ hybridization (FISH) techniques like interphase FISH (IP-FISH) and hypermetaphase FISH (HM-FISH) increases the sensitivity of cytogenetic analysis to detect the BCR-ABL fusion gene in Ph-positive cells at the molecular level. However, even these tests became negative in a significant proportion of CML patients after CCR [15, 29, 30]. The use of FISH with probes mapping to BCR and ABL enables the detection of cells with the typical Ph translocation on interphase or metaphase nuclei [31]. FISH analysis is not dependent on the appearance of the typical Ph chromosome and therefore rare BCR-ABL fusions and variant Ph translocations can be visualized in interphases as well as on metaphases [31–34]. We observed that IP-FISH per-

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formed on bone marrow (BM) specimens still revealed BCR-ABL-positive cells in patients with CCR on imatinib [15]. IP-FISH does not require dividing cells, but the variant proportion of false-positive cells, i.e. the rate with which BCR and ABL signals randomly colocalize in normal cells, limits the quantitative assessment of residual disease [35–37]. In clinical practice the limit of detection of CML cells by IP-FISH ranges typically between 1 and 5% and depends on the probe selection system, the size of the nucleus, the precise position of the breakpoint within ABL gene and criteria used to define colocalization [35]. We observed a median proportion of 3.3% BCR-ABL+ cells in samples obtained during CCR after imatinib treatment. Therefore, IP-FISH did not provide further information for this group of patients because the lab-specific cutoff for false-positive results was 5% [15]. The sensitivity of IP-FISH can be improved by introducing an additional probe that permits identification of both Ph chromosome and the derivative chromosome 9 in Ph-positive cells, thus lowering the rate of false-positive cells, or by choosing breakpoint spanning probes that result in two fusion signals. Definition of more stringent colocalization criteria (at a cost of increasing the false-negative rate) can reduce the false-positivity rate. New FISH strategies that use probes spanning the breakpoint region [38, 39] or the use of probes that additionally detect the ABL-BCR fusion at the derivative 9q+ chromosome may help to reduce the rate of false-positive results [40–42]. Large deletions of the ABL-BCR gene and flanking regions on the 9q+ chromosome prevent the application of more sensitive FISH techniques in a significant minority of patients [43–45]. HM-FISH combines FISH with long-term colcemid exposure of BM cultures [46] and is highly effective in monitoring of low levels of leukemic cells [15, 29, 30]. This method makes it possible to evaluate 400–500 metaphases, which minimizes the sampling error and the rate of false-positive results because of analyzing metaphases instead of interphases. One limitation of HM-FISH is the inability to detect cytogenetic clonal evolution, because it focuses on scoring the single molecular-cytogenetic event (juxtaposition of BCR and ABL) and the highly contracted metaphases produced by this technique are not suitable for cytogenetic analysis. HM-FISH was reported to detect in CML patients BCR-ABL+ metaphases in 20% of cases that showed no Ph-positive metaphases in chromosome banding analysis [30]. Our experience with imatinib-treated patients shows that HM-FISH is able to detect BCR-ABL+ in about 31% when chromosome banding remains negative [15].

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Reverse-Transcriptase Polymerase Chain Reaction Since 1989, when MRD testing by PCR in CML patients after allogeneic BM transplantation was invented, PCR applications have been further developed and optimized [4–7, 9–12, 47–51]. In CML, genomic breakpoints, particularly within the ABL gene, are too widely dispersed to allow a simple genomic DNA-based amplification. The combination of RNA transcription and PCR (RT-PCR) enables the amplification of individual RNA after cDNA synthesis [52] and is by far the most sensitive method for detection and surveillance of MRD in CML. Quantitative PCR techniques were routinely implemented for monitoring of CML patients in treatment protocols with the novel tyrosine kinase inhibitor imatinib [13, 15–17]. Qualitative RT-PCR The standard method to screen for BCR-ABL transcripts at diagnosis is a robust single step multiplex RTPCR, which is able to detect all types of BCR-ABL transcripts and BCR transcripts as an internal control by using three BCR and one ABL primers [1]. This method has a low sensitivity but high specificity with a low risk of falsepositive results due to contamination. For patients on treatment, the sensitivity level of qualitative PCR assays can be improved up to 10 –6 using a two-step (‘nested’) RTPCR [53]. This qualitative method had considerably improved the MRD detection and has become widely used for MRD detection after allogeneic stem cell transplantation. With extremely powerful RT-PCR techniques based on the analysis of large blood volumes (400 ml), BCR-ABL mRNA can be even detected with a sensitivity of 10 –8, but these results must be interpreted carefully, bearing in mind that BCR-ABL transcripts could even be detected in normal individuals at this level of sensitivity [54, 55]. Because of the lack of quantitative information, positive detection of BCR-ABL transcripts provides only restricted information about MRD in CML patients, as some patients, who have been tested PCR-positive, maintain their MRD state and even become PCR-negative [24]. Quantitative RTPCR-like competitive RT-PCR and real-time RT-PCR were developed and used to estimate the amount of residual leukemic mass in CML [4–12].

The equivalence point, at which competitor and sample bands are of equal intensity, can be determined by densitometry, which allows an estimation of the amount of transcripts in the unknown sample. An endogenous control gene, e.g. ABL, serves as a measure for the sample quality and is being quantified likewise. Results are expressed as the ratio BCR-ABL/ABL or as number of chimeric transcripts per microgram of RNA [4, 8]. Real-Time Quantitative RT-PCR Q-PCR permits, in contrast to the classical PCR endpoint analysis, an accurate quantification of PCR products during the exponential phase of the PCR amplification process. Because of the real-time detection of fluorescent signals during and/or after each subsequent PCR cycle, quantitative PCR data can be obtained in a short period of time and no post-PCR processing is needed, which reduces the risk of PCR product contamination. The two commonly used detection systems are fluorescence resonance energy transfer (FRET) and TaqMan. LightCycler monitoring of PCR amplification is based on the concept of FRET between two adjacent hybridization probes carrying donor and acceptor fluorophores. Excitation of a donor fluorophore (fluorescein) with an emission spectrum that overlaps the excitation spectrum of an acceptor fluorophore (LC Red 640) results in a nonradioactive energy transfer to the acceptor. This results in the emission of fluorescence signals which can be detected during the annealing phase and the first part of the extension phase of the PCR reaction. The fluorescence signal is plotted against the cycle number for the investigated samples and external standards consisting of serial plasmid dilutions. Using the LightCycler technology the CML cells could be detected between normal white blood cells with a sensitivity of 10 –5 [51]. The TaqMan assay uses the targetspecific dual-labeled fluorogenic hybridization probe, where one dye serves as fluorescent reporter (FAM, i.e. 6-carboxyfluoresceine) and one as quencher (TAMRA, i.e. 6-carboxy-tetramethyl-rhodamine) at its opposite ends. The probes are cleaved through the 5'-nuclease activity of Taq-DNA polymerase, leading to an increasing fluorescence emission of the reporter dye that can be detected during reaction [7, 9].

Competitive RT-PCR In principle, competitive RT-PCR is based on a titration assay. Serial dilutions of an artificially constructed BCR-ABL competitor are added to the same volume of the patients’ cDNA. Both, target and competitor, are amplified with the same primers, but the product sizes are different, and can be separated using gel electrophoresis.

Other Techniques Alternative techniques to assess residual leukemia like Southern or Western blot analysis, which provide quantitative results, are increasingly replaced by the techniques described above. Southern blot does not require dividing cells and PB can be used for analysis, but its sensitivity is

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comparable only with that achieved by conventional cytogenetic analysis and a major drawback is the need of autoradiography. Western blot analysis can detect BCR-ABL proteins in PB or BM specimens. The maximum sensitivity, which was determined by dilution experiments, is 0.2– 0.4%. This assay requires highly toxic reagents and extreme precaution in order to prevent protein degradation.

Clinical Implications of MRD Monitoring in Imatinib-Treated CML Patients

Considering the high proportion of patients in CCR after imatinib therapy, the need for sensitive monitoring methods to assess residual disease becomes obvious. RTPCR is the most sensitive and rapid method to monitor patients with a good response to imatinib therapy. Several working groups found a good correlation between conventional cytogenetic analyses and molecular data obtained by Q-PCR in imatinib-treated patients [13, 14, 56]. However, one of these groups, where only patients with CML in CP after IFN failure were treated with imatinib, reported about 10% major discordances when cytogenetic response groups (none, minor, partial, and complete) were compared to molecular response groups defined by cutoffs of PCR ratios BCR-ABL/ABL of 110, 2– 10 and !2% [57]. In patients with CP after failure of IFN therapy and with advanced stages of CML, who achieved a major cytogenetic remission after imatinib therapy, we observed in general a good correlation of HM-FISH results and QPCR results obtained from PB and BM. However, in 69% of CCR cases, HM-FISH was also negative and the only method to reveal and quantify residual disease was RTPCR [15]. In CCR patients, Q-PCR revealed BCR-ABL mRNA in 80% of cases in PB and 83% of cases in BM even when HM-FISH was negative. Most of the samples, which were negative by Q-PCR, still remained positive in the qualitative nested RT-PCR, which underlines the importance of this qualitative assay in detection of BCRABL transcripts below the detection level of Q-PCR [15]. For patients in CP after IFN failure who are treated with imatinib, Q-PCR results from PB and BM are comparable [15]. Q-PCR from PB is less invasive for a patient than cytogenetic analysis, which requires the aspiration of BM, and allows a more frequent monitoring of the response to therapy. Early molecular analysis at 2 months of imatinib therapy was found to be predictive for the cytogenetic response in this group of patients. The probability of major cytogenetic response after 6 months of

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imatinib therapy was significantly higher in patients having achieved a BCR-ABL/ABL ratio !20% within 2 months [13]. This result is supported by a recent report obtained from patients with CP and accelerated phase, where the 3-month Q-PCR values were found to predict a subsequent CCR. The median BCR-ABL/ABL ratio at 3 months in patients with a CCR at 6 months was with 0.53% significantly lower compared to 27% in patients who failed to achieve a CCR by 6 months [58]. In patients who achieve a CCR by imatinib the measurement of residual disease over time may help to identify patients who are likely to maintain their CCR or to relapse. Analyzing samples of nonrelapsing patients continuing with the imatinib therapy after CCR for a median period of 13 months shows that BCR-ABL levels decline over time. This suggests an ongoing process of quantitative disease depletion by imatinib [15]. Patients who achieve BCRABL/ABL levels !0.1% have a significantly lower risk of relapse as compared to patients with a best molecular response 10.1% [15]. Another study did not find that after a median follow-up of 26 months high PCR values predict a higher rate of relapse within the different cytogenetic response categories [57]. The risk of relapse in CML patients treated with imatinib is significantly higher in advanced stages of CML [19, 20, 59] than in CP [18]. In patients with advanced disease the loss of CCR has been observed despite having achieved BCR-ABL/ABL levels !0.1% [15]. Molecular results obtained from German patients with newly diagnosed CML, who were treated with imatinib as first-line therapy in the ‘International Randomized Study of Interferon and STI571’ (IRIS study) show that response to imatinib was independent of the level of BCR-ABL transcripts prior to therapy. Using a cutoff point of 10% for the BCR-ABL/ABL ratio after 3 months of therapy a CCR after 12 months could be predicted with a positive predictive value of 71% and a negative predictive value of 82%. In only 4 patients, who were treated with imatinib as firstline or as second-line therapy after crossover from IFN/ Ara-C, BCR-ABL became undetectable by both, Q-PCR and qualitative nested PCR. Patients with ongoing CCR achieved significantly lower levels of residual disease as compared to patients with a subsequent relapse. None of the patients with a best ratio BCR-ABL/ABL !0.1% relapsed, whilst about 10% of the patients with a ratio BCR-ABL/ABL 10.1% lost CCR. In imatinib- as well as in IFN/Ara-C-treated patients a gradual decline of residual disease after CCR was observed, but within an observation period of 12 months the decline was faster in imatinib-treated patients [17]. The molecular data from the

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international IRIS study examined as one objective the probability of progression in relation to reduction of BCR-ABL transcript levels in patients after imatinib treatment. All patients with CCR, who showed at least a 3-log reduction (comparable with a ratio BCR-ABL/ABL !0.1%) of BCR-ABL transcript levels at 12 months remained progression free at 24 months. In contrast, the probability of remaining progression free was 96% at month 24, when the patients achieved a less than 3-log reduction of BCR-ABL transcript levels at month 12 [16]. Quantitative assessment of residual disease will help to modify and optimize treatment with imatinib for individual patients over time. Standardized parameters should be used to measure and express residual disease levels within clinical trials, which is the goal of a recently launched International Study on Standardization of BCRABL quantification [60].

Mechanisms and Monitoring of Resistance to Imatinib

[69, 71–75] and Ph-positive ALL [69, 75, 76]. Mutations in the ABL tyrosine kinase domain may lead to various biological consequences: (1) a disturbed function of BCRABL that would lead to death of the individual cell and would not be detectable, (2) impaired binding of imatinib but retained binding of ATP, resulting in restoration of BCR-ABL function and clonal selection of mutated cells, (3) impaired binding of imatinib and ATP with reduced kinase activity that is sufficient to allow cellular survival with imatinib resistance, and (4) mutations of the activation loop may result in an activated conformation that is insensitive to inhibition by imatinib, as suggested previously [75]. Serial Q-PCR testing could help to identify patients who are going to become resistant to therapy in good time. The analysis of BCR-ABL kinase activity can be determined by assessment of tyrosine phosphorylation of the CRK-oncogene-like protein (CRKL) in leukocytes [77–81].

Conclusion

Particularly in patients with advanced phases of disease short-term relapses occurred despite the considerable proportion of initial responses to the therapy [19, 20, 61]. The mechanisms of resistance are heterogenous ranging from pharmacologic effects to specific molecular events. Increased levels of ·1-acid glycoprotein (AGP) have been reported to bind imatinib preventing the drug from inhibiting the BCR-ABL tyrosine kinase [62, 63]. Treatment of mice with drugs, which bind AGP, e.g. erythromycin, was able to overcome such resistance [62]. In vitro models using imatinib-resistant cell lines of BCR-ABL-transformed murine hematopoietic cells and BCR-ABL-positive human cell lines were developed to study the mechanisms of resistance. Mechanisms of imatinib resistance identified from these in vitro studies include reduction in the uptake of the drug by overexpression of the multidrug resistance P-glycoprotein, which is known to affect the uptake of a drug by pumping it out through the plasma membrane, the severalfold increase in the amount of BCR-ABL protein, and the amplification of the BCRABL gene [64–68]. Sensitivity to imatinib may be regained by its withdrawal from the cultures [66]. Furthermore, novel cytogenetic aberrations were found in resistant patients. In these patients aneuploidy, which is known to be associated with chromosomal instability, was observed in most cases [69, 70]. Mutations affecting imatinib binding have been described by several groups within the last 2 years in CML

Molecular analysis of residual disease in CML patients treated with imatinib may be achieved by several methods. Q-PCR, which is a reliable and sensitive method for monitoring of patients with a good response to imatinib, has been shown to have therapeutic implications, especially in CP, for predicting probability of response and of relapse to therapy. Moreover, the frequency of BM aspirations for cytogenetic analyses can be reduced and the measurements may be more acceptable to patients, because they are less invasive when PB is used. Frequent serial Q-PCR monitoring is important because it allows early detection of molecular relapse and optimizing treatment for this group of patients. Due to the high specificity and sensitivity of nested PCR methods, these are still of importance when Q-PCR remains negative. A longer follow-up is required to determine whether imatinib is capable of inducing durable molecular remissions. The standardization of RT-PCR methods and interpretation of results will help to reduce the interlaboratory variations and to improve the evaluation of the efficacy of treatment in clinical trials.

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Acknowledgement The work was supported by the competence network ‘Acute and chronic leukemias’, sponsored by the German Bundesministerium für Bildung und Forschung (BMBF), Projektträger Gesundheitsforschung e.V. – DLR, 01 GI9980/6.

