Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme that catalyses the S-methylation of thiopurines (eg 6-mercaptopurine (6-. MP), 6-thioguanine ...
Correspondence
1422
A novel TPMT missense mutation associated with TPMT deficiency in a 5-year-old boy with ALL
Leukemia (2003) 17, 1422–1424. doi:10.1038/sj.leu.2402981
TO THE EDITOR Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme that catalyses the S-methylation of thiopurines (eg 6-mercaptopurine (6MP), 6-thioguanine (6-TG) and azathioprine).1 6-MP is a component of essentially all modern treatment protocols for acute lymphoblastic leukaemia (ALL) in childhood (eg Berlin–Frankfurt–Mu¨nster ALL regimen, St Jude Children’s Research Hospital ALL regimen), since successful therapy has been associated with a long period of maintenance treatment with standard doses of 6-MP (ie 50–75 mg/ m2 per day) in combination with methotrexate.2 Moreover, thiopurines are also used in the treatment of autoimmune diseases, chronic inflammatory bowel diseases and as immunosuppressants following solid organ transplantation. Thiopurines are prodrugs that undergo metabolic activation by a multistep pathway to form 6-TG nucleotides, which are thought to be the major cytotoxic compounds, by triggering cell cycle arrest and apoptosis. This process is in competition with direct inactivation of 6-MP by TPMT. TPMT exhibits a common genetic polymorphism, and to date nine alleles (TPMT*2–*10) have been identified, which are associated with decreased activity compared to TPMT wild type (Figure 1).1,3 Several clinical studies as well as case reports have shown that patients with exceptionally very low TPMT activity (approximately 1 in 300 individuals) are carriers of two mutant TPMT alleles. These patients are at a high risk of developing severe and potentially fatal haematopoietic toxicity after treatment with standard doses of thiopurines caused by an accumulation of 6-TGN metabolites.3–5 Furthermore, children with ALL heterozygous for TPMT, constituting about 10% of Caucasian and AfricanAmerican populations, are also at greater risk of thiopurine haematotoxicity.4 Prospective measurement of erythrocyte TPMT activity is therefore emerging as a routine safety measure prior to therapy to avoid toxicity. However, there are a number of limitations with respect to the determination of the constitutive TPMT enzyme activity. If a TPMT-deficient or heterozygous patient has received a transfusion with red blood cells (RBC) from a homozygous wild-type individual, which is a rather likely case in children with ALL, TPMT activity cannot be reliably determined within 30–90 days after transfusion.5 Additionally, thiopurine administration itself as well as a comedication (eg 5-aminosalicylates) may alter TPMT activity in RBCs resulting in a possible misclassification especially for heterozygous patients.3 Therefore, genotyping of TPMT has been considered to be an alternative. Since several mutant alleles have been described, it has been suggested that molecular diagnosis appears to be a feasible approach to predict the correct TPMT phenotype in up to 95%.1,6 However, additional mutant alleles hold promise to increase the percentage of patients who can be molecularly diagnosed. A 5-year and 2-month-old boy of Spanish origin developed common ALL (FAB classification L1) with initial white blood cells of 5600/ml (13% leukaemic blasts) and haemoglobin concentration and platelet count at diagnosis of 4.9 g/dl and 276 000/ml, respectively. At admisson before transfusion of RBC, EDTA blood Correspondence: Dr Matthias Schwab, Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Auerbachstrasse 112, Stuttgart, 70376 Germany; Fax: +49 711 85 92 95 Received 13 January 2003; accepted 10 March 2003 Leukemia
was taken for the determination of TPMT activity and TPMT genotype. TPMT phenotype was assigned on the basis of TPMT activity in RBC using an HPLC method,3 and genotyping for the TPMT*2,*3A,*3B,*3C and *3D alleles was performed by a DHPLC method.7 Exons 3–10 of the TPMT gene and flanking intronic regions were amplified by PCR as described previously.8 In the 50 flanking region a fragment spanning the region 843 to +13 was amplified using the following oligonucleotides: 50 -GAAGTCGGAGAAGCAACTGCC-30 ; 50 -GCCTCCGCCACCAATGAC-30 . Automated DNA sequencing was performed with the ABI BigDye Terminator sequencing kit and samples were analysed on an ABI310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Phenotyping