Bone Marrow Transplantation (2014) 49, 726–727 & 2014 Macmillan Publishers Limited All rights reserved 0268-3369/14 www.nature.com/bmt
LETTER TO THE EDITOR
MTHFR C677T/A1298C genotype: a possible risk factor for liver sinusoidal obstruction syndrome Bone Marrow Transplantation (2014) 49, 726–727; doi:10.1038/ bmt.2014.16; published online 3 March 2014
High-dose BU has become a mainstay in conditioning regimens for hematopoietic SCT (HSCT), despite its narrow therapeutic window and high toxicity. BU areas under the concentration time curves (AUC) higher than 1500 mM min are associated with increased risk of liver sinusoidal obstruction syndrome (SOS),1 which still contributes to significant treatment-related morbidity and mortality. Therapeutic and toxic responses to BU are largely unpredictable, and occur in patients receiving identical treatment schedules. An AUC of 900–1500 mM min has been defined as the target exposure range in adults,1 and controlling patient AUC by therapeutic drug monitoring (TDM) has become mandatory in many transplant centers, especially with oral administration of BU.2 Despite TDM, SOS still presents a serious challenge in HSCT, suggesting other predisposing factors to liver toxicity likely exist in patients who develop this complication. BU, like many chemotherapeutic drugs associated with liver toxicity, is metabolized in the liver via conjugation of glutathione by members of the glutathione S transferase (GST) family of phase II detoxification enzymes.3 Srivastava et al.4 assessed the impact of polymorphisms in GST genes on the risk of SOS in patients with b-thalassemia major undergoing HSCT and concluded that the GSTM1-null genotype predisposes to SOS. We did not find this association in thalassemic children of Israeli descent.5 An increasing body of evidence indicates that elevated levels of plasma homocysteine (hyperhomocysteinemia) are associated with various vascular diseases, including cerebral, coronary, peripheral artery and venous thrombosis,6 whereas hypohomocysteinemia limits production of sulfate, taurine and glutathione, all involved in phase II liver detoxification.7 Methylenetetrahydrofolate reductase (MTHFR) is one of the main regulatory enzymes involved in homocysteine metabolism.6 The C677T gene polymorphism in MTHFR was shown to correlate with reduced MTHFR enzyme activity. Consequently, homozygotes for the variant allele were found to have significantly higher plasma homocysteine concentrations. There was no difference in total homocysteine concentration between heterozygotes and subjects not carrying the C677T variant allele. Nevertheless, other reports have shown little or no impact of MTHFR C677T on the risk of vascular disease.6 A second polymorphism in MTHFR, A1298C, like C677T, results in decreased MTHFR activity. Goekkurt et al.3 found an association between A1298C and SOS but not C677T, in patients treated with BU. In this referred study the majority of the included subjects suffered from CML. In the current study, we recruited 62 adult AML patients, referred for HSCT. Myeloablative conditioning regimen was based on high-dose oral BU (16 mg/kg total dose) and i.v. CY (120 mg/kg total daily dose). Patients also received CYA and a short course of MTX for prophylaxis of GVHD. One patient also received TBI. None of the patients received prior getsuzumab treatment. Prophylaxis against SOS included low-dose sodium heparin (i.v. 100 U/Kg in continuous infusion) and oral ursodeoxycholic acid. Despite TDM
and prophylaxis, nine patients developed SOS, diagnosed according to the McDonald criteria8 (that is, modified Seattle criteria); namely, the occurrence of two of the following events within 30 days of transplantation: unexplained weight gain of 410% from baseline values due to fluid accumulation, jaundice, hepatomegaly or upper right quadrant pain of liver origin. Of these, eight patients matched the more stringent Baltimore criteria for SOS. SOS was not biopsy-proven. One patient with SOS developed multi organ failure (MOF). Treatment for SOS consisted of diuretics and one patient received defibrotide. No statistically significant demographic or pharmacokinetic differences were found between individuals who developed SOS and those who did not (Table 1). Furthermore, BU AUC values were similar and within the recommended therapeutic range, as previously published.9 Patient DNA samples, isolated from whole blood taken before HSCT, were genotyped for MTHFR C677T and A1298C (rs1801133 and rs1801131, respectively). Our results show that MTHFR C677T ancestral allele (C) and A1298C variant allele (C) were significant risk factors for SOS (Table 2). MTHFR haplotype 677CC/1298CC appeared to be significantly associated with SOS. These results are in partial agreement with those published by Goekkurt et al.,3 possibly due to the fact that our cohort consisted of AML patients only, whereas the patients in Goekkurt’s cohort were mainly affected by CML, which is an entirely different disease.10 Somewhat counter-intuitively, a study on 377 Israelis of Jewish descent found that subjects with the MTHFR 677CC/1298CC haplotype had significantly lower total homocysteine concentrations compared with subjects with the ancestral non variant 677CC/1298AA haplotype,6 thus leading to reduced production of glutathione, sulfate and taurine. As BU is eliminated via conjugation to glutathione,3 reduced levels of homocysteine could impair the liver’s ability to eliminate BU. The involvement of glutathione exhaustion in SOS is further suggested by findings that low constitutive levels of glutathione in liver cells in vitro, together with further glutathione exhaustion by cytotoxic drugs or radiation, contribute to SOS development.11 If this proposed model is proven accurate, it may suggest that MTHFR C677T/A1298C genotyping along with measurement of plasma homocysteine concentration could be of clinical utility when using high-dose BU for myeloablation.
