The Pharmacogenomics Journal (2014) 14, 432–438 & 2014 Macmillan Publishers Limited All rights reserved 1470-269X/14 www.nature.com/tpj
ORIGINAL ARTICLE
Association between methylenetetrahydrofolate reductase polymorphisms and the relapse of acute lymphoblastic leukemia: a meta-analysis H-R He1, S-Y Chen1, H-S You1, S-S Hu, J-Y Sun, Y-L Dong and J Lu Relapse is a threat in patients treated for acute lymphoblastic leukemia (ALL). Methylenetetrahydrofolate reductase (MTHFR) activity may affect the sensitivity of patients to folate-based chemotherapeutic drugs, thus influencing the relapse risk. Two polymorphisms of the gene encoding MTHFR, C677T and A1298C, alter MTHFR enzyme activity and may be associated with ALL relapse. The aim of this meta-analysis was to clarify the correlation between the C677T and A1298C polymorphisms and ALL relapse. To this end, data were collected from studies of the association between these two polymorphisms and ALL relapse. Analysis of the data revealed a serious contradiction among the results. A recessive model demonstrated that the ALL relapse risk was significantly increased in carriers of the 677 TT genotype, especially for pediatric ALL, but was unaffected by the A1298C polymorphism. These findings confirm that the MTHFR C677T polymorphism could be considered as a good marker of the pediatric ALL relapse risk. The Pharmacogenomics Journal (2014) 14, 432–438; doi:10.1038/tpj.2014.10; published online 18 March 2014
INTRODUCTION Despite remarkable progress in the cure rate of acute lymphoblastic leukemia (ALL), most adult patients ultimately die from this disease. The most common reason for ALL treatment failure is relapse.1 In addition, there is also much heterogeneity in the pharmacological response in the treatment of childhood ALL. Folate is essential for nucleotide synthesis. Some clinical and in vitro studies indicate that polymorphisms in genes encoding key enzymes involved in folate metabolism could affect the sensitivity of patients to folate-based chemotherapeutic drugs, such as methotrexate (MTX). These may either directly or indirectly result in shifts in intracellular folate distribution.2 In addition, several studies have shown that in addition to drug-related toxicity, the patient’s response to chemotherapeutic agents can also vary with polymorphism frequency.3–5 Certain prognostic factors have been used to stratify ALL patients according to the risk of relapse,6,7 including clinical and biological characteristics that are evaluable at diagnosis. The influence of polymorphisms of a variety of genes involved in the metabolism of chemotherapeutic agents has been regarded as a prognostic factor in recent years, and has been studied widely in relation to therapeutic responses in ALL.8 MTX is the main chemotherapeutic agent used in the treatment of ALL, and acts as an antifolate. Its toxicity may be not only lifethreatening, but also one of the main reasons for the interruption or discontinuation of chemotherapy, which may in turn increase the relapse rate for the disease.9 Methylenetetrahydrofolate reductase (MTHFR) is an important enzyme in the folate/MTX metabolism pathway. It catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methylenetetrahydrofolate in the folic acid cycle.10 The activity of MTHFR may exert a substantial impact on the occurrence and relapse of ALL via the following mechanism. Decreased MTHFR activity may increase the availability of
5,10-methylenetetrahydrofolate and reduce the concentration of 5-methylenetetrahydrofolate. All that can influence DNA hypomethylation, which can alter gene expression (oncogenes) and increase the number of breaks in DNA strands, leading to an increased risk of carcinogenesis.11 MTHFR gene has two common functional polymorphisms: C677T and A1298C, and both of them have been investigated intensively.12 The C677T (rs1801133) polymorphism occurs in exon 4 and results in an alanine-to-valine substitution at codon 222. This substitution lies at the binding site for the flavin adenine dinucleotide, which is an important cofactor for MTHFR.13 The MTHFR 677 T allele encodes proteins with decreased enzymatic activity in comparison with those encoded by the 677C allele.14 With respect to the A1298C polymorphism (rs1801131), it occurs in exon 7 and results in glutamate-to-alanine substitution at codon 429, which represents the S-adenosylmethionine regulatory domain of MTHFR.13 The enzyme activity is 40% lower for the 1298CC genotype than the 1298AA genotype.15 Both polymorphisms produce a thermolabile enzyme with reduced functional activity, and a consequent altered normal intracellular distribution of folate substrates.13 These data demonstrated that the two single-nucleotide polymorphisms (SNPs) are associated with various aspects of ALL treatment. Numerous published studies have investigated the association between the two SNPs and the relapse risk in ALL, with conflicting results:11,12,16–24 some studies have found no association, while others have found both negative and positive associations. The possible reasons for these discrepancies include differences in ethnicity, treatment protocols, sample sizes and the age of the patients. It is clear, therefore, that the clarification of this putative association is necessary. In the present study, data were collected from all published studies on the relationship between the MTHFR C677T, A1298C SNPs and ALL relapse. A meta-analysis was then applied to all
Department of Pharmacy, The First Affiliated Hospital, College of Medicine, Xi’an Jiaotong University, Xi’an, China. Correspondence: Dr J Lu and Dr Y Dong, Department of Pharmacy, The First Affiliated Hospital, College of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi 710061, People’s Republic of China. E-mail: [email protected] and [email protected] 1 These authors contributed equally to this work. Received 1 October 2013; revised 9 January 2014; accepted 31 January 2014; published online 18 March 2014
MTHFR SNPs in relapse risk of ALL H-R He et al
433 eligible studies that had been selected based on strict criteria to demonstrate the role of MTHFR polymorphisms on the relapse risk in ALL patients. MATERIALS AND METHODS Literature and search strategy An exhaustive literature search of PubMed (www.ncbi.nlm.nih.gov/ pubmed) and Web of Knowledge (http://isiknowledge.com/) was conducted using the following keywords and subject terms: ‘acute lymphoblastic leukemia and relapse and polymorphism (variant)’ and ‘methylenetetrahydrofolate reductase (MTHFR) and acute lymphoblastic leukemia and relapse.’ Only journal articles were included in the analysis, and the publication language was restricted to English. All references within the identified studies were then reviewed to identify additional relevant work. The literature search was last updated on 20 September 2013.
