Abstract
Purpose
Recurrence and toxicity occur commonly among rectal cancer patients treated with 5-fluorouracil (5-FU). We hypothesized that genetic variation in folate-metabolizing genes could play a role in the inter-individual variability. Our objective was to evaluate the associations between genetic variants in folate-metabolizing genes and clinical outcomes among rectal cancer patients treated with 5-FU.
Experimental Design
We investigated eight functionally significant polymorphisms in six genes (MTHFR (C667T, A1298C), SLC191A (G80A), SHMT1 (C1420T), DHFR (Del19bp)), TS 1494del, TSER) involved in folate metabolism in n=745 stage II or III rectal cancer patients enrolled in a phase III adjuvant clinical trial of three regimens of 5-FU and radiation therapy (INT-0144; SWOG 9304).
Results
There were no statistically significant associations between polymorphisms in any of the genes and overall survival (OS), disease free survival (DFS) and toxicity in the overall analyses. Nevertheless, there was a trend towards worse DFS among patients with the variant allele of MTHFR C677T compared to wildtype, particularly in treatment arm 2 where patients with the MTHFR C677T TT genotype had worse OS (HR=1.76; 95% CI: 1.06-2.93; p-value=0.03) and DFS (HR=1.84; 95% CI: 1.12-3.03; p-value=0.02) than those with homozygous wildtype. In addition, there was a trend towards reduced hematological toxicity among variants of SLC19A1 G80A in treatment arm 1 (p-trend=0.06) and reduced esophagitis/stomatitis among variants of TSER in treatment arm 3 (p-trend=0.06).
Conclusions
Genetic variability in folate-metabolizing enzymes was only to a limited degree associated with clinical outcomes among rectal cancer patients treated with 5-FU.
Keywords: rectal cancer, folate, MTHFR polymorphism, 5-fluorouracil, survival, toxicity, clinical trial, pharmacogenetics
Introduction
Colorectal cancer (CRC) is a major cause of cancer-related death. Among patients with stages II and III rectal cancer, 5-fluorouracil (5-FU)-based chemotherapy regimens with radiotherapy before and/or after surgery are the standard care 1, 2. The major mechanism of action of 5-FU is the inhibition of thymidylate synthase (TS) through an active metabolite, which forms a complex with TS and 5,10-methylenetetrahydrofolate (5,10-MTHF)3, 4. Intracellular MTHF levels are controlled by methylene-tetrahydrofolate reductase (MTHFR), a key enzyme in folate-related one carbon metabolism (FOCM)3, 4. The solute carrier family 19 member 1 encoded by the SLC19A1 gene, and dihydrofolate reductase (DHFR) also play essential roles in FOCM pathway by providing tetrahydrofolate (THF) and MTHF for the cells5-7.
There are large inter-individual differences in therapeutic response to 5-FU and the attendant toxicities 2. Polymorphisms in genes involved in FOCM could explain some of the inter-individual differences in clinical response and toxicity to 5-FU 8, 9. Two common single nucleotide polymorphisms (SNPs) in the MTHFR gene (C677T and A1298C) confer decreased MTHFR enzyme activity 10, 11 and alter intracellular folate intermediates 12. Some studies have reported associations between MTHFR gene polymorphisms and clinical outcome in CRC patients, 13-19 while others have not 20-27. However, very little is known of the associations of polymorphisms in the MTHFR gene, other FOCM genes and clinical outcomes in rectal cancer patients. Because clinical response to 5-FU and toxicity differ substantially among rectal cancer patients 2, identification of predictive markers of clinical outcome and chemotherapy-related toxicity is needed in order to deliver targeted therapies 8.
In order to address these gaps in knowledge, we performed a comprehensive analysis of the associations between functional significant polymorphisms in MTHFR (C667T and A1298C), SLC19A1 (G80A), SHMT1 (C1420T), DHFR (IVS1 del19bp), TS 1494del, TSER and clinical outcomes (overall survival, disease-free survival and toxicity) in rectal cancer patients (stage II or III) enrolled in a phase III adjuvant clinical trial (GI INT-0144; SWOG 9304) [1]. Our hypotheses were that polymorphisms in these genes would be associated with clinical outcomes in these patients, e.g. that the variant MTHFR C677T genotype would confer increased toxicity. Patients were randomly assigned to three different treatment arms: arm 1, with bolus FU in two 5-day cycles every 28 days before and after radiotherapy (XRT) plus FU via protracted venous infusion (PVI) 225 mg/m2/d during XRT; arm 2 (PVI-only arm), with PVI 42 days before and 56 days after XRT PVI; or arm 3 (bolus-only arm), with bolus FU + leucovorin (LV) in two 5-day cycles before and after XRT, plus bolus FU + LV (levamisole was administered each cycle before and after XRT) [1].