Table 1.
Baseline characteristics of the SOS and No SOS cohorts
Disease stage at HSCT: 39 (73.5%) 8 (15%) 1 (1.9%) AUC/kg Median Range AST (IU/L)
No SOS (n ¼ 53)
SOS (n ¼ 9)
3 (33.3%) 6 (66.6%) 0
CR Not in CR Relapse
17.09 10.55–33.36 22.9±13.3
18.03 9.07–33.36 36.1±40.4
Data regarding the disease stage at HSCT of 5 patients with No SOS were not available.
Letter to the Editor
727 2
Table 2.
MTHFR polymorphisms and SOS
MTHFR C677T Variant homozygote Heterozygote wt MTHFR A1298C Variant homozygote Heterozygote
SOS (n ¼ 9)
No SOS (n ¼ 53)
P-value
0 (0%) 2 (22.2%) 7 (77.7%)
9 (16.9%) 30 (56.6%) 14 (26.4%)
0.0096a (wt vs non wt)
REFERENCES a
5 (55.5%) 3 (33.3%)
3 (5.6%) 26 (49.1%)
1 (11.2%)
24 (45.3%)
MTHFR C677T Ancestral allele C Variant allele T
0.888 0.111
0.547 0.452
0.0080b
MTHFR A1298C Ancestral allele A Variant allele C
0.277 0.722
0.698 0.302
P ¼ 0.0030b
5 4
4 48
0.0021b
wt
MTHFR 677CC þ 1298CC Non (677CC þ 1298CC)
Clinical Pharmacology Institute, Haifa, Israel; 3 Rambam Health Care Campus, Hematology Institute, Haifa, Israel and 4 Technion-Israel Institute of Technology, B. Rappaport Faculty of Medicine, Haifa, Israel E-mail: [email protected]
0.0002 (homozygote vs non homozygote)
a 2
w square test. bFisher’s exact test.
CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS This study was funded by the Israel Ministry of Health, Chief Scientist Grant (number 3-00000-6030).