Inclusion criteria The inclusion criteria for the meta-analysis required that each study must (1) be an independent association study investigating the relationship between the C677T and A1298C polymorphisms and the ALL relapse risk, (2) provide sufficient data on relapse by genotype or sufficient information for such data to be calculated and (3) have a retrospective design and include a population of ALL patients.
Genetic model A recessive model for the MTHFR C677T (TT versus CC þ CT) and A1298C (CC versus AA þ AC) polymorphisms was assumed, based on the majority of the research performed. The use of this model maximized the number of included studies.
Data extraction The following information was gathered for each article included in the study: name of the first author, year of publication, number of patients, ethnicity of the study population, median age at ALL diagnosis, median follow-up duration, data on relapse by genotype and main findings of the research.
Figure 1. Flow chart of study selection.
Statistical analysis The overall pooled relative risk (RR) and corresponding 95% confidence interval of the relapse risk were estimated using the Mantel–Haenszel method with a random-effects model. Random-effect model accounts for the heterogeneity in the data that undoubtedly exists due to within- and between-study variations and thus provides more conservative estimated effect values.25 The significance of RR was determined by a z-test. The level of statistical significance was set at Po0.05. The presence of heterogeneity was assessed using Cochran’s Q-statistic (with a significance cutoff of Po0.05) and quantified using the I2-statistic, which is proportional to the degree of heterogeneity; an I2-value above 50% denotes a very high degree of heterogeneity. Sensitivity analysis was performed by excluding individual studies when substantial heterogeneity was present. Outlying studies were identified and excluded, and the I2-estimates for these different sets of studies examined.
Meta-analysis On the basis of the influence of age on the development and prognosis of ALL, the association between the two SNPs and ALL relapse was evaluated in childhood ALL patients, adult ALL patients and all ALL patients (regardless of their age), respectively.
RESULTS Overview of the study characteristics The flow chart for the study selection process is depicted in Figure 1. In total, 204 articles were selected using the two groups of keywords described earlier. Checking for duplicates resulted in the removal of 26 articles. Of the remaining 178 articles, 76 did not report the association between SNPs and the relapse of ALL patients, 70 did not focus on MTHFR polymorphisms and 16 were & 2014 Macmillan Publishers Limited
reviews. After exclusion of these studies, only 16 remained, which investigated the relationship between MTHFR SNPs and the relapse risk of ALL patients. However, eight of these did not provide sufficient data and were thus also excluded. Therefore, ultimately, only eight of the studies qualified for inclusion in this meta-analysis. The basic data for every eligible study were extracted and are listed in Table 1. Two of the studies were performed on adults; although Chiusolo et al.16 studied ALL patients of all ages, the median age of their subjects was 31.5 years, and adult patients accounted for a higher proportion than all others. That article was therefore regarded as a study performed on adult patients. The other studies involved children. The results of the included studies were contradictory. Meta-analysis results MTHFR SNPs and relapse risk in childhood ALL. Six of the studies investigated the association between the MTHFR C677T polymorphism and the relapse risk in childhood ALL, and provided sufficient data to be included in the analysis.17–22 A meta-analysis of the six studies was conducted, involving data from 1553 patients. The z-test verified a significant association between the C677T polymorphism and the relapse risk in childhood ALL, and the RR value demonstrated that the relapse risk in pediatric ALL was higher for the 677TT genotype than for the CT or CC genotype. The I2-value denoted the degree of heterogeneity was not high among the six studies (Figure 2). The Pharmacogenomics Journal (2014), 432 – 438
MTHFR SNPs in relapse risk of ALL H-R He et al
434 Table 1.
List of seven studies that analyzed the association between the MTHFR SNPs and ALL relapse
Study
Year
Aplenc et al.20 Chiusolo et al.16 Tantawy et al.19 D’Angelo et al.17 Pietrzyk et al.22 EL-Khodary et al.18 Sepe et al.21 Eissa et al.12
2005 2007 2010 2011 2011 2012 2012 2013
Sample size
520 81 40 151 319 40 483 50
Ethnicity
Median age at diagnosis (range)
Mixed European Caucasian Egyptian Caucasian European origin European Egyptian Mixed Egyptian
1–10 31.5 (12–75) 6.1±1.2 (3–15) 5 (0.25–15) (1–18) (2–16) 4 47.5 (22–74)
Median follow-up (months; range)
120 21 (1–240) 60 (37.2–78) 65 72 (60–84) 2-year relapse — 29 (10–36)
Results C677T
A1298C
þ NA þ þ NA A þ þ
NA NA NA NA — — —
Abbreviations: A: the SNP is associated with ALL relapse; ALL, acute lymphoblastic leukemia; NA, no association between the SNP and toxicity; þ , the SNP is associated with increased relapse risk; –, the SNP is associated with decreased relapse risk.
Figure 2.
Results of meta-analysis of association between the two SNPs and relapse risk in pediatric ALL.
