Correlation between MTHFR 677C > T polymorphism and response of pemetrexed-based chemotherapy in advanced NSCLC: A meta-analysis


 
 
 The aim of this study was to investigate the correlation between MTHFR 677C > T polymorphism and response of pemetrexed-based chemotherapy in advanced non-small-cell lung cancer (NSCLC) by pooling the open published relevant studies.
 
 
 
 Clinical studies associated with MTHFR 677C > T polymorphism and response of pemetrexed-based chemotherapy in advanced NSCLC were systematically searched in databases of Pubmed, Embase, Cochrance Library, China national knowledge infrastructure (CNKI) and Wanfang. The correlation was expressed by odds ratio (OR) and corresponding 95% confidence interval (95% CI). The publication bias of the included studies was evaluated through Begg’s funnel plot and Egger’s line regression test.
 
 
 
 Ten prospective clinical studies relevant to MTHFR 677C > T polymorphism and response of pemetrexed-based chemotherapy in NSCLC were included in the present meta-analysis. The pooled results indicated that the partial response in NSCLC patients with TT or CT genotype was inferior to CC genotype in a dominant gene model (TT + CT vs CC) (OR = 0.16, 95% CI: 0.06–0.41, P = 0.001). NSCLC cases with T genotype were inferior to C genotype in the objective response rate treated with pemetrexed-based chemotherapy for dominant (OR = 0.28, 95% CI: 0.18–0.45, P = 0.001), recessive (OR = 0.43, 95% CI: 0.19–0.94, P = 0.03) and homozygous models (OR = 0.30, 95% CI: 0.13–0.67, P = 0.003). However, there was no statistical difference in disease control rate, progressive disease between different genotypes of different gene models (P
 all > 0.05).
 
 
 
 The pemetrexed-based chemotherapy response was decreased in NSCLC cases with T genotype, which can be applied as a potential pemetrexed-based chemotherapy response marker.



Introduction
Lung cancer, especially NSCLC, is known as the leading cause of tumor-associated mortality globally. It was estimated that 131,880 new lung cancer cases will be diagnosed in the year 2021, which accounts for almost one-quarter of all cancers diagnosed in the same year [1]. About 75-80% of NSCLC cases were at advanced stages and lost the opportunity of operation when was first diagnosed. Most of the patients who receive tumor resection also need adjuvant chemotherapy after operation except for stage Ia NSCLC cases. Therefore, chemotherapy is one of the major treatment methods for NSCLC. Pemetrexedbased chemotherapy is extensively applied clinically in NSCLC especially for lung adenocarcinoma [2][3][4]. Pemetrexed is a multitarget folate antagonist, which has been proved to be effective in the first-line treatment, secondline treatment and maintenance treatment of advanced NSCLC [5]. It plays an antitumor role by inhibiting the activities of several key enzymes in the folate metabolism pathway. MTHFR is a key enzyme in folate metabolism, which had been reported to be associated with the response of pemetrexed-based chemotherapy for NSCLC [6]. 677C > T polymorphism of MTHFR gene is a main single-nucleotide polymorphism, which is considered to be correlated with the pemetrexed-based chemotherapy response [7]. However, the correlation between MTHFR677C > T polymorphism and pemetrexed-based chemotherapy response is not conclusive. Therefore, we performed this meta-analysis by pooling the relevant open published studies to further investigate the correlation.

Search of relevant studies in the electronic databases
Prospective clinical studies relevant to the correlation between MTHFR 677C > T polymorphism and response of pemetrexed-based chemotherapy in advanced NSCLC were systematically searched in the electronic databases of Pubmed, Embase, Cochrance Library, CNKI and Wanfang. The prospective clinical studies on MTHFR 677C > T polymorphism and response of pemetrexed-based chemotherapy in advanced NSCLC published in English or Chinese were electronically searched through the following text words: pemetrexed, pemetrexed disodium, disodium, alimta, MTHFR, methylenetetrahydrofolate reductase, methylenetetrahydrofolate reductase (NADPH), methylene-THF reductase (NADPH), 5,10-methylenetetrahydrofolate reductase (NADPH), methylene tetrahydrofolate reductase, tetrahydrofolate reductase and methylene. The references of the included studies were also further screened to identify the potentially suitable publications, which were not identified in the electronic databases.

