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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

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ONTEMPORARY TREATMENT

strategies for childhood acute l y mphobl asti c l eukemi a (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 2002. 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, 0.13-0.86). 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. www.jama.com

JAMA. 2005;293:1485-1489

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-FischerBosch Institute of Clinical Pharmacology, Stuttgart, Germany (Drs Schaeffeler, Zanger, Eichelbaum, and Schwab); Robert-Rössle Clinic, Department of

©2005 American Medical Association. All rights reserved.

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-NeubergStrasse 1, 30625 Hannover, Germany (Stanulla.Martin @MH-Hannover.de).

(Reprinted) JAMA, March 23/30, 2005—Vol 293, No. 12

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THIOPURINE METHYLTRANSFERASE GENOTYPE AND CHILDHOOD LEUKEMIA

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.4-10 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 cy tosolic enzy me ubiquitously expressed in the human body and catalyzes the S-methylation of thiopurines. The TPMT locus is subject to genetic polymorphism, with heterozy-

gous 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. 13-17 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 d efecti v e T P MT acti v i ty 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 colleagues18 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-FrankfurtMünster (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.

1486 JAMA, March 23/30, 2005—Vol 293, No. 12 (Reprinted)

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 10 000 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 cy tarabine, 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-

©2005 American Medical Association. All rights reserved.

THIOPURINE METHYLTRANSFERASE GENOTYPE AND CHILDHOOD LEUKEMIA

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.

tively, and genotype frequencies were TPMT*3A, 2.95%; TPMT*3C, 0.56%; in Hardy-Weinberg equilibrium. Al- TPMT*9, 0.06%; and TPMT*11, 0.06%. In the TABLE, patient characterislele frequencies w ere as follow s: TPMT *1, 96.12%; TPMT *2, 0.25%; tics are depicted by TPMT genotype. ExTable. Patient Characteristics and Response to Treatment According to TPMT Genotype in 814 Patients With Childhood Acute Lymphoblastic Leukemia TPMT Genotype, No. (%)

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), respec-

Homozygous Wild-Type vs Mutation Wild-Type Heterozygous (TPMT Deficient) Heterozygous (n = 755) (n = 55) (n = 4) P Value* Sex Male Female Age at diagnosis, y 1-6 7-10 11-18 Presenting WBC count, cells/µL ⬍10 000

425 (56.3)

35 (63.6)

2 (50.0)

330 (43.7)

20 (36.4)

2 (50.0)

423 (56.0) 145 (19.2) 187 (24.8)

38 (69.1) 8 (14.5) 9 (16.4)

2 (50.0) 1 (25.0) 1 (25.0)

347 (46.0)

21 (38.2)

2 (50.0)

10 000 to ⬍50 000 50 000 to ⬍100 000 100 000 ⱖ Immunophenotype B

247 (32.7) 81 (10.7) 80 (10.6)

21 (38.2) 4 (7.3) 9 (16.4)

1 (25.0) 1 (25.0)

625 (82.8)

36 (65.5)

3 (75.0)

T Biphenotypic Unknown DNA index† ⬍1.16

115 (15.2) 2 (0.3) 13 (1.7)

14 (25.5) 0 5 (9.1)

1 (25.0) 0 0

469 (62.1)

31 (56.4)

ⱖ.16 1 No result

77 (10.2) 209 (27.7)

5 (9.1) 19 (34.5)

TEL/AML1 Positive Negative Unknown

174 (23.0) 520 (68.9) 61 (8.1)

13 (23.6) 40 (72.7) 2 (3.6)

1 (25.0) 3 (75.0) 0

BCR/ABL Positive Negative Unknown

16 (2.1) 716 (94.8) 23 (3.1)

0 55 (100) 0

0 4 (100) 0

MLL/AF4 Positive Negative

1 (0.1) 691 (91.5)

0 53 (96.4)

0 4 (100)

63 (8.4)

2 (3.6)

671 (88.9) 79 (10.5) 5 (0.7)

51 (92.7) 2 (3.6) 2 (3.6)

Unknown Prednisone response‡ Good Poor Unknown

Minimal residual disease Day 33, before mercaptopurine Negative 378 (50.1) ⱖ0−4 residual blasts 1 377 (49.9) Day 78, after mercaptopurine Negative 583 (77.2) 10−4 residual blasts ⱖ 172 (22.8)

29 (52.7) 26 (47.3) 50 (90.9) 5 (9.1)

.29

.16

.35

0

2 (50.0) 0

.07

.97

2 (50.0) .93

.62

.99

0 3 (75.0) 1 (25.0) 0

3 (75.0) 1 (25.0) 3 (75.0) 1 (25.0)

.15

.70

.02

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 G0/G1 cells to normal diploid lymphocytes. ‡Good: ⬍1000 leukemic blood blasts/µL on treatment day 8; poor: ⱖ1000/µL.

©2005 American Medical Association. All rights reserved.

(Reprinted) JAMA, March 23/30, 2005—Vol 293, No. 12

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THIOPURINE METHYLTRANSFERASE GENOTYPE AND CHILDHOOD LEUKEMIA

cept for immunophenotype, no major differences with regard to characteristics known to be associated with treatment response w ere 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 activ ity w ere 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, 0.13-0.86; 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: ⱖ1000/µL) (RR, 0.30; 95% CI, 0.10-0.88; 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, 60-201] days; heterozygous, childhood ALL. Because this rationale median of 90 [range, 76-116] days). will affect TPMT wild-type individuals, it could have an impact on the majority COMMENT of patients and, therefore, substantially Our results indicate that TPMT geno- influence overall treatment results. In addition, our data support a role type has a substantial impact on minimal residual disease after admin- for combining analysis of genetic variaistration of mercaptopurine during tion in drug-metabolizing enzymes and induction consolidation treatment in minimal residual disease in the assessthe early course of childhood ALL, most ment of treatment response to specific likely through modulation of mercap- drugs in multiagent chemotherapeutic treatment regimens. topurine dose intensity. Several studies have shown that paContributions: Drs Stanulla and Schwab had tients with homozygous mutant TPMT Author full access to all of the data in the study and take realleles conferring very low enzyme sponsibility for the integrity of the data and the acof the data analysis. Drs Stanulla and Schaefactivity are at high risk of developing curacy feler contributed equally to the study. severe hematopoietic toxicity after treat- Study concept and design: Stanulla, Eichelbaum, ment with standard doses of thiopu- Schrappe, Schwab. Acquisition of data: Stanulla, Schaeffeler, Flohr, rines.13,14 However, whether heterozy- Schrauder, Welte, Ludwig, Bartram, Zanger, Schwab. gous patients need dose reductions as Analysis and interpretation of data: Stanulla, Schaeffeler, Flohr, Cario, Bartram, Eichelbaum, Schwab, well is less clear; moreover, the require- Zimmermann. ment of dose adjustment most likely Drafting of the manuscript: Stanulla, Schrappe, Schwab. revision of the manuscript for important depends on the thiopurine dose and Critical intellectual content: Stanulla, Schaeffeler, Flohr, Cario, Schrauder, Welte, Ludwig, Bartram, Zanger, concurrently administered chemoSchrappe, Schwab, Zimmermann. therapy.23,24 Based on the data available Eichelbaum, Statistical analysis: Stanulla, Cario, Schwab, Zimmerfor our study, hematopoietic toxicity did mann. funding: Bartram, Eichelbaum, Schrappe. not differ between heterozygous pa- Obtained Administrative, technical, or material support: Schaeftients and those homozygous wild- feler, Welte, Ludwig, Bartram, Zanger, Eichelbaum, type for TPMT. Although these data...