Only three studies surveyed the influence of the A1298C polymorphism on the relapse risk in childhood ALL, and no one found an association.17,19,20 A meta-analysis performed on these three studies, based on the data from 711 patients, verified that there was no association. The I2-value showed that there was almost no heterogeneity among the three studies (Figure 2). MTHFR SNPs and relapse risk in adult ALL. Two studies analyzed the association between the two SNPs and the relapse risk in adult ALL; the results were inconsistent for each SNP.12,16 A metaanalysis using the data from 131 patients found no association between the C677T polymorphism and the relapse risk in adult ALL. Cochran’s Q-statistic and I2-value denoted a high degree of heterogeneity between the two studies; however, the inclusion of only two studies precluded the performance of a sensitivity analysis. Therefore, more studies are needed before reliable conclusions can be drawn regarding the influence of the C677T polymorphism on relapse in adult ALL. The Pharmacogenomics Journal (2014), 432 – 438
The findings of a meta-analysis based on the data from 131 patients also did not support an association between the A1298C polymorphism and the relapse risk in adult ALL. The degree of heterogeneity between the studies was small (Figure 3). MTHFR SNPs and relapse risk in all ALL cases. A meta-analysis was performed using the data from all ALL cases, regardless of their age. All eight of the studies analyzed the association between the C677T polymorphism and the relapse risk of ALL.12,16–22 The metaanalysis involving data from 1684 patients found a significant association between the C677T polymorphism and the relapse risk. The RR value revealed that the relapse risk was higher in patients with the 677 TT genotype than in those with the CT or CC genotype. As the I2-value revealed a high degree of heterogeneity among the included studies, a sensitivity analysis was undertaken, which identified the study by Eissa et al.12 as an outlier. Removal of the data of that study from the meta-analysis reduced the heterogeneity; however, a significant association was still observed. & 2014 Macmillan Publishers Limited
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Figure 3.
Results of meta-analysis of association between the two SNPs and relapse risk in adult ALL.
Moreover, both the RR value and its 95% confidence intervals were greater than 1, suggesting that the relapse risk is higher for ALL patients with the 677 TT genotype (Figure 4). For the A1298C polymorphism, five studies provided sufficient data for inclusion in the meta-analysis.12,16,17,19,20 On the basis of the data from 842 patients, no association was observed between the A1298C polymorphism and the relapse risk of ALL. The I2-value revealed a small degree of heterogeneity among the five studies (Figure 4). DISCUSSION This study followed a strict screening methodology to collect data from eight published articles in which the association between the MTHFR C677T and A1298C polymorphisms and the relapse risk of ALL was researched.12,16–22 All of the included articles were of high quality and presented a rigorous scientific design, accurate data and clear results. The results of these studies were inconsistent. For the C677T SNP, most of the studies found an association between the 677 TT genotype and an increased relapse risk of ALL,12,17–21 but that association was not observed in two of the studies.16,22 The A1298C polymorphism was evaluated in five of the studies, four of which found no association between the SNP and the relapse risk of ALL,16,17,19,20 whereas one concluded that patients with the 1298CC genotype tended to have a decreased relapse risk relative to wild-type or heterozygote carriers.12 Of the eight studies, two focused on adults and six on children. The present study applied a meta-analysis to review and summarize the relapse risk according to genotype in childhood ALL patients, adult ALL patients and all ALL patients, with the aim of obtaining an objective and consistent conclusion. The main conclusion was that there appears to be an association between the MTHFR 677 TT genotype and an increased relapse risk of pediatric ALL, whereas the A1298C polymorphism does not appear to be associated with the relapse risk of ALL. Of the eight studies, the population within the same study was homogenous in the race in six studies,12,16–19,22 whereas the population in the left two studies was mixed.20,21 In the study by Sepe et al.,21 authors addressed the issue of population stratification by including ethnicity in the Cox proportional hazards models. Aplenc et al.20 found the impact of the MTHFR T677 variant allele remains significant after controlling for ethnicity, thus indicating an effect of the variant allele that is unlikely to be & 2014 Macmillan Publishers Limited
confounded by ethnicity. In addition, several authors have shown that population stratification is unlikely to cause significant bias in studies with multiple admixed populations.26–28 Above all, including these two studies did not cause serious heterogeneity in our meta-analyses. We are therefore reasonable to include these two studies. Reasons for the discrepant findings Four main reasons could explain the discrepancies among the included studies: differences in patient ages, patient ethnicities, toxicity of treatment and follow-up times. Age. Clinically, several prognostic factors have been used to stratify ALL patients, and one of the main ones is age. Although chemotherapy regimens cure nearly 80% of pediatric patients with ALL, the death rate of adult patients remains high. There is a large discrepancy in the sensitivity to drug treatments among the different patient age groups, and patients with a lower sensitivity would have an increased relapse risk.6,29,30 In other words, age may influence the relapse risk of ALL by affecting the sensitivity to drug treatment. Ethnicity. The genotype distribution of the two SNPs differs according to race. Some studies have showed different genotype frequency of the two SNPs in people of different races.31,32 Karas et al.11 also pointed out that the activities of the folate metabolism enzymes could vary in different populations, ethnic groups, and races, thus creating differing metabolic profiles. Moreover, the nutritional status varies between countries, and the nutritional status at diagnosis may be a significant prognostic factor in the long-term treatment outcome of pediatric patients with ALL.33 All of these observations demonstrate that ethnic factors could indirectly affect the relapse risk of ALL. The races of the patients evaluated differ between the studies included in the present meta-analysis, which could at least partially explain the discrepant results. Toxicity. Treatment-related toxicity could lead to interruption or discontinuation of chemotherapy, which may reduce the effects of chemotherapy drugs and result in tumor cells not being killed completely, thus increasing the relapse risk of ALL.9 Most of our eligible studies researched the toxicity of patients, and different The Pharmacogenomics Journal (2014), 432 – 438
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Figure 4.