Inclusion and exclusion criteria
The inclusion criteria of the studies were as follows: (1) clinical studies relevant to MTHFR 677C > T polymorphism and response of pemetrexed-based chemotherapy; (2) the patients included in the original publication should be diagnosed with NSCLC; (3) genotype of CC, CT, and TT frequency of the included cases can be extracted or calculated from the original study; (4) the genotyping methods were PCR or Taqman;  about small cell lung cancer; (5) genotype and/or treatment response cannot be extracted or calculated from the original study.

Data extraction from the original study
The studies were first screened by two reviewers (HF and XWG) independently. The publications were included according to the inclusion and exclusion criteria. When there was a controversy for inclusion or exclusion criteria of a certain study, the discussion was first adapted and then a third reviewer was consulted. The main characteristics of the included studies such as the author name, year of publication, region, genotyping methods and outcomes were extracted and shown in a summary table. The data of TT, CT and CC genotype frequency, treatment response such as complete response, partial response, objective response rate, disease control rate and progressive disease were extracted or calculated from each original included study and cross-checked by two reviewers.

Studies quality evaluation
The general quality of the 10 included studies was evaluated by Newcastle-Ottawa Scale (NOS). The highest score of NOS is 9 points. The high, moderate, and low quality of the original studies were considered as score ≥6 points, 3-5 points and <3 points [8].

Publication bias evaluation
Begg's funnel plot and Egger's line regression test were applied for publication bias evaluation for the correlation between MTHFR 677C > T polymorphism and response of pemetrexed-based chemotherapy in advanced NSCLC.

Statistical analysis
Stata/SE 11.0 (StataCorp LP, http://www.stata.com) statistical software was used for the data analysis. The correlation between MTHFR 677C > T polymorphism and  response of pemetrexed-based chemotherapy in advanced NSCLC was demonstrated by the odds ratio (OR) and its 95% CI. The statistical heterogeneity across the 10 included publications was evaluated by I 2 text. Two-tailed P < 0.05 was considered as statistically significant.
Ethical approval: The conducted research is not related to either human or animal use.

Main characteristics of the included original studies
Ten prospective studies [6,7,[9][10][11][12][13][14][15][16] relevant to MTHFR 677C > T polymorphism and response of pemetrexed-based chemotherapy in advanced NSCLC were included in the present meta-analysis ( Figure 1). Of the 10 included studies, six were from China, two were from Poland, one was from South Korea and one was from Italy. The sample size ranges from 25 to 208. The general quality of the included studies was relatively high. The NOS score ranges from 5 to 7 with the median NOS score of 6.0. The main characteristics including the study region, age of the subject, the sample size, chemotherapy regimen, the outcome and others were presented in Table 1. The number of responses in each of the included studies is presented in Table 2.

Correlation between MTHFR 677C > T polymorphism and partial response
The statistical heterogeneity was not significant in dominant, recessive and homozygous models (P all > 0.05). Therefore, the data were pooled in a fixed effect model.

Correlation between MTHFR 677C > T polymorphism and objective response rate
Statistical heterogeneity was found in a dominant gene model (P = 0.038), but not in recessive and homozygous gene models (P > 0.05). The data were pooled by randomand fixed-effect models in dominant and recessive models and homozygous gene model, respectively. The combined results showed that NSCLC cases with T genotype were inferior to C genotype in the objective response rate when treated with pemetrexed-

Correlation between MTHFR 677C > T polymorphism and disease control rate
The data were pooled in the fixed-effect model in the disease control rate due to nonsignificant statistical heterogeneity (P > 0.05). The pooled results indicated that the disease control rates were not statistically different in different genotypes in dominant, recessive and homozygous gene models (P all > 0.05), Figure 4.

Correlation between MTHFR 677C > T polymorphism and progressive disease
The data were pooled by the fixed-effect model for the dominant gene model and the random-effect model for recessive and homozygous gene models. The combined results showed that progressive disease was not statistically different in different genotypes (CC, CT or TT) in dominant, recessive and homozygous gene models (P all > 0.05). However, NSCLC cases with T genotype had a trend of an increased risk for disease progression, Figure 5.

Progression-free survival and overall survival analysis
The progression-free survival and overall survival time for CC, CT and TT allele were presented in Table 3. There was no statistical difference in progression-free survival and overall survival time for NSCLC patients with CC, CT or TT alleles treated with pemetrexed-based chemotherapy (P all > 0.05), Figure 6.

Publication bias evaluation
Begg's funnel plots of objective response rate and disease control rate were in general left and right symmetrical, which indicated no obviously publications bias (Figure 7). Egger's line regression test also showed that the publication bias was not significant (P > 0.05).