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37 Seong DC, Song MY, Henske EP, Zimmerman SO, Champlin RE, Deisseroth AB, Siciliano MJ: Analysis of interphase cells for the Philadelphia translocation using painting probe made by inter-Alu-polymerase chain reaction from a radiation hybrid. Blood 1994;83:2268– 2273. 38 Buno I, Wyatt WA, Zinsmeister AR, DietzBand J, Silver RT, Dewald GW: A special fluorescent in situ hybridization technique to study peripheral blood and assess the effectiveness of interferon therapy in chronic myeloid leukemia. Blood 1998;92:2315–2321. 39 Dewald GW, Wyatt WA, Juneau AL, Carlson RO, Zinsmeister AR, Jalal SM, Spurbeck JL, Silver RT: Highly sensitive fluorescence in situ hybridization method to detect double BCR/ ABL fusion and monitor response to therapy in chronic myeloid leukemia. Blood 1998;91: 3357–3365. 40 Sinclair PB, Green AR, Grace C, Nacheva EP: Improved sensitivity of BCR-ABL detection: A triple-probe three-color fluorescence in situ hybridization system. Blood 1997;90:1395– 1402. 41 Dewald G, Stallard R, Alsaadi A, Arnold S, Blough R, Ceperich TM, Elejalde BR, Fink J, Higgins JV, Higgins RR, Hoeltge GA, Hsu WT, Johnson EB, Kronberger D, McCorquodale DJ, Meisner LF, Micale MA, Oseth L, Payne JS, Schwartz S, Sheldon S, Sophian A, Storto P, van Tuinen P, Wenger GD, Wiktor A, Willis LA, Yung JF, Zenger HJ: A multicenter investigation with D-FISH BCR/ABL1 probes. Cancer Genet Cytogenet 2000;116:97–104. 42 Grand F, Kulkarni S, Chase A, Goldman JM, Gordon MY, Cross NCP: Frequent deletion of hSNF5/INI1, a component of the SWI/SNF complex, during progression of chronic myeloid leukemia. Cancer Res 1999;59:3870–3874. 43 Sinclair PB, Nacheva EP, Leversha M, Telford N, Chang J, Reid A, Bench A, Champion K, Huntly B, Green AR: Large deletions at the t(9;22) breakpoint are common and may identify a poor-prognosis subgroup of patients with chronic myeloid leukemia. Blood 2000;95: 738–744. 44 Huntly BJ, Reid AG, Bench AJ, Campbell LJ, Telford N, Shepherd P, Szer J, Prince HM, Turner P, Grace C, Nacheva EP, Green AR: Deletions of the derivative chromosome 9 occur at the time of the Philadelphia translocation and provide a powerful and independent prognostic indicator in chronic myeloid leukemia. Blood 2001;98:1732–1738. 45 Kolomietz E, Al Maghrabi J, Brennan S, Karaskova J, Minkin S, Lipton J, Squire JA: Primary chromosomal rearrangements of leukemia are frequently accompanied by extensive submicroscopic deletions and may lead to altered prognosis. Blood 2001;97:3581–3588. 46 Seong DC, Kantarjian HM, Ro JY, Talpaz M, Xu J, Robinson JR, Deisseroth AB, Champlin RE, Siciliano MJ: Hypermetaphase fluorescence in situ hybridization for quantitative monitoring of Philadelphia chromosome-positive cells in patients with chronic myelogenous leukemia during treatment. Blood 1995;86: 2343–2349.

47 Morgan GJ, Hughes T, Janssen JW, Gow J, Guo AP, Goldman JM, Wiedemann LM, Bartram CR: Polymerase chain reaction for detection of residual leukaemia. Lancet 1989;i:928– 929. 48 Malinge MC, Mahon FX, Delfau MH, Daheron L, Kitzis A, Guilhot F, Tanzer J, Grandchamp B: Quantitative determination of the hybrid Bcr-Abl RNA in patients with chronic myelogenous leukaemia under interferon therapy. Br J Haematol 1992;82:701–707. 49 Thompson JD, Brodsky I, Yunis JJ: Molecular quantification of residual disease in chronic myelogenous leukemia after bone marrow transplantation. Blood 1992;79:1629–1635. 50 Nagel S, Schmidt M, Thiede C, Huhn D, Neubauer A: Quantification of Bcr-Abl transcripts in chronic myelogenous leukemia (CML) using standardized, internally controlled, competitive differential PCR (CD-PCR). Nucleic Acids Res 1996;24:4102–4103. 51 Emig M, Saussele S, Wittor H, Weisser A, Reiter A, Willer A, Berger U, Hehlmann R, Cross NCP, Hochhaus A: Accurate and rapid analysis of residual disease in patients with CML using specific fluorescent hybridization probes for real time quantitative RT-PCR. Leukemia 1999;13:1825–1832. 52 Chelly J, Kaplan JC, Maire P, Gautron S, Kahn A: Transcription of the dystrophin gene in human muscle and non-muscle tissue. Nature 1988;333:858–860. 53 van Dongen JJM, MacIntyre EA, Gabert JA, Delabesse E, Rossi V, Saglio G, Gotardi E, Rambaldi A, Dotti G, Giesinger F, Parreira A, Gameiro P, Gonzalez Diaz M, Malec M, Langerak AW, San Miguel JF, Biondi A: Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease. Leukemia 1999;12:1901–1928. 54 Biernaux C, Loos M, Sels A, Huez G, Stryckmans P: Detection of major bcr-abl gene expression at a very low level in blood cells of some healthy individuals. Blood 1995;88: 3118–3122. 55 Bose S, Deininger M, Gora-Tybor J, Goldman JM, Melo JV: The presence of typical and atypical BCR-ABL fusion genes in leukocytes of normal individuals: Biological significance and implications for the assessment of minimal residual disease. Blood 1998;92:3362–3367. 56 Kantarjian HM, Talpaz M, Cortes J, O’Brien S, Faderl S, Thomas D, Giles F, Rios MB, Shan J, Arlinghaus R: Quantitative polymerase chain reaction monitoring of BCR-ABL during therapy with imatinib mesylate (STI571; Gleevec) in chronic-phase chronic myelogenous leukemia. Clin Cancer Res 2003;9:160–166. 57 Kantarjian HM, O’Brien S, Cortes JE, Shan J, Giles FJ, Rios MB, Faderl SH, Wierda WG, Ferrajoli A, Verstovsek S, Keating MJ, Freireich EJ, Talpaz M: Complete cytogenetic and molecular responses to interferon-alpha-based therapy for chronic myelogenous leukemia are associated with excellent long-term prognosis. Cancer 2003;97:1033–1041.

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58 Hughes T, Branford S: Molecular monitoring of chronic myeloid leukemia. Semin Hematol 2003;40:62–68. 59 Ottmann OG, Druker BJ, Sawyers CL, Goldman JM, Reiffers J, Silver RT, Tura S, Fischer T, Deininger MW, Schiffer CA, Baccarani M, Gratwohl A, Hochhaus A, Hoelzer D, Fernandes-Reese S, Gathmann I, Capdeville R, O’Brien SG: A phase 2 study of imatinib in patients with relapsed or refractory Philadelphia chromosome-positive acute lymphoid leukemias. Blood 2002;100:1965–1971. 60 van der Velden VHJ, Hochhaus A, Cazzaniga G, Szczepanski T, Gabert J, van Dongen JJ: Detection of minimal residual disease in hematologic malignancies by real-time quantitative PCR: Principles, approaches, and laboratory aspects. Leukemia 2003;17:1013–1034. 61 Druker BJ, Talpaz M, Resta DJ, Peng B, Buchdunger E, Ford JM, Lydon NB, Kantarjian H, Capdeville R, Ohno-Jones S, Sawyers CL: Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med 2001;344:1031–1037. 62 Gambacorti-Passerini C, Barni R, Le Coutre P, Zucchetti M, Cabrita G, Cleris L, Rossi F, Gianazza E, Brueggen J, Cozens R, Pioltelli P, Pogliani E, Corneo G, Formelli F, D’Incalci M: Role of alpha1 acid glycoprotein in the in vivo resistance of human BCR-ABL(+) leukemic cells to the abl inhibitor STI571. J Natl Cancer Inst 2000;92:1641–1650. 63 Gambacorti-Passerini C, Le Coutre P, Zucchetti M, D’Incalci M: Binding of imatinib by alpha(1)-acid glycoprotein. Blood 2002;100: 367–368. 64 Mahon FX, Deininger MW, Schultheis B, Chabrol J, Reiffers J, Goldman JM, Melo JV: Selection and characterization of BCR-ABL positive cell lines with differential sensitivity to the tyrosine kinase inhibitor STI571: Diverse mechanisms of resistance. Blood 2000;96: 1070–1079.

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65 Vossebeld PJ, Sonneveld P: Reversal of multidrug resistance in hematological malignancies. Blood Rev 1999;13:67–78. 66 Tipping AJ, Mahon FX, Lagarde V, Goldman JM, Melo JV: Restoration of sensitivity to STI571 in STI571-resistant chronic myeloid leukemia cells. Blood 2001;98:3864–3867. 67 Weisberg E, Griffin JD: Mechanism of resistance to the ABL tyrosine kinase inhibitor STI571 in BCR/ABL-transformed hematopoietic cell lines. Blood 2000;95:3498–3505. 68 Le Coutre P, Tassi E, Varella-Garcia M, Barni R, Mologni L, Cabrita G, Marchesi E, Supino R, Cambarcorti-Passerini C: Induction of resistance to the Abelson inhibitor STI571 in human leukemic cells through gene amplification. Blood 2000;95:1758–1766. 69 Hochhaus A, Kreil S, Corbin AS, La Rosée P, Müller MC, Lahaye T, Hanfstein B, Schoch C, Cross NC, Berger U, Gschaidmeier H, Druker BJ, Hehlmann R: Molecular and chromosomal mechanisms of resistance to imatinib (STI571) therapy. Leukemia 2002;16:2190–2196. 70 Duesberg P, Stindl R, Hehlmann R: Origin of multidrug resistance in cells with and without multidrug resistance genes: Chromosome reassortments catalyzed by aneuploidy. Proc Natl Acad Sci USA 2001;98:11283–11288. 71 Barthe C, Cony-Makhoul P, Melo JV, Mahon JR: Roots of clinical resistance to STI-571 cancer therapy. Science 2001;293:2163a. 72 Branford S, Rudzki Z, Walsh S, Grigg A, Arthur C, Taylor K, Herrmann R, Lynch KP, Hughes TP: High frequency of point mutations clustered within the adenosine triphosphatebinding region of BCR/ABL in patients with chronic myeloid leukemia or Ph-positive acute lymphoblastic leukemia who develop imatinib (STI571) resistance. Blood 2002;99:3472– 3475. 73 Gorre ME, Mohammed M, Ellwood K, Hsu N, Paquette R, Rao PN, Sawyers CL: Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science 2001;293:876–880.

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74 Shah NP, Nicoll JM, Nagar B, Gorre ME, Paquette RL, Kuriyan J, Sawyers CL: Multiple BCR-ABL kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib (STI571) in chronic phase and blast crisis chronic myeloid leukemia. Cancer Cell 2002;2:117–125. 75 von Bubnoff N, Schneller F, Peschel C, Duyster J: BCR-ABL gene mutations in relation to clinical resistance of Philadelphia-chromosomepositive leukaemia to STI571:A prospective study. Lancet 2002;359:487–491. 76 Hofmann WK, Jones LC, Lemp NA, de Vos S, Gschaidmeier H, Hoelzer D, Ottmann OG, Koeffler HP: Ph(+) acute lymphoblastic leukemia resistant to the tyrosine kinase inhibitor STI571 has a unique BCR-ABL gene mutation. Blood 2002;99:1860–1862. 77 Oda T, Heaney C, Hagopian JR, Okuda K, Griffin JD, Druker BJ: Crkl is the major tyrosine-phosphorylated protein in neutrophils from patients with chronic myelogenous leukemia. J Biol Chem 1994;269:22925–22928. 78 Nichols GL, Raines MA, Vera JC, Lacomis L, Tempst P, Golde DW: Identification of CRKL as the constitutively phosphorylated 39-kD tyrosine phosphoprotein in chronic myelogenous leukemia cells. Blood 1994;84:2912–2918. 79 ten Hoeve J, Arlinghaus RB, Guo JQ, Heisterkamp N, Groffen J: Tyrosine phosphorylation of CRKL in Philadelphia+ leukemia. Blood 1994;84:1731–1736. 80 Heaney C, Kolibaba K, Bhat A, Oda T, Ohno S, Fanning S, Druker BJ: Direct binding of CRKL to BCR-ABL is not required for BCRABL transformation. Blood 1997;89:297–306. 81 Senechal K, Heaney C, Druker B, Sawyers CL: Structural requirements for function of the Crkl adapter protein in fibroblasts and hematopoietic cells. Mol Cell Biol 1998;18:5082– 5090.

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Review Acta Haematol 2004;112:93–104 DOI: 10.1159/000077564

Clinical Implications of Minimal Residual Disease Monitoring for Stem Cell Transplantation after Reduced Intensity and Nonmyeloablative Conditioning Avichai Shimoni Arnon Nagler Division of Hematology and Bone Marrow Transplantation, Chaim Sheba Medical Center, Tel Hashomer, Israel

Key Words Lymphocyte infusion, donor W Minimal residual disease W Nonmyeloablative conditioning W Stem cell transplantation

more effective for MRD than at frank relapse. Timing and dosing of therapy are not yet well established and depend on aggressiveness of the disease, type of conditioning, level and kinetics of MRD. Copyright © 2004 S. Karger AG, Basel

Abstract Allogeneic stem cell transplantation (SCT) is a potentially curative therapy for a variety of hematological malignancies; however, relapse and treatment-related toxicities are major obstacles to cure. Nonmyeloablative and reduced-intensity conditioning regimens were designed not to eradicate the malignancy completely, but rather to be immunosuppressive enough to allow engraftment, and to serve as a platform for additional cellular immunotherapy. Minimal residual disease (MRD) typically persists after SCT, and is gradually eliminated with different kinetics typical of each disease. Significant progress has been achieved with technologies for MRD assessment. Quantitative PCR tests are very sensitive in detecting tumor-associated transcripts, allowing serial monitoring. Threshold levels have been established for some malignancies, above which relapse is imminent. Persistent negative tests, a low level or a decreasing MRD level are consistent with continuous remission, whereas high-level MRD or increasing levels predict an incipient relapse. Patients at high risk of relapse are candidates for additional cellular or targeted therapy. Immunotherapy is

ABC

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Accessible online at: www.karger.com/aha

Introduction

Allogeneic stem cell transplantation (SCT) is an effective, potentially curative treatment of advanced or highrisk hematological malignancies [1, 2]. However, treatment-related toxicities and disease recurrence are major obstacles to cure. Nonmyeloablative and reduced-intensity conditioning regimens have been designed to reduce toxicity in an attempt to improve outcome, yet relapse remains a major cause for treatment failure. Patients who are in morphological remission after prior therapy may still harbor a significant tumor load that may contribute to disease recurrence. For example, patients with acute leukemia have approximately 1012 leukemia cells at presentation. A tumor load of as much as 1010 leukemia cells may still be present in a patient in morphological remission. The study of minimal residual disease (MRD) is directed to identify patients who are at risk of disease recurrence after standard chemotherapy, high-dose chemotherapy, and immunotherapy, such that these patients may be candidates for further therapy.

Avichai Shimoni, MD Department of Bone Marrow Transplantation Chaim Sheba Medical Center Tel Hashomer (Israel) Tel. +972 3 530 5830, Fax +972 3 530 5377, E-Mail [email protected]

Fig. 1. NST program: the initial NST regimen induces MC with persistence of both donor and recipient hematopoietic cells. The underlying malignancy (m) is suppressed but not completely eliminated. In the second phase, immune-therapeutic interventions, e.g. withdrawal of immunosuppressive therapy (IST) supplemented if necessary by DLI induce graft-versus-hematopoietic tissue and graft-versus-tumor effects eliminating recipient hematopoiesis and the underlying malignancy and converting to CC.

In this review, we discuss the rationale for nonmyeloablative stem cell transplantation (NST), the methods to detect MRD, the kinetics and clinical implications of MRD detection, and how they may differ after standard myeloablative SCT and NST.