Results of meta-analysis of association between the two SNPs and relapse risk in all ALL.
incidences were found among them. For example, the hematopoietic toxicity, hepatic toxicity and hepatic plus hematopoietic toxicity were assessed in the paper of Chiusolo et al.16 and 40% incidence of toxicity was found. The study performed by D’Angelo et al.17 also recorded MTX-induced toxicity, including hematological and non-hematological (hepatic, gastrointestinal and nervous system) toxicities, and the incidence of toxicity was 68.3%. Likewise, Eissa et al.12 researched hematologic and/or nonhematologic (hepatic and gastrointestinal) toxicity induced by MTX and they summarized that 82% patients developed hematologic and/ or nonhematologic toxicity. Such differences in the incidence of toxicity could be partially attributed to the use of different treatment protocols, and would influence the drug effects and lead to different relapse risks. Follow-up duration. The follow-up duration differed among the included studies (see Table 1), and the relapse risk may vary with The Pharmacogenomics Journal (2014), 432 – 438
time. In brief, the differences in follow-up may also be at least partially responsible for the contradictory results of the seven studies evaluated herein. Scientific method In this study, meta-analysis was adopted to review whether the two MTHFR SNPs influence the relapse risk of ALL and, if so, what that impact is. Meta-analysis is a statistical method for analyzing and summarizing all studies relevant to the research topic that are collected based on strict criteria. The results of these studies are pooled to estimate the efficacy of interventions.34 It is also recognized as one of the best methods of secondary research. Our analytical process involved a scientific method for optimizing the pooled results. First, a recessive model was adopted, assuming a recessive effect of the minor allele, in order to maximize the & 2014 Macmillan Publishers Limited
MTHFR SNPs in relapse risk of ALL H-R He et al
437 number of included studies. This model is consistent with the use of recessive models in previous studies.22 Second, a randomeffects model was used to integrate the results data. This model combined the clinical and methodological differences between the studies and adequately considered the heterogeneity between them, thus ensuring that the conclusions drawn were both conservative and as accurate as possible.
Summary The results of this meta-analysis indicate that the C677T polymorphism is a good marker of the relapse risk in pediatric ALL; however, the A1298C polymorphism has no effect on recurrence risk in ALL. Early preventive measures should therefore be applied in clinical settings according to the patient’s genotype.
Clinical significance of the findings The present meta-analysis is the first to summarize the relationship between the MTHFR C677T and A1298C polymorphisms and the relapse risk of ALL, and has significant clinical relevance. Many studies have investigated the influence of MTHFR polymorphisms on the treatment of ALL because of its special pharmacological mechanism of action.35–38 Studies analyzing the impact of MTHFR polymorphisms on the relapse risk of ALL have produced inconsistent findings. The results of the present meta-analysis demonstrate that pediatric ALL patients with the 677 TT genotype suffer an increased relapse risk relative to carriers of other genotypes. This finding can be explained by people with the 677 TT genotype exhibiting only 30% of the normal MTHFR activity.39 Decreased MTHFR activity may lead to increased availability of 5,10methylenetetrahydrofolate; thus, it may cause two results: Firstly, it may counteract the folate-depleting effect of MTX; secondly, the increased availability of 5,10-methylenetetrahydrofolate also facilitates thymidylate synthase action, which could increase intracellular thymine. Both of the two sides oppose MTX efficacy, enhance DNA synthesis and shorten patients’ survival.12,40 Rosenblatt et al.41 also pointed out that the reduced folate pools derived from inherited changes in MTHFR activity may significantly affect the response of malignant and nonmalignant cells to MTX, whose activity depends upon the cellular composition of folate. Moreover, although our results indicate that the MTHFR C677T polymorphism does not affect the recurrence rate of adult ALL, the included research exhibited significant heterogeneity, and the number of studies included was insufficient for statistical analysis. Similar conclusions can be drawn if the age factor is removed, namely that the recurrence rate is higher for patients carrying the 677TT genotype than for carriers of other genotypes. However, this conclusion should be treated with caution as only two of the eight included studies investigated adult ALL, and the study of Eissa et al.12 was identified as an outlier and hence excluded. Only one of the seven studies that were finally included focused on adults; this represents a serious imbalance among the studies, which makes this conclusion unreliable. Thus, the final result of this meta-analysis is that the C677T polymorphism is a good marker of the relapse risk in children ALL. This analysis also revealed that the A1298C polymorphism does not affect the relapse risk among either children or adults patients with ALL. The result is still negative even if the age factor is removed. This is consistent with the findings of Toffoli et al.42, who reported that although the 1298 A-C variant can reduce MTHFR activity, the levels of both homocysteine and folic acid did not change for carriers of either the 677 TT or 677CT genotypes. Therefore, the A1298C genotype cannot accurately predict the relapse risk of patients with ALL. Further research is therefore needed to clarify the underlying reason for this conclusion.
The authors declare no conflict of interest.
CONFLICT OF INTEREST
Limitations Some limitations should be considered when interpreting the results of this study. Although all of the information in the previously published articles was integrated in an optimal manner, there were few studies of adult ALL and they exhibited a high degree of heterogeneity. Thus, more such studies are needed before more accurate conclusions can be drawn. & 2014 Macmillan Publishers Limited
ACKNOWLEDGMENTS This work was supported by the Fundamental Research Funds for the Central Universities (No. 08143047) of China.