Discussion
Ten prospective clinical studies relevant to MTHFR 677C > T polymorphism and response of pemetrexed-based chemotherapy in advanced NSCLC were included in the present meta-analysis. The general quality of the 10 publications was relatively high with the median NOS score of 6.0. The original data of the ten included studies were pooled and indicated that the partial response in NSCLC patients with T genotype was inferior to C genotype in the dominant gene model (TT + CT vs CC; OR = 0.16, 95% CI: 0.06-0.41, P = 0.001). The objective response rate in cases with T genotype was inferior to C genotype in dominant (OR = 0.28, 95% CI: 0.18-0.45, P = 0.001), recessive (OR = 0.43, 95% CI: 0.19-0.94, P = 0.03) and homozygous models (OR = 0.30, 95% CI: 0.13-0.67, P = 0.003). However, the progression-free survival and overall survival were not statistically different in NSCLC patients with CC, CT or TT alleles treated with pemetrexed-based chemotherapy. The results indicated that NSCLC with T allele only had 0.16 odds of partial response in pemetrexed-based chemotherapy response compared to C allele in the dominant gene model. For the objective response rate, the chemotherapy odds ranges from 0.28 to 0.43 for T allele compared with C allele in NSCLC treated with pemetrexed-based chemotherapy. Therefore, NSCLC cases with T genotype of MTHFR gene may have decreased response when treated with pemetrexed-based chemotherapy, which indicated that T allele of MTHFR 677 SNP was a contraindication for pemetrexed-based chemotherapy regimen in NSCLC. Pemetrexed is a multitarget folate antagonist. Its main anticancer mechanism is inhibiting cell proliferation by destroying the key folate-dependent metabolic process of cell replication [17][18][19]. MTHFR is a key enzyme in folate metabolism, which can irreversibly catalyze the reduction of 5,10-methylenetetrahydrofolate to 5-methylenetetrahydrofolate, which is the main form of folate in plasma [20,21]. Therefore, the activity of MTHFR can affect the metabolism of folic acid and further affect the response of pemetrexed-based chemotherapy. Currently, the study on MTHFR activity mainly focuses on its SNP, and C677T is the most-studied SNP site. However, the relationship between MTHFR677C > T polymorphism and the response of pemetrexed-based chemotherapy in patients with advanced NSCLC is not consistent according to the previous publications [6,7]. Zhao et al. [10] found that patients with CC allele of MTHFR gene had better chemotherapy response and longer progressionfree survival than patients with CT + TT genotype. However, Tiseo et al. [6] found that patients with TT genotype of MTHFR gene had longer progression-free survival and overall survival time compared to CC genotype. Therefore, whether MTHFR 677C > T polymorphism is correlated with the response of pemetrexed-based chemotherapy in advanced NSCLC is not conclusive. Our meta-analysis results showed pemetrexed-based chemotherapy response was decreased in NSCLC cases with T genotype. The reason of the inferior pemetrexed-based chemotherapy response in NSCLC cases with T genotype may be due to the decreased MTHFR activity and folic acid metabolism of the T genotypes. In addition, it has also been  pointed out that the increase of folic acid in the serum of NSCLC cases can increase the pemetrexed chemotherapy response, which validates the aforementioned point of view [22].
Our results showed that pemetrexed-based chemotherapy response was decreased in NSCLC cases with T genotype, which can be applied as a potential pemetrexed-based chemotherapy response marker. However, the meta-analysis also had its own limitations: First, only 10 studies were included in the meta-analysis with a small sample size. Therefore, the statistical power is limited. Second, the studies screened and included in the meta-analysis were limited to English or Chinese publications. Third, the long-term survival such as overall survival and progression-free survival were not pooled due to inadequate data. Fourth, clinical heterogeneity such as mutations in EGFR or the presence of the ROS1 or EML4-ALK fusion oncogenes might affect the treatment response and outcome.
However, the information relevant to the aforementioned molecular characteristics was not mentioned in the original study. Therefore, how much the molecular characteristics affects the results of the meta-analysis was not clear.

Conclusion
The pemetrexed-based chemotherapy response was decreased in NSCLC cases with T genotype, which can be applied as a potential pemetrexed-based chemotherapy response marker. However, due to the aforementioned limitations, large-scale well-designed prospective clinical trials relevant to this topic are still necessary to further investigate the correlation between MTHFR 677C > T polymorphism and response of pemetrexed-based chemotherapy in advanced NSCLC to provide more reliable evidence.  Funding information: The authors state that no funding was involved.