Material and Methods
Study Population and Sample Collection
Patients in this study were a subset of participants recruited into the clinical trial SWOG 9304 for whom DNA was available. The detailed description of the trial has been reported [1]. Briefly, patients were required to have had curatively resected nonmetastatic rectal adenocarcinoma. Extension through the muscularis propria and/or nodal spread (T3-4, N0 or T1-4, N1-3) was required. Patients with involved radial margins were ineligible with the exception of the extraperitoneal serosal margin (T4a). Rectal cancer was defined as tumor noted intraoperatively below the peritoneal reflection or ≤ 2 cm from the anal verge. Patients with dentate involvement were eligible. Patients were older than 18 years, had 0 to 2 performance score, and were not pregnant or lactating. No prior chemotherapy or radiation therapy for rectal cancer, or prior history of rectal cancer (with the exception of previously resected T1-2, N0, M0 tumors) was allowed. Satisfactory pretreatment laboratory parameters and the absence of serious illness were required. Chest x-ray and abdominopelvic computed tomography scans were required within 56 days of registration. Computed tomography scans were mandatory, unless bilirubin, AST, and alkaline phosphatase were within institutional normal limits, and the operative report specifically described the liver as normal. Registration was between days 20 to 70, and institutional review board approval and written informed consent were obtained.
Patients were randomly assigned to arm 1, with bolus FU in two 5-day cycles every 28 days before and after radiotherapy (XRT) plus FU via protracted venous infusion (PVI) 225 mg/m2/d during XRT; arm 2 (PVI-only arm), with PVI 42 days before and 56 days after XRT PVI; or arm 3 (bolus-only arm), with bolus FU + leucovorin (LV) in two 5-day cycles before and after XRT, plus bolus FU + LV (levamisole was administered each cycle before and after XRT) 1.
Genotyping
Seven hundred and fifty four (754) cases were successfully genotyped and included in the analyses. Samples were genotyped for the following: MTHFR C667T (rs1801133) and MTHFR A1298C (rs1801131) were genotyped by Taqman [32], in the Fred Hutchinson Cancer Research Center Molecular Epidemiology Laboratory (Dr. Makar) using 2x Universal Master Mix (Applied Biosystems, Foster City, CA) in 10μl reactions. SLC19A1 G80A (rs1051266) was genotyped by Taqman [33] using 2x SNP Genotyping Master Mix (Applied Biosystems) in 10μl reactions. SHMT1 C1420T (rs1979277) was genotyped by Taqman [34] in 10μl reactions. Taqman PCR reactions were run for 50 cycles on an ABI 7900HT sequence detection system, and real-time qPCR data was collected in addition to endpoint reads for allelic discrimination. The 19bp deletion polymorphism in intron 1 of DHFR was genotyped by size-dependent separation of fluorescently tagged PCR products as described [29] on an ABI 3130xl genetic analyzer and analyzed using GeneMapper software (Applied Biosystems). All assays were validated by genotyping 30 CEPH family trios from HapMap and comparing the results to known genotypes of these individuals, with no discrepancies or mendelian inheritance errors. Due to the poor quality of DNA isolated from laser capture microdissected (LCM) tissue; all samples were assayed at least twice for each polymorphism. Genotyping success rates ranged from 92.5% for MTHFR A1298C to 84.8% for MTHFR C677T. 5% blinded duplicates were included in every assay and duplicate concordance rates ranged from 100% for MTHFR C677T, 95.2% for cSHMT1 C1420T, 91.5% for MTHFR A1298C to 83.3% for DHFR del. Hardy-Weinberg equilibrium calculations showed some divergence, largely for DHFR. While some disequilibrium is possible in patients, there may also be some biased allele dropout. The low HWE p-value for the DHFR deletion may be due to the preferential loss of the longer allele as it requires a 20% longer PCR amplicon and the DNA from FFPE tissue may be degraded.
