Thiopurine Methyltransferase (TPMT) Genotype and Early Treatment Response to Mercaptopurine in Childhood Acute Lymphoblastic Leukemia

Transcription

1 BRIEF REPORT Thiopurine Methyltransferase (TPMT) Genotype and Early Treatment Response to Mercaptopurine in Childhood Acute Lymphoblastic Leukemia Martin Stanulla, MD, MSc Elke Schaeffeler, PhD Thomas Flohr, PhD Gunnar Cario, MD André Schrauder, MD Martin Zimmermann, PhD Karl Welte, MD Wolf-Dieter Ludwig, MD Claus R. Bartram, MD Ulrich M. Zanger, PhD Michel Eichelbaum, MD Martin Schrappe, MD Matthias Schwab, MD CONTEMPORARY TREATMENT strategies for childhood acute lymphoblastic leukemia (ALL) are based on essential therapeutic elements that are consecutively applied over 2 to 3 years and lead to an overall long-term survival of approximately 80%. 1-3 These therapeutic elements include the induction of remission ( 5% leukemic blasts in the bone marrow) to restore normal hematopoiesis, extracompartment therapy to treat leukemic cells in the central nervous system and testes, an induction consolidation and reinduction phase to further intensify treatment to prevent emergence of a drug-resistant clone, and a maintenance phase for eradication of residual leukemic cells. Another common feature in the current clinical management of children with ALL is the adjustment of therapy Context Early response to multiagent chemotherapy, including mercaptopurine, as measured by minimal residual disease is an important prognostic factor for children with acute lymphoblastic leukemia (ALL). Thiopurine methyltransferase (TPMT) is involved in the metabolism of mercaptopurine and subject to genetic polymorphism, with heterozygous individuals having intermediate and homozygous mutant individuals having very low TPMT activity. Objective To assess the association of TPMT genotype with minimal residual disease load before and after treatment with mercaptopurine in the early treatment course of childhood ALL. Design, Setting, and Patients TPMT genotyping of childhood ALL patients (n=814) in Germany consecutively enrolled in the ALL-BFM (Berlin-Frankfurt-Münster) 2000 study from October 1999 to September Minimal residual disease was analyzed on treatment days 33 and 78 for risk-adapted treatment stratification. A 4-week cycle of mercaptopurine was administered between these 2 minimal residual disease measurements. Patients (n=4) homozygous for a mutant TPMT allele, and consequently deficient in TPMT activity, were treated with reduced doses of mercaptopurine and, therefore, not included in the analyses. Main Outcome Measures Minimal residual disease load before (day 33) and after (day 78) mercaptopurine treatment. Loads smaller than 10 4 were defined as negative. Results Patients (n=55) heterozygous for allelic variants of TPMT conferring lower enzyme activity had a significantly lower rate of minimal residual disease positivity (9.1%) compared with patients (n=755) with homozygous wild-type alleles (22.8%) on day 78 (P=.02). This translated into a 2.9-fold reduction in risk for patients with wild-type heterozygous alleles (relative risk, 0.34; 95% confidence interval, ). Conclusions TPMT genotype has a substantial impact on minimal residual disease after administration of mercaptopurine in the early course of childhood ALL, most likely through modulation of mercaptopurine dose intensity. Our findings support a role for minimal residual disease analyses in the assessment of genotype-phenotype associations in multiagent chemotherapeutic trials. JAMA. 2005;293: Author Affiliations: Department of Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany (Drs Stanulla, Cario, Schrauder, Zimmermann, and Welte); Institute of Human Genetics, Ruprecht-Karls University, Heidelberg, Germany (Drs Flohr and Bartram); Margarete-Fischer- Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (Drs Schaeffeler, Zanger, Eichelbaum, and Schwab); Robert-Rössle Clinic, Department of Hematology, Oncology and Tumor Immunology, HELIOS Clinic Berlin, Berlin, Germany (Dr Ludwig); and University Children s Hospital, Kiel, Germany (Dr Schrappe). Corresponding Author: Martin Stanulla, MD, MSc, Department of Pediatric Hematology and Oncology, Hannover Medical School, Carl-Neuberg- Strasse 1, Hannover, Germany 2005 American Medical Association. All rights reserved. (Reprinted) JAMA, March 23/30, 2005 Vol 293, No

2 intensity according to the risk of treatment failure conferred by different prognostic factors. 1-3 These prognostic factors include clinical and biological characteristics that are assessable at diagnosis (eg, age at diagnosis, presenting white blood cell count, cytogenetic aberrations of the leukemic clone) as well as a variety of estimates of early response to treatment. Measures of early response to treatment were traditionally based on cytomorphologic evaluation of peripheral blood or bone marrow smears for leukemic cells at specific points during the first 2 weeks of ALL treatment. 