SYNOPSIS Detection of minimal residual disease in acute ... - Nature

Leukemia (2001) 15, 278–279  2001 Nature Publishing Group All rights reserved 0887-6924/01 $15.00 www.nature.com/leu

SYNOPSIS Detection of minimal residual disease in acute lymphoblastic leukemia: the St Jude experience After many years of laboratory and preclinical studies, assessment of minimal residual disease (MRD) during treatment is now widely accepted as a method of determining the effectiveness of therapy for ALL. The use of MRD assays to guide therapy promises to further increase cure rates by preventing overtreatment of patients with highly responsive disease and undertreatment of those with more aggressive leukemia. Although they are clinically informative, current MRD assays have not been perfected, and their optimal clinical use has not yet been established. The most reliable methods of MRD detection in ALL include flow cytometric profiling of aberrant immunophenotypes, polymerase chain reaction (PCR) amplification of antigenreceptor genes, and PCR amplification of fusion transcripts. At St Jude, all three methods are used to study MRD. Conventional karyotyping and fluorescence in situ hybridization, with a sensitivity of 1% to 5%, are occasionally useful for clarifying the nature of morphologically suspect blast cells, but cannot reliably detect submicroscopic leukemia. Detection of MRD presents some unique technical challenges that go beyond those of the individual method used. Therefore, investigators who have technical expertise, but little experience in MRD detection, should exercise caution by following well-established methodological protocols or by extensively testing newly developed approaches. If results obtained in different centers are to be comparable, the uniformity of MRD measurements must be ensured. In this regard, an international collaboration between St Jude and other centers will use modern Internet-based communication methods to exchange and review flow cytometry files used in MRD studies.1 The efforts of the Berlin–Frankfurt–Mu¨nster (BFM) Consortium to standardize PCR assays among participating centers are also commendable. Current strategies for flow cytometric detection of MRD rely on combinations of leukocyte markers that do not normally occur in cells of the peripheral blood and bone marrow. We identify such leukemia-associated phenotypes by quadruplecolor staining techniques and include in our panels only marker combinations that allow the detection of one leukemic cell among 104 or more normal cells.2 We can currently study approximately 90% of ALL cases with this level of sensitivity. We have pioneered the use of DNA microarrays to identify new leukemia markers,3 with the aim of increasing the number of patients that can be studied and simplify the complex antibody panels currently used. In this regard, investigators should beware that simplification without extensive testing of remission samples is likely to lead to an unacceptable number of false-negative results. The greatest obstacle to the use of PCR amplification as a routine MRD assay is that it requires the identification of Ig

Correspondence: D Campana, Department of Hematology-Oncology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis TN 38105–2794, USA; Fax: 901 495 3749 Received 27 March 2000; accepted 11 October 2000

or T cell receptor (TCR) gene rearrangements in each patient’s malignant cells at the time of diagnosis. In many cancer centers this procedure has been simplified by automation. At St Jude, for example, antigen-receptor gene configuration is identified in ALL samples at the time of diagnosis by using a relatively small panel of consensus primers and then performing direct automated sequencing of the PCR products (in the institution’s DNA chemistry core facility). After the leukemia-specific sequences are determined, the corresponding probes are synthesized, also in the core facility. This process requires approximately 5 days. During the subsequent 2 to 10 days, the optimal PCR conditions for specific amplification of the leukemic rearrangement are identified. When the remission sample arrives in the laboratory, assessment of MRD requires only 5 to 6 h. There has been some debate about the optimal method of quantifying MRD with PCR studies of antigen-receptor genes. We prefer limiting-dilution PCR based on detection of a single leukemic gene rearrangement by using two rounds of PCR amplification. At the sample dilution at which both positive and negative results occur, the percentage of leukemic cells can be estimated by using Poisson statistics. Limiting-dilution PCR is relatively rapid (5 to 6 h), has a uniform sensitivity, and provides quantitative assessment of MRD. The main drawback to this method is the need for a relatively large number of dilution replicates for sample analysis. Real-time PCR technology has great potential for MRD assessment. It ensures quantification of samples during the exponential phase of PCR and eliminates processing of samples after amplification. There are, however, some disadvantages associated with this technology. The cost of synthesizing a junction-specific fluorogenic probe is approximately 20 times that of synthesizing an unmodified oligonucleotide. This cost could be reduced through alternative PCR strategies, such as those using a limited number of consensus probes, positioned at the 3⬘ end of V regions or J regions. In addition, at present there are rather strict limitations on the nucleotide composition of the probes and amplification primers which may restrict the sensitivity of the procedure. Limited applicability and difficult quantitation of residual leukemia are the main limitations of MRD assays based on RT-PCR amplification of fusion transcripts. Only approximately 40% of ALL cases have specific chromosomal aberrations with well-defined breakpoint-fusion regions. Quantification of PCR products has been significantly improved by the development of real-time PCR technology. Nevertheless, the relationship between the number of transcripts and the number of residual tumor cells may be difficult to establish, because it is not yet clear whether the levels of transcripts fluctuate during therapy. Moreover, the susceptibility of mRNA to degradation remains a potential problem. At our institution, MRD studies are performed primarily by flow cytometry and by PCR amplification of IgH genes. To determine how well measurements obtained by these techniques were correlated, we studied serial dilutions of normal and leukemic cells by both flow cytometry and PCR amplifi-