Rationale for NST

SCT was initially developed as a means to deliver highdose chemotherapy and radiation for the elimination of the underlying disorder. Escalation of treatment doses results in better tumor kill but leads to irreversible myelosuppression. SCT was viewed as a supportive-care modality to restore hematopoiesis after treatment. However, it has subsequently become apparent that high-dose chemoradiotherapy does not eradicate the disease in many patients and that much of the therapeutic benefit of SCT relates to an associated immune-mediated graft-versusleukemia (GVL) or graft-versus-malignancy (GVM) effect. High-dose chemoradiotherapy with allogeneic SCT is associated with significant morbidity and mortality due to the toxicity of the preparative regimen, graft-versushost disease (GVHD), and the immunodeficient state that accompanies the procedure. The risk of regimen-related toxicity and GVHD increases with advanced age, limiting standard SCT to younger patients who are in good general condition. Many hematological malignancies are more common and have a worse prognosis in the elderly and disease and prior therapy may result in comorbidities precluding further intensive therapy. Extensive research has been directed towards the development of safer and less toxic approaches to allogeneic transplantation and thus to allowing the application of a potentially curative thera-

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peutic approach to a much wider patient population. The discovery of the curative potential of the immune-mediated GVL/GVM effect has led to a novel therapeutic approach. Low-dose, relatively nontoxic and tolerable conditioning regimens have been designed, not to eradicate the malignancy, but rather to provide sufficient immunosuppression to achieve engraftment and to allow induction of GVL as the primary treatment [3–6]. NST does not eliminate all host hematopoiesis and commonly leads to a state of mixed chimerism (MC). MC describes persistence of donor cells with either benign host hematopoietic cells and/or cells of the underlying malignancy (fig. 1). Stable long-lived MC has been reported in animal models and in patients having NST for nonmalignant disorders. However, in patients with malignancies, MC is most often transient and conversion to complete chimerism (CC), autologous reconstitution, or relapse occurs either spontaneously or following immune manipulations within the first few months following NST [7]. The initial nonmyeloablative treatment is expected to produce only transient suppression of the underlying malignancy, but this is expected to allow time for the immune GVM effect to develop. MC is considered to induce a state of bilateral transplantation tolerance and temporarily protect from GVHD. However, GVL is also more prominent among patients after conversion to CC, in most but not all studies. Thus progression of residual disease to frank relapse is more common in MC and in the absence of GVHD. The ultimate goal of NST is achievement of CC and eradication of residual disease. Patients with benign MC or with detectable residual malignancy post-NST may respond to additional immunotherapeutic approaches. Immunosuppressive therapy given posttransplant for the

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prevention of GVHD can also suppress the GVL effect. Early withdrawal of immunosuppressive therapy allows the occurrence of potent graft-versus-hematopoietic tissue effect that can potentially eliminate both residual disease and host hematopoiesis producing CC (fig. 1) [3, 8, 9]. If this does not occur donor lymphocyte infusion (DLI) may harness this effect and switch the balance towards CC and also complete eradication of the underlying malignancy. The initial NST and achievement of engraftment thus serve as a platform for additional allogeneic cellular therapy. NST regimens comprise a spectrum of regimens with different immunosuppressive and myelosuppressive properties. The kinetics of engraftment, chimerism, and eradication of residual disease differ accordingly. Conditioning regimens have been referred to as nonmyeloablative if they do not completely eradicate host hematopoiesis and immunity. A few of these regimens have been given as chemotherapeutic regimens with no stem cell support and allow relatively prompt hematological recovery. Autologous reconstitution of hematopoiesis is expected if the allograft is rejected. These nonmyeloablative regimens have potent immunosuppressive effects. They are only mildly myelosuppressive and commonly result in the induction of MC. The Seattle regimen consisting of lowdose total body irradiation (200 cGy) with (or initially without) fludarabine and intensive pre- and posttransplant immunosuppression is such a regimen [6]. Other examples are the combinations of fludarabine and cyclophosphamide (FC) and the Flag/Ida regimen developed initially at the MD Anderson [5]. These are very tolerable regimens, allowing in some cases ambulatory treatment, and treatment of elderly patients. More intensive regimens have also been developed. These regimens have been referred to as reduced intensity conditioning regimens (RIC). They have not been given without stem cell support and autologous recovery following treatment may be slow if taking place at all. These regimens usually combine immunosuppressive agents (such as fludarabine with or without serotherapy with ATG or Campath) with agents with moderate myelosuppressive effects (such as busulfan or melphalan) [4, 7, 10]. However dose intensity is reduced compared to the standard ablative regimen allowing a reduction of toxicity. Reduced intensity regimens rapidly induce CC and antitumor responses, but are more toxic, and associated with a higher risk of GVHD. The selection of the appropriate regimen for a patient depends on several factors including age, general medical condition, immune competence of the recipient, genetic disparity between the patient and donor, and center expe-

rience. Perhaps the most important determining factor is the aggressiveness and chemosensitivity of the underlying malignancy. The reduced intensity regimens are a more appropriate approach for aggressive malignancies such as acute leukemia, especially when not in remission. In this setting rapid achievement of CC and transient disease control is needed to induce GVL. However, in indolent malignancies GVM may occur slowly, even in mixed chimeras, and toxicity may be reduced further using nonmyeloablative regimens. GVHD is one of the major causes of posttransplant morbidity and mortality. It was hoped when these regimens were introduced that the GVHD incidence would decrease. As discussed above MC allows bilateral transplantation tolerance with graft acceptance and some protection from GVHD [11]. Acute GVHD results at least partially from tissue injury and cytokine release secondary to the toxicity of the preparative regimen, amplified by donor immune cells. The use of less toxic conditioning should theoretically limit tissue injury and cytokine release and reduce the incidence and severity of GVHD [12, 13]. However, host antigen-presenting cells that have a major role in the initiation of the GVHD reaction may persist after NST [14]. The duration of immunosuppressive therapy is usually shorter after NST and immune manipulations are often incorporated into NST programs increasing the likelihood of GVHD although delayed immune manipulations, once the toxicity of conditioning and cytokine release are already resolved, are less likely to produce severe GVHD [15]. The net effects of these differences between NST and ablative SCT is that in contrary to what was hoped, GVHD is only delayed, but not reduced in incidence [16] and is a major contributor to treatment-related mortality after NST. Thus, immune manipulations after NST should be limited to those destined to relapse rather than given to all patients as initially when incorporated in NST protocols.

MRD following Reduced Intensity Transplants

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Methods for Evaluation of MRD

Various methods have been developed for the detection of MRD [17]. An ideal MRD test should be sensitive for the detection of malignant cells (usually detecting 1 cell in more than 104 normal cells). It should be specific, detecting only those patients destined to relapse. It should be reproducible, readily available, and results should be available within a short period after sampling. Testing peripheral blood is preferable to allow frequent testing if needed. As will be discussed below it has become appar-

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ent that quantitative tests are superior to those only allowing qualitative assessment even though with high sensitivity. Not all relapses can be identified by monitoring MRD. This can be related to low sensitivity, too long an interval between testing, extramedullary relapse, or phenotypic switch where the tumor loses the marker or where relapse arises from a subclone that does not present the marker. Morphological analysis of the marrow can identify residual disease occupying more than 5% of marrow space and thus lacks the necessary sensitivity for early detection. Also, it is often difficult to differentiate a small leukemia blast population from normal donor-derived hematopoietic blasts repopulating the marrow without using additional markers. Specific tests can detect MRD when there is a specific tumor marker such as a specific cytogenetic abnormality, a specific immunophenotype that can be identified by flow cytometry, or specific DNA or RNA sequences that can be amplified by PCR. Cytogenetic abnormalities such as fusion genes, and changes in chromosome number, are common in acute and chronic leukemia. Standard band cytogenetics has been used at diagnosis to detect clonal abnormalities and also to follow response to therapy and MRD. However, this method is limited by the study of a limited number of metaphases. This results in a sensitivity of 1–5%, which is several orders of magnitude less than required of a sensitive MRD test. This method also requires dividing cells and is, therefore, performed most often in bone marrow rather than peripheral blood. It is cumbersome with a low turnover. Its major advantage is the ability to detect new abnormalities acquired with clonal evolution. Standard cytogenetics has largely been replaced by FISH. FISH has the advantage of studying a larger number of cells, of a study of interphase cells rather than dividing cells, and of a rapid turnover. The sensitivity of FISH is in the range of 1%. With standard FISH false-positive scoring may occur due to nonspecific hybridization of the probe. False-positive scoring for chromosomal translocations may occur due to random spatial association and optical fusion [18]. Molecular methods are increasingly used for the detection of MRD. Southern blot analysis of genomic DNA changes and Western blot analysis of abnormal fusion proteins lost popularity due to their relatively low sensitivity, cost and complexity. Monitoring MRD with PCR methods has become the technique of choice [19]. Several PCR targets are available for MRD detection: (1) rearrangements of immunoglobulin gene or T cell receptor gene; these are patient-specific targets, used mostly in acute

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lymphoblastic leukemia (ALL); (2) breakpoint fusion regions of chromosome aberrations (DNA targets) amplified by PCR and fusion gene transcripts (RNA targets) amplified by reverse transcription (RT)-PCR (e.g., BCR/ ABL), and (3) aberrant genes or aberrantly expressed genes. The applicability of these MRD-PCR targets varies with disease category. In chronic myeloid leukemia (CML) and ALL, a specific MRD-PCR target can be found in virtually all patients, whereas in acute myelogenous leukemia (AML) such a target can be found in only 30–50% of patients [20]. The Wilms’ tumor gene (WT1) belongs to the third category. It has been suggested as a ‘panleukemic MRD marker’. WT1 is highly expressed in various types of acute leukemia and its expression was found to increase significantly at relapse compared to diagnosis. Thus, WT1 levels can be followed after SCT using RT-PCR. The level of expression and its doubling time have been shown to predict relapse and response to therapy [21, 22]. FLT3 internal tandem duplication (FLT3-ITD) mutations have recently been recognized as the most common mutations in de novo AML. They are present in 20–30% of patients at diagnosis, and confer poor prognosis. There are initial reports of using FLT3-ITD determinations by genomic or RT-PCR to follow MRD. However, FLT3-ITD mutations are unstable, they may be lost or are sometimes acquired at relapse and, therefore, their use as markers of MRD is probably limited [23, 24]. PCR tests were initially only qualitative. The sensitivity of multiplex PCR is 10 –3 to 10 –4. Nested PCR techniques increase the sensitivity to 10 –5 to 10 –6. It was then apparent (see below) that the qualitative MRD tests give only limited information and do not enable accurate assessment of tumor load and kinetics. A few groups developed quantitative tests, such as competitive PCR and more recently, real-time PCR. This is a rapid, automated, reliable technique with a fast turnover that is becoming the technology of choice in MRD research. The sensitivity is similar to nested PCR, in the range of 10 –5 to 10 –6. Quantitative results are given, as the number of transcripts per microgram RNA or as the ratio of target transcript number to that of a housekeeping gene. These results can be followed to assess the kinetics of tumor load. Multiparameter flow cytometry is an additional quantitative method for the detection of MRD [25, 26]. Leukemic blasts frequently display an aberrant or uncommon immune phenotype, which allows their distinction from normal hematopoietic cells. Cells are stained simultaneously with multiple antibodies labeled with different

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fluorochromes allowing the identification of the aberrant phenotype with flow cytometry. The sensitivity is in the range of 10 –2 to 10 –4. This is a quantitative test with rapid results. However, there may be a significant background of aberrant phenotype in normal cells limiting the specificity of the test. A phenotype switch during relapse is also a known limitation. These disadvantages can be minimized by the use of a large panel of antibodies. In the absence of a specific tumor marker for MRD, chimerism testing can be used to detect recipient-derived cells [7]. Donor and recipient cells can be distinguished by testing informative genetic markers that are determined prior to the transplant [17]. In sex-mismatched transplant different methods are usually implied to detect the proportion of female and male cells by analysis of sex chromosome markers. Regular cytogenetics for chimerism testing has largely been replaced by FISH techniques. The sensitivity and specificity may be markedly improved by the simultaneous use of X and Y probes labeled with dual fluorescent colors. Assessment of a variable number of tandem repeats (VNTR) or short tandem repeats (STR) polymorphism has become the most valuable method for determining chimerism in sex-matched transplants. Certain core DNA sequences are tandemly repeated in the genome and the number of tandem repeats varies among different individuals. These repeats differ in base pair length to form microsatellites (2–8 base pairs) or minisatellites [8–50 base pair TG(n) repeats]. There are a very large number of loci available for testing, and multiple alleles in each site, allowing identification of informative markers in almost all donor-recipient pairs using a limited panel of loci. VNTR can be identified by Southern blot assays or preferably by DNA amplification of these highly polymorphic sequences. With these PCR tests a minor population of DNA can be detected even when its concentration is as low as 1–5% of the total. More recently multiplex PCR amplification of STR markers has been applied to chimerism testing [27]. This method uses a commercially available kit originally designed for forensic purposes (genetic fingerprinting) that coamplifies up to 9 STR markers. Whereas the majority of PCR techniques allow only semiquantitative information, this technique is able to provide reproducible quantitative results. The sensitivity of the molecular techniques can be increased by applying them to a subset of leukocytes enriched for the minor population. For example, immunophenotyping of an original leukemic clone can be used for FACS sorting [28]. Application of chimerism testing analysis to the sorted population results in a two-log increase in the sensitivity of identifying leukemic cells up

to 1:103 to 1:104. Leukemia cells can also be identified with higher sensitivity within CD34+ selected cells of peripheral blood leukocytes. Chimerism changes in subset population may precede that detected in total leukocytes by weeks to months allowing an earlier application of therapeutic interventions. However, the detection of recipient-derived cells may not always correlate with the risk of relapse. Recipient cells contributing to MC belong to the malignant clone or may be normal hematopoietic cells, or even stromal cells. During the first few weeks after standard allogeneic transplantation recipient cells can still be detected when sensitive tests are used. These are mostly T cells surviving the conditioning regimen. Host cells may be detected at low levels (!1%) even later in the posttransplant course. MC is detected more often after nonmyeloablative conditioning. Thus, tumor-specific marker probes are probably superior and more accurate than sex mismatch probes for the detection of MRD [29]. We have used a novel system, DuetTM (BioView, Israel) to overcome this problem [30]. This system provides combined simultaneous morphological and cytogenetical analysis on the same cells. In brief, slides for Duet are prepared by density gradient centrifugation and cytospin. The slides are stained with MGG and scanned. The system saves the coordinates and images of all cells found on the slides for future reference during the next phases of analysis. The MGG staining is removed and FISH is applied to the same slides. Hybridization can then be removed and a second FISH with another probe can be applied to the same cells. After the FISH procedure, the slides are investigated either manually or automatically for target cells containing the fluorescence signals. When the underlying malignancy is associated with a specific cytogenetic abnormality a specific FISH probe is used to identify the MRD population and the system relocates the morphology of cells harboring this abnormality. For sex-mismatched transplants, recipient cells presenting XY signals or XX signals are searched for and targeted. The system allows rapid automatic scanning of a large number of cells, thus increasing the sensitivity of the detection of a small recipient population to 10 –4 compared to 1% of FISH. The clinical significance of MRD detection is improved by identifying the morphology of recipient cells (identified by gender or underlying cytogenetic abnormality). Identification of recipient characteristics within blasts rather than mature hematopoietic cells or stromal cells predicts clinically significant MRD. The detection of MRD with tumor-specific tests may not always be correlated with the risk of relapse. Positive PCR tests for BCR/ABL and bcl2 rearrangements have

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been reported in healthy individuals. There may be a threshold for MRD to be clinically significant (see below), such that extrasensitive qualitative tests may not always be significant. Residual malignant cells may be in a dormant state lacking the potential to contribute to relapse, because of a lack or gain of a second genetic alteration. In some diseases partial differentiation to more mature cells can occur and these cells may lack the potential to proliferate. MRD may be under immune surveillance, or the MRD cells may be in an apoptotic phase rendering them irrelevant for disease recurrence.