1-3 In comparison with cytomorphologic evaluation, minimal residual disease analysis through polymerase chain reaction (PCR) based detection of leukemic clone-specific immunoglobulin and T-cell receptor gene rearrangements or by flow cytometry provides a more sensitive approach to response evaluation. Recent studies showed that measuring minimal residual disease at times during induction and consolidation treatment was highly predictive of disease recurrence Since their introduction to leukemia treatment in the 1950s, the thiopurines mercaptopurine and thioguanine have played an essential role in treatment protocols for ALL. 11,12 Several contemporary treatment protocols for childhood ALL apply consecutive cycles of either mercaptopurine or thioguanine starting as early as during induction consolidation treatment and continue administration during maintenance therapy for up to 36 months after diagnosis. 1-3 As prodrugs, thiopurines require bioactivation by a multistep pathway to form thioguanine nucleotides, which are thought to be the major cytotoxic compounds through triggering cell cycle arrest and apoptosis. 13,14 This process is in competition with direct inactivation of thiopurines or their metabolites by thiopurine S-methyltransferase (TPMT). TPMT is a cytosolic enzyme ubiquitously expressed in the human body and catalyzes the S-methylation of thiopurines. The TPMT locus is subject to genetic polymorphism, with heterozygous individuals (6%-11% of white individuals) having intermediate TPMT activity and homozygous mutant individuals (0.2%-0.6% of white individuals) having very low TPMT activity To date 20 variant alleles (TPMT*2-*18) have been identified, which are associated with decreased activity compared with the TPMT*1 wild-type allele. More than 95% of defective TPMT activity can be explained by the most frequent mutant alleles TPMT*2 and TPMT*3(A-D).In several independent studies, TPMT genotype showed excellent concordance with TPMT phenotype. 13,14 With regard to treatment outcome in childhood ALL, Lennard and colleagues 18 described in 1990 a higher relapse rate in children with lower thioguanine nucleotide concentrations measured in erythrocytes and suggested a substantial role for genetically determined TPMT activity in the predisposition to the cytotoxic effects of mercaptopurine and, consequently, ALL outcome. Their hypothesis is supported by the work of Relling and colleagues, 19 who demonstrated in a study of 182 children with ALL that mercaptopurine dose intensity was the strongest predictor of outcome. In that study a tendency toward better event-free survival was described for children with intermediate and low TPMT activity compared with that of homozygous wild-type TPMT phenotypes. Although the prognostic impact of early response to treatment is well known and thiopurines are applied as early as during induction consolidation treatment, the impact of TPMT genotype on mercaptopurine-mediated antileukemic effects in the early course of childhood ALL therapy has not yet been determined. To derive a better understanding of a potential prognostic role for TPMT genotype in childhood ALL in the Berlin-Frankfurt- Münster (BFM) based protocols, we analyzed the association of TPMT genotype with minimal residual disease levels before and after application of a 4-week cycle of mercaptopurine during induction consolidation treatment. METHODS Patients The ongoing BFM trial on treatment of childhood ALL (ALL-BFM 2000) enrolls patients from age 1 to 18 years at diagnosis and uses minimal residual disease analysis on treatment days 33 and 78 for risk-adapted treatment stratification. From October 1999 to September 2002, 956 patients enrolled in our ongoing trial were monitored for minimal residual disease at 2 follow-up points (days 33 and 78) with at least 1 marker having a minimum sensitivity of 10 4 (detection of 1 leukemic cell per cells). Of these 956 patients, 814 patients (85.1% of the entire patient population) had additional DNA available and could be prospectively genotyped at the TPMT locus. The 142 patients not available for TPMT analysis did not differ from the included 814 patients with regard to characteristics known to be associated with early treatment response (data not shown). Within the ALL-BFM strategy, remission induction is initiated through 7-day monotherapy with orally administered prednisone and 1 dose of intrathecal methotrexate on treatment day 1. From day 8 onward, treatment is complemented by intravenous application of 3 additional drugs: vincristine, daunorubicin, and L-asparaginase. In addition, from day 8 onward, patients are randomly assigned to corticosteroid treatment with either prednisone or dexamethasone. This induction strategy leads to cytomorphological remission ( 5% leukemic blasts in the bone marrow) in more than 97% of patients on treatment day 33. Remission induction is followed by consolidation treatment with intravenous cyclophosphamide and cytarabine, intrathecal methotrexate, and oral mercaptopurine. Routine bone marrow aspirates are taken at diagnosis and after completion of induction (treatment day 33) and consolidation (treatment day 78). Minimal residual disease for analysis of leukemic cell dynamics is measured on treatment days 33 and 78. There were no imbalances with regard to TPMT genotype and randomiza JAMA, March 23/30, 2005 Vol 293, No. 12 (Reprinted) 2005 American Medical Association. All rights reserved.