of TPMT in RBC with ALL showed TPMT deficiency with a very low enzyme activity of 2 U ( ¼ nmol 6-methylthioguanine/g Hb/h; p3 ¼ deficient, 4–23 ¼ intermediate, X24 ¼ normal/ high activity) using a nonradiochemical HPLC method and 6-TG as substrate3,5 in contrast to the classical method from Weinshilboum et al.9 The higher cutoff values observed for 6-TG in comparison to those obtained with 6-MP as substrate in the original radiochemical assay (o5 ¼ deficient, 5–10 ¼ intermediate, 410 ¼ normal/high activity)10 are caused by higher TPMT activities with 6-TG. Subsequent genotyping for the common alleles TPMT*2 and *3AD revealed a heterozygous genotype with one mutant allele (TPMT*1/*3A), which was in discrepancy with the patients’ very low TPMT activity. To further elucidate the genetic basis of TPMT deficiency, the entire coding region of TPMT was completely sequenced including exon–intron boundaries. None of the presently known rare TPMT alleles (TPMT*4–*10) was detected, but we identified a novel missense mutation (G395A) in exon 6 resulting in an amino-acid exchange C132Y (Figure 2a). Additionally, to identify splice variants we isolated, amplified and sequenced the patients’ TPMT cDNA, and also a systematic mutation search in the 5’flanking sequence (843 to +13) was performed. However, no further mutations, deletions or anomalously spliced variants could be detected. Thus, the phenotypically defined complete TPMT deficiency in the present case is the consequence of compound heterozygosity consisting of the frequent TPMT*3A allele and the identified mutation G395A in exon 6, which defines a novel allele termed as TPMT*11. This finding is corroborated by the fact that intermediate TPMT activities were found in both parents (mother: 20 U; father: 22 U) (Figure 2b). Whereas the intermediate TPMT activity of the mother
Novel mutant allele
Known mutant alleles
*11
*5 *2 *3D
*10
*6
*3A,*3B
*4
*3A,*3C
*8 *7
Figure 1 Summary of the currently known allelic variations of the TPMT gene. Black boxes represent exons that encode open-reading frame sequences; white boxes represent exons out of the ORF. Exons but not intron sizes are proportional to their relative lengths. Allele nomenclature of TPMT is according to McLeod et al1 and Schwab et al.3
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1423 molecular diagnosis for the three most common alleles TPMT*2, *3A and *3C is able to identify 80–95% of heterozygous and homozygous mutant patients.1 The lack of a higher concordance rate between TPMT genotype and phenotype may be caused by the existence of further rare so far unknown mutations at the human TPMT locus. In the present case, we did not find this new mutation again in more than 1200 healthy individuals tested for TPMT using phenotyping and genotyping strategy. Thus, we suppose that the newly identified mutation is rare, similar to the TPMT alleles *4– *10. However, this case demonstrates clearly that also rare mutations should be kept in mind to avoid severe drug toxicity especially in patients (eg children with ALL) who will be treated with additional cytotoxic agents. This is, for example, corroborated by a recent observation in a patient with autoimmune hepatitis who rapidly developed severe azathioprine-related myelosuppression. In this case, undetectable TPMT activity was in discordance to a heterozygous TPMT genotype *1/*3A.12 In addition, 15 patients (predominately children with ALL) who were intolerant to treatment with thiopurines showed TPMT deficiency (n ¼ 6), as predicted by genotype, or intermediate TPMT activity (n ¼ 9).4 Five of these nine TPMT intermediate patients did not have one of the three most common TPMT mutant alleles (TPMT*2, *3A, *3C). To this end, our case demonstrates that additional deficient alleles can be identified and may be present in TPMT discordant patients. Large-scale genotype–phenotype correlation studies are needed to evaluate the predictive power of TPMT genotyping before a sole genotype-guided approach of thiopurine medication will become a clinical reality.
Acknowledgements Figure 2 Pedigree and phenotype of a family with reduced TPMT activity and sequence analysis of the G395A mutation. (a) Electropherogram of the TPMT gene sequence at nucleotides 379–408: there is a heterozygous G–A substitution seen for the patient. (b) Pedigree: phenotype and genotype of family members (unit ¼ nmol 6methylthioguanine/g Hb/h), ’ patient.