Significance and Kinetics of MRD in Different Malignancies

Chronic Myeloid Leukemia CML has been the paradigm for many facets of molecular diagnosis and monitoring of MRD after SCT. Many patients harbor MRD as shown by positive qualitative PCR tests during the first 6 months post-SCT, but may later clear MRD and be cured of the disease without further interventions. However, following standard ablative SCT, a positive PCR test 6–12 months after SCT is highly associated with relapse, in a median of 200 days after the first positive test [31]. Persistently negative tests are associated with a low risk. The risk is extremely high when a positive PCR test follows T cell-depleted SCT and is lower after unrelated donor SCT, due to an absence or persistence of GVL, respectively. The relative risk of relapse associated with positive PCR steadily decreases after 1 year. This may result from a selection of patients with slow kinetics of disease recurrence or with a GVL effect that controls MRD [32]. The introduction of quantitative PCR tests revealed three patterns of MRD within the first months after ablative SCT [33]: patients with negative PCR, low-level MRD (BCR-ABL/ABL !0.02%), and high-level MRD (BCR-ABL/ABL 10.02%). Patients who are destined to remain in remission have undetected or low MRD or falling levels on sequential analyses. Most are negative 1 year post-SCT. Patients who have increasing levels or a persistently high BCR-ABL transcript number are most likely to progress to cytogenetic and hematological relapse within a few months. The doubling time of MRD level is also predictive of relapse and response to therapy [34]. The precise definition of molecular relapse remains controversial in the absence of a standardization of the tests depending on the center protocol and other variables. The kinetics of MRD with sequential testing is more important than the absolute parameters. The Inter-

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national Bone Marrow Transplant Registry (IBMTR) recommends testing MRD with a quantitative PCR every 3– 6 months during the first 2 years, then every 6 months up to 5 years after SCT, and then yearly [35]. Finding a rising or high level (BCR-ABL/ABL 10.05%) requires more frequent testing, and assessment of the indication for therapy (see below). Bone marrow and peripheral blood give the same information. It is now established that CML can probably be cured with NST [6, 9, 36]. However, there is only limited data regarding the kinetics of MRD and its significance after reduced intensity conditioning. Uzunel et al. [37] reported that all NST recipients had detectable MRD during the first 3 months, with a median of 0.2%, which is significantly more compared with 0.01% in recipients of ablative conditioning, reflecting the use of reduced doses of chemoradiotherapy. Only 20% remained positive 12 months after SCT and the median time to complete molecular remission was 3.5 months (range, 1–7 months). Eradication of MRD most often followed conversion to CC, as also reported by other groups [38] but could occur in the absence of GVHD. From this limited set of data it seems that the kinetics of MRD in CML after NST is similar to that after ablative therapy. The levels of MRD may be higher, and sequential testing is needed to predict outcome and determine the indication for intervention. Acute Leukemia ALL is the first hematological malignancy where MRD testing has been routinely incorporated into chemotherapy protocols for the evaluation of response and risk stratification. MRD is most often evaluated using immunoglobulin or TCR rearrangement or by multiparameter flow cytometry, and when appropriate by RT-PCR for fusion gene transcripts such as BCR-ABL in Philadelphiapositive ALL. The value of MRD has been extensively studies in children and less so in adults. MRD status prior to SCT is a major determinant of post-SCT outcome. The presence of high-level MRD prior to ablative T celldepleted SCT is almost invariably associated with relapse [39]. High-level MRD prior to conditioning may be overcome in a selected group of children with replete T cell SCT and the occurrence of chronic GVHD [40]. This association is less prominent in adults [41]. Miglino et al. [42] described four patterns of MRD kinetics in 23 patients with ALL. Four patients had negative PCR before SCT and all remained in remission after SCT (–/–– pattern), supporting the good prognosis in patients with negative MRD pre-SCT. Eleven patients had persistent MRD after SCT and 60% relapsed (+/++ pattern). Three

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patients had early resolution of MRD after high-dose chemotherapy (+/–– pattern) and 5 had late resolution (+/+– pattern), most of whom after the onset of chronic GVHD, and none relapsed. Radich et al. [43] reported that MRD detected within 100 days after SCT was associated with a 6-fold increase in the relapse rate with a median of 30 days or 1–6 months in other studies [40, 44–45]. Most patients destined to remain in remission have no MRD detected at any point after SCT. However, low-level MRD within the first 6 months after SCT is compatible with continuous remission [40]. Using quantitative PCR Lee et al. [46] showed a rapid decrease of tumor-related transcripts with GVHD and an increase of copy numbers 2–4 months prior to relapse. Thus, quantitative methods have a significant advantage over the qualitative, and they may be able to define the threshold level of MRD associated with clinical relapse. MRD in AML has been studied to a lesser degree. Quantitative tests have shown that threshold levels can be established, above and below which the risk of relapse is high or low. These thresholds are different among different AML subtypes [47]. It is well known with the use of qualitative tests that in AML with t(8;21), MRD as determined by RT-PCR for AML1-ETO transcripts may be detected years after remission induction, and even after allogeneic SCT [48] and therefore this test is not useful to follow up MRD. However, there are emerging data that a rise in the transcript number heralds the incipient relapse in 3–6 months [49]. The same concept has recently been shown in AML with inv(16) using quantitative PCR for CBFbeta-MYH11 [50, 51]. In acute promyelocytic leukemia MRD detected even with a qualitative PCR test for PML-RARA is highly predictive of relapse. There are over 70 other fusion genes or aberrant genes expressed by leukemia cells (such as WT1, see above) that have been followed as MRD and correlated with relapse [20, 47]. We have shown with a system of combined morphological and cytogenetic analysis (see above) that in the absence of specific cytogenetic abnormality, detection of blast cells with host gender with a sensitivity of 10 –3 to 10 –4 is almost invariably associated with relapse within 2 months of testing, whereas negative tests ensure continuous remission for at least 2 months [52]. Thus, although this is a quantitative test, the detection with lower sensitivity, and therefore of a larger tumor load than when first detected with more sensitive tests does not allow time for serial documentation of rising tumor load before relapse. Most of the patients included in this study had myeloablative conditioning.

MRD following Reduced Intensity Transplants

There is only limited data on the kinetics of MRD in acute leukemia after NST or RIC transplants, as there are no large disease-specific studies approaching this issue. Perez-Simon et al. [53] have shown that MRD assessment on days +21 to +56 after RIC SCT for various hematological malignancies allowed the identification of patients at risk of relapse; 58% of patients with MRD relapsed compared to none of those remaining persistently MRD negative. In 7 of 9 patients immune intervention employed allowed clearance of MRD and prevention of relapse. MRD clearance was associated with conversion to CC and GVHD. In particular, 5 of 11 patients with acute leukemia had MRD detected early after SCT, 3 relapsed before day 100, and 1 relapsed later; only 1 had prophylactic immune intervention and he did not relapse. None of the others relapsed. In another study of patients with ALL having RIC SCT, all 8 patients with MRD after SCT progressed, while 7 of 8 patients with at least two consecutive negative tests did not progress [54]. GVHD was associated in that study, similarly to studies in myeloablative conditioning, with a reduced relapse risk. CLL and Follicular Lymphoma Similar to CML, MRD is detected in most patients in the first few months after allogeneic SCT, either ablative or with reduced intensity conditioning [55]. Molecular remission may be achieved late, up to 2 years after SCT, with a median of 3–9 months in different studies [56, 57] and may be associated with the onset of GVHD. The predictive value of MRD is different after autologous and allogeneic SCT. Using quantitative MRD testing, such as PCR for IgH rearrangement or multiparameter flow cytometry, it has been shown that high-dose chemoradiotherapy followed by autologous SCT may reduce the tumor load to below 10 –4 [58]; however, the persistence of MRD invariably leads to relapse. NST reduces tumor load to a lesser level, 10 –1 to 10 –2. Persistent MRD can be detected with no progression, and often decreases after cyclosporine withdrawal. An increase in MRD level may herald relapse. Similar kinetics is also typical of follicular lymphoma [59, 60]. Multiple Myeloma Reduced intensity regimens are increasingly used in the treatment of patients with multiple myeloma to exploit the graft-versus-myeloma effect. The kinetics of response, even after high-dose chemotherapy and autologous SCT, is remarkable for slow clearance of disease within months after SCT [61]. A few groups have shown that molecular remission, as evidenced by PCR tests for

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patient-specific complementarity determining region 3 of the rearranged IgH gene, or multiparameter flow cytometry, can be achieved following myeloablative therapy. In the largest study [62] clone-specific molecular marker could be generated for 48 of 70 patients having myeloablative SCT. Three patterns were documented: (1) 16 patients (33%) remained MRD negative with all tests, and none of them relapsed, (2) 13 patients (27%) had persistent PCR positivity and all relapsed, and (3) 19 (40%) had a mixed pattern, with some tests that were positive and others negative. Those with positive tests who later became negative had a good prognosis. Only about 50% of patients with clinical CR also had molecular CR, which was associated with a very low relapse rate [63]. The time period of gradual resolution of MRD to the level of molecular CR was 6–12 months, with no other interventions. There is only limited data on the occurrence of molecular CR after NST and its kinetics.

Treatment of MRD: When and How

The treatment of relapsed malignancy after myeloablative SCT usually involves rapid withdrawal of immunosuppressive therapy and DLI trying to exploit the GVL effect [64, 65]. CML is the disease most sensitive to immune manipulations [66], but responses are less prominent in acute leukemia [64, 65], myeloma [67] and other malignancies. CML may have an intrinsic exquisite sensitivity to the GVL effect; however, other explanations contribute to this observation. CML has been monitored closely with MRD testing and relapse is treated at an early stage. Patients in molecular or cytogenetic relapse respond better, and require fewer lymphocytes to achieve a response than patients with hematological relapse [66, 68, 69]. Among hematological relapses, the relapse in the chronic phase responds better than in the accelerated phase, and the blast crisis is mostly resistant to DLI. The interval from SCT to relapse and to DLI is also an important factor in predicting response [70]. Patients with a rapid rise in their tumor load are less likely to respond [34]. The relative ineffectiveness of DLI in the treatment of acute leukemia is probably related to the rapid progression of the malignancy outpacing the building of effective immunity against the tumor with DLI. Therefore, transient suppression of the malignancy with chemotherapy prior to DLI may improve the results [71]. There are initial reports suggesting that administration of DLI at MRD or impending relapse may prevent a relapse more effectively [72, 73].

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Most of the data on the safety and efficacy of DLI comes from myeloablative SCT. DLI has also been administered after NST in a variety of indications [74]. Initially DLI was incorporated into NST protocols for conversion of MC to CC [7]. As discussed above MC may be associated with an increased risk of relapse, especially in aggressive malignancies, and may also be associated with MRD. However, as experience with NST was gained, the role of DLI in this setting becomes more controversial. High-grade MC (more than 50–60% donor chimerism) usually spontaneously converts to CC, whereas patients with low-grade donor chimerism (less than 20–40%) often reject the graft despite DLI [6, 75]. Second, DLI has been given for persistent or progressive disease. The factors predicting response are similar to those discussed above. There is experimental data suggesting that DLI may even be more potent in mixed chimera due to persistence of host antigen-presenting cells [76]. DLI has also been explored as prophylactic therapy after NST [76–80]. Although this approach may reduce relapse risk, responses are often associated with GVHD. DLI is associated with significant morbidity and mortality, mostly due to complications related to GVHD and marrow aplasia. Marks et al. [74] reported in the largest series of DLI after NST that the rates of severe GVHD were 15%, treatment-related mortality was 9%, and marrow aplasia was rare, suggesting that DLI after NST was safer than what is reported after standard myeloablative SCT. This may represent advances in DLI administration, such as administration in incremental dosing and at MRD. However, DLI is still associated with a substantial risk and should be limited only to those destined to relapse, avoiding unnecessary toxicity in those destined to remain in remission. The timing of prophylactic DLI is complicated. DLI administered late after SCT has a lower risk of complications [10]; however, the window of opportunities for administration of DLI for the prevention of relapse may be short and missed while waiting for a safer time point. Even in programs planning early DLI, on days 60–100 post-SCT, DLI is only administered after cyclosporine withdrawal and many patients are ineligible for prophylactic DLI because of GVHD or rapid progression already occurring prior to DLI. The optimal time point and cell dose of DLI have not been established. The decision to administer DLI can be based on several factors: the aggressiveness of the underlying malignancy and the risk of rapid progression, the sensitivity of the test employed to determine MRD, the expected kinetics of MRD and the trend of MRD in serial quantitative testing, the level of MRD, as well as the SCT

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regimen used. In indolent malignancies such as CML, chronic lymphocytic leukemia (CLL), follicular lymphoma, and to a lesser extent multiple myeloma MRD is often detected after SCT, both after ablative conditioning, and even more so after reduced intensity conditioning. MRD can be followed and no intervention is indicated unless progression or a plateau in response is observed or quantitative MRD is rising. The doubling time of MRD is also important in predicting response and planning dosing and timing of DLI. MC per se is not an absolute indication for DLI in this setting unless for the prevention of graft rejection in the face of declining chimerism [7]. In aggressive malignancies such as acute leukemia and CML in blast crisis, and especially when not in remission at SCT, timing is crucial. There may not be sufficient time to follow quantitative MRD since the doubling time of MRD may be short and a relapse may occur within weeks, while an effective DLI response may take 2–3 months. Thus, the sensitivity of the test is important. When using very sensitive tests, such as quantitative PCR, when applicable, one can follow MRD very closely, every 1–2 weeks. If MRD is decreasing no intervention is needed. The kinetics of MRD in this setting after NST has not been established as well as after ablative conditioning. The same level of MRD may not necessarily have the same significance. MRD surviving high-dose chemotherapy and to a lesser extent reduced intensity conditioning

represent highly resistant malignancy, whereas MRD is expected after NST. MRD remaining after T cell depletion SCT or the use of Campath in NST is also highly predictive of a relapse. Quantitative results are emerging to determine levels of MRD that are below or above the threshold for relapse, and which can be used to direct intervention. MC may also direct a more aggressive approach in aggressive malignancies, not only as a marker for MRD, but also to rapidly induce CC and GVL. Targeted therapy is another option for treatment or control of MRD. Imatinib mesylate (formerly STI571) is an effective therapy for CML. Data is emerging that imatinib may be effective in salvaging patients with a relapse after SCT, either frontline, or as second-line therapy after failure of DLI [81]. Imatinib may also have a synergistic effect with DLI [82]. It has been explored as an adjuvant to reduced intensity conditioning both pretransplant allowing reduction of conditioning intensity and posttransplant to eliminate MRD. Rituximab is another example. We used rituximab after SCT in patients with aggressive lymphoma [83]. The reduced risk of relapse in very highrisk patients suggested that rituximab may have eliminated MRD. It may have synergized with the donor immune system providing effectors for antibody-dependent cytotoxicity. Future studies may identify other methods to target MRD trying to reduce the relapse risk after SCT.

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30 Shimoni A, Nagler A, Kaplinsky C, Reichart M, Avigdor A, Hardan I, Yeshurun M, Daniely M, Zilberstein Y, Amariglio N, Brok-Simoni F, Rechavi G, Trakhtenbrot L: Chimerism testing and detection of minimal residual disease after allogeneic hematopoietic transplantation using the bioView (Duet) combined morphological and cytogenetical analysis. Leukemia 2002;16: 1413–1418. 31 Radich JP, Gehly G, Gooley T, Bryant E, Clift RA, Collins S, Edmands S, Kirk J, Lee A, Kessler P, Schoch G, Buckner CD, Sullivan KM, Appelbaum FR, Thomas ED: Polymerase chain reaction detection of the BCR-ABL fusion transcript after allogeneic marrow transplantation for chronic myeloid leukemia: Results and implications in 346 patients. Blood 1995;85:2632–2638. 32 Radich JP, Gooley T, Bryant E, Chauncey T, Clift R, Beppu L, Edmands S, Flowers ME, Kerkof K, Nelson R, Appelbaum FR: The significance of bcr-abl molecular detection in chronic myeloid leukemia patients ‘late’, 18 months or more after transplantation. Blood 2001;98:1701–1707. 33 Olavarria E, Kanfer E, Szydlo R, Kaeda J, Rezvani K, Cwynarski K, Pocock C, Dazzi F, Craddock C, Apperley JF, Cross NC, Goldman JM: Early detection of BCR-ABL transcripts by quantitative reverse transcriptase-polymerase chain reaction predicts outcome after allogeneic stem cell transplantation for chronic myeloid leukemia. Blood 2001;97:1560–1565. 34 Lin F, van Rhee F, Goldman JM, Cross NC: Kinetics of increasing BCR-ABL transcript numbers in chronic myeloid leukemia patients who relapse after bone marrow transplantation. Blood 1996;87:4473–4478. 35 Giralt SA, Goldman J, Anasetti C, Cervantes F, Champlin R, Cross N, Hochhaus A, Klein JP, Papadopoulos E, Sobocinski K, Weisdorf D, Horwitz M, on behalf of the Chronic Leukemia Working Committee of the IBMTR: Recommendations for assessment and definitions of response and relapse in CMK: A report from the Chronic Leukemia Working Committee of the IBMTR; in Carella AM, Daley DQ, Eaves CJ, Goldman JM, Hehlmann R (eds): Chronic Myeloid Leukemia: Biology and Treatment. London, Dunitz, 2001, pp 357–368. 36 Or R, Shapira MY, Resnick I, Amar A, Ackerstein A, Samuel S, Aker M, Naparstek E, Nagler A, Slavin S: Nonmyeloablative allogeneic stem cell transplantation for the treatment of chronic myeloid leukemia in first chronic phase. Blood 2003;101:441–445. 37 Uzunel M, Mattsson J, Brune M, Johansson JE, Aschan J, Ringden O: Kinetics of minimal residual disease and chimerism in patients with chronic myeloid leukemia after nonmyeloablative conditioning and allogeneic stem cell transplantation. Blood 2003;101:469–472. 38 Childs R, Epperson D, Bahceci E, Clave E, Barrett J: Molecular remission of chronic myeloid leukaemia following a non-myeloablative allogeneic peripheral blood stem cell transplant: In vivo and in vitro evidence for a graft-versusleukaemia effect. Br J Haematol 1999;107: 396–400.

