<i>MTHFR</i> Glu429Ala and <i>ERCC5</i> His46His Polymorphisms Are Associated with Prognosis in Colorectal Cancer Patients: Analysis of Two Independent Cohorts from Newfoundland

<div><p>Introduction</p><p>In this study, 27 genetic polymorphisms that were previously reported to be associated with clinical outcomes in colorectal cancer patients were investigated in relation to overall survival (OS) and disease free survival (DFS) in colorectal cancer patients from Newfoundland.</p><p>Methods</p><p>The discovery and validation cohorts comprised of 532 and 252 patients, respectively. Genotypes of 27 polymorphisms were first obtained in the discovery cohort and survival analyses were performed assuming the co-dominant genetic model. Polymorphisms associated with disease outcomes in the discovery cohort were then investigated in the validation cohort.</p><p>Results</p><p>When adjusted for sex, age, tumor stage and microsatellite instability (MSI) status, four polymorphisms were independent predictors of OS in the discovery cohort <i>MTHFR</i> Glu429Ala (HR: 1.72, 95%CI: 1.04–2.84, p = 0.036), <i>ERCC5</i> His46His (HR: 1.78, 95%CI: 1.15–2.76, p = 0.01), <i>SERPINE1 −</i>675indelG (HR: 0.52, 95%CI: 0.32–0.84, p = 0.008), and the homozygous deletion of <i>GSTM1</i> gene (HR: 1.4, 95%CI: 1.03–1.92, p = 0.033). In the validation cohort, the <i>MTHFR</i> Glu429Ala polymorphism was associated with shorter OS (HR: 1.71, 95%CI: 1.18–2.49, p = 0.005), although with a different genotype than the discovery cohort (CC genotype in the discovery cohort and AC genotype in the validation cohort). When stratified based on treatment with 5-Fluorouracil (5-FU)-based regimens, this polymorphism was associated with reduced OS only in patients not treated with 5-FU. In the DFS analysis, when adjusted for other variables, the TT genotype of the <i>ERCC5</i> His46His polymorphism was associated with shorter DFS in both cohorts (discovery cohort: HR: 1.54, 95%CI: 1.04–2.29, p = 0.032 and replication cohort: HR: 1.81, 95%CI: 1.11–2.94, p = 0.018).</p><p>Conclusions</p><p>In this study, associations of the <i>MTHFR</i> Glu429Ala polymorphism with OS and the <i>ERCC5</i> His46His polymorphism with DFS were identified in two colorectal cancer patient cohorts. Our results also suggest that the <i>MTHFR</i> Glu429Ala polymorphism may be an adverse prognostic marker in patients not treated with 5-FU.</p></div

Polymorphisms investigated in the discovery cohort.

<p>na: not available, MAF: minor allele frequency, VNTR: variable number of tandem repeats, 2R: 2 VNTR repeats, 3R: 3 VNTR repeats. The <i>EGFR</i> rs2227983 polymorphism is also known as rs11543848. The <i>PTGS2</i> c.3618A/G excluded from analysis due to its low minor allele frequency.</p>*<p>genotyped by MassArray® technology,</p>**<p>genotyped by gel electrophoresis of PCR-amplified fragments,</p>***<p>genotyped by TaqMan® SNP genotyping assays.</p>****<p>MAF information was retrieved from the dbSNP database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061469#pone.0061469-Sherry1" target="_blank">[75]</a>.</p>*****<p>MAFs are as reported in a published report <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061469#pone.0061469-Lin1" target="_blank">[76]</a>. The chromosomal locations of polymorphisms are extracted from the dbSNP database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061469#pone.0061469-Sherry1" target="_blank">[75]</a> (Genome Reference Consortium Human Build 37 patch release 5).</p

The <i>MTHFR</i> Glu429Ala polymorphism and overall survival in the discovery cohort patients (stratified by treatment with 5-Fluorouracil).

<p>5-FU: 5-fluorouracil, CI: confidence interval, HR: hazard ratio, n: number of patients, vs: versus.</p><p>#The referent categories are underlined. Please note that reflecting the small numbers of patients, the CIs for the effect estimate in stage IV patients are quite wide and should not be interpreted as an accurate estimation.</p

Multivariable model for disease-free survival in the discovery and validation cohorts.