Statistical analysis
The primary end-points of this study were overall survival (OS), disease-free survival (DFS), and toxicity. Overall survival was measured as the date from registration to death from any cause. Patients known to be alive were censored at last known date of contact. Disease-free survival was defined as time from registration to the first development of recurrent disease, or of death from any cause, whichever came first. Patients still known to be alive without recurrence at the time of analysis were considered to be censored at their last follow-up time. OS and DFS curves were generated using the Kaplan-Meier method. Toxicities defined as National Cancer Institute Common Toxicity Criteria (CTC Version 2.0) were categorized by maximum grade, evaluating at Grades 0-2 vs Grades 3-5. The maximum grade of any toxicity was identified, as well as the maximum grade of specific toxicities including hematologic, gastrointestinal, pain, and stomatitis/esophagitis.
The associations of polymorphisms with OS and DFS were analyzed using the Cox proportional hazards model (univariately and adjusted for variables used in the stratification at the time of randomization: type of surgery, nodal involvement, primary tumor stage, and time from surgery to registration). To obtain tests for trend, the genotypes were treated as continuous variables and we compared the likelihood ratio of a model without the variable using a χ2 test with one degree of freedom. The association between the polymorphisms and toxicity was tested using univariate logistic regression and logistic regression analysis adjusted for age, gender, and treatment arm. Genotypes were modeled using indicator variables for the heterozygous and the homozygous variant genotypes (unrestricted or co-dominant model). Given the investigations of functional candidate polymorphisms with prior hypotheses, no formal multiple comparisons adjustments were applied.
To address joint effects of two functional variants, MTHFR A1298C and C677T were also a priori combined into a single variable, reflecting combined genotypes coded as: 0 variants (AA, CC), one variant (AC/CC or AA/CT), or two or more variants (AA/ TT, AC/CT, AC/TT, CC/CC, CC/CT or CC/TT).
Results
Seven hundred and fifty-four (754) eligible patients were evaluated in the study of which 482 were men and 272 were women (Table 1). The patients were predominantly Caucasian (92%) and median age was 61 years (range; 19-86). The proportion of patients randomly assigned to three different arms was similar. Patients’ characteristics included in this correlative study were similar to the remaining 1100 patients in the original study for whom specimens were not available (data not shown).
Table 1.
Patient characteristics and genotype frequencies in the study population
| Characteristics | Number of patients (N=754) |
|---|---|
| Gender | |