was in agreement with the heterozygous genotype TPMT*1/*3A, in the father none of the so far known TPMT alleles associated with decreased TPMT activity could be detected. Again, complete sequencing of the open-reading frame TPMT confirmed the presence of the novel mutation G395A in a heterozygous manner (TPMT*1/*11) and no further mutations were found. After diagnosis of TPMT deficiency in the boy with ALL, doseadjusted treatment of 6-MP during the induction period and maintenance therapy in the BFM ALL2000 protocol was performed with about 15% of standard dosage. No signs of 6-MP-related toxicity were observed. In accordance with TPMT deficiency, no methylated metabolites (eg 6-methylmercaptopurine) could be measured by drug monitoring of plasma levels during 6-MP treatment. Taken together, the in vivo data indicate a major functional consequence of the TPMT*11 allele, although the molecular mechanism for loss of catalytic activity has not been elucidated. However, it can be assumed that similar to TPMT*2 and *3A a posttranscriptional mechanism with enhanced proteolysis of the mutant protein seems to be the most likely explanation.11 Although there are limitations with respect to the correct determination of the constitutive TPMT enzyme activity as mentioned above, this case report demonstrates likewise how important it is to recognize the limitations of genotyping. A knowledge of almost all occurring mutations in a defined population is necessary when genotyping is considered to be the method of choice to predict the correct patient’s phenotype. To date,
The work was supported by the BMBF Grant 01GG 9894 and the Robert Bosch Foundation, Stuttgart, Germany. 1 E Schaeffeler1 Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany; M Stanulla2 2 Department of Paediatric Haematology and J Greil3 Oncology, Hannover Medical School, Hannover, M Schrappe2 Germany; M Eichelbaum1,4 3 Department of Paediatric Haematology and UM Zanger1 Oncology, Children’s Hospital, Eberhard-KarlsM Schwab1
University,Tuebingen,Germany; Division of Clinical Pharmacology, EberhardKarls-University, Tuebingen, Germany
4
References 1 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: 567–572. 2 Pui CH, Evans WE. Acute lymphoblastic leukemia. N Engl J Med 1998; 339: 605–615. 3 Schwab M, Schaffeler E, Marx C, Fischer C, Lang T, Behrens C et al. Azathioprine therapy and adverse drug reactions in patients with inflammatory bowel disease: impact of thiopurine S-methyltransferase polymorphism. Pharmacogenetics 2002; 12: 429–436. 4 Evans WE, Hon YY, Bomgaars L, Coutre S, Holdsworth M, Janco R et al. Preponderance of thiopurine S-methyltransferase deficiency and heterozygosity among patients intolerant to mercaptopurine or azathioprine. J Clin Oncol 2001; 19: 2293–2301. 5 Schwab M, Schaeffeler E, Marx C, Zanger U, Aulitzky W, Eichelbaum M. Shortcoming in the diagnosis of TPMT deficiency in a patient with Crohn’s disease using phenotyping only. Gastroenterology 2001; 121: 498–499. 6 Yates CR, Krynetski EY, Loennechen T, Fessing MY, Tai HL, Pui CH et al. Molecular diagnosis of thiopurine S-methyltransferase deficiency: genetic basis for azathioprine and mercaptopurine intolerance. Ann Intern Med. 1997; 126: 608–614. Leukemia
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1424 7 Schaeffeler E, Lang T, Zanger UM, Eichelbaum M, Schwab M. Highthroughput genotyping of thiopurine S-methyltransferase by denaturing HPLC. Clin Chem 2001; 47: 548–555. 8 Otterness D, Szumlanski C, Lennard L, Klemetsdal B, Aarbakke J, Park-Hah JO et al. Human thiopurine methyltransferase pharmacogenetics: gene sequence polymorphisms. Clin Pharmacol Ther 1997; 62: 60–73. 9 Weinshilboum RM, Raymond FA, Pazmino PA. Human erythrocyte thiopurine methyltransferase: radiochemical microassay and biochemical properties. Clin Chim Acta 1978; 85: 323–333.
10 Weinshilboum RM, Sladek SL. Mercaptopurine pharmacogenetics: monogenic inheritance of erythrocyte thiopurine methyltransferase activity. Am J Hum Genet 1980; 32: 651–662. 11 Tai HL, Krynetski EY, Schuetz EG, Yanishevski Y, Evans WE. Enhanced proteolysis of thiopurine S-methyltransferase (TPMT) encoded by mutant alleles in humans (TPMT*3A, TPMT*2): mechanisms for the genetic polymorphism of TPMT activity. Proc Natl Acad Sci USA 1997; 94: 6444–6449. 12 Langley P, Underhill J, Tredger J, Norris S, McFarlane I. Thiopurine methyltransferase phenotype and genotype in relation to azathioprine therapy in autoimmune hepatitis. J Hepatol 2002; 37: 441–447.