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ORIGINAL ARTICLE
Association between methylenetetrahydrofolate reductase polymorphisms and the relapse of acute lymphoblastic leukemia: a meta-analysis H-R He1, S-Y Chen1, H-S You1, S-S Hu, J-Y Sun, Y-L Dong and J Lu Relapse is a threat in patients treated for acute lymphoblastic leukemia (ALL). Methylenetetrahydrofolate reductase (MTHFR) activity may affect the sensitivity of patients to folate-based chemotherapeutic drugs, thus influencing the relapse risk. Two polymorphisms of the gene encoding MTHFR, C677T and A1298C, alter MTHFR enzyme activity and may be associated with ALL relapse. The aim of this meta-analysis was to clarify the correlation between the C677T and A1298C polymorphisms and ALL relapse. To this end, data were collected from studies of the association between these two polymorphisms and ALL relapse. Analysis of the data revealed a serious contradiction among the results. A recessive model demonstrated that the ALL relapse risk was significantly increased in carriers of the 677 TT genotype, especially for pediatric ALL, but was unaffected by the A1298C polymorphism. These findings confirm that the MTHFR C677T polymorphism could be considered as a good marker of the pediatric ALL relapse risk. The Pharmacogenomics Journal (2014) 14, 432–438; doi:10.1038/tpj.2014.10; published online 18 March 2014
INTRODUCTION Despite remarkable progress in the cure rate of acute lymphoblastic leukemia (ALL), most adult patients ultimately die from this disease. The most common reason for ALL treatment failure is relapse.1 In addition, there is also much heterogeneity in the pharmacological response in the treatment of childhood ALL. Folate is essential for nucleotide synthesis. Some clinical and in vitro studies indicate that polymorphisms in genes encoding key enzymes involved in folate metabolism could affect the sensitivity of patients to folate-based chemotherapeutic drugs, such as methotrexate (MTX). These may either directly or indirectly result in shifts in intracellular folate distribution.2 In addition, several studies have shown that in addition to drug-related toxicity, the patient’s response to chemotherapeutic agents can also vary with polymorphism frequency.3–5 Certain prognostic factors have been used to stratify ALL patients according to the risk of relapse,6,7 including clinical and biological characteristics that are evaluable at diagnosis. The influence of polymorphisms of a variety of genes involved in the metabolism of chemotherapeutic agents has been regarded as a prognostic factor in recent years, and has been studied widely in relation to therapeutic responses in ALL.8 MTX is the main chemotherapeutic agent used in the treatment of ALL, and acts as an antifolate. Its toxicity may be not only lifethreatening, but also one of the main reasons for the interruption or discontinuation of chemotherapy, which may in turn increase the relapse rate for the disease.9 Methylenetetrahydrofolate reductase (MTHFR) is an important enzyme in the folate/MTX metabolism pathway. It catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methylenetetrahydrofolate in the folic acid cycle.10 The activity of MTHFR may exert a substantial impact on the occurrence and relapse of ALL via the following mechanism. Decreased MTHFR activity may increase the availability of
5,10-methylenetetrahydrofolate and reduce the concentration of 5-methylenetetrahydrofolate. All that can influence DNA hypomethylation, which can alter gene expression (oncogenes) and increase the number of breaks in DNA strands, leading to an increased risk of carcinogenesis.11 MTHFR gene has two common functional polymorphisms: C677T and A1298C, and both of them have been investigated intensively.12 The C677T (rs1801133) polymorphism occurs in exon 4 and results in an alanine-to-valine substitution at codon 222. This substitution lies at the binding site for the flavin adenine dinucleotide, which is an important cofactor for MTHFR.13 The MTHFR 677 T allele encodes proteins with decreased enzymatic activity in comparison with those encoded by the 677C allele.14 With respect to the A1298C polymorphism (rs1801131), it occurs in exon 7 and results in glutamate-to-alanine substitution at codon 429, which represents the S-adenosylmethionine regulatory domain of MTHFR.13 The enzyme activity is 40% lower for the 1298CC genotype than the 1298AA genotype.15 Both polymorphisms produce a thermolabile enzyme with reduced functional activity, and a consequent altered normal intracellular distribution of folate substrates.13 These data demonstrated that the two single-nucleotide polymorphisms (SNPs) are associated with various aspects of ALL treatment. Numerous published studies have investigated the association between the two SNPs and the relapse risk in ALL, with conflicting results:11,12,16–24 some studies have found no association, while others have found both negative and positive associations. The possible reasons for these discrepancies include differences in ethnicity, treatment protocols, sample sizes and the age of the patients. It is clear, therefore, that the clarification of this putative association is necessary. In the present study, data were collected from all published studies on the relationship between the MTHFR C677T, A1298C SNPs and ALL relapse. A meta-analysis was then applied to all
Department of Pharmacy, The First Affiliated Hospital, College of Medicine, Xi’an Jiaotong University, Xi’an, China. Correspondence: Dr J Lu and Dr Y Dong, Department of Pharmacy, The First Affiliated Hospital, College of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi 710061, People’s Republic of China. E-mail: [email protected] and [email protected] 1 These authors contributed equally to this work. Received 1 October 2013; revised 9 January 2014; accepted 31 January 2014; published online 18 March 2014
MTHFR SNPs in relapse risk of ALL H-R He et al
433 eligible studies that had been selected based on strict criteria to demonstrate the role of MTHFR polymorphisms on the relapse risk in ALL patients. MATERIALS AND METHODS Literature and search strategy An exhaustive literature search of PubMed (www.ncbi.nlm.nih.gov/ pubmed) and Web of Knowledge (http://isiknowledge.com/) was conducted using the following keywords and subject terms: ‘acute lymphoblastic leukemia and relapse and polymorphism (variant)’ and ‘methylenetetrahydrofolate reductase (MTHFR) and acute lymphoblastic leukemia and relapse.’ Only journal articles were included in the analysis, and the publication language was restricted to English. All references within the identified studies were then reviewed to identify additional relevant work. The literature search was last updated on 20 September 2013.
