Unlabelled: THE AIM of this study was to evaluate the impact of genomic polymorphisms of methylene-tetrahydrofolate-reductase (MTHFR-C677T, MTHFR-A1298C) and various glutathione S-transferases (GSTP1-Ilel05Val, GSTA1*a/b, GSTM1, GSTT1) on the occurrence of liver toxicity in patients receiving allogeneic hematopoietic stem cell transplantation (HSCT). Patients and methods: Eighty-four adult patients were enrolled in this retrospective study. All patients were treated with busulfan/cyclophosphamide as a conditioning regimen and received cyclosporine and short-course MTX for GvHD prophylaxis. Genotyping was performed using PCR based restriction-fragment-length-polymorphism (RFLP) techniques. Results: Multivariate analysis identified the MTHFR-A1298C polymorphism as an independent predictor for maximum levels of bilirubin (p=0.0025) and duration of hyperbilirubinaemia (p=0.013). Furthermore, there was an association between this polymorphism and the occurrence of the sinusoidal obstruction syndrome (SOS) (p=0.048). No significant associations between the MTHFR-C677T or the various GST polymorphisms and liver toxicity were observed. Conclusion: The MTHFR-A1298C polymorphism might be associated with liver toxicity in patients receiving allogeneic HSCT

Ehninger, Gerhard

Eiermann, Thomas

Goekkurt, Eray

Iacobelli, Simona

Kröger, Nicolaus

Stoehlmacher, Jan

Stueber, Christian

Wolschke, Christine

Zander, Axel R

English

Abstract. The aim of this study was to evaluate the impact ofgenomic polymorphisms of methylene-tetrahydrofolate-reductase(MTHFR-C677T, MTHFR-A1298C) and various glutathione S-transferases (GSTP1-Ile105Val, GSTA1*a/b, GSTM1, GSTT1)on the occurrence of liver toxicity in patients receiving allogeneichematopoietic stem cell transplantation (HSCT). Patients andMethods: Eighty-four adult patients were enrolled in thisretrospective study. All patients were treated withbusulfan/cyclophosphamide as a conditioning regimen andreceived cyclosporine and short-course MTX for GvHDprophylaxis. Genotyping was performed using PCR basedrestriction-fragment-length-polymorphism (RFLP) techniques.Results: Multivariate analysis identified the MTHFR-A1298Cpolymorphism as an independent predictor for maximum levelsof bilirubin (p=0.0025) and duration of hyperbilirubinaemia(p=0.013). Furthermore, there was an association between thispolymorphism and the occurrence of the sinusoidal obstructionsyndrome (SOS) (p=0.048). No significant associations betweenthe MTHFR-C677T or the various GST polymorphisms and livertoxicity were observed. Conclusion: The MTHFR-A1298Cpolymorphism might be associated with liver toxicity in patientsreceiving allogeneic HSCT. Allogeneic hematopoietic stem cell transplantation (HSCT) isstill associated with a high incidence of therapy-relatedmortality (TRM) due to various complications such as graft-versus-host-disease (GvHD) and infectious diseases, and alsotoxic side-effects following high-dose chemotherapy. Livertoxicity including sinusoidal obstruction syndrome (SOS), alsoknown as veno-occlusive disease (VOD), caused by theconditioning regimen, is very frequent and has an impact ontherapy outcome. Interestingly, there is a remarkableinterindividual variability in the observed occurence of livertoxicity in patients receiving identical treatment schedules.Pharmacogenetic approaches have potential to give anexplanation for this observed interindividual variability. Methylene-tetrahydrofolate-reductase (MTHFR) plays acentral role in normal DNA synthesis and methylation,important steps in the repair of cell damages caused bydrugs. MTHFR catalyses the reduction of 5,1,-methyleneTHF to 5-methylTHF, thereby providing one-carbon group for methionine synthesis, and regulating folatemetabolism. Polymorphisms of MTHFR (C677T andA1298C) have been shown to alter enzyme activity (1). TheC677T polymorphism has been shown to be associated withtoxicity (such as mucositis) in HSCT (2, 3). Additionally, many chemotherapeutics (such as busulfan andmetabolites of cyclophosphamide) that have been linked to livertoxicity are metabolised by glutathione S-transferases (GSTs).GSTs catalyze the conjugation of busulfan, as well as the activemetabolite of cyclophosphamide with glutathione, the maincellular antioxidant in the liver. Several tissue-specific and non-tissue-specific isoforms of GSTs are known and functionalpolymorphisms of these isoforms have been described. A nullgenotype polymorphism of one of these isoforms (GSTM1)could be linked to SOS in thalassemic children undergoingHSCT after busulfan/cyclophosphamide conditioning (4). An4377*Both authors contributed equally to this work.Correspondence to: Eray Goekkurt, MD, Department of InternalMedicine I, University Hospital Carl Gustav Carus, Fetscherstr. 74,01307 Dresden, Germany. Tel: +49 0 351 458 37 57, Fax: +49 0351 458 43 94, e-mail: eray.goekkurt@uniklinikum-dresden.deKey Words: Allogeneic hematopoietic stem cell transplantation,glutathione S-tranferases, methylene-tetrahydrofolate-reductase,sinusoidal obstruction syndrome. ANTICANCER RESEARCH 27: 4377-4380 (2007)Pharmacogenetic Analysis of Liver Toxicity afterBusulfan/Cyclophosphamide-based Allogeneic Hematopoietic Stem Cell TransplantationERAY GOEKKURT1*, JAN STOEHLMACHER1*, CHRISTIAN STUEBER2, CHRISTINE WOLSCHKE2, THOMAS EIERMANN3, SIMONA IACOBELLI4, AXEL R. ZANDER2, GERHARD EHNINGER1 and NICOLAUS KRÖGER21Department of Internal Medicine I, University Hospital Carl Gustav Carus, University of Dresden, Fetscherstr. 74, 01307 Dresden;2Bone Marrow Transplantation and 3Transfusion Medicine, University Hospital Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany;4University La Sapienza, Rome, Italy0250-7005/2007 $2.00+.40A/G single nucleotide polymorphism (SNP) within thesubstrate-binding domain of GSTP1 decreases enzyme activity(5). The main GSTs present in the liver are GSTA1 and A2.Promoter polymorphisms of GSTA1 (GSTA1*a/b) have beenshown to have a major impact on promoter activity (6). The aim of our study was to evaluate the impact ofMTHFR genomic polymorphisms and various GSTs on livertoxicity in patients receiving busulfan/cyclophoshamide-based allogeneic HSCT.Patients and MethodsEighty-four adult patients were enrolled in this retrospective study.DNA was isolated from leucocytes of stem cell recipients collectedprior to HSCT using the QiaAmp kit (Qiagen, Valencia, CA,USA). The GSTP1-Ile105Val, GSTA1*a/b and MTHFR (C677T,A1298C) polymorphisms were detected by PCR-based restriction-fragment-length-polymorphism (RFLP) techniques and the nullgenotypes GSTM1 and GSTT1 were detected by multiplex-PCR asdescribed previously (1, 6-9). For quality purposes a random 20%of samples was repeated and showed 100% concordance. All patients were treated at the Bone Marrow TransplantationUnit of the University Hospital Hamburg, Germany, with standardbusulfan (16mg/kg)/cyclophosphamide (2x60 mg/kg) as theconditioning regimen and received cyclosporine and short-coursemethotrexate (MTX) (day1:15 mg/m2, days 3+6:10 mg/m2) forGvHD prophylaxis. Busulfan was given orally at a fixed dose. Sixtyseven patients (80%) additionally received anti-thymocyte globulin(ATG). Assessment of liver toxicity was based on the maximumbilirubin levels within 20 days after HSCT, the duration ofhyperbilirubinaemia >2 mg/dl and the occurrence of SOS accordingto the Baltimore criteria. The bilirubin levels were measured dailyright from the beginning of the conditioning regimen. In addition tothe measurement of bilirubin levels, evaluation of SOS occurrencewas performed by daily clinical examination including abdominalultrasound (hepatomegaly, ≥5% weight gain, ascites). All patientswere tested negative for previous hepatitis B or C infection. Inaddition, none of the patients had initial bilirubin levels >2.4 mg/dl. Univariate and multivariate analyses were performed to assessassociations between genotypes and toxicity parameters. Univariateanalyses were performed for