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39 Knechtli CJ, Goulden NJ, Hancock JP, Grandage VL, Harris EL, Garland RJ, Jones CG, Rowbottom AW, Hunt LP, Green AF, Clarke E, Lankester AW, Cornish JM, Pamphilon DH, Steward CG, Oakhill A: Minimal residual disease status before allogeneic bone marrow transplantation is an important determinant of successful outcome for children and adolescents with acute lymphoblastic leukemia. Blood 1998;92:4072–4079. 40 Knechtli CJ, Goulden NJ, Hancock JP, Harris EL, Garland RJ, Jones CG, Grandage VL, Rowbottom AW, Green AF, Clarke E, Lankester AW, Potter MN, Cornish JM, Pamphilon DH, Steward CG, Oakhill A: Minimal residual disease status as a predictor of relapse after allogeneic bone marrow transplantation for children with acute lymphoblastic leukaemia. Br J Haematol 1998;102:860–871. 41 Mortuza FY, Papaioannou M, Moreira IM, Coyle LA, Gameiro P, Gandini D, Prentice HG, Goldstone A, Hoffbrand AV, Foroni L: Minimal residual disease tests provide an independent predictor of clinical outcome in adult acute lymphoblastic leukemia. J Clin Oncol 2002;20:1094–1104. 42 Miglino M, Berisso G, Grasso R, Canepa L, Clavio M, Pierri I, Pietrasanta D, Gatto S, Varaldo R, Ballerini F, Verdiani S, Casarino L, DeStefano F, Sessarego M, Dominietto A, Raiola AM, Bregante S, di Grazia C, Gobbi M, Bacigalupo A: Allogeneic bone marrow transplantation (BMT) for adults with acute lymphoblastic leukemia (ALL): Predictive role of minimal residual disease monitoring on relapse. Bone Marrow Transplant 2002;30:579– 585. 43 Radich J, Ladne P, Gooley T: Polymerase chain reaction-based detection of minimal residual disease in acute lymphoblastic leukemia predicts relapse after allogeneic BMT. Biol Blood Marrow Transplant 1995;1:24–31. 44 Uzunel M, Jaksch M, Mattsson J, Ringden O: Minimal residual disease detection after allogeneic stem cell transplantation is correlated to relapse in patients with acute lymphoblastic leukaemia. Br J Haematol 2003;122:788–794. 45 Sanchez J, Serrano J, Gomez P, Martinez F, Martin C, Madero L, Herrera C, Garcia JM, Casano J, Torres A: Clinical value of immunological monitoring of minimal residual disease in acute lymphoblastic leukaemia after allogeneic transplantation. Br J Haematol 2002; 116:686–694. 46 Lee S, Kim DW, Cho B, Kim YJ, Kim YL, Hwang JY, Park YH, Shin HJ, Park CY, Min WS, Kim HK, Kim CC: Risk factors for adults with Philadelphia-chromosome-positive acute lymphoblastic leukaemia in remission treated with allogeneic bone marrow transplantation: The potential of real-time quantitative reversetranscription polymerase chain reaction. Br J Haematol 2003;120:145–153. 47 Yin JA, Grimwade D: Minimal residual disease evaluation in acute myeloid leukaemia. Lancet 2002;360:160–162.

MRD following Reduced Intensity Transplants

48 Jurlander J, Caligiuri MA, Ruutu T, Baer MR, Strout MP, Oberkircher AR, Hoffmann L, Ball ED, Frei-Lahr DA, Christiansen NP, Block AM, Knuutila S, Herzig GP, Bloomfield CD: Persistence of the AML1/ETO fusion transcript in patients treated with allogeneic bone marrow transplantation for t(8;21) leukemia. Blood 1996;88:2183–2191. 49 Tobal K, Newton J, Macheta M, Chang J, Morgenstern G, Evans PA, Morgan G, Lucas GS, Liu Yin JA: Molecular quantitation of minimal residual disease in acute myeloid leukemia with t(8;21) can identify patients in durable remission and predict clinical relapse. Blood 2000;95:815–819. 50 Laczika K, Mitterbauer G, Mitterbauer M, Knobl P, Schwarzinger I, Greinix HT, Rabitsch W, Fonatsch C, Mannhalter C, Lechner K, Jaeger U: Prospective monitoring of minimal residual disease in acute myeloid leukemia with inversion(16) by CBFbeta/MYH11 RTPCR: Implications for a monitoring schedule and for treatment decisions. Leuk Lymphoma 2001;42:923–931. 51 Buonamici S, Ottaviani E, Testoni N, Montefusco V, Visani G, Bonifazi F, Amabile M, Terragna C, Ruggeri D, Piccaluga PP, Isidori A, Malagola M, Baccarani M, Tura S, Martinelli G: Real-time quantitation of minimal residual disease in inv(16)-positive acute myeloid leukemia may indicate risk for clinical relapse and may identify patients in a curable state. Blood 2002;99:443–449. 52 Shimoni A, Nagler A, Bielorai B, Reichart M, Rothman R, Toren A, Rechavi G, Amariglio N, Trakhtenbrot L: Detection of host blasts but not mature hematopoietic cells among minute host populations remaining after allogeneic transplantation, using combined morphological and cytogenetic analysis, predicts imminent relapse. Blood 2002;100:631a. 53 Perez-Simon JA, Caballero D, Diez-Campelo M, Lopez-Perez R, Mateos G, Canizo C, Vazquez L, Vidriales B, Mateos MV, Gonzalez M, San Miguel JF: Chimerism and minimal residual disease monitoring after reduced intensity conditioning (RIC) allogeneic transplantation. Leukemia 2002;16:1423–1431. 54 Martino R, Giralt S, Caballero MD, Mackinnon S, Corradini P, Fernandez-Aviles F, San Miguel J, Sierra J: Allogeneic hematopoietic stem cell transplantation with reduced-intensity conditioning in acute lymphoblastic leukemia: A feasibility study. Haematologica 2003; 88:555–560. 55 Esteve J, Villamor N, Colomer D, Montserrat E: Different clinical value of minimal residual disease after autologous and allogenic stem cell transplantation for chronic lymphocytic leukemia. Blood 2002;99:1873–1874.

56 Schetelig J, Thiede C, Bornhäuser M, Schwerdtfeger R, Kiehl M, Beyer J, Sayer HG, Kroger N, Hensel M, Scheffold C, Held TK, Höffken K, Ho AD, Kienast J, Neubauer A, Zander AR, Fauser AA, Ehninger G, Siegert W; Cooperative German Transplant Study Group: Evidence of a graft-versus-leukemia effect in chronic lymphocytic leukemia after reducedintensity conditioning and allogeneic stem-cell transplantation: The Cooperative German Transplant Study Group. J Clin Oncol 2003; 21:2747–2753. 57 Dreger P, Brand R, Hansz J, Milligan D, Corradini P, Finke J, Deliliers GL, Martino R, Russell N, Van Biezen A, Michallet M, Niederwieser D; Chronic Leukemia Working Party of the EBMT: Treatment-related mortality and graftversus-leukemia activity after allogeneic stem cell transplantation for chronic lymphocytic leukemia using intensity-reduced conditioning. Leukemia 2003;17:841–848. 58 Ritgen M, Dreger P, Humpe A, Stilgenbauer S, Döhner H, Kneba M: Quantitative PCR demonstrates effective graft-versus-leukemia activity after allogeneic stem cell transplantation using reduced intensity conditioning in patients with chronic lymphocytic leukemia (CLL). Blood 2002;100,854a. 59 Khouri IF, Saliba RM, Giralt SA, Lee MS, Okoroji GJ, Hagemeister FB, Korbling M, Younes A, Ippoliti C, Gajewski JL, McLaughlin P, Anderlini P, Donato ML, Cabanillas FF, Champlin RE: Nonablative allogeneic hematopoietic transplantation as adoptive immunotherapy for indolent lymphoma: Low incidence of toxicity, acute graft-versus-host disease, and treatment-related mortality. Blood 2001;98: 3595–3599. 60 Mitterbauer M, Neumeister P, Kalhs P, Brugger S, Fischer G, Dieckmann K, Hoecker P, Hinterberger W, Linkesch W, Simonitsch I, Jaeger U, Lechner K, Mannhalter C, Mitterbauer G, Greinix HT: Long-term clinical and molecular remission after allogeneic stem cell transplantation (SCT) in patients with poor prognosis non-Hodgkin’s lymphoma. Leukemia 2001;15:635–641. 61 Shimoni A, Smith TL, Aleman A, Weber D, Dimopoulos M, Anderlini P, Andersson B, Claxton D, Ueno NT, Khouri I, Donato M, Korbling M, Alexanian R, Champlin R, Giralt S: Thiotepa, busulfan, cyclophosphamide (TBC) and autologous hematopoietic transplantation: An intensive regimen for the treatment of multiple myeloma. Bone Marrow Transplant 2001;27:821–828. 62 Corradini P, Cavo M, Lokhorst H, Martinelli G, Terragna C, Majolino I, Valagussa P, Boccadoro M, Samson D, Bacigalupo A, Russell N, Montefusco V, Voena C, Gahrton G; Chronic Leukemia Working Party of the European Group for Blood and Marrow Transplantation (EBMT): Molecular remission after myeloablative allogeneic stem cell transplantation predicts a better relapse-free survival in patients with multiple myeloma. Blood 2003;102: 1927–1929.

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70 Dazzi F: Monitoring of minimal residual disease after allografting: A requirement to guide DLI treatment? Ann Hematol 2002;81(suppl 2):S29–S30. 71 Levine JE, Braun T, Penza SL, Beatty P, Cornetta K, Martino R, Drobyski WR, Barrett AJ, Porter DL, Giralt S, Leis J, Holmes HE, Johnson M, Horowitz M, Collins RH Jr: Prospective trial of chemotherapy and donor leukocyte infusions for relapse of advanced myeloid malignancies after allogeneic stem-cell transplantation. J Clin Oncol 2002;20:405–412. 72 Bader P, Klingebiel T, Schaudt A, TheurerMainka U, Handgretinger R, Lang P, Niethammer D, Beck JF: Prevention of relapse in pediatric patients with acute leukemias and MDS after allogeneic SCT by early immunotherapy initiated on the basis of increasing mixed chimerism: A single center experience of 12 children. Leukemia 1999;13:2079–2086. 73 Shimoni A, Trakhtenbrot L, Bielorai B, Reichart M, Rothman R, Toren A, Rechavi G, Nagler A: Application of immune-therapeutic interventions after allogeneic transplantation based on MRD detection with combined morphological and cytogenetic analysis. Biol Bone Marrow Transplant 2003;9:68a. 74 Marks DI, Lush R, Cavenagh J, Milligan DW, Schey S, Parker A, Clark FJ, Hunt L, Yin J, Fuller S, Vandenberghe E, Marsh J, Littlewood T, Smith GM, Culligan D, Hunter A, Chopra R, Davies A, Towlson K, Williams CD: The toxicity and efficacy of donor lymphocyte infusions given after reduced-intensity conditioning allogeneic stem cell transplantation. Blood 2002;100:3108–3114. 75 Dey BR, McAfee S, Colby C, Sackstein R, Saidman S, Tarbell N, Sachs DH, Sykes M, Spitzer TR: Impact of prophylactic donor leukocyte infusions on mixed chimerism, graft-versushost disease, and antitumor response in patients with advanced hematologic malignancies treated with nonmyeloablative conditioning and allogeneic bone marrow transplantation. Biol Blood Marrow Transplant 2003;9:320– 329. 76 Mapara MY, Kim YM, Wang SP, Bronson R, Sachs DH, Sykes M: Donor lymphocyte infusions mediate superior graft-versus-leukemia effects in mixed compared to fully allogeneic chimeras: A critical role for host antigen-presenting cells. Blood 2002;100:1903–1909.

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77 Massenkeil G, Nagy M, Lawang M, Rosen O, Genvresse I, Geserick G, Dörken B, Arnold R: Reduced intensity conditioning and prophylactic DLI can cure patients with high-risk acute leukaemias if complete donor chimerism can be achieved. Bone Marrow Transplant 2003; 31:339–345. 78 Corradini P, Tarella C, Olivieri A, Gianni AM, Voena C, Zallio F, Ladetto M, Falda M, Lucesole M, Dodero A, Ciceri F, Benedetti F, Rambaldi A, Sajeva MR, Tresoldi M, Pileri A, Bordignon C, Bregni M: Reduced-intensity conditioning followed by allografting of hematopoietic cells can produce clinical and molecular remissions in patients with poor-risk hematologic malignancies. Blood 2002;99:75–82. 79 de Lima M, Bonamino M, Vasconcelos Z, Colares M, Diamond H, Zalcberg I, Tavares R, Lerner D, Byington R, Bouzas L, da Matta J, Andrade C, Carvalho L, Pires V, Barone B, Maciel C, Tabak D: Prophylactic donor lymphocyte infusions after moderately ablative chemotherapy and stem cell transplantation for hematological malignancies: High remission rate among poor prognosis patients at the expense of graft-versus-host disease. Bone Marrow Transplant 2001;27:73–78. 80 Badros A, Barlogie B, Siegel E, Cottler-Fox M, Zangari M, Fassas A, Morris C, Anaissie E, Van Rhee F, Tricot G: Improved outcome of allogeneic transplantation in high-risk multiple myeloma patients after nonmyeloablative conditioning. J Clin Oncol 2002;20:1295–1303. 81 Olavarria E, Ottmann OG, Deininger M, Clark RE, Bandini G, Byrne J, Lipton J, Vitek A, Michallet M, Siegert W, Ullmann A, Wassmann B, Niederwieser D, Fischer T: Response to imatinib in patients who relapse after allogeneic stem cell transplantation for chronic myeloid leukemia. Leukemia 2003;17:1707– 1712. 82 Shimoni A, Kröger N, Zander AR, Rowe JM, Hardan I, Avigdor A, Yeshurun M, Ben-Bassat I, Nagler A: Imatinib mesylate (STI571) in preparation for allogeneic hematopoietic stem cell transplantation and donor lymphocyte infusions in patients with Philadelphia-positive acute leukemias. Leukemia 2003;17:290–297. 83 Shimoni A, Hardan I, Avigdor A, Yeshurun M, Raanani P, Ben-Bassat I, Nagler A: Rituximab reduces relapse risk after allogeneic and autologous stem cell transplantation in patients with high-risk aggressive non-Hodgkin’s lymphoma. Br J Haematol 2003;122:457–464.

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Review Acta Haematol 2004;112:105–110 DOI: 10.1159/000077565

Molecular and Clinical Follow-Up after Treatment of Multiple Myeloma Thomas Rasmussen Lene M. Knudsen Tuan K. Huynh Hans E. Johnsen Department of Hematology L 54P4, Herlev Hospital, University of Copenhagen, Herlev, Denmark

Key Words Allele-specific oligonucleotide PCR W Flow cytometry W Multiple myeloma

Abstract Multiple myeloma (MM) is a B cell malignancy characterized by accumulation of plasma cells (PCs) in the bone marrow. Traditional methods for the detection of minimal residual disease (MRD) measure the presence of monoclonal immunoglobulin protein secreted by the malignant PCs. However, changes in the level of MRD in MM may span 6 logs, and methods with a high sensitivity and dynamic range are necessary for quantitating MRD in MM. The two main technologies used in MRD detection are flow cytometry and patient-specific reverse transcription (RT) PCR. Patient-specific RT-PCR has high sensitivity and may be beneficial in monitoring patients receiving allogeneic transplantation. However, for the MRD evaluation of autotransplants, where few patients achieve molecular remission, flow cytometry monitoring seems to be sufficient. Copyright © 2004 S. Karger AG, Basel

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© 2004 S. Karger AG, Basel 0001–5792/04/1122–0105$21.00/0

Fax + 41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/aha

Introduction

Multiple myeloma (MM) is an incurable B cell malignancy characterized by an uncontrolled growth of plasma cells (PCs) usually in the bone marrow (BM). Myeloma PCs secrete monoclonal immunoglobulin (Ig) in serum, referred to as the M component or M protein. In western industrialized countries the incidence of MM is approximately 4 new cases/100,000 each year with a male/female ratio of 3:2. The median age of the patients is around 65 years and the incidence increases with age [1]. Even though myeloma cells respond to standard chemotherapy, it seems impossible to cure MM. All patients will inevitably relapse, with a median survival time of 30 months, when conventional chemotherapy is used [2]. To improve the treatment of MM patients, high-dose chemotherapy in combination with peripheral blood stem cell transplantation (PBSCT) is now commonly used [1]. Although patients receiving high-dose chemotherapy and PBSCT show a better complete remission (CR) rate, event-free survival, and overall survival, relapse remains a major problem [3]. Evidence exists that achievement of CR improves the outcome and attaining a CR may be a first step toward a cure. Thus, there is a need for monitoring minimal residual disease (MRD) with sensitive and specific methods to be able to study tumor cell kinetics and to define response with more accurate and stringent criteria than the traditional criteria used today. The aim of this

Thomas Rasmussen, PhD Department of Hematology L 54P4 Herlev Hospital, University of Copenhagen DK–2730 Herlev (Denmark) Tel. +45 44 88 34 15, Fax +45 44 53 01 76, E-Mail [email protected]

review is to describe methods with a potential for the detection of MRD in the clinical scene. The utility of the methods is illustrated by examples of MRD monitoring in relation to tumor cell mobilization, purging and novel therapies. Response criteria and correlation with outcome and a rational for selection of methods for clinical studies will be discussed.