| Male | 482 (64%) |
| Female | 272 (36%) |
| Median Age (range) | 61 (19 – 86) |
| Race | |
| White | 695 (92%) |
| Black | 27 (4%) |
| Asian | 18 (2%) |
| Pacific Islander | 5 (<1%) |
| Native American | 4 (<1%) |
| Unknown | 5 (<1%) |
| Spanish/Hispanic Origin | |
| No | 708 (94%) |
| Yes | 32 (4%) |
| Unknown | 14 (2%) |
| N Stage | |
| N0 | 227 (30%) |
| N1 | 320 (42%) |
| N2-3 | 207 (27%) |
| T Stage | |
| T1 - 2 | 111 (15%) |
| T3 – T4a | 596 (79%) |
| T4b | 47 (6%) |
| Treatment Arm | |
| Arm 1-Bolus 5-FU + XRT | 257 (34%) |
| Arm 2-PVI 5-FU + XRT | 237 (31%) |
| Arm 3-Bolus 5-FU + LV + LEV + XRT | 260 (34%) |
| Performance Status | |
| 0-1 | 735 (97%) |
| 2 | 19 (3%) |
| Genotype | |
| MTHFR / A1298C (N=693) | |
| AA | 318 (46%) |
| AC | 320 (46%) |
| CC | 55 (8%) |
| MTHFR / C677T (N=643) | |
| CC | 301 (47%) |
| CT | 253 (39%) |
| TT | 89 (14%) |
| MTHFR A1298C, C677T (N=609) | |
| 0 variant | 94 (15%) |
| 1 variant | 250 (41%) |
| >1 variant | 265 (44%) |
| SLC19A1 / G80A (N=669) | |
| GG | 211 (31%) |
| GA | 300 (45%) |
| AA | 158 (24%) |
| DHFR / 19bp del (N=648) | |
| wt/wt | 185 (28%) |
| wt/del | 268 (41%) |
| del/del | 195 (30%) |
| SHMT1 / C1420T (N=666) | |
| CC | 332 (50%) |
| CT | 240 (36%) |
| TT | 94 (14%) |
| TS 1494del (N=719) | |
| 6bp+/6bp+ | 236 (33%) |
| 6bp+/6bp− | 287 (40%) |
| 6bp−/6bp− | 196 (27%) |
| TSER (N=716) | |
| 2R/2R | 134 (19%) |
| 2R/3R | 391 (54%) |
| 3R/3R | 191 (27%) |
| TSER Functional Status* (N=692) | |
| Low | 375 (54%) |
| Intermediate | 274 (40%) |
| High | 43 (6%) |
TSER Functional Status definitions:
Low: 2R/2R, 2R/3RC, 3RC/3RC
Intermediate: 2R/3RG, 3RC/3RG
High: 3RG/3RG
R= repeat
Evaluating all genotypes, including the combined genotypes in MTHFR, there were overall no statistically significant associations between any polymorphism and either OS or DFS (with or without adjusting for treatment assignment and/or stratification factors) (Table 2). Nevertheless, there was a marginal trend towards worse OS (p-trend=0.08) and DFS (p-trend=0.05) among variants of MTHFR C677T and better DFS (p-trend=0.09) among variants of the TS 1494del. Analyses restricted to the Caucasian population yielded similar results (data not shown). Although there was no overall statistically significant trend in OS related to the DHFR 19 bp deletion polymorphism, heterozygous (wt/del) had a HR= 0.76 (95%CI 0.59-0.99) compared to the homozygous (wt/wt) genotype.
Table 3.
Polymorphisms in folate metabolism in relation to survival by treatment arm
| Gene / Variant | Treatment Arm | Genotype | Reference | Overall Survival* | Disease-free Survival* | ||
|---|---|---|---|---|---|---|---|
| HR (95% CI) | P-value | HR (95% CI) | P-value | ||||
| MTHFR / A1298C | Arm 1 | AC | AA | 0.99 (0.67,1.45) | 0.94 | 1.00 (0.69,1.46) | 1.00 |
| CC | AA | 0.83 (0.45,1.53) | 0.55 | 0.87 (0.48,1.58) | 0.65 | ||