3 tion groups or central nervous system involvement at diagnosis (data not shown). Toxicity data were collected using the National Cancer Institute s Common Toxicity Criteria. 20 Informed consent was obtained from patients or their legal guardians and the study was approved by the local ethics committee. Minimal Residual Disease Analysis and TPMT Genotyping Minimal residual disease was analyzed using allele-specific oligonucleotide PCR protocols for quantitative detection of leukemic clone-specific immunoglobulin and T-cell receptor gene rearrangements, and TAL1 deletions on a LightCycler instrument (Roche Diagnostics, Mannheim, Germany). 6,21 Genotyping for TPMT (eg, *2 and *3 alleles) was performed using a denaturing high-performance liquid chromatography method using DNA prepared from either leukemic or remission bone marrows. 14,22 Investigators performing TPMT genotyping were blinded with regard to a patient s minimal residual disease status. Statistical Analysis Frequencies of characteristics and common factors known to be associated with treatment response were obtained in the beginning of the analysis. Proportional differences between groups were analyzed by 2 or Fisher exact tests. The association between TPMT genotype and minimal residual disease was examined by use of unconditional logistic regression analysis to calculate relative risks (RRs) and their 95% confidence intervals (CIs). Minimal residual disease loads smaller than 10 4 were defined as negative. Statistical significance was set a priori at P.05. Analyses were computed using SPSS version 12.0 (SPSS Inc, Chicago, Ill). RESULTS Genotyping of 814 patients with childhood ALL revealed 755 (92.8%) patients with TPMT wild-type, 55 (6.8%) with heterozygous, and 4 (0.5%) with homozygous mutant genotype (*2/ *3A, *3A/*3A [n=2], *3A/*11), respectively, and genotype frequencies were in Hardy-Weinberg equilibrium. Allele frequencies were as follows: TPMT*1, 96.12%; TPMT*2, 0.25%; TPMT*3A, 2.95%; TPMT*3C, 0.56%; TPMT*9, 0.06%; and TPMT*11, 0.06%. In the TABLE, patient characteristics are depicted by TPMT genotype. Ex- Table. Patient Characteristics and Response to Treatment According to TPMT Genotype in 814 Patients With Childhood Acute Lymphoblastic Leukemia TPMT Genotype, No. (%) Wild-Type (n = 755) Heterozygous (n = 55) Homozygous Mutation (TPMT Deficient) (n=4) Wild-Type vs Heterozygous P Value* Sex Male 425 (56.3) 35 (63.6) 2 (50.0) Female 330 (43.7) 20 (36.4) 2 (50.0).29 Age at diagnosis, y (56.0) 38 (69.1) 2 (50.0) (19.2) 8 (14.5) 1 (25.0) (24.8) 9 (16.4) 1 (25.0) Presenting WBC count, cells/µl (46.0) 21 (38.2) 2 (50.0) to (32.7) 21 (38.2) 1 (25.0) to (10.7) 4 (7.3) 1 (25.0) (10.6) 9 (16.4) 0 Immunophenotype B 625 (82.8) 36 (65.5) 3 (75.0) T 115 (15.2) 14 (25.5) 1 (25.0).07 Biphenotypic 2 (0.3) 0 0 Unknown 13 (1.7) 5 (9.1) 0 DNA index (62.1) 31 (56.4) 2 (50.0) (10.2) 5 (9.1) 0.97 No result 209 (27.7) 19 (34.5) 2 (50.0) TEL/AML1 Positive 174 (23.0) 13 (23.6) 1 (25.0) Negative 520 (68.9) 40 (72.7) 3 (75.0) Unknown 61 (8.1) 2 (3.6) 0 BCR/ABL Positive 16 (2.1) 0 0 Negative 716 (94.8) 55 (100) 4 (100) Unknown 23 (3.1) 0 0 MLL/AF4 Positive 1 (0.1) 0 0 Negative 691 (91.5) 53 (96.4) 4 (100) Unknown 63 (8.4) 2 (3.6) 0 Prednisone response Good 671 (88.9) 51 (92.7) 3 (75.0) Poor 79 (10.5) 2 (3.6) 1 (25.0) Unknown 5 (0.7) 2 (3.6) 0 Minimal residual disease Day 33, before mercaptopurine Negative 378 (50.1) 29 (52.7) 3 (75.0) 10 4 residual blasts 377 (49.9) 26 (47.3) 1 (25.0) Day 78, after mercaptopurine Negative 583 (77.2) 50 (90.9) 3 (75.0) 10 4 residual blasts 172 (22.8) 5 (9.1) 1 (25.0) Abbreviations: TPMT, thiopurine methyltransferase; WBC, white blood cell. * 2 or Fisher exact test comparing homozygous wild-type and heterozygous. Ratio of DNA content of leukemic G 0 /G 1 cells to normal diploid lymphocytes. Good: 1000 leukemic blood blasts/µl on treatment day 8; poor: 1000/µL American Medical Association. All rights reserved. (Reprinted) JAMA, March 23/30, 2005 Vol 293, No