Detection of MRD in MM

Traditional Methods The traditional methods to determine MRD in MM are based on the detection of the M protein in serum or urine either by protein electrophoresis or immunofixation, the latter being the most sensitive. Today immunofixation is recommended in response evaluation by the European bone marrow transplantation group (EBMT) whereas others used the less sensitive electrophoretically based methods. The traditional methods have been described extensively elsewhere [4]. Flow Cytometry MM PCs are distinguished from their normal counterparts by the expression of a variable set of cell surface antigens. Myeloma PCs may show an aberrant expression of a promiscuous array of cell surface markers as the expression of CD28 and CD117, overexpression of CD56 and downregulation of CD19 [5–8]. These markers may be regarded as ‘tumor-specific antigens’ for myeloma cells. The phenotype and frequency of a specific cellular subset is easily determined by flow cytometry using monoclonal antibodies specific for the cell surface molecules of interest. The identification of aberrant MM PCs cells can in the majority of cases be based on a multiparametric analysis of four-color staining with CD38 or CD138 together with CD45, CD19 and CD56. The major advantages of flow cytometry are the use of a simple set of markers applicable to 195% of patients. It is quantitative and performed rapidly with a high throughput allowing a day-to-day answer and gives additional information on the immunological status. The major disadvantage is the sensitivity which is 1:104. Polymerase Chain Reaction The VDJ gene rearrangement with different V gene mutations, unique for the individual B cell and its progeny, is an ideal marker for clonality [9]. The production of a unique Ig molecule has provided a clonal marker for myeloma cells at the DNA, RNA and protein level. With

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the advent of polymerase chain reaction (PCR), Ig heavy chain (IgH) PCR techniques using PCR primers annealing to the conserved regions of the IgH gene (referred to as consensus IgH PCR) became widely used for identifying myeloma cells [10–13]. Sequencing of VDJ rearrangements identifying the complementary-determining regions facilitates the design of allele-specific oligonucleotides (ASO) to be used as myeloma patient-specific PCR primers (referred to as ASO IgH PCR), enhancing the specificity and sensitivity of myeloma cell detection. Today, the ASO IgH PCR recognizing the complementarydetermining regions is a well-established method to identify clonal cells in MM [14–17]. The high production of Ig mRNA in myeloma cells makes mRNA the preferred target for identifying MRD, whereas for quantitation of MRD only DNA can be used (one copy of MM-specific IgH DNA/MM cell). Intraclonal variation and clonal progression are generally not observed in MM [18–20], although clonal heterogeneity has been reported in a single MM patient [21]. The most sensitive method for the detection of MRD is nested ASO RT-PCR with a sensitivity of 1:106 and the most clinically applicable PCR-based method for quantitation of MRD is real-time ASO PCR with a sensitivity of 1:105 [22]. The major advantages of ASO PCR-based methods are the high sensitivity. However, there are several disadvantages: a clonal VDJ gene rearrangement can be identified in less than 80% of patients, an assay must be designed and optimized for each patient and a diagnostic BM sample must be available for assay design. Further, ASO PCR-based assays identify all cells belonging to the myeloma clone, including B lymphocytes with an unknown malignant potential.

MRD and Transplantation

Introducing PBSCT in MM has improved and prolonged survival from 36 to 60 months [3, 23]. However, it is still an incurable disease and all patients will inevitably relapse. Several studies have shown a higher CR rate after PBSCT compared to standard chemotherapy when standard methods of monitoring the remission state are used, i.e. electrophoresis and immunofixation [3, 4, 23]. As all patients relapse over time it is reasonable to assume that residual disease exists. Several studies have shown that tumor cells exist in peripheral blood (PB) and BM after PBSCT [24, 25]. Clonally related cells remain in the PB after autologous transplantation and this cell population in PB is quite stable [24, 26, 27]. In a study by Rawstron et al. [28] immunophenotypic aberrant PCs were detected

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by flow cytometry in 42% of patients 3 months posttransplant, and even in 27% of patients whose immunofixation was negative. The presence of abnormal PCs despite negative immunofixation seems to predict a shortened survival [28, 29]. Tumor cells were detected posttransplant with PCR methods. The usage of the consensus primer PCR method provided no additional prognostic information [4]. When the more labor-intensive and sensitive ASO PCR method is used tumor cells can be detected even in immunofixation-negative patients [4, 30]. However, the studies are small and only in a low number of patients can a VDJ rearrangement be detected. The importance and prognostic relevance of ASO PCR positivity posttransplant is thus unknown [4, 30]. Tumor cell contamination of PB stem cell products and reinfusion of tumor cells are major concerns and may contribute to relapse after autologous transplantation. Several studies have shown that tumor cells are mobilized to the PB along with CD34+ stem cells [31–33]. It has been shown that the detection of monoclonal PCs in the PB stem cell graft is associated with a shortened relapsefree survival [34]. Several studies have also shown that virtually all stem cell grafts contain tumor cells as measured by PCR [16], however often in a small amount [35]. In order to reduce the tumor cell content of autografts the CD34+ cell selection has been used. However, transplantation with CD34+ selected cells offers no survival advantage to transplantation with unmanipulated products [35]. In the attempt to improve the purging strategy, a two-step procedure consisting of CD34+ enrichment followed by CD19 depletion was studied [36]. A flow-cytometric analysis documented the expected reduction of CD34– B cells and PCs, in most cases to a level below the sensitivity of flow cytometry. CD19 real-time RT-PCR documented a CD19 mRNA-relative reduction to 0.042 (range 0.01–0.21). ASO IgH primers were designed for 5 patients. All products were positive for clonal myeloma cells before processing and only 1 of 5 was negative after the procedure. This implies that the tumor burden in the patient may be more relevant than the tumor burden in the stem cell product as a source of relapse. The only curative treatment option in MM today is the allogeneic approach either as standard allogeneic transplantation or the newly developed miniallogeneic transplantation. Comparisons between auto- and standard allografted patients have shown that a higher molecular remission rate is achieved in the latter group measured with consensus PCR [37] and ASO PCR [37–39]. Molecular remission can be achieved as late as 1 year posttransplant [39].

Minimal Residual Disease in Multiple Myeloma

MRD Quantitation as an End Point in Evaluation of New Trials

Ideally, the therapeutic outcome has to be judged by overall survival. However, initial and fast evaluation of new strategies may benefit from surrogate markers associated with outcome. For example, it has been demonstrated that survival time is predicted by obtaining a welldefined CR, which, however, correlates to the level of myeloma cells in BM and PB. Consequently, quantitation of the myeloma clone may act as a surrogate end point for clinical phase II–III trials evaluating the potential effect of new treatments. Of course, this only allows to identify new strategies, which should move into classical phase III trials with proper end points. Evaluation of Vaccination Therapy Clinical studies of the vaccination of patients with B cell disease follicular lymphomas have shown evidence of eradication of molecular MRD [40]. However, in MM, vaccination as treatment option to induce immunological response and antitumor effect has not been convincingly proved. The idiotype (Id) of the myeloma Ig is regarded a tumor-specific antigen and a target for immunotherapy. Naturally occurring MHC-restricted myeloma Id-specific T cells have been identified. Such T cells may target Id Ig peptide/MHC complexes on myeloma cells and might lyse tumor cells [41, 42]. Idiotype vaccination may therefore be a novel therapeutic approach to induce an idiotype-specific T cell response. Recently monitoring of MRD by an ASO real-time PCR method in a cohort of MM receiving Id vaccination showed a reduction in PB tumor cells. Sequential analyses of circulating blood myeloma cells in 6 patients with detectable clonal cells and stage I disease revealed 1 patient who achieved a complete molecular remission, 3 patients with a reduction of 92, 79 and 54%, respectively, and 2 patients with unchanged levels [43]. Evaluation of Targeted Induction Therapy The classical MM tumor cell is a malignant PC. Nevertheless, cells of the earlier B cell hierarchy have been demonstrated to belong to the myeloma clone [44–47]. This cell population has been reported to include resting multidrug-resistant cells with replenished potential [48]. The hypothesis is that such cells may be the origin for disease recurrence following remission. Some preclinical data and clinical studies indicated a higher activity when fludarabine was given in combination with suitable chemotherapeutic agents. A combination of conventional

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VAD with fludarabine may, therefore, in theory eliminate a greater fraction of the myeloma hierarchy, especially the CD19+ compartment of cells resistant to cycle dependent drugs. In a phase II pilot study 19 patients with newly diagnosed MM were randomized to receive induction treatment either with four cycles of the VAD regimen (arm A) or with two cycles of VAD followed by two cycles of VAD combined with fludarabine (25 mg/m2/day in 5 days; arm B). At 3 months posttherapy follow-up 9 of the arm B-treated patients responded with 2 in CR and 7 patients in partial response (PR) compared with 5 responders (all PR) in the arm A. Assessment of MRD by flow cytometry revealed a significant reduction in MM PCs in the fludarabine arm, in parallel with a significant reduction of the CD19+ compartment. However, when patients in CR by flow cytometry were analyzed by nested ASO RT-PCR, none of the patients achieved a molecular remission [49].

Discussion

Changes in the level of residual disease in patients with MM may span 6 logs, and methods with high sensitivity and reproducibility over a broad dynamic range are thus necessary for quantitation of MRD in MM. Cell-based technologies such as flow cytometry and ASO PCR-based assays are extensively used to study MRD in MM. However, whereas methods such as nested ASO RT-PCR are superior in the detection of MRD, all cell-based MRD detection assays have a potential problem attempting to determine the actual number of PCs in BM aspirates. This problem is related to the nature of the disease, a heterogeneous infiltration of the marrow, and the unknown degree of PB contamination in the BM sampling procedure. The degree of tumor cell variations in BM samples sequential-

ly aspirated during the same procedure has been addressed demonstrating large sampling variations determined both by ASO real-time quantitative PCR and flow cytometry [22]. However, a more homogeneous infiltration is expected for patients without detectable osteolytic processes [50]. Thus, with standardized BM sampling procedures, flow cytometry and real-time ASO PCR may be used for semiquantitative evaluation of MRD in BM from patients considered to be in CR.

Choosing Methods for Monitoring MRD in Clinical Protocols

For clinical trials including a large cohort of patients the labor-intensive and expensive ASO PCR-based method is not an option for the majority of centers. It has been shown that molecular remission is rarely attained after autografting neither after single nor double autotransplants [37, 39]. Thus MRD monitoring using ASO PCR methods in this setting is not worthwhile. However, the high sensitivity of molecular monitoring of MRD may be important in the follow-up of selected patient groups, for example patients receiving either standard or mini-allogeneic stem cell transplantation [38] or in the evaluation of new treatment strategies where achievement of molecular remission may be expected. With all the advantages of flow-cytometric monitoring of MRD it will be beneficial in the evaluation of autotransplants and in the majority of clinical trials [28, 29].

Acknowledgments This work was supported in part by the Danish Cancer Society, grant No. DP 00 006 to T.R. and grant No. 98 100 09 to H.E.J. and by the Danish Research Agency grant No. 99 00 771.

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19 Ralph QM, Brisco M, Joshua DE, Brown R, Gibson J, Morley AA: Advancement of multiple myeloma from diagnosis through plateau phase to progression does not involve a new Bcell clone: Evidence from the Ig heavy chain. Blood 1993;82/1:202–206. 20 Vescio RA, Cao J, Hong CH, Lee JC, Wu CH, Der DM, Wu V, Newman R, Lichtenstein AK, Berenson JR: Myeloma Ig heavy chain V region sequences reveal prior antigenic selection and marked somatic mutation but no intraclonal diversity. J Immunol 1995;155:2487– 2497. 21 Biggs DD, Kraj P, Goldman J, Jefferies L, Carchidi C, Anderson K, Silberstein LE: Immunoglobulin gene sequence analysis to further assess B-cell origin of multiple myeloma. Clin Diagn Lab Immunol 1995;2/1:44–52. 22 Rasmussen T, Poulsen TS, Honore L, Johnsen HE: Quantitation of minimal residual disease in multiple myeloma using an allele specific real-time PCR assay. Exp Hematol 2000;28: 1039–1045. 23 Lenhoff S, Hjorth M, Holmberg E, Turesson I, Westin J, Nielsen JL, Wisloff F, Brinch L, Carlson K, Carlsson M, Dahl IM, Gimsing P, Hippe E, Johnsen H, Lamvik J, Lofvenberg E, Nesthus I, Rodjer S: Impact on survival of high-dose therapy with autologous stem cell support in patients younger than 60 years with newly diagnosed multiple myeloma: A population-based study. Nordic Myeloma Study Group. Blood 2000;95/1:7–11. 24 Rasmussen T, Jensen L, Honoré L, Johnsen HE: Frequency and kinetics of polyclonal and clonal B-cells in the peripheral blood of patients being treated for multiple myeloma. Blood 2000;96:4357–4359. 25 Kiel K, Cremer FW, Rottenburger C, Kallmeyer C, Ehrbrecht E, Atzberger A, Hegenbart U, Goldschmidt H, Moos M: Analysis of circulating tumor cells in patients with multiple myeloma during the course of high-dose therapy with peripheral blood stem cell transplantation. Bone Marrow Transplant 1999;23:1019– 1027. 26 Billadeau D, Prosper F, Verfaillie C, Weisdorf D, Van Ness B: Sequential analysis of bone marrow and peripheral blood after stem cell transplant for myeloma shows disparate tumor involvement. Leukemia 1997;11:1565–1570. 27 Rasmussen T, Jensen L, Johnsen HE: The CD19 compartment in myeloma includes a population of clonal cells persistent after highdose treatment. Leuk Lymphoma 2002;43: 1075–1077. 28 Rawstron AC, Davies FE, DasGupta R, Ashcroft AJ, Patmore R, Drayson MT, Owen RG, Jack AS, Child JA, Morgan GJ: Flow cytometric disease monitoring in multiple myeloma: The relationship between normal and neoplastic plasma cells predicts outcome after transplantation. Blood 2002;100:3095–3100.

29 San Miguel JF, Almeida J, Mateo G, Blade J, Lopez-Berges C, Caballero D, Hernandez J, Moro MJ, Fernandez-Calvo J, Diaz-Mediavilla J, Palomera L, Orfao A: Immunophenotypic evaluation of the plasma cell compartment in multiple myeloma: A tool for comparing the efficacy of different treatment strategies and predicting outcome. Blood 2002;99:1853– 1856. 30 Lipinski E, Cremer FW, Ho AD, Goldschmidt H, Moos M: Molecular monitoring of the tumor load predicts progressive disease in patients with multiple myeloma after high-dose therapy with autologous peripheral blood stem cell transplantation. Bone Marrow Transplant 2001;28:957–962. 31 Lemoli RM, Cavo M, Fortuna A: Concomitant mobilization of plasma cells and hematopoietic progenitors into peripheral blood of patients with multiple myeloma. J Hematother 1996;5: 339–349. 32 Gazitt Y, Tian E, Barlogie B, Reading CL, Vesole DH, Jagannath S, Schnell J, Hoffman R, Tricot G: Differential mobilization of myeloma cells and normal hematopoietic stem cells in multiple myeloma after treatment with cyclophosphamide and granulocyte-macrophage colony-stimulating factor. Blood 1996; 87:805–811. 33 Knudsen LM, Rasmussen T, Nikolaisen K, Johnsen HE: Mobilisation of tumour cells along with CD34+ cells to peripheral blood in multiple myeloma. Eur J Haematol 2001;67/5– 6:289–295. 34 Gertz MA, Witzig TE, Pineda AA, Greipp PR, Kyle RA, Litzow MR: Monoclonal plasma cells in the blood stem cell harvest from patients with multiple myeloma are associated with shortened relapse-free survival after transplantation. Bone Marrow Transplant 1997;19:337– 342. 35 Stewart AK, Vescio R, Schiller G, Ballester O, Noga S, Rugo H, Freytes C, Stadtmauer E, Tarantolo S, Sahebi F, Stiff P, Meharchard J, Schlossman R, Brown R, Tully H, Benyunes M, Jacobs C, Berenson R, White M, Dipersio J, Anderson KC, Berenson J: Purging of autologous peripheral-blood stem cells using CD34 selection does not improve overall or progression-free survival after high-dose chemotherapy for multiple myeloma: Results of a multicenter randomized controlled trial. J Clin Oncol 2001;19:3771–3779. 36 Rasmussen T, Bjorkstrand B, Andersen H, Gaarsdal E, Johnsen HE: Efficacy and safety of CD34-selected and CD19-depleted autografting in multiple myeloma patients: A pilot study. Exp Hematol 2002;30/1:82–88. 37 Corradini P, Voena C, Tarella C, Astolfi M, Ladetto M, Palumbo A, Van Lint MT, Bacigalupo A, Santoro A, Musso M, Majolino I, Boccadoro M, Pileri A: Molecular and clinical remissions in multiple myeloma: Role of autologous and allogeneic transplantation of hematopoietic cells. J Clin Oncol 1999;17/1:208– 215.