E Efrati1,2, T Zuckerman3,4, E Ben-Ami1 and N Krivoy1,2,4 Rappaport-Rambam Center for Translational Genetics, B. Rappaport Institute for Research in the Medical Sciences and Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel;
1
& 2014 Macmillan Publishers Limited
1 Slattery JT, Sanders JE, Buckner CD, Schaffer RL, Lambert KW, Langer FP et al. Graft-rejection and toxicity following bone marrow transplantation in relation to busulfan pharmacokinetics. Bone Marrow Transplant 1995; 16: 31–42. 2 McCune JS, Holmberg LA. Busulfan in hematopoietic stem cell transplant setting. Expert Opin Drug Metab Toxicol 2009; 5: 957–969. 3 Goekkurt E, Stoehlmacher J, Stueber C, Wolschke C, Eiermann T, Iacobelli S et al. Pharmacogenetic analysis of liver toxicity after busulfan/cyclophosphamidebased allogeneic hematopoietic stem cell transplantation. Anticancer Res 2007; 27: 4377–4380. 4 Srivastava A, Poonkuzhali B, Shaji RV, George B, Mathews V, Chandy M et al. Glutathione S-transferase M1 polymorphism: a risk factor for hepatic venoocclusive disease in bone marrow transplantation. Blood 2004; 104: 1574–1577. 5 Elhasid R, Krivoy N, Rowe JM, Sprecher E, Adler L, Elkin H et al. Influence of glutathione S-transferase A1, P1, M1, T1 polymorphisms on oral busulfan pharmacokinetics in children with congenital hemoglobinopathies undergoing hematopoietic stem cell transplantation. Pediatr Blood Cancer 2010; 55: 1172–1179. 6 Friedman G, Goldschmidt N, Friedlander Y, Ben-Yehuda A, Selhub J, Babaey S et al. A common mutation A1298C in human methylenetetrahydrofolate reductase gene: association with plasma total homocysteine and folate concentrations. J Nutr 1999; 129: 1656–1661. 7 Vitvitsky V, Mosharov E, Tritt M, Ataullakhanov F, Banerjee R. Redox regulation of homocysteine-dependent glutathione synthesis. Redox Rep 2003; 8: 57–63. 8 McDonald GB, Sharma P, Matthews DE, Shulman HM, Thomas ED. Venocclusive disease of the liver after bone marrow transplantation: diagnosis, incidence, and predisposing factors. Hepatology 1984; 4: 116–122. 9 Krivoy N, Zuckerman T, Elkin H, Froymovich L, Rowe JM, Efrati E. Pharmacokinetic and pharmacogenetic analysis of oral busulfan in stem cell transplantation: prediction of poor drug metabolism to prevent drug toxicity. Curr Drug Safety 2012; 7: 211–217. 10 Song JH, Kim HJ, Lee CH, Kim SJ, Hwang SY, Kim TS. Identification of gene expression signatures for molecular classification in human leukemia cells. Int J Oncol 2006; 29: 57–64. 11 DeLeve LD, Wang X. Role of oxidative stress and glutathione in busulfan toxicity in cultured murine hepatocytes. Pharmacology 2000; 60: 143–154.
Bone Marrow Transplantation (2014) 726 – 727
LETTER TO THE EDITOR
MTHFR C677T/A1298C genotype: a possible risk factor for liver sinusoidal obstruction syndrome Bone Marrow Transplantation (2014) 49, 726–727; doi:10.1038/ bmt.2014.16; published online 3 March 2014
High-dose BU has become a mainstay in conditioning regimens for hematopoietic SCT (HSCT), despite its narrow therapeutic window and high toxicity. BU areas under the concentration time curves (AUC) higher than 1500 mM min are associated with increased risk of liver sinusoidal obstruction syndrome (SOS),1 which still contributes to significant treatment-related morbidity and mortality. Therapeutic and toxic responses to BU are largely unpredictable, and occur in patients receiving identical treatment schedules. An AUC of 900–1500 mM min has been defined as the target exposure range in adults,1 and controlling patient AUC by therapeutic drug monitoring (TDM) has become mandatory in many transplant centers, especially with oral administration of BU.2 Despite TDM, SOS still presents a serious challenge in HSCT, suggesting other predisposing factors to liver toxicity likely exist in patients who develop this complication. BU, like many chemotherapeutic drugs associated with liver toxicity, is metabolized in the liver via conjugation of glutathione by members of the glutathione S transferase (GST) family of phase II detoxification enzymes.3 Srivastava et al.4 assessed the impact of polymorphisms in GST genes on the risk of SOS in patients with b-thalassemia major undergoing HSCT and concluded that the GSTM1-null genotype predisposes to SOS. We did not find this association in thalassemic children of Israeli descent.5 An increasing body of evidence indicates that elevated levels of plasma homocysteine (hyperhomocysteinemia) are associated with