genotypes and adjustment factors(gender, age, ATG treatment, donor type, previous treatment withimatinib, hydroxyurea and/or interferon). Analyses with respect tothe occurrence of SOS were done using the Chi-square test orFisher’s exact test. In the case of continuous toxicity outcomes thenon-parametric Wilcoxon (2 groups) or Kruskall-Wallis tests (>2groups) were used. Multivariate analysis was performed using thelogistic-regression-model (SOS) and linear-regression-model(maximum bilirubin levels, duration of hyperbilirubinaemia)considering candidate factors (genotypes and adjustment factors)with p-values below 0.2 from the univariate analysis. All patientsagreed to the genotype analysis in this study.ResultsPatient characteristics are given in Table I. The genotypedistributions were as follows: MTHFR-677CC 33%, 677CT50%, 677TT 17%; MTHFR-1298AA 46%, 1298AC 45%,1298CC 9%; GSTP1-105Ile/Ile 55%, Ile/Val 34%, Val/Val11%; GSTA1*a/a 45%, a/b 46%, b/b 9%, GSTM1-null 57%,GSTT1-null 18%. The distribution of genotypes among ourstudy population was comparable to previous reports ofother groups. There was no deviation from Hardy-Weinberg-Equilibrium. As expected, there was a highly significantlinkage disequilibrium between the two MTHFRpolymorphisms (p<0.001). None of the patients washomozygous for both variants (677TT + 1298CC) in ourstudy population, which was consistent with previous reports. Univariate analysis revealed a statistically significantassociation between the MTHFR-A1298C polymorphismand liver toxicity. The patients carrying the MTHFR-1298CCgenotype demonstrated higher median maximum levels ofbilirubin with 6.8 mg/dl than the MTHFR-1298AC andMTHFR-1298AA patients (3.0 mg/dl and 3.4 mg/dl,respectively) (p=0.004) (Figure 1). Furthermore, elevationof bilirubin levels >2 mg/dl lasted longer in the MTHFR-1298CC patients with a median of 14 days compared to 3and 6 days in patients harbouring the MTHFR-1298AC andMTHFR-1298AA genotypes (p=0.004), respectively (Figure1). There was a significantly higher incidence of SOS with86% in patients that were homozygous for the C allelecompared to 39% in patients harbouring at least one A allele(odds ratio 9.4; 95%CI 1.1;81.9) (p=0.043). Multivariateanalysis confirmed the statistical independent impact of theMTHFR-A1298C polymorphism on the maximum elevationof bilirubin (p=0.0025), duration of hyperbilirubinaemia(p=0.013) and appearance of SOS (p=0.048). No significantassociations between MTHFR-C677T, GSTP1-Ile105Val,GSTA1*a/b, GSTM1 and GSTT1 polymorphisms and livertoxicity were found in this study. Furthermore, none of theanalyzed polymorphisms was linked to GvHD, overallsurvival, therapy-related-mortality or relapse.DiscussionOur data suggested that the MTHFR-A1298C polymorphismmight be associated with SOS in patients treated withbusulfan/cyclophosphamide as a conditioning regimenfollowed by allogeneic HSCT. The MTHFR-A1298Cpolymorphism is located on exon 7 which codes for the C-terminal regulatory region. The observed effect of theA1298C polymorphism might be due to its location, sincethis region is known to regulate enzyme activity dependingon adenosylmethionine levels. Our suggestion is that thispolymorphism may lead to imbalances in MTHFR reactivityon cell damages. A study by Mathews et al. also demonstrated anassociation between the MTHFR-A1298C polymorphismand liver complications in patients with acute promyelocyticleukemia treated with single agent As2O3 as induction,consolidation and maintenance chemotherapy (10). TheANTICANCER RESEARCH 27: 4377-4380 (2007)4378MTHFR-C677T polymorphism was not related to livercomplications, which is in agreement with our results. Thedata suggest that there might be a general effect of theMTHFR-A1298C polymorphism on drug-induced celltoxicity in the liver. This effect may be a consequence ofinadequate cell reactivity (lack of DNA-repair) to toxicinfluences. Such an imbalance could lead to a tendency forapoptosis of the injured cells and