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38 Cavo M, Terragna C, Martinelli G, Ronconi S, Zamagni E, Tosi P, Lemoli RM, Benni M, Pagliani G, Bandini G, Tura S: Molecular monitoring of minimal residual disease in patients in long-term complete remission after allogeneic stem cell transplantation for multiple myeloma. Blood 2000;96:355–357. 39 Martinelli G, Terragna C, Zamagni E, Ronconi S, Tosi P, Lemoli RM, Bandini G, Motta MR, Testoni N, Amabile M, Ottaviani E, Vianelli N, de Vivo A, Gozzetti A, Tura S, Cavo M: Molecular remission after allogeneic or autologous transplantation of hematopoietic stem cells for multiple myeloma. J Clin Oncol 2000; 18:2273–2281. 40 Bendandi M, Gocke CD, Kobrin CB, Benko FA, Sternas LA, Pennington R, Watson TM, Reynolds CW, Gause BL, Duffey PL, Jaffe ES, Creekmore SP, Longo DL, Kwak LW: Complete molecular remissions induced by patientspecific vaccination plus granulocyte-monocyte colony-stimulating factor against lymphoma. Nat Med 1999;5:1171–1177. 41 Yi Q, Dabadghao S, Osterborg A, Bergenbrant S, Holm G: Myeloma bone marrow plasma cells: Evidence for their capacity as antigenpresenting cells. Blood 1997;90:1960–1967.

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42 Osterborg A, Yi Q, Henriksson L, Fagerberg J, Bergenbrant S, Jeddi-Tehrani M, Ruden U, Lefvert AK, Holm G, Mellstedt H: Idiotype immunization combined with granulocytemacrophage colony-stimulating factor in myeloma patients induced type I, major histocompatibility complex-restricted, CD8- and CD4specific T-cell responses. Blood 1998;91:2459– 2466. 43 Rasmussen T, Hansson L, Osterborg A, Johnsen HE, Mellstedt H: Idiotype vaccination in multiple myeloma induced a reduction of circulating clonal tumor B cells. Blood 2003;101: 4607–4610. 44 Rasmussen T, Jensen L, Johnsen HE: The clonal hierarchy in multiple myeloma. Acta Oncol 2001;39:765–770. 45 Billadeau D, Ahmann G, Greipp P, Van Ness B: The bone marrow of multiple myeloma patients contains B cell populations at different stages of differentiation that are clonally related to the malignant plasma cell. J Exp Med 1993;178:1023–1031. 46 Corradini P, Boccadoro M, Voena C, Pileri A: Evidence for a bone marrow B cell transcribing malignant plasma cell VDJ joined to C mu sequence in immunoglobulin (IgG)- and IgAsecreting multiple myelomas. J Exp Med 1993; 178:1091–1096.

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47 Bakkus MH, Van Riet I, Van Camp B, Thielemans K: Evidence that the clonogenic cell in multiple myeloma originates from a preswitched but somatically mutated B cell. Br J Haematol 1994;87/1:68–74. 48 Pilarski LM, Cass CE, Tsuro T, Belch AR: Multidrug resistance of a continuously differentiating monoclonal B lineage in the blood and bone marrow of patients with multiple myeloma. Curr Top Microbiol Immunol 1992;182:177– 185. 49 Bjorkstrand B, Rasmussen T, Remes K, Gruber A, Pelliniemi TT, Johnsen HE: Feasibility of fludarabine added to VAD during induction therapy in multiple myeloma: A randomised phase II-study. Eur J Haematol 2003;70:379– 383. 50 Billadeau D, Blackstadt M, Greipp P, Kyle RA, Oken MM, Kay N, Van Ness B: Analysis of Blymphoid malignancies using allele-specific polymerase chain reaction: A technique for sequential quantitation of residual disease. Blood 1991;78:3021–3029.

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Review Acta Haematol 2004;112:111–119 DOI: 10.1159/000077566

Significance of Minimal Residual Disease in Lymphoid Malignancies Monika Brüggemann Christiane Pott Matthias Ritgen Michael Kneba II. Medizinische Klinik, Universitätsklinikum Schleswig-Holstein, Kiel, Germany

Key Words Minimal residual disease W Chronic lymphocytic leukemia W Acute lymphoblastic leukemia W Non-Hodgkin’s lymphoma W Quantitative PCR

Abstract Modern treatment protocols lead to complete remission (CR) in a considerable proportion of patients with lymphoproliferative disorders. However, many of these patients ultimately relapse, implying that achievement of a clinical CR is compatible with significant amounts of residual malignant cells. Cytogenetic, molecular and immunological techniques that are more sensitive than morphology are increasingly used to assess and quantify minimal residual disease (MRD). Immunological marker analysis allows the detection of aberrant or unusual immunophenotypes, PCR techniques target fusion regions of chromosome aberrations and clone-specific immunoglobulin and T-cell receptor gene rearrangements. The rationale underlying MRD studies is to improve measurement of treatment response, to provide independent prognostic information and to optimise therapeutic strategies. In acute lymphoblastic leukemia (ALL), the MRD based evaluation of initial response to front-line therapy emerged as a highly relevant diagnostic tool, particularly in childhood ALL, where MRD has been shown to be an independent prognostic factor allowing a precise risk group classification. In patients with chronic lymphocytic leukemia (CLL) and non-Hodgkin’s lymphoma (NHL) the prognostic significance of MRD is still a matter of debate,

ABC

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as the majority of patients remain MRD positive after conventional treatment. This phenomenon has changed with the implementation of new treatment modalities, such as application of monoclonal antibodies, where a significant proportion of patients with NHL converts to MRD negativity and experiences prolonged remission. Whether this molecular remission will translate into a superior overall survival is currently the goal of ongoing prospective studies. Copyright © 2004 S. Karger AG, Basel

Introduction

Although a considerable proportion of patients with lymphoid malignancies achieve complete clinical remission with current treatment protocols, the majority of them will ultimately relapse. The source of relapses are residual malignant cells that are below the limit of detection using standard diagnostic techniques. With the development of more sensitive techniques for the detection of malignant cells, the presence of minimal residual disease (MRD) in patients in complete clinical remission has clearly been demonstrated. The critical issue is to determine whether such sensitive detection of MRD will identify patients with a high or low risk of relapse. In childhood acute lymphoblastic leukemia (ALL) the relevance of quantitative MRD detection has been proven in several large prospective studies, so that molecular biological techniques already became an important part of staging, follow-up and risk stratification in ALL.

Prof. Dr. med. Michael Kneba II. Medizinische Klinik, Universitätsklinikum Schleswig-Holstein Campus Kiel Chemnitzstrasse 33 DE–24116 Kiel (Germany) Tel. +49 431 16971200, Fax +49 431 16971202, E-Mail [email protected]

Currently clinical relevant MRD information is obtained mainly using three techniques: genetic analyses detecting structural or numerical chromosomal abnormalities, flow-cytometric immunophenotyping using aberrant or lymphoma-/leukemia-associated phenotypes and polymerase chain reactions (PCR) using chromosomal aberrations or clonally rearranged immune genes as targets. This review outlines the use of immunophenotypic analysis and PCR for MRD detection. We discuss the application and significance of these techniques to the diagnosis, staging, and subsequent clinical management of patients with lymphoid malignancies.

MRD Techniques

Each approach is characterized by advantages and limitations, mainly related to its sensitivity and specificity, which should be taken into account when large-scale multicenter clinical MRD studies are planned. Immunological Analysis Flow cytometry represents a rapid and reliable option for investigating MRD. Targets mainly concern leukemiaassociated phenotypes representing combinations of cellular antigens which are rare or absent among normal hematopoietic cells. In mature lymphoid leukemias restriction of light chain expression or monotypic Vß, VÁ or V‰ can additionally be measured. New approaches base on multiparametric detection of multiple surface antigens and can reach sensitivities up to 10 –4. However, in addition to relatively low sensitivity in a significant proportion of cases immunological methods have their limitations in phenotypic switches, which can lead to false-negative results especially in immature lymphoid malignancies. PCR Analysis Chromosomal translocations as well as immune gene rearrangements have been used extensively as targets for MRD detection in lymphoid malignancies. Structural chromosomal aberrations are ideal PCR targets, which remain stable during the disease course and can reach excellent sensitivities of 10 –4 to 10 –6. Two main disadvantages hamper the application as MRD targets: the chance of false-positive results and applicability only in a minority of patients with lymphoid malignancies. In contrast, the majority of malignant lymphoproliferations display clonal rearrangements of the antigen recep-

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tor genes. Their junctional regions represent fingerprintlike sequences varying enormously due to the fact that different variable (V), diversity (D) and joining (J) gene segments are combined and various numbers of nucleotides are randomly inserted and deleted at the joining sites (fig. 1a). The immunoglobulin heavy chain (IgH) gene rearrangements have been used as MRD target in different subtypes of lymphoid malignancies, such as chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL) and ALL. They can be identified in about 90% of B cell malignancies but also in about 10–15% of lymphoblastic T cell malignancies, whereas their frequency in the more mature T cell lymphomas is much lower [1]. The vast majority of T cell malignancies display clonally rearranged T cell receptor (TCR) ß and TCR Á genes which are, therefore, applicable for MRD assessment in these entities. Clonal TCR gene rearrangements also occur in a relative high frequency in precursor B-ALL, but their frequency is much lower or absent in B-ALL and mature B cell malignancies. Clonality can be assessed using primers directed against the conserved region of the V, D and J segments of the different antigen receptor and immune genes. With the use of fluorescent dye-labeled primers PCR products can easily be detected by automated fluorescence fragment analysis (gene-scanning) with a size discrimination down to 1 base pair (fig. 1b). By this ‘consensus primer – PCR’ approach a clonal rearrangement can be detected with a sensitivity of one single tumor cell in the background of 20–1,000 polyclonal lymphoid cells. The sensitivity and specificity of the gene-scanning method with consensus primers depends on the size of the clone-specific PCR product in relation to the polyclonal background pattern: clone-specific PCR fragments located in the middle of the Gaussian size-distribution curve might be detected with lower sensitivity compared to clonotypic products with sizes outside the main peak area (fig. 1b). To reach a higher level of sensitivity, DNA sequencing of the junctional regions is required in order to design tumor-specific primers and probes. Conventional PCR analyses with end point quantification can lead to major difficulties of quantification. Thanks to the development of real-time quantitative (RQ)-PCR techniques, precise quantification of target sequences is possible during the early exponential phase of PCR amplification (fig. 2, 3). This technology eliminates biases taking place during late postexponential phases of PCR reaction and during postPCR manipulation of samples.

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Fig. 1. Scheme of quantitation strategy by IgH-CDR3 TaqMan PCR. a IgH consensus PCR with a fluorescence dye-labeled primer directed towards conserved areas of the IGVH and JH gene segments. b Genescan analysis of the PCR products and subsequent sequencing of the monoclonal clone-specific junctions. c Primer design of allele-

specific oligonucleotide (ASO) primers that match the clonotypic region of individual rearranged immune genes. Resulting in a high specificity of the assays ASO primers were used with specific TaqMan probes complementary to the junctional region in combination with allele-specific upstream V-N-(D) and downstream germline J primers for the individual JH family gene.

A major drawback of using rearranged immune genes as MRD-PCR targets is the possible occurrence of continuing rearrangements during the course of therapy and during follow-up, which can lead to false-negative PCR results. This phenomenon takes place in more immature disorders like ALL but is rare in mature lymphoid tumors such as indolent lymphomas and CLL.

Minimal Residual Disease in Lymphoid Malignancies

Clinical Importance of MRD in ALL

Residual Disease and Clinical Outcome in Childhood ALL Several large-scale studies in childhood ALL have demonstrated that MRD analysis by molecular or highly sensitive immunological methods can predict the clinical

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outcome and that this MRD information is independent of classical prognostic factors at diagnosis such as age, sex, white blood cell count, immunophenotype and chromosome aberrations [2–6]. The most significant application of MRD assessment is the evaluation of the initial response to front-line therapy, which has been shown to be a strong prognostic factor. Depending on the technique of MRD monitoring and the treatment protocol, 42–75% of childhood patients are MRD negative at the end of induction. These children have an excellent outcome (relapse rate 2–8%). On the other hand, high level MRD at this time point is associated with a high relapse rate of 50–100% [2–4, 6, 7]. The International Berlin-FrankfurtMünster Study Group (I-BFM-SG) identified three different MRD-based risk groups according to the MRD kinetics at two time points before consolidation treatment: a low-risk group, defined by MRD negativity at time points one and two, comprised 43% of patients and had a 3-year relapse rate of only 2%. In contrast, the high-risk group (15% of patients) with MRD levels 610 –3 at both time points relapsed in 75% of cases; the intermediate risk group, accounting for 43% of patients, had a relapse rate of 23% [6]. Significant differences in the kinetics of MRD have been reported between T-ALL and B cell precursor ALL [8]. Frequency of MRD-positive patients mainly during the first 1.5 years was higher in the T-ALL group compared to precursor B-ALL. Also, distribution between the MRD-based risk groups differed: more than twice as many patients with T-ALL were classified in the high-risk group compared to precursor B-ALL (28 vs. 11%), whereas only half of the T-ALL patients belonged to the low-risk group (23 vs. 46%). With 5-year relapse-free survival rates of 100% (high-risk group) and 0% (low-risk group) the prognostic value of MRD in T-ALL was even higher than in B lineage ALL. The predictive impact of MRD is also shown for childhood patients with relapsed ALL [9]. High level MRD 610 –3 during the first stages of therapy (day 36) was highly predictive of relapse, whereas lower MRD levels were associated with a high probability of relapse-free survival. However, whether patients with a slow reduction of tumor load benefit from more intensive treatment remains unclear. Results of MRD Evaluation in Adult ALL Compared to childhood ALL, information on the value of MRD in adult patients is rare. The frequency of MRD positivity and MRD levels seem to be higher than in children with ALL [10, 11]. Brisco et al. [10] reported

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detectable MRD in 67% of cases (18/27) between day 29 and 68 with MRD levels 610 –3 strongly correlating with subsequent relapse. Another immunophenotypic evaluation considered early response to therapy and identified a small subset of patients with negative or low level MRD by day 14 with an excellent prognosis, whereas patients with a slow decrease of MRD (MRD 110 –3 after day 35) represented a high-risk group [12]. MRD kinetics during later blocks of therapy were also analyzed and demonstrated the association of MRD positivity [13] or MRD levels 110 –4 [14] with a dismal outcome at all investigated time points. Only few MRD studies focused on T-ALL [15, 16], because this type only constitutes about 20% of adult ALL cases. Immunophenotypic data also suggest the relapsepredicting value of MRD for this patient group: MRD positivity before consolidation (MRD+: 38% of patients), before the third reinduction (MRD+: 34%) and before reinduction cycle 6 (MRD+: 17%) was associated with a high relapse rate of 81.5, 54.5 and 50.0%, respectively, whereas MRD negativity at these time points predicted a more favorable outcome (2-year relapse rate 38.9, 15.8 and 16.4%) [16]. In contrast, preliminary PCR data did not render stringent results [15], and particularly failed to predict the outcome on the basis of discrete testing time points. In case of Philadelphia chromosome/BCR-ABL-positive (Ph+) ALL, RT-PCR analysis of BCR-ABL fusion transcript is also a useful tool for monitoring the response to treatment and particularly for predicting the risk of relapse after bone marrow transplantation. It appears that evidence of MRD after allogeneic BMT for Ph+ patients is a sign of poor prognosis [17, 18]. Conclusions MRD has turned out to be an independent prognostic factor in ALL allowing a precise risk group classification mainly adapted from the evaluation of early tumor kinetics. Most MRD studies focused on children, but currently comparable results are being obtained in adult ALL. As patients may profit from risk-adapted strategies, several study groups have adopted an MRD-based risk group classification for treatment stratification in their ongoing clinical studies.

Clinical Importance of MRD in CLL

CLL predominates in the elderly and is known to have a variable prognosis depending on certain chromosomal aberrations (deletions in 11q, 13q and 17p, trisomy 12)

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Fig. 2. Amplification plot of a quantitative

RQ-PCR assay. The fluorescence intensity (RN) is plotted versus time (cycle number) based on a real time measurement of RN due to free fluorescence reporter molecules. Sequence detection software (SDS; PE Biosystems) averages peak normalized reporter fluorescence intensity (fig. 1) for each cycle and plots it versus cycle number.