Synopses

cation of IgH genes.4 We found the two methods to be highly concordant (r2 = 0.962). We then examined 62 bone marrow samples collected from children with ALL during clinical remission.4 In 12 samples, both techniques detected MRD levels ⭓1 in 104. The percentages of leukemic cells measured by the two methods correlated well (r2=0.978). Of the remaining 50 samples, 48 had MRD levels ⬍1 in 104. Results were discordant in only two samples: PCR detected two in 104 leukemic cells in one sample and five in 104 in the other, whereas the flow cytometric assay was negative. Both patients remain in remission by clinical, flow cytometric, and molecular criteria, one 18 months and one 28 months after testing. In childhood ALL, results of studies based on flow cytometry or PCR amplification of antigen receptor genes are remarkably concordant,5–7 suggesting that MRD studies with these methods should be incorporated into treatment protocols. A single measurement at an informative time-point during therapy may be sufficient for most patients. For example, patients with B-lineage ALL who show no MRD at the end of remission induction have only a 5.8% 5-year cumulative incidence of relapse.8 The outlook for patients who do have MRD at the end of induction therapy depends on the quantity and the persistence of MRD.8 The presence of 1% or more leukemic cells is strongly associated with subsequent relapse and indicates high-risk ALL. Measurements at subsequent time-points may also help to evaluate risk. In our series, patients whose MRD disappeared by week 14 of continuation therapy had a cumulative incidence of relapse similar to that of patients who had no MRD at any time after induction therapy. By contrast, if MRD persisted during continuation chemotherapy, the risk of subsequent relapse continued to increase as time elapsed. Thus, patients who have positive MRD findings at the end of remission induction therapy may benefit from more frequent MRD testing during clinical remission. Clearly, the potential benefits of MRD monitoring should be extended to all patients. Because we have found no single MRD detection technique that can be used for all patients, multiple methods must be used for universal monitoring of MRD. At St Jude, we use flow cytometry and PCR amplification of antigen-receptor genes simultaneously. Since we started using this strategy, we have been able to conduct MRD studies in 80 consecutive ALL cases. This approach should also prevent false-negative results caused by changes in immunophenotype or predominant antigen-receptor gene clones during the course of the disease. It remains to be decided how MRD assays should be used to guide treatment. Should the results of these assays outweigh traditional prognostic features? Because prognostic features predict response to therapy, whereas MRD assays measure it, it would be logical to give more weight to the latter. However, the association between response to treatment and clinical and biologic parameters such as age, leukocyte count, and genetic features is well established, even with different treatment protocols, whereas the available information about MRD

is less extensive. Therefore, oncologists may be reluctant to abandon the established prognostic factors. At present, a prudent course would be to combine MRD with clinical and biological parameters for comprehensive risk assignment of children with ALL.

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Acknowledgements This work was supported by grants CA60419, CA52259, CA21765 and CA20180 from the National Cancer Institute, and by the American Lebanese Syrian Associated Charities (ALSAC). D Campana GAM Neale E Coustan-Smith C-H Pui

Departments of Hematology-Oncology and Pathology, St Jude Children’s Research Hospital, and University of Tennessee, Memphis, Tennessee, USA

References 1 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. 2 Campana D, Coustan-Smith E. Detection of minimal residual disease in acute leukemia by flow cytometry. Cytometry 1999; 38: 139–152. 3 Chen J-S, Coustan-Smith E, Suzuki T, Neale GA, Pui CH, Campana D. Identification of novel leukemia markers by comparative analysis of gene expression with DNA microarrays. Blood 1999; 94: 657a. 4 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. 5 Coustan-Smith E, Behm FG, Sanchez J, Boyett JM, Hancock ML, Raimondi SC, Rubnitz JE, Rivera GK, Sandlund JT, Pui CH, Campana D. Immunological detection of minimal residual disease in children with acute lymphoblastic leukaemia. Lancet 1998; 351: 550–554. 6 Cave H, van der Werff ten Bosch, 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. 7 van Dongen JJ, Seriu T, Panzer-Grumayer ER, Biondi A, PongersWillemse MJ, Corral L, Stolz F, Schrappe M, Masera G, Kamps WA, Gadner H, Van Wering ER, Ludwig W-D, Basso G, de Bruijn MAC, Cazzaniga G, Hettinger K, van der Does-van den Berg A, Hop WCJ, Riehm H, Bartram CR. Prognostic value of minimal residual disease in acute lymphoblastic leukaemia in childhood. Lancet 1998; 352: 1731–1738. 8 Coustan-Smith E, Sancho J, Hancock ML, Boyett JM, Behm FG, Raimondi SC, Sandlund JT, Rivera GK, Rubnitz JE, Ribeiro RC, Pui CH, Campana D. Clinical importance of minimal residual disease in childhood acute lymphoblastic leukemia. Blood 2000; 96: 2691–2696.

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