Vaccination of chronic myeloid leukemia patients with autologous in vitro cultured leukemic dendritic cells
Leukemia (2003) 17, 1424–1426. doi:10.1038/sj.leu.2402979
TO THE EDITOR T-cell immunity plays a pivotal role in chronic myelogenous leukemia (CML), as has become clear from clinical experience with allogeneic stem cell transplantation (SCT).1 The most striking and direct evidence for the immune-mediated graft-versus-leukemia concept stems from the observation that patients with relapsed CML after allogeneic SCT can be reinduced to complete remission by infusing donor-derived peripheral blood lymphocytes.2 In the autologous setting, leukemia reactive T cells are present.3 However, antileukemic responses are lacking because of T-cell anergy to the CML cells. T-cell anergy to leukemic cells in CML occurs most probably because of the lack of appropriate immune costimulatory molecules. It has recently been shown that the amino-acid sequences at the fusion point of the bcr/abl translocation characteristic for CML are processed within the cell and presented by HLA molecules on the surface of leukemic cells to T cells.4 This implies that vaccination has a sound basis and deserves further exploration. Dendritic cells (DCs) are the most potent antigen-presenting cells, possessing the unique ability to prime naı¨ve CTL and Th cells and to break immunological tolerance.5 The ability to generate leukemic DC from patients with CML with the potency to induce autologous T-cell proliferation in vitro, while conserving the CML-specific antigenic profile, has been shown.6 Here, we report on the use of in vitro-generated leukemic DC for in vivo stimulation and generation of effector T cells by intradermal vaccination of IFN-resistant CML patients with autologous CML/DC. After informed consent, three IFN-a-resistant CML patients without an HLA-identical donor were included in this protocol, which was approved by the medical ethical committee of the VU University medical center. The clinical characteristics are listed in
Table 1 Patient 1 2 3
Clinical characteristics of CML patients treated with leukemic DC vaccination Age m/f
Sokala
Diagnosisb
Cytogeneticsc
Bcr/abl breakpoint
WBCd
Blasts (%)
Plateletsd
Previous treatment
63 m 60 m 49 f
0.67 0.38 0.78
60 20 22
t(9;22) t(9;22) t(9;22)
b2a2 b3a2/b2a2 b3a2
88 14.5 5.8
4 2 1
345 102 627
IFN/ARA-C Hydroxyurea IFN/ ARA-C Hydroxyurea IFN/ARA-C Hydroxyurea
a
At diagnosis; bMonths before vaccination; cAt vaccination; d 109/l at vaccination
Correspondence: Dr GJ Ossenkoppele, Dept of Hematology, Vrije Universiteit Medical Center, de Boelelaan 1117(Br250), Amsterdam 1081 HV, The Netherlands; Fax: +31 20 444 26 01 Leukemia
Table 1. Treatment was stopped 14 days before harvesting of leukocytes for vaccine preparation. Pheripheral blood mononuclear cells (PBMCs) of patients were isolated by Lymphoprep (Nycomed, Oslo, Norway) centrifugation from a total of 160 (2 80) ml of blood. CML/DC were generated by culturing PBMC (2 106/ml) at 371C in 5% CO2 in 175 cm2 tissue culture flasks (Corning Inc., Corning, NY, USA) in 25 ml of RPMI1640 medium (GIBCO, Grand Island, NY, USA); 10% fetal calf serum (FCS) (Life Technologies, Eggenstein, Germany); 2 mM Lglutamine and 50 U/ml penicillin/streptomycin, supplemented with 100 ng/ml GM-CSF (Schering-Plough ), 10 ng/ml IL-4 (ScheringPlough ) and 2.5 ng/ml TNFa (CLB, Amsterdam, the Netherlands) for 14–21 days. The CML/DC phenotype was confirmed by flowcytometric analysis of the following markers: CD1a, CD40, CD54, CD80, CD83, CD86, and HLA-DR, as previously described. Differentiated CML/DC were harvested and concentrated to 1 107 viable CML/DC per ml. Vaccines were not cryopreserved, but immediately administered. The cells were pulsed with keyhole limpet hemocyanin (KLH) (50 mg/ml Perimmune, Seattle, WA, USA) for 2 h at room temperature. KLH-pulsed CML/DCs were washed once and resuspended in 0.3 ml of Hanks’ balanced salt solution containing 100 ng/ml GMCSF (Schering-Plough). The first and second vaccines were supplemented with 107 viable BCG particles (a kind gift of Dr A Claessen, VUMC, Amsterdam, the Netherlands). Aliquots of freshly obtained CML cells as well as cultured CML/DC were frozen for in vivo skin testing and in vitro immunomonitoring. Skin testing preparations consisted of KLH, uncultured CML, and CML-DC or monocyte-derived DC (MoDC), generated either in FCS-containing or serum-free medium (Stemspan H2000, Stem cell Technologies, Meylan, France). All vaccine and skin testing preparations were irradiated (60 Gy) prior to administration. A total of four CML/DC