Inclusion criteria The inclusion criteria for the meta-analysis required that each study must (1) be an independent association study investigating the relationship between the C677T and A1298C polymorphisms and the ALL relapse risk, (2) provide sufficient data on relapse by genotype or sufficient information for such data to be calculated and (3) have a retrospective design and include a population of ALL patients.
Genetic model A recessive model for the MTHFR C677T (TT versus CC þ CT) and A1298C (CC versus AA þ AC) polymorphisms was assumed, based on the majority of the research performed. The use of this model maximized the number of included studies.
Data extraction The following information was gathered for each article included in the study: name of the first author, year of publication, number of patients, ethnicity of the study population, median age at ALL diagnosis, median follow-up duration, data on relapse by genotype and main findings of the research.
Figure 1. Flow chart of study selection.
Statistical analysis The overall pooled relative risk (RR) and corresponding 95% confidence interval of the relapse risk were estimated using the Mantel–Haenszel method with a random-effects model. Random-effect model accounts for the heterogeneity in the data that undoubtedly exists due to within- and between-study variations and thus provides more conservative estimated effect values.25 The significance of RR was determined by a z-test. The level of statistical significance was set at Po0.05. The presence of heterogeneity was assessed using Cochran’s Q-statistic (with a significance cutoff of Po0.05) and quantified using the I2-statistic, which is proportional to the degree of heterogeneity; an I2-value above 50% denotes a very high degree of heterogeneity. Sensitivity analysis was performed by excluding individual studies when substantial heterogeneity was present. Outlying studies were identified and excluded, and the I2-estimates for these different sets of studies examined.
Meta-analysis On the basis of the influence of age on the development and prognosis of ALL, the association between the two SNPs and ALL relapse was evaluated in childhood ALL patients, adult ALL patients and all ALL patients (regardless of their age), respectively.
RESULTS Overview of the study characteristics The flow chart for the study selection process is depicted in Figure 1. In total, 204 articles were selected using the two groups of keywords described earlier. Checking for duplicates resulted in the removal of 26 articles. Of the remaining 178 articles, 76 did not report the association between SNPs and the relapse of ALL patients, 70 did not focus on MTHFR polymorphisms and 16 were & 2014 Macmillan Publishers Limited
reviews. After exclusion of these studies, only 16 remained, which investigated the relationship between MTHFR SNPs and the relapse risk of ALL patients. However, eight of these did not provide sufficient data and were thus also excluded. Therefore, ultimately, only eight of the studies qualified for inclusion in this meta-analysis. The basic data for every eligible study were extracted and are listed in Table 1. Two of the studies were performed on adults; although Chiusolo et al.16 studied ALL patients of all ages, the median age of their subjects was 31.5 years, and adult patients accounted for a higher proportion than all others. That article was therefore regarded as a study performed on adult patients. The other studies involved children. The results of the included studies were contradictory. Meta-analysis results MTHFR SNPs and relapse risk in childhood ALL. Six of the studies investigated the association between the MTHFR C677T polymorphism and the relapse risk in childhood ALL, and provided sufficient data to be included in the analysis.17–22 A meta-analysis of the six studies was conducted, involving data from 1553 patients. The z-test verified a significant association between the C677T polymorphism and the relapse risk in childhood ALL, and the RR value demonstrated that the relapse risk in pediatric ALL was higher for the 677TT genotype than for the CT or CC genotype. The I2-value denoted the degree of heterogeneity was not high among the six studies (Figure 2). The Pharmacogenomics Journal (2014), 432 – 438
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434 Table 1.
List of seven studies that analyzed the association between the MTHFR SNPs and ALL relapse
Study
Year
Aplenc et al.20 Chiusolo et al.16 Tantawy et al.19 D’Angelo et al.17 Pietrzyk et al.22 EL-Khodary et al.18 Sepe et al.21 Eissa et al.12
2005 2007 2010 2011 2011 2012 2012 2013
Sample size
520 81 40 151 319 40 483 50
Ethnicity
Median age at diagnosis (range)
Mixed European Caucasian Egyptian Caucasian European origin European Egyptian Mixed Egyptian
1–10 31.5 (12–75) 6.1±1.2 (3–15) 5 (0.25–15) (1–18) (2–16) 4 47.5 (22–74)
Median follow-up (months; range)
120 21 (1–240) 60 (37.2–78) 65 72 (60–84) 2-year relapse — 29 (10–36)
Results C677T
A1298C
þ NA þ þ NA A þ þ
NA NA NA NA — — —
Abbreviations: A: the SNP is associated with ALL relapse; ALL, acute lymphoblastic leukemia; NA, no association between the SNP and toxicity; þ , the SNP is associated with increased relapse risk; –, the SNP is associated with decreased relapse risk.
Figure 2.
Results of meta-analysis of association between the two SNPs and relapse risk in pediatric ALL.