Fig. 3. Standard calibration curve obtained from amplification of specific tumor DNA from the sample at primary diagnosis diluted into polyclonal DNA. This makes it possible to calculate a threshold cycle (CT) defined as cycle number at which normalized reporter fluorescence (RN) passes a fixed threshold baseline. For distinct amplification targets, the amount of target molecules in the reaction tube determines CT. Quantification results from comparing CT of the follow-up samples with a standard curve established by serially diluting, for example, from DNA of diagnostic bone marrow into polyclonal DNA and subsequent RQ-PCR analysis. Sensitivity threshold is defined as the last dilution with a specific fluorescence signal. To equalize differences in amount and quality of the DNA, albumin copies were quantitated as internal reference in all samples and used for subsequent normalization of MRD values.

and IGVH mutational status. As a consequence, symptom palliation is a reasonable goal of conventional therapy in many patients with advanced age. Purine analogues are the most effective conventional agents in CLL therapy to induce remissions; however, most responses are partial, and even complete remission (CR) patients likely harbor a significant level of residual disease [19, 20]. That is the reason why MRD measurement in conventionally treated

CLL has not been considered to be very important. Nevertheless, one recently published study demonstrated conversion to MRD negativity in 17% of patients with resistant or relapsed CLL treated with fludarabine, cyclophosphamide and mitoxantrone [21]. It is of note that there was no difference between the duration of response in CR cases reaching MRD negativity compared to those in CR with persistent MRD.

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In the group of younger patients new therapeutic strategies, such as hematopoietic stem cell transplantation (SCT) and monoclonal antibodies, have resulted in a significant proportion of patients attaining much more profound responses. In these cases, the evaluation of MRD might constitute an important issue as outcome predictor. Recent studies monitoring MRD during and after high-dose chemotherapy with autologous stem cell support demonstrated a significant decrease of MRD after stem cell transplantation. Nevertheless, a considerable number of patients remained MRD positive, which was associated with a poor prognosis, whereas patients who achieved an MRD-negative status had a more favorable outcome [22]. However, most of the patients with autologous transplants ultimately relapse with a recurrence rate of about 50% 4 years after transplantation [22]. Reappearance of MRD after autologous stem cell transplantation seems to anticipate an overt clinical relapse [20, 22– 24]. For this reason additional strategies such as application of monoclonal antibodies, which seem to have the potential to eradicate MRD [25], are warranted. As a result of the growing doubt of the curable potency of autologous stem cell transplantation, allogeneic transplantation is coming into focus in CLL. Although this therapy is associated with a high treatment-related mortality, several studies suggest that a fraction of CLL patients can be cured by allotransplantation. This is often associated with eradication of detectable MRD. Esteve et al. [22] and our group [28] demonstrated conversion to MRD negativity in 7 out of 8 patients achieving CR after allotransplantation. All these patients remained in CR. Provan et al. [24] have reported a correlation between the persistence of detectable MRD and subsequent relapse. However, in contrast with what can be seen in autografts, clearance of MRD in a considerable number of cases with allogeneic transplants is delayed, taking up to more than 1 year after transplant [22, 26–28]. Even cases with detectable MRD more than 3 years post-SCT are described [26], most likely being explained by the modulation of the graft-versus-tumor effect on the residual leukemic cells. Mattsson et al. [26] reported a correlation between the degree of tumor burden pre-SCT and MRD positivity post-SCT. Whether MRD detection provides a basis for the decision of additional immunotherapy remains unclear. Conclusion Monitoring MRD in CLL after high-dose chemotherapy is currently being done in a growing number of studies.

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Quantitative MRD assessment in patients with clinical CR allows to determine the efficacy of new therapy protocols. The relevance of the detection of subclinical molecular relapse is the subject of ongoing prospective studies. At present first results suggest that after autologous SCT the majority of patients still harbor the malignant clone although in rare cases long lasting molecular remissions are possible. Whether these patients are definitely cured and evaluation of MRD will influence further therapeutic decisions in CLL is presently unknown.

Clinical Importance of MRD in Non-Hodgkin’s Lymphoma

Current treatment strategies for patients with indolent malignant lymphoma range from ‘watch and wait’ procedures to aggressive treatment protocols with a curative intention. However, even patients achieving CRs might still harbor 108 to 1010 tumor cells that are missed by conventional staging procedures. These residual lymphoma cells are proven to be of prognostic relevance for the individual patient because they are the suspected source of relapse. Their quantitative determination seems to be necessary to determine new prognostic parameters and the efficacy of current treatment protocols. As discussed above, the vast majority of B cell malignancies are characterized by clonal IgH rearrangements. In addition, specific PCR-detectable chromosomal translocations, particularly t(11;14) and t(14;18) translocation, are present in specific non-Hodgkin’s lymphoma entities. The t(14;18) translocation is a major pathogenetic mechanism of follicular lymphoma (FL) causing deregulation of the bcl-2 protooncogene which induces prolonged cell survival and inhibition of apoptosis. Three main breakpoint regions on chromosome 18 require different forward primer sets, whereas the JH region of the IgH gene on chromosome 14 can be covered by a single JH consensus primer. This strategy allows PCR detection of the t(14;18) in about 60% of FL. Seminested and nested PCR procedures are extremely sensitive and can routinely detect one lymphoma cell in 105 to 106 normal cells. The t(11;14) translocation fuses the bcl-1 locus with the IgH locus on chromosome 14 and is the characteristic translocation for mantle cell lymphoma (MCL). Breakpoints on chromosome 11 scatter over a region of 100 kb. Mainly for this reason about two third of t(11;14) translocations escape PCR detection. Both translocations as well as clonal IgH rearrangements serve as targets for quantitative MRD assessment in lymphomas. Recently our

Brüggemann/Pott/Ritgen/Kneba

group could demonstrate that immune modulation by donor lymphocyte infusion (DLI) or stop of immunosuppression can induce molecular remission in patients with CLL after allogeneic SCT [28]. Conventional Treatment An important application of MRD assays is the molecular monitoring of patients achieving CR. Sequential MRD analysis has provided novel insights into the behavior of residual neoplastic lymphoma cell populations and also allowed the identification of different prognostic subgroups after conventional chemotherapy protocols. Lopez-Guillermo et al. [29] reported a high rate of molecular remissions (66%) in 194 de novo FL after conventional chemotherapy. The investigators demonstrated a correlation between molecular response during the first year of therapy and failure-free survival. Remarkably, a t(14;18) translocation is also detectable by PCR on low levels in 10–25% of healthy individuals [31, 32]. This fact underlines the necessity of quantitative PCR approaches in the setting of clinical MRD studies. With the introduction of the monoclonal anti-CD20chimeric antibody rituximab into conventional treatment protocols, sustained clinical and molecular remissions can be achieved in approximately 50% of patients with FL [33]. Furthermore, assessment of a durable PCR-negative status after treatment with rituximab turned out to be associated with a better clinical outcome in this entity [34]. However, in MCL rituximab in combination with CHOP is associated with favorable clinical and molecular response rates which do not translate into prolonged progression-free survival [35]. Therefore, careful long-term monitoring is required to ascertain the clinical relevance of molecular data in the setting of different lymphoma entities.

In MCL molecular follow-up data are available only for smaller groups of patients and the vast majority of patients showed persistent PCR positivity after transplantation with purged or unpurged autografts [39, 40]. However, a recent investigation in 18 MCL patients demonstrated that molecular remission after peripheral blood SCT can be achieved predicting prolonged progressionfree survival in a significant proportion of MCL [41]. The use of in vitro purging in indolent lymphoma is still a matter of controversy. After induction therapy only 30% of stem cell grafts are PCR negative; by immunological purging procedures with monoclonal antibodies 40– 50% of the grafts of FL become PCR negative. In several studies it could be demonstrated that reinfusion of PCRnegative grafts was correlated with a better disease-free survival in patients with FL [42, 43]. In MCL immunological purging was not effective and had no influence on relapse [39, 44]. Notably a considerable effect of rituximab as an in vivo purging method for FL and MCL could be demonstrated by Magni et al. [45] resulting in 80% PCR-negative autografts; however, these results were statistically not significant. Allogeneic transplantation has gained considerable interest as salvage treatment for patients with relapsed FL or MCL, but clinical and molecular data are rare and prospective studies are lacking. A significant proportion of these patients seem to experience molecular remission with prolonged disease-free survival [46]. PCR monitoring of MRD will be extremely helpful to evaluate efficiency of this treatment approach and will allow programmed infusion of donor lymphocytes depending on MRD status.

Conclusions and Future Perspectives

High-Dose Treatment and Peripheral Blood Stem Cell Transplantation High-dose chemoradiotherapy protocols followed by autologous or allogeneic stem cell transplantation are increasingly used for younger patients with indolent lymphoma and MCL. However, autologous stem cell grafts are often contaminated by occult lymphoma cells that may contribute to relapse. MRD status before autografting and the PCR status assessed after SCT are significant prognostic parameters for relapse-free survival in patients with FL [36]. In FL patients after autologous bone marrow transplantation disease-free survival was markedly increased in patients achieving a PCR-negative status after autologous bone marrow transplantation [37, 38].

Minimal Residual Disease in Lymphoid Malignancies

The clinical impact of MRD detection in different lymphoproliferative disorders is not the same. While MRD has proven to be an independent prognostic factor in ALL in several large-scale studies, the clinical relevance of MRD assessment in NHL and CLL is still unclear. Further studies are required to obtain additional MRD information particularly for new treatment settings like application of monoclonal antibodies or SCT.

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28 Ritgen M, Stilgenbauer S, von Neuhoff N, Humpe A, Brüggemann M, Pott C, Raff T, Kröber A, Bunjes D, Schlenk R, Schmitz N, Döhner H, Kneba M, Dreger P: Graft-versusleukemia activity may overcome therapeutic resistance of chronic lymphocytic leukemia with unmutated immunoglobulin variable heavy chain gene status: implications of minimal residual disease measurement with quantitative PCR. Blood 2004, in press. 29 Lopez-Guillermo A, Cabanillas F, McLaughlin P, Smith T, Hagemeister F, Rodriguez MA, Romaguera JE, Younes A, Sarris AH, Preti HA, Pugh W, Lee MS: The clinical significance of molecular response in indolent follicular lymphomas. Blood 1998;91:2955–2960. 30 Leonard BM, Hetu F, Busque L, Gyger M, Belanger R, Perreault C, Roy DC: Lymphoma cell burden in progenitor cell grafts measured by competitive polymerase chain reaction: Less than one log difference between bone marrow and peripheral blood sources. Blood 1998;91: 331–339. 31 Summers KE, Goff LK, Wilson AG, Gupta RK, Lister TA, Fitzgibbon J: Frequency of the Bcl-2/IgH rearrangement in normal individuals: Implications for the monitoring of disease in patients with follicular lymphoma. J Clin Oncol 2001;19:420–424. 32 Ladetto M, Drandi D, Compagno M, Astolfi M, Volpato F, Voena C, Novarino A, Pollio B, Addeo A, Ricca I, Falco P, Cavallo F, Vallet S, Corradini P, Pileri A, Tamponi G, Palumbo A, Bertetto O, Boccadoro M, Tarella C: PCRdetectable nonneoplastic Bcl-2/IgH rearrangements are common in normal subjects and cancer patients at diagnosis but rare in subjects treated with chemotherapy. J Clin Oncol 2003; 21:1398–1403. 33 Czuczman MS, Grillo-Lopez AJ, McLaughlin P, White CA, Saleh M, Gordon L, LoBuglio AF, Rosenberg J, Alkuzweny B, Maloney D: Clearing of cells bearing the bcl-2 [t(14;18)] translocation from blood and marrow of patients treated with rituximab alone or in combination with CHOP chemotherapy. Ann Oncol 2001;12:109–114.

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34 Rambaldi A, Lazzari M, Manzoni C, Carlotti E, Arcaini L, Baccarani M, Barbui T, Bernasconi C, Dastoli G, Fuga G, Gamba E, Gargantini L, Gattei V, Lauria F, Lazzarino M, Mandelli F, Morra E, Pulsoni A, Ribersani M, Rossi-Ferrini PL, Rupolo M, Tura S, Zagonel V, Zaja F, Zinzani P, Reato G, Foa R: Monitoring of minimal residual disease after CHOP and rituximab in previously untreated patients with follicular lymphoma. Blood 2002;99:856– 862. 35 Howard OM, Gribben JG, Neuberg DS, Grossbard M, Poor C, Janicek MJ, Shipp MA: Rituximab and CHOP induction therapy for newly diagnosed mantle-cell lymphoma: Molecular complete responses are not predictive of progression-free survival. J Clin Oncol 2002;20: 1288–1294. 36 Moos M, Schulz R, Martin S, Benner A, Haas R: The remission status before and the PCR status after high-dose therapy with peripheral blood stem cell support are prognostic factors for relapse-free survival in patients with follicular non-Hodgkin’s lymphoma. Leukemia 1998; 12:1971–1976. 37 Gribben JG, Neuberg D, Freedman AS, Gimmi CD, Pesek KW, Barber M, Saporito L, Woo SD, Coral F, Spector N: Detection by polymerase chain reaction of residual cells with the bcl-2 translocation is associated with increased risk of relapse after autologous bone marrow transplantation for B-cell lymphoma. Blood 1993;81:3449–3457. 38 Zwicky CS, Maddocks AB, Andersen N, Gribben JG: Eradication of polymerase chain reaction detectable immunoglobulin gene rearrangement in non-Hodgkin’s lymphoma is associated with decreased relapse after autologous bone marrow transplantation. Blood 1996;88:3314–3322. 39 Andersen NS, Donovan JW, Borus JS, Poor CM, Neuberg D, Aster JC, Nadler LM, Freedman AS, Gribben JG: Failure of immunologic purging in mantle cell lymphoma assessed by polymerase chain reaction detection of minimal residual disease. Blood 1997;90:4212– 4221. 40 Corradini P, Astolfi M, Cherasco C, Ladetto M, Voena C, Caracciolo D, Pileri A, Tarella C: Molecular monitoring of minimal residual disease in follicular and mantle cell non-Hodgkin’s lymphomas treated with high-dose chemotherapy and peripheral blood progenitor cell autografting. Blood 1997;89:724–731.

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Author Index Vol. 112, No. 1–2, 2004

Amariglio, N. 24 Ben-Bassat, I. 40 Bornhäuser, M. 16 Brüggemann, M. 111 Campana, D. 8 Cilloni, D. 79 Coustan-Smith, E. 8 Ehninger, G. 16 Grimwade, D. 55 Haferlach, T. 68 Hehlmann, R. 30 Hiddemann, W. 68 Hochhaus, A. 5, 30, 85

Hördt, T. 30 Huynh, T.K. 105 Izraeli, S. 34 Johnsen, H.E. 105 Kern, W. 68 Kneba, M. 111 Knudsen, L.M. 105 La Rosée, P. 30 Lengfelder, E. 55 Merx, K. 30, 85 Müller, M.C. 30 Nagler, A. 93 Paschka, P. 30, 85

Pott, C. 111 Raanani, P. 5, 40 Rasmussen, T. 105 Rechavi, G. 24 Reiter, A. 55 Ritgen, M. 111 Saglio, G. 79 Schnittger, S. 68 Schoch, C. 68 Shimoni, A. 93 Thiede, C. 16 Trakhtenbrot, L. 24 Waldman, D. 34

Subject Index Vol. 112, No. 1–2, 2004

Acute lymphoblastic leukemia 111 Allele-specific oligonucleotide PCR, multiple myeloma 105 BCR-ABL-positive chronic myelogenous leukemia 30 Childhood, acute lymphoblastic leukemia 34 Chimerism 16 Chronic lymphocytic leukemia 111 Combined analyses, minimal residual disease 24 Cytogenetics 40, 85

ABC Fax + 41 61 306 12 34 E-Mail [email protected] www.karger.com

© 2004 S. Karger AG, Basel

Accessible online at: www.karger.com/aha

Flow cytometry 8, 105 FLT3 gene, length mutations 68 Fluorescence-activated cell sorter 34 Fluorescence in situ hybridization 24, 40, 85 Follow-up studies, FLT3 gene length mutations 68 Imatinib mesylate 85 Leuk(a)emias 8, 16, 30, 34, 40, 55, 68, 79, 85 Lymphocyte infusion, donor 93 Minimal residual disease 16, 111

Multiple myeloma 105 Non-Hodgkin’s lymphoma 111 Nonmyeloablative conditioning, cellular immunotherapy 93 PCR 16 Polymerase chain reaction technology 30, 34, 40, 85, 105 Quantitative PCR 111 Retinoic acid receptor alpha 55 RNA stabilization 30 Stem cell transplantation 16, 93 Wilm’s tumor gene 79