various vascular diseases, including cerebral, coronary, peripheral artery and venous thrombosis,6 whereas hypohomocysteinemia limits production of sulfate, taurine and glutathione, all involved in phase II liver detoxification.7 Methylenetetrahydrofolate reductase (MTHFR) is one of the main regulatory enzymes involved in homocysteine metabolism.6 The C677T gene polymorphism in MTHFR was shown to correlate with reduced MTHFR enzyme activity. Consequently, homozygotes for the variant allele were found to have significantly higher plasma homocysteine concentrations. There was no difference in total homocysteine concentration between heterozygotes and subjects not carrying the C677T variant allele. Nevertheless, other reports have shown little or no impact of MTHFR C677T on the risk of vascular disease.6 A second polymorphism in MTHFR, A1298C, like C677T, results in decreased MTHFR activity. Goekkurt et al.3 found an association between A1298C and SOS but not C677T, in patients treated with BU. In this referred study the majority of the included subjects suffered from CML. In the current study, we recruited 62 adult AML patients, referred for HSCT. Myeloablative conditioning regimen was based on high-dose oral BU (16 mg/kg total dose) and i.v. CY (120 mg/kg total daily dose). Patients also received CYA and a short course of MTX for prophylaxis of GVHD. One patient also received TBI. None of the patients received prior getsuzumab treatment. Prophylaxis against SOS included low-dose sodium heparin (i.v. 100 U/Kg in continuous infusion) and oral ursodeoxycholic acid. Despite TDM
and prophylaxis, nine patients developed SOS, diagnosed according to the McDonald criteria8 (that is, modified Seattle criteria); namely, the occurrence of two of the following events within 30 days of transplantation: unexplained weight gain of 410% from baseline values due to fluid accumulation, jaundice, hepatomegaly or upper right quadrant pain of liver origin. Of these, eight patients matched the more stringent Baltimore criteria for SOS. SOS was not biopsy-proven. One patient with SOS developed multi organ failure (MOF). Treatment for SOS consisted of diuretics and one patient received defibrotide. No statistically significant demographic or pharmacokinetic differences were found between individuals who developed SOS and those who did not (Table 1). Furthermore, BU AUC values were similar and within the recommended therapeutic range, as previously published.9 Patient DNA samples, isolated from whole blood taken before HSCT, were genotyped for MTHFR C677T and A1298C (rs1801133 and rs1801131, respectively). Our results show that MTHFR C677T ancestral allele (C) and A1298C variant allele (C) were significant risk factors for SOS (Table 2). MTHFR haplotype 677CC/1298CC appeared to be significantly associated with SOS. These results are in partial agreement with those published by Goekkurt et al.,3 possibly due to the fact that our cohort consisted of AML patients only, whereas the patients in Goekkurt’s cohort were mainly affected by CML, which is an entirely different disease.10 Somewhat counter-intuitively, a study on 377 Israelis of Jewish descent found that subjects with the MTHFR 677CC/1298CC haplotype had significantly lower total homocysteine concentrations compared with subjects with the ancestral non variant 677CC/1298AA haplotype,6 thus leading to reduced production of glutathione, sulfate and taurine. As BU is eliminated via conjugation to glutathione,3 reduced levels of homocysteine could impair the liver’s ability to eliminate BU. The involvement of glutathione exhaustion in SOS is further suggested by findings that low constitutive levels of glutathione in liver cells in vitro, together with further glutathione exhaustion by cytotoxic drugs or radiation, contribute to SOS development.11 If this proposed model is proven accurate, it may suggest that MTHFR C677T/A1298C genotyping along with measurement of plasma homocysteine concentration could be of clinical utility when using high-dose BU for myeloablation.
Table 1.
Baseline characteristics of the SOS and No SOS cohorts
Disease stage at HSCT: 39 (73.5%) 8 (15%) 1 (1.9%) AUC/kg Median Range AST (IU/L)
No SOS (n ¼ 53)
SOS (n ¼ 9)
3 (33.3%) 6 (66.6%) 0
CR Not in CR Relapse
17.09 10.55–33.36 22.9±13.3
18.03 9.07–33.36 36.1±40.4
Data regarding the disease stage at HSCT of 5 patients with No SOS were not available.
Letter to the Editor
727 2
Table 2.