in consequence toincreased toxicity. Interestingly, Nuckel et al. reportedincreased spontaneous apoptosis in B cells from chroniclymphocytic leukaemia (CLL) patients with MTHFR-1298CC genotypes (11). This might also imply a tendencyfor increased apoptosis in MTHFR-1298CC carrying normalcells (such as hepatocytes and hepatic sinusoidal endothelialcells) as a reaction to drug induced cell damage. Thissuggestion provides a possible explanation for the observedassociation of this genotype with SOS in this study sinceSOS is a consequence of toxic injury of hepatic sinusoidalendothelial cells. However, the exact molecular mechanismunderlying this hypothesis remains unclear at this point andshould be evaluated in the future. The lack of a significant association between theMTHFR-C677T polymorphism and SOS or hyperbiliru-binaemia in this study is in concordance with previousstudies in allogeneic HSCT (3, 12). To date, the MTHFR-C677T polymorphism has been linked mainly to oralmucositis in allogeneic HSCT (2, 3). Mucositis was notanalyzed in this study since documentation of mucositisGoekkurt et al: Pharmacogenetics of Liver Toxicity in Allogeneic HSCT4379Figure 1. Association between MTHFR A1298C and liver toxicity.Toxicities were assessed by median max. bilirubin levels (mg/dl) andmedian duration of hyperbilirubinaemia (days). Patients harbouring theless active MTHFR-1298CC genotype demonstrated higher median max.bilirubin levels (p=0.004) and prolonged hyperbilirubinaemia (p=0.004)compared to patients possessing at least one A allele.Table I. Patient characteristics.Patient characteristics n (%)Demographic informationAge (years)Median 39.5 -Range 16-59 -GenderMale 49 58Female 35 42DiseaseCML 79 95CML in 1st chronic phase 63 75AML (M4) 2 2MDS 2 2Farber’s Disease 1 1Transplant related informationDonor typeRelated 42 50Unrelated 42 50ATG treatmentYes 67 80No 17 20Previous imatinib treatmentYes 11 13No 73 87Previous interferon treatmentYes 31 37No 53 63Previous hydroxyurea treatmentYes 77 92No 7 8Survival statusAlive 56 67Dead 28 33Relapse status (only CML patients)Relapse 5 6No relapse 74 94Acute GvHDGrade 0-2 68 81Grade 3-4 16 19Sinusoidal obstruction syndrome (SOS)Yes 36 43No 48 57Maximum bilirubin levels (mg/dl) Median 3.4 -Range 0.7-25.4 -Duration of hyperbilirubinaemia >2 mg/dl (days)Median 5 -Range 0-32 -CML: Chronic myelogenous leukaemia, AML: acute myeloidleukaemia, MDS: myelodysplastic syndrome, ATG: anti-thymocyteglobulin, GvHD: graft-versus-host disease.scores was not standardized during the evaluation period.Both MTHFR polymorphisms are linked to alteredphenotypes and differing impacts of both MTHFRpolymorphisms on clinical outcomes are common in theliterature (13, 14). While the GSTM1 null genotype has been identified as arisk factor for SOS after bone marrow transplantation inthalassemic children (4), it nevertheless, failed to besignificantly associated with SOS in our study. One reasonmight be the fact that our patient population consisted onlyof adults which might imply a different impact of theGSTM1 null genotype with respect to age. In a study byBredschneider et al. GSTA1 promoter polymorphisms(GSTA1*a/b) had no effect on GSTA1 protein expressionor on GSTA1 function and the authors postulated thatGSTA1 promoter polymorphisms were therefore unlikely tobe associated with SOS (15). In fact, no association betweenGSTA1*a/b and SOS could be observed in our study.To the best of our knowledge this is the first reportlinking the MTHFR-A1298C polymorphism to liver toxicityincluding the occurrence of SOS in allogeneic HSCT. Alongwith previous reports, our data provide evidence thatpharmacogenetic approaches have potential for identifyingpatients who are at a higher risk of experiencing toxic side-effects in HSCT.References1 Weisberg I, Tran P, Christensen B, Sibani S and Rozen R: Asecond genetic polymorphism in methylenetetrahydrofolatereductase (MTHFR) associated with decreased enzyme activity.Mol Genet Metab 64: 