<p>The multivariable model contained location, stage and MSI status in addition to the <i>ERCC5</i> His46His, <i>OGG1</i> Ser326Cys, <i>ERCC5</i> Asn118Asn, <i>TYMS</i> indel 6 bp, and <i>GSTM1</i> gene deletion genotypes in the discovery cohort and the <i>ERCC5</i> His46His and <i>GSTM1</i> gene deletion genotypes in the validation cohort as covariates. The genotypes for the <i>OGG1</i> Ser326Cys, <i>ERCC5</i> Asn118Asn, and <i>TYMS</i> indel 6 bp polymorphisms were not available in the validation cohort. CI: confidence interval, HR: hazard ratio, n: number of patients, nd: not done, vs: versus. Event refers to recurrence, metastasis or death in the patient, whichever had occurred earlier.</p><p>#The referent categories are underlined. Please note that reflecting the small numbers of patients in the validation cohort, the CIs for the effect estimate in stage IV patients are quite wide and should not be interpreted as an accurate estimation.</p

Multivariable Cox regression analysis results for overall survival in the discovery and validation cohorts.

*<p>The multivariable Cox regression model assuming the co-dominant genetic model contained the <i>MTHFR</i> Glu429Ala, <i>ERCC5</i> His46His, <i>SERPINE1 −</i>675indelG, <i>GSTM1</i> gene deletion genotypes as well as sex, age, stage and MSI status as covariates.</p>**<p>Only the multivariable Cox regression analysis result for the <i>MTHFR</i> Glu429Ala polymorphism when adjusted for sex, age, stage and MSI status is shown (assuming the dominant genetic model). The complete multivariable models for the dominant genetic model can be found in <b>Tables S4</b> and <b>S6</b> in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061469#pone.0061469.s001" target="_blank">File S1</a></b> for the discovery and validation cohorts.</p>***<p>The multivariable Cox regression analysis result for the <i>MTHFR</i> Glu429Ala polymorphism when adjusted for sex, age, stage and MSI status is shown (assuming the recessive genetic model). The complete multivariable models can be found in <b>Tables S5</b> and <b>S7</b> in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061469#pone.0061469.s001" target="_blank">File S1</a></b> for the discovery and validation cohorts. CI: confidence interval, HR: hazard ratio, n: number of patients, vs: versus.</p><p>#The major homozygote genotypes and other referent categories are underlined.</p

Kaplan-Meier survival curves for the <i>MTHFR</i> Glu429Ala (overall survival) and the <i>ERCC5</i> His46His polymorphisms (disease-free survival) assuming the co-dominant genetic model. P-values are based on log-rank test.

<p>Kaplan-Meier survival curves for the <i>MTHFR</i> Glu429Ala (overall survival) and the <i>ERCC5</i> His46His polymorphisms (disease-free survival) assuming the co-dominant genetic model. P-values are based on log-rank test.</p

Region of chromosome 3 examined with genes and 3 SNPs.

<p>A total of 99 polymorphisms were examined in the Ontario samples across a 500kb region of chromosome 3 surrounding the <i>MLH1</i> gene. Genes in this region are outlined (top panel) along with their transcriptional directionality (bottom panel). The three polymorphisms of interest are indicated. Modified from Ensembl (<a href="http://www.ensembl.org" target="_blank">www.ensembl.org</a>).</p

Proposed model for genetic susceptibility to DNA methylation in sporadic MSI-H CRCs.

<p>Specific SNPs predispose the region, including the <i>MLH1</i> gene promoter, to methylation, which results in promoter silencing and loss of <i>MLH1</i> gene expression that is measured by immunohistochemical staining. Loss of the <i>MLH1</i> gene expression leads to genome-wide microsatellite instability and MSI-H colorectal cancer.</p

Logistic regression model results for MSI status with various predictor combinations in the combined data.

<p>Age at diagnosis, sex, and location are covariates common to all the models described above. IHC refers to the MLH1 immunohistochemical staining variable, CH3 refers to the <i>MLH1</i> promoter methylation variable, AIC  =  Akaike's information criterion. Logistic regression models for each SNP per study population and for the combined data are shown in <b>Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013314#pone.0013314.s003" target="_blank">File S3</a></b>.</p><p>The role of three SNPs of interest, rs1800734, rs749072, and rs13098279, is explored.</p

Single marker analysis in the combined data for 3 SNPs for CRC cases versus controls, <i>MLH1</i> promoter methylation, MLH1 IHC staining and MSI tumor status.

<p>Analyses of CRC cases versus controls are adjusted for age, sex, and site.</p><p>OR  =  odds ratio, CI  =  confidence interval.</p><p>Single marker results for the above SNPs for each study population are shown in <b>Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013314#pone.0013314.s002" target="_blank">File S2</a></b>.</p