Only three studies surveyed the influence of the A1298C polymorphism on the relapse risk in childhood ALL, and no one found an association.17,19,20 A meta-analysis performed on these three studies, based on the data from 711 patients, verified that there was no association. The I2-value showed that there was almost no heterogeneity among the three studies (Figure 2). MTHFR SNPs and relapse risk in adult ALL. Two studies analyzed the association between the two SNPs and the relapse risk in adult ALL; the results were inconsistent for each SNP.12,16 A metaanalysis using the data from 131 patients found no association between the C677T polymorphism and the relapse risk in adult ALL. Cochran’s Q-statistic and I2-value denoted a high degree of heterogeneity between the two studies; however, the inclusion of only two studies precluded the performance of a sensitivity analysis. Therefore, more studies are needed before reliable conclusions can be drawn regarding the influence of the C677T polymorphism on relapse in adult ALL. The Pharmacogenomics Journal (2014), 432 – 438
The findings of a meta-analysis based on the data from 131 patients also did not support an association between the A1298C polymorphism and the relapse risk in adult ALL. The degree of heterogeneity between the studies was small (Figure 3). MTHFR SNPs and relapse risk in all ALL cases. A meta-analysis was performed using the data from all ALL cases, regardless of their age. All eight of the studies analyzed the association between the C677T polymorphism and the relapse risk of ALL.12,16–22 The metaanalysis involving data from 1684 patients found a significant association between the C677T polymorphism and the relapse risk. The RR value revealed that the relapse risk was higher in patients with the 677 TT genotype than in those with the CT or CC genotype. As the I2-value revealed a high degree of heterogeneity among the included studies, a sensitivity analysis was undertaken, which identified the study by Eissa et al.12 as an outlier. Removal of the data of that study from the meta-analysis reduced the heterogeneity; however, a significant association was still observed. & 2014 Macmillan Publishers Limited
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Figure 3.
Results of meta-analysis of association between the two SNPs and relapse risk in adult ALL.
Moreover, both the RR value and its 95% confidence intervals were greater than 1, suggesting that the relapse risk is higher for ALL patients with the 677 TT genotype (Figure 4). For the A1298C polymorphism, five studies provided sufficient data for inclusion in the meta-analysis.12,16,17,19,20 On the basis of the data from 842 patients, no association was observed between the A1298C polymorphism and the relapse risk of ALL. The I2-value revealed a small degree of heterogeneity among the five studies (Figure 4). DISCUSSION This study followed a strict screening methodology to collect data from eight published articles in which the association between the MTHFR C677T and A1298C polymorphisms and the relapse risk of ALL was researched.12,16–22 All of the included articles were of high quality and presented a rigorous scientific design, accurate data and clear results. The results of these studies were inconsistent. For the C677T SNP, most of the studies found an association between the 677 TT genotype and an increased relapse risk of ALL,12,17–21 but that association was not observed in two of the studies.16,22 The A1298C polymorphism was evaluated in five of the studies, four of which found no association between the SNP and the relapse risk of ALL,16,17,19,20 whereas one concluded that patients with the 1298CC genotype tended to have a decreased relapse risk relative to wild-type or heterozygote carriers.12 Of the eight studies, two focused on adults and six on children. The present study applied a meta-analysis to review and summarize the relapse risk according to genotype in childhood ALL patients, adult ALL patients and all ALL patients, with the aim of obtaining an objective and consistent conclusion. The main conclusion was that there appears to be an association between the MTHFR 677 TT genotype and an increased relapse risk of pediatric ALL, whereas the A1298C polymorphism does not appear to be associated with the relapse risk of ALL. Of the eight studies, the population within the same study was homogenous in the race in six studies,12,16–19,22 whereas the population in the left two studies was mixed.20,21 In the study by Sepe et al.,21 authors addressed the issue of population stratification by including ethnicity in the Cox proportional hazards models. Aplenc et al.20 found the impact of the MTHFR T677 variant allele remains significant after controlling for ethnicity, thus indicating an effect of the variant allele that is unlikely to be & 2014 Macmillan Publishers Limited
confounded by ethnicity. In addition, several authors have shown that population stratification is unlikely to cause significant bias in studies with multiple admixed populations.26–28 Above all, including these two studies did not cause serious heterogeneity in our meta-analyses. We are therefore reasonable to include these two studies. Reasons for the discrepant findings Four main reasons could explain the discrepancies among the included studies: differences in patient ages, patient ethnicities, toxicity of treatment and follow-up times. Age. Clinically, several prognostic factors have been used to stratify ALL patients, and one of the main ones is age. Although chemotherapy regimens cure nearly 80% of pediatric patients with ALL, the death rate of adult patients remains high. There is a large discrepancy in the sensitivity to drug treatments among the different patient age groups, and patients with a lower sensitivity would have an increased relapse risk.6,29,30 In other words, age may influence the relapse risk of ALL by affecting the sensitivity to drug treatment. Ethnicity. The genotype distribution of the two SNPs differs according to race. Some studies have showed different genotype frequency of the two SNPs in people of different races.31,32 Karas et al.11 also pointed out that the activities of the folate metabolism enzymes could vary in different populations, ethnic groups, and races, thus creating differing metabolic profiles. Moreover, the nutritional status varies between countries, and the nutritional status at diagnosis may be a significant prognostic factor in the long-term treatment outcome of pediatric patients with ALL.33 All of these observations demonstrate that ethnic factors could indirectly affect the relapse risk of ALL. The races of the patients evaluated differ between the studies included in the present meta-analysis, which could at least partially explain the discrepant results. Toxicity. Treatment-related toxicity could lead to interruption or discontinuation of chemotherapy, which may reduce the effects of chemotherapy drugs and result in tumor cells not being killed completely, thus increasing the relapse risk of ALL.9 Most of our eligible studies researched the toxicity of patients, and different The Pharmacogenomics Journal (2014), 432 – 438
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Figure 4.