4 cept for immunophenotype, no major differences with regard to characteristics known to be associated with treatment response were observed between patients homozygous for the wild-type allele or heterozygous. All patients homozygous for a mutant TPMT allele, and consequently deficient in TPMT activity, were treated with an approximately 10-fold reduced dose of mercaptopurine to prevent hematopoietic toxicity (dose adjustments were not performed for heterozygous patients). Therefore, the 4 patients with deficient TPMT activity were not included in further analyses. In heterozygous patients and those homozygous for the wild-type allele, minimal residual disease levels on treatment day 33 were equally distributed between the groups (Table). However, when minimal residual disease levels were assessed on treatment day 78, after administration of induction consolidation treatment, including a 4-week cycle of mercaptopurine (60 mg/m 2 per day), significant differences with regard to clearance of minimal residual disease were observed between wild-type and heterozygous patients (Table). For heterozygous patients, this distribution translated into a 2.9-fold reduction in risk of having measurable minimal residual disease after induction consolidation treatment (RR, 0.34; 95% CI, ; P=.02). This point estimate did not significantly change in multivariate analysis including variables known to be associated with treatment response: sex, age at diagnosis, presenting white blood cell count, immunophenotype, and prednisone response (good: 1000 leukemic blood blasts/µl on treatment day 8; poor: 1000/µL) (RR, 0.30; 95% CI, ; P=.03). Data on hematopoietic and hepatic toxicity were available for 75% of heterozygous patients and homozygous wild-type for TPMT and did not differ between the groups (data not shown). Similarly, there was no difference detectable in the total group of patients when analyzing time to treatment day 78 (wild-type patients, median of 90 [range, ] days; heterozygous, median of 90 [range, ] days). COMMENT Our results indicate that TPMT genotype has a substantial impact on minimal residual disease after administration of mercaptopurine during induction consolidation treatment in the early course of childhood ALL, most likely through modulation of mercaptopurine dose intensity. Several studies have shown that patients with homozygous mutant TPMT alleles conferring very low enzyme activity are at high risk of developing severe hematopoietic toxicity after treatment with standard doses of thiopurines. 13,14 However, whether heterozygous patients need dose reductions as well is less clear; moreover, the requirement of dose adjustment most likely depends on the thiopurine dose and concurrently administered chemotherapy. 23,24 Based on the data available for our study, hematopoietic toxicity did not differ between heterozygous patients and those homozygous wildtype for TPMT. Although these data were available for only 75% of the patients, the time to treatment day 78 (2 weeks after completion of the 4-week cycle of mercaptopurine) was available for all patients and as a surrogate marker for toxicity did not differ between the groups. Therefore, it seems unlikely that childhood ALL patients with heterozygous mutant TPMT alleles treated in the BFM protocols would benefit from dose reductions in induction consolidation treatment, as suggested for other protocols. 