169-172, 1998.2 Robien K, Schubert MM, Bruemmer B, Lloid ME, Potter JDand Ulrich CM: Predictors of oral mucositis in patients receivinghematopoietic cell transplants for chronic myelogenousleukemia. J Clin Oncol 22: 1268-1275, 2004.3 Ulrich CM, Yasui Y, Storb R, Schubert MM, Wagner JL, BiglerJ et al: Pharmacogenetics of methotrexate: toxicity among marrowtransplantation patients varies with the methylenetetrahydrofolatereductase C677T polymorphism. Blood 98: 231-234, 2001.4 Srivastava A, Poonkuzhali B, Shaji RV, George B, Mathews V,Chandy M et al: Glutathione S-transferase M1 polymorphism: arisk factor for hepatic venoocclusive disease in bone marrowtransplantation. Blood 104: 1574-1577, 2004.5 Zimniak P, Nanduri B, Pikula S, Bandorowicz-Pikula J, SinghalSS, Srivastava SK et al: Naturally occurring human glutathioneS-transferase GSTP1-1 isoforms with isoleucine and valine inposition 104 differ in enzymic properties. Eur J Biochem 224:893-899, 1994.6 Coles BF, Morel F, Rauch C, Huber WW, Yang M, Teitel CHet al: Effect of polymorphism in the human glutathione S-transferase A1 promoter on hepatic GSTA1 and GSTA2expression. Pharmacogenetics 11: 663-669, 2001.7 Arand M, Muhlbauer R, Hengstler J, Jager E, Fuchs J, WinklerL et al: A multiplex polymerase chain reaction protocol for thesimultaneous analysis of the glutathione S-transferase GSTM1and GSTT1 polymorphisms. Anal Biochem 236: 184-186, 1996.8 Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA,Matthews RG et al: A candidate genetic risk factor for vasculardisease: a common mutation in methylenetetrahydrofolatereductase. Nat Genet 10: 111-113, 1995.9 Stoehlmacher J, Park DJ, Zhang W, Yang D, Groshen S, ZahedyS et al: A multivariate analysis of genomic polymorphisms:prediction of clinical outcome to 5-FU/oxaliplatin combinationchemotherapy in refractory colorectal cancer. Br J Cancer 91:344-354, 2004.10 Mathews V, Salamun DE, George B, Viswabandya A, LakshmiK, Bajel A et al: Hepatotoxicity profile of single agent As2O3in the management of newly diagnosed cases of acutepromyelocytic leukemia: Impact on outcome and correlationwith genetic polymorphisms. Blood 106: Abstract 893, 2005.11 Nuckel H, Frey UH, Durig J, Duhrsen U and Siffert W:Methylenetetrahydrofolate reductase (MTHFR) gene 677C>Tand 1298A>C polymorphisms are associated with differentialapoptosis of leukemic B cells in vitro and disease progression inchronic lymphocytic leukemia. Leukemia 18: 1816-1823, 2004.12 Kalayoglu-Besisik S, Caliskan Y, Sargin D, Gurses N andOzbek U: Methylenetetrahydrofolate reductase C677Tpolymorphism and toxicity in allogeneic hematopoietic celltransplantation. Transplantation 76: 1775-1777, 2003.13 Evans WE: Differing effects of methylenetetrahydrofolatereductase single nucleotide polymorphisms on methotrexateefficacy and toxicity in rheumatoid arthritis. Pharmacogenetics12: 181-182, 2002.14 Urano W, Taniguchi A, Yamanaka H, Tanaka E, Nakajima H,Matsuda Y et al: Polymorphisms in the methylenetetrahy-drofolate reductase gene were associated with both the efficacyand the toxicity of methotrexate used for the treatment ofrheumatoid arthritis, as evidenced by single locus and haplotypeanalyses. Pharmacogenetics 12: 183-190, 2002.15 Bredschneider M, Klein K, Murdter TE, Marx C, EichelbaumM, Nussler AK et al: Genetic polymorphisms of glutathione S-transferase A1, the major glutathione S-transferase in humanliver: consequences for enzyme expression and busulfanconjugation. Clin Pharmacol Ther 71: 479-487, 2002.Received June 5, 2007Revised August 22, 2007Accepted October 8, 2007ANTICANCER RESEARCH 27: 4377-4380 (2007)4380

Pharmacogenetic analysis of liver toxicity after busulfan/cyclophosphamide-based allogeneic hematopoietic stem cell transplantation

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Pharmacogenetic analysis of liver toxicity after busulfan/cyclophosphamide-based allogeneic hematopoietic stem cell transplantation

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