MTHFR polymorphisms and SOS
MTHFR C677T Variant homozygote Heterozygote wt MTHFR A1298C Variant homozygote Heterozygote
SOS (n ¼ 9)
No SOS (n ¼ 53)
P-value
0 (0%) 2 (22.2%) 7 (77.7%)
9 (16.9%) 30 (56.6%) 14 (26.4%)
0.0096a (wt vs non wt)
REFERENCES a
5 (55.5%) 3 (33.3%)
3 (5.6%) 26 (49.1%)
1 (11.2%)
24 (45.3%)
MTHFR C677T Ancestral allele C Variant allele T
0.888 0.111
0.547 0.452
0.0080b
MTHFR A1298C Ancestral allele A Variant allele C
0.277 0.722
0.698 0.302
P ¼ 0.0030b
5 4
4 48
0.0021b
wt
MTHFR 677CC þ 1298CC Non (677CC þ 1298CC)
Clinical Pharmacology Institute, Haifa, Israel; 3 Rambam Health Care Campus, Hematology Institute, Haifa, Israel and 4 Technion-Israel Institute of Technology, B. Rappaport Faculty of Medicine, Haifa, Israel E-mail: [email protected]
0.0002 (homozygote vs non homozygote)
a 2
w square test. bFisher’s exact test.
CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS This study was funded by the Israel Ministry of Health, Chief Scientist Grant (number 3-00000-6030).
E Efrati1,2, T Zuckerman3,4, E Ben-Ami1 and N Krivoy1,2,4 Rappaport-Rambam Center for Translational Genetics, B. Rappaport Institute for Research in the Medical Sciences and Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel;
1
& 2014 Macmillan Publishers Limited
1 Slattery JT, Sanders JE, Buckner CD, Schaffer RL, Lambert KW, Langer FP et al. Graft-rejection and toxicity following bone marrow transplantation in relation to busulfan pharmacokinetics. Bone Marrow Transplant 1995; 16: 31–42. 2 McCune JS, Holmberg LA. Busulfan in hematopoietic stem cell transplant setting. Expert Opin Drug Metab Toxicol 2009; 5: 957–969. 3 Goekkurt E, Stoehlmacher J, Stueber C, Wolschke C, Eiermann T, Iacobelli S et al. Pharmacogenetic analysis of liver toxicity after busulfan/cyclophosphamidebased allogeneic hematopoietic stem cell transplantation. Anticancer Res 2007; 27: 4377–4380. 4 Srivastava A, Poonkuzhali B, Shaji RV, George B, Mathews V, Chandy M et al. Glutathione S-transferase M1 polymorphism: a risk factor for hepatic venoocclusive disease in bone marrow transplantation. Blood 2004; 104: 1574–1577. 5 Elhasid R, Krivoy N, Rowe JM, Sprecher E, Adler L, Elkin H et al. Influence of glutathione S-transferase A1, P1, M1, T1 polymorphisms on oral busulfan pharmacokinetics in children with congenital hemoglobinopathies undergoing hematopoietic stem cell transplantation. Pediatr Blood Cancer 2010; 55: 1172–1179. 6 Friedman G, Goldschmidt N, Friedlander Y, Ben-Yehuda A, Selhub J, Babaey S et al. A common mutation A1298C in human methylenetetrahydrofolate reductase gene: association with plasma total homocysteine and folate concentrations. J Nutr 1999; 129: 1656–1661. 7 Vitvitsky V, Mosharov E, Tritt M, Ataullakhanov F, Banerjee R. Redox regulation of homocysteine-dependent glutathione synthesis. Redox Rep 2003; 8: 57–63. 8 McDonald GB, Sharma P, Matthews DE, Shulman HM, Thomas ED. Venocclusive disease of the liver after bone marrow transplantation: diagnosis, incidence, and predisposing factors. Hepatology 1984; 4: 116–122. 9 Krivoy N, Zuckerman T, Elkin H, Froymovich L, Rowe JM, Efrati E. Pharmacokinetic and pharmacogenetic analysis of oral busulfan in stem cell transplantation: prediction of poor drug metabolism to prevent drug toxicity. Curr Drug Safety 2012; 7: 211–217. 10 Song JH, Kim HJ, Lee CH, Kim SJ, Hwang SY, Kim TS. Identification of gene expression signatures for molecular classification in human leukemia cells. Int J Oncol 2006; 29: 57–64. 11 DeLeve LD, Wang X. Role of oxidative stress and glutathione in busulfan toxicity in cultured murine hepatocytes. Pharmacology 2000; 60: 143–154.
Bone Marrow Transplantation (2014) 726 – 727