24 Moreover, with the prognostic power of minimal residual disease, the observed differences in tumor cell clearance depending on TPMT genotype suggest an important role for mercaptopurine in the induction consolidation phase and could be a relevant determinant of treatment outcome for childhood ALL treated according to BFM protocols. Finally, after long-term outcome data become available, our results may provide a rationale for increasing mercaptopurine dosing according to TPMT genotype in the early course of childhood ALL. Because this rationale will affect TPMT wild-type individuals, it could have an impact on the majority of patients and, therefore, substantially influence overall treatment results. In addition, our data support a role for combining analysis of genetic variation in drug-metabolizing enzymes and minimal residual disease in the assessment of treatment response to specific drugs in multiagent chemotherapeutic treatment regimens. Author Contributions: Drs Stanulla and Schwab had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Drs Stanulla and Schaeffeler contributed equally to the study. Study concept and design: Stanulla, Eichelbaum, Schrappe, Schwab. Acquisition of data: Stanulla, Schaeffeler, Flohr, Schrauder, Welte, Ludwig, Bartram, Zanger, Schwab. Analysis and interpretation of data: Stanulla, Schaeffeler, Flohr, Cario, Bartram, Eichelbaum, Schwab, Zimmermann. Drafting of the manuscript: Stanulla, Schrappe, Schwab. Critical revision of the manuscript for important intellectual content: Stanulla, Schaeffeler, Flohr, Cario, Schrauder, Welte, Ludwig, Bartram, Zanger, Eichelbaum, Schrappe, Schwab, Zimmermann. Statistical analysis: Stanulla, Cario, Schwab, Zimmermann. Obtained funding: Bartram, Eichelbaum, Schrappe. Administrative, technical, or material support: Schaeffeler, Welte, Ludwig, Bartram, Zanger, Eichelbaum, Schrappe. Study supervision: Stanulla, Schrappe, Schwab. Financial Disclosures: None reported. Funding/Support: This work was supported in part by the Deutsche Krebshilfe, the Bundesministerium für Bildung und Forschung, the Robert-Bosch-Stiftung, the Madeleine-Schickedanz-Kinderkrebsstiftung, and the Verein zur Förderung der Behandlung krebskranker Kinder Hannover ev. Role of the Sponsors: The sponsors had no role in the conduct of the study; in the collection, analysis, and interpretation of the data; or in the data analysis. The sponsors had no role regarding the decision to publish, did not see the submitted manuscript, and are not aware of the data obtained as the result of our analysis. Acknowledgment: We acknowledge all participants of ALL-BFM REFERENCES 1. Silverman LB, Sallan SE. Newly diagnosed childhood acute lymphoblastic leukemia: update on prognostic factors and treatment. Curr Opin Hematol. 2003; 10: Pui CH, Campana D, Evans WE. Childhood acute lymphoblastic leukaemia-current status and future perspectives. Lancet Oncol. 2001;2: Schrappe M, Reiter A, Zimmermann M, et al. Longterm results of four consecutive trials in childhood ALL performed by the ALL-BFM study group from 1981 to Leukemia. 2000;14: Cave H, van der Werff ten Bosch J, Suciu S, et al; European Organization for Research and Treatment of Cancer Childhood Leukemia Cooperative Group. Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia. N Engl J Med. 1998;339: JAMA, March 23/30, 2005 Vol 293, No. 12 (Reprinted) 2005 American Medical Association. All rights reserved.