Results of meta-analysis of association between the two SNPs and relapse risk in all ALL.
incidences were found among them. For example, the hematopoietic toxicity, hepatic toxicity and hepatic plus hematopoietic toxicity were assessed in the paper of Chiusolo et al.16 and 40% incidence of toxicity was found. The study performed by D’Angelo et al.17 also recorded MTX-induced toxicity, including hematological and non-hematological (hepatic, gastrointestinal and nervous system) toxicities, and the incidence of toxicity was 68.3%. Likewise, Eissa et al.12 researched hematologic and/or nonhematologic (hepatic and gastrointestinal) toxicity induced by MTX and they summarized that 82% patients developed hematologic and/ or nonhematologic toxicity. Such differences in the incidence of toxicity could be partially attributed to the use of different treatment protocols, and would influence the drug effects and lead to different relapse risks. Follow-up duration. The follow-up duration differed among the included studies (see Table 1), and the relapse risk may vary with The Pharmacogenomics Journal (2014), 432 – 438
time. In brief, the differences in follow-up may also be at least partially responsible for the contradictory results of the seven studies evaluated herein. Scientific method In this study, meta-analysis was adopted to review whether the two MTHFR SNPs influence the relapse risk of ALL and, if so, what that impact is. Meta-analysis is a statistical method for analyzing and summarizing all studies relevant to the research topic that are collected based on strict criteria. The results of these studies are pooled to estimate the efficacy of interventions.34 It is also recognized as one of the best methods of secondary research. Our analytical process involved a scientific method for optimizing the pooled results. First, a recessive model was adopted, assuming a recessive effect of the minor allele, in order to maximize the & 2014 Macmillan Publishers Limited
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437 number of included studies. This model is consistent with the use of recessive models in previous studies.22 Second, a randomeffects model was used to integrate the results data. This model combined the clinical and methodological differences between the studies and adequately considered the heterogeneity between them, thus ensuring that the conclusions drawn were both conservative and as accurate as possible.
Summary The results of this meta-analysis indicate that the C677T polymorphism is a good marker of the relapse risk in pediatric ALL; however, the A1298C polymorphism has no effect on recurrence risk in ALL. Early preventive measures should therefore be applied in clinical settings according to the patient’s genotype.
Clinical significance of the findings The present meta-analysis is the first to summarize the relationship between the MTHFR C677T and A1298C polymorphisms and the relapse risk of ALL, and has significant clinical relevance. Many studies have investigated the influence of MTHFR polymorphisms on the treatment of ALL because of its special pharmacological mechanism of action.35–38 Studies analyzing the impact of MTHFR polymorphisms on the relapse risk of ALL have produced inconsistent findings. The results of the present meta-analysis demonstrate that pediatric ALL patients with the 677 TT genotype suffer an increased relapse risk relative to carriers of other genotypes. This finding can be explained by people with the 677 TT genotype exhibiting only 30% of the normal MTHFR activity.39 Decreased MTHFR activity may lead to increased availability of 5,10methylenetetrahydrofolate; thus, it may cause two results: Firstly, it may counteract the folate-depleting effect of MTX; secondly, the increased availability of 5,10-methylenetetrahydrofolate also facilitates thymidylate synthase action, which could increase intracellular thymine. Both of the two sides oppose MTX efficacy, enhance DNA synthesis and shorten patients’ survival.12,40 Rosenblatt et al.41 also pointed out that the reduced folate pools derived from inherited changes in MTHFR activity may significantly affect the response of malignant and nonmalignant cells to MTX, whose activity depends upon the cellular composition of folate. Moreover, although our results indicate that the MTHFR C677T polymorphism does not affect the recurrence rate of adult ALL, the included research exhibited significant heterogeneity, and the number of studies included was insufficient for statistical analysis. Similar conclusions can be drawn if the age factor is removed, namely that the recurrence rate is higher for patients carrying the 677TT genotype than for carriers of other genotypes. However, this conclusion should be treated with caution as only two of the eight included studies investigated adult ALL, and the study of Eissa et al.12 was identified as an outlier and hence excluded. Only one of the seven studies that were finally included focused on adults; this represents a serious imbalance among the studies, which makes this conclusion unreliable. Thus, the final result of this meta-analysis is that the C677T polymorphism is a good marker of the relapse risk in children ALL. This analysis also revealed that the A1298C polymorphism does not affect the relapse risk among either children or adults patients with ALL. The result is still negative even if the age factor is removed. This is consistent with the findings of Toffoli et al.42, who reported that although the 1298 A-C variant can reduce MTHFR activity, the levels of both homocysteine and folic acid did not change for carriers of either the 677 TT or 677CT genotypes. Therefore, the A1298C genotype cannot accurately predict the relapse risk of patients with ALL. Further research is therefore needed to clarify the underlying reason for this conclusion.
The authors declare no conflict of interest.
CONFLICT OF INTEREST
Limitations Some limitations should be considered when interpreting the results of this study. Although all of the information in the previously published articles was integrated in an optimal manner, there were few studies of adult ALL and they exhibited a high degree of heterogeneity. Thus, more such studies are needed before more accurate conclusions can be drawn. & 2014 Macmillan Publishers Limited
ACKNOWLEDGMENTS This work was supported by the Fundamental Research Funds for the Central Universities (No. 08143047) of China.
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