5 5. Brisco MJ, Condon J, Hughes E, et al. Outcome prediction in childhood acute lymphoblastic leukaemia by molecular quantification of residual disease at the end of induction. Lancet. 1994;343: van Dongen JJ, Seriu T, Panzer-Grümayer ER, et al. Prognostic value of minimal residual disease in acute lymphoblastic leukaemia in childhood. Lancet. 1998; 352: Nyvold C, Madsen HO, Ryder LP, et al. Precise quantification of minimal residual disease at day 29 allows identification of children with acute lymphoblastic leukemia and an excellent outcome. Blood. 2002;99: Coustan-Smith E, Sancho J, Hancock ML, et al. Clinical importance of minimal residual disease in childhood acute lymphoblastic leukemia. Blood. 2000;96: Ciudad J, San Miguel JF, Lopez-Berges MC, et al. Prognostic value of immunophenotypic detection of minimal residual disease in acute lymphoblastic leukemia. J Clin Oncol. 1998;16: Dworzak MN, Froschl G, Printz D, et al. Prognostic significance and modalities of flow cytometric minimal residual disease detection in childhood acute lymphoblastic leukemia. Blood. 2002;99: Elion GB, Burgi E, Hitchings GH. Studies on condensed pyrimidine systems, IX: the synthesis of some 6-substituted purines. J Am Chem Soc. 1952;74: Burchenal JH, Murphy ML, Ellison RR, et al. Clinical evaluation of a new antimetabolite, 6-mercaptopurine, in the treatment of leukemia and allied diseases. Blood. 1953;8: McLeod HL, Krynetski EY, Relling MV, Evans WE. Genetic polymorphism of thiopurine methyltransferase and its clinical relevance for childhood acute lymphoblastic leukemia. Leukemia. 2000;14: Schaeffeler E, Fischer C, Brockmeier D, et al. Comprehensive analysis of thiopurine S-methyltransferase phenotype-genotype correlation in a large population of German-Caucasians and identification of novel TPMT variants. Pharmacogenetics. 2004;14: Indjova D, Atanasova S, Shipkova M, Armstrong VW, Oellerich M, Svinarov D. Phenotypic and genotypic analysis of thiopurine s-methyltransferase polymorphism in the Bulgarian population. Ther Drug Monit. 2003;25: Ganiere-Monteil C, Medard Y, Lejus C, et al. Phenotype and genotype for thiopurine methyltransferase activity in the French Caucasian population: impact of age. Eur J Clin Pharmacol. 2004;60: Kurzawski M, Gawronska-Szklarz B, Drozdzik M. Frequency distribution of thiopurine S-methyltransferase alleles in a Polish population. Ther Drug Monit. 2004; 26: Lennard L, Lilleyman JS, Van Loon J, Weinshilboum RM. Genetic variation in response to 6-mercaptopurine for childhood acute lymphoblastic leukaemia. Lancet. 1990;336: Relling MV, Hancock ML, Boyett JM, Pui C-H, Evans WE. Prognostic importance of 6-mercaptopurine dose intensity in acute lymphoblastic leukemia. Blood. 1999; 93: National Cancer Institute. Common Toxicity Criteria Manual. Version Available at: http: //ctep.cancer.gov/forms/ctcmanual_v4_ pdf. Accessibility verified March 2, van der Velden VH, 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: Schaeffeler E, Lang T, Zanger UM, Eichelbaum M, Schwab M. High-throughput genotyping of thiopurine S-methyltransferase by denaturing HPLC. Clin Chem. 2001;47: McLeod H, Coulthard SA, Thomas AE, et al. Analysis of thiopurine methyltransferase variant alleles in childhood acute lymphoblastic leukaemia. Br J Haematol. 1999;105: Relling MV, Hancock ML, Rivera GK, et al. Mercaptopurine therapy intolerance and heterozygosity at the thiopurine S-methyltransferase gene locus. J Natl Cancer Inst. 1999;91: If you find it in your heart to care for somebody else, you will have succeeded. Maya Angelou (1928- ) 2005 American Medical Association. All rights reserved. (Reprinted) JAMA, March 23/30, 2005 Vol 293, No