| Arm 2 | AC | AA | 1.01 (0.68,1.49) | 0.98 | 0.95 (0.65,1.40) | 0.80 | |
| CC | AA | 0.92 (0.42,2.02) | 0.83 | 0.77 (0.35,1.69) | 0.51 | ||
| Arm 3 | AC | AA | 0.83 (0.57,1.21) | 0.34 | 0.75 (0.52,1.08) | 0.12 | |
| CC | AA | 0.39 (0.12,1.25) | 0.12 | 0.50 (0.18,1.38) | 0.18 | ||
| MTHFR / C677T | Arm 1 | CT | CC | 1.26 (0.85,1.86) | 0.29 | 1.21 (0.82,1.78) | 0.33 |
| TT | CC | 1.08 (0.57,2.08) | 0.81 | 0.99 (0.52,1.89) | 0.97 | ||
| Arm 2 | CT | CC | 1.07 (0.68,1.69) | 0.77 | 1.14 (0.73,1.76) | 0.57 | |
| TT | CC | 1.76 (1.06,2.93) | 0.03 | 1.84 (1.12,3.03) | 0.02 | ||
| Arm 3 | CT | CC | 1.17 (0.78,1.76) | 0.44 | 1.17 (0.79,1.74) | 0.44 | |
| TT | CC | 1.04 (0.59,1.81) | 0.90 | 1.17 (0.69,2.01) | 0.55 | ||
| SLC19A1 | Arm 1 | GA | GG | 0.95 (0.62,1.46) | 0.82 | 1.02 (0.67,1.55) | 0.94 |
| AA | GG | 1.02 (0.62,1.69) | 0.92 | 0.97 (0.59,1.60) | 0.91 | ||
| Arm 2 | GA | GG | 1.33 (0.82,2.17) | 0.25 | 1.28 (0.81,2.04) | 0.30 | |
| AA | GG | 1.51 (0.87,2.62) | 0.14 | 1.40 (0.82,2.38) | 0.21 | ||
| Arm 3 | GA | GG | 0.89 (0.58,1.38) | 0.62 | 0.73 (0.48,1.12) | 0.15 | |
| AA | GG | 1.06 (0.65,1.73) | 0.82 | 0.90 (0.56,1.44) | 0.66 | ||
| DHFR del | Arm 1 | Het | wt | 0.78 (0.50,1.22) | 0.26 | 0.89 (0.57,1.38) | 0.59 |
| Mut | wt | 1.11 (0.70,1.76) | 0.65 | 1.15 (0.73,1.83) | 0.54 | ||
| Arm 2 | Het | wt | 0.82 (0.52,1.29) | 0.39 | 0.79 (0.50,1.23) | 0.30 | |
| Mut | wt | 0.61 (0.37,1.03) | 0.06 | 0.73 (0.45,1.19) | 0.21 | ||
| Arm 3 | Het | wt | 0.72 (0.45,1.13) | 0.15 | 0.88 (0.56,1.37) | 0.56 | |
| Mut | wt | 0.94 (0.58,1.52) | 0.80 | 0.89 (0.55,1.44) | 0.63 | ||
| SHMT1 / C1420T | Arm 1 | CT | CC | 1.00 (0.67,1.50) | 0.99 | 0.96 (0.65,1.43) | 0.86 |
| TT | CC | 1.24 (0.77,2.02) | 0.40 | 1.28 (0.80,2.05) | 0.31 | ||
| Arm 2 | CT | CC | 0.99 (0.64,1.53) | 0.96 | 1.05 (0.68,1.61) | 0.83 | |
| TT | CC | 0.54 (0.27,1.11) | 0.09 | 0.75 (0.40,1.40) | 0.37 | ||
| Arm 3 | CT | CC | 0.90 (0.60,1.35) | 0.62 | 0.99 (0.67,1.46) | 0.96 | |
| TT | CC | 1.59 (0.91,2.78) | 0.10 | 1.63 (0.95,2.80) | 0.08 | ||
| TS 1494del | Arm 1 | 6bp+/6bp− | 6bp+/6bp+ | 1.11 (0.74,1.65) | 0.61 | 1.05 (0.71,1.57) | 0.80 |
| 6bp−/6bp− | 6bp+/6bp+ | 0.80 (0.49,1.30) | 0.37 | 0.87 (0.55,1.39) | 0.57 | ||
| Arm 2 | 6bp+/6bp− | 6bp+/6bp+ | 1.18 (0.74,1.87) | 0.49 | 1.07 (0.69,1.67) | 0.75 | |
| 6bp−/6bp− | 6bp+/6bp+ | 1.16 (0.71,1.91) | 0.55 | 1.11 (0.69,1.79) | 0.66 | ||
| Arm 3 | 6bp+/6bp− | 6bp+/6bp+ | 0.65 (0.42,0.99) | 0.04 | 0.66 (0.44,0.99) | 0.05 | |
| 6bp−/6bp− | 6bp+/6bp+ | 0.61 (0.39,0.98) | 0.04 | 0.55 (0.35,0.87) | 0.01 | ||
| TSER | Arm 1 | 2/3 | 2/2 | 0.93 (0.60,1.43) | 0.74 | 0.89 (0.59,1.36) | 0.60 |
| 3/3 | 2/2 | 0.65 (0.38,1.12) | 0.12 | 0.61 (0.36,1.03) | 0.07 | ||
| Arm 2 | 2/3 | 2/2 | 0.81 (0.49,1.33) | 0.40 | 0.77 (0.47,1.24) | 0.27 | |
| 3/3 | 2/2 | 0.91 (0.53,1.58) | 0.74 | 0.81 (0.47,1.37) | 0.43 | ||
| Arm 3 | 2/3 | 2/2 | 1.12 (0.66,1.90) | 0.69 | 1.09 (0.65,1.82) | 0.75 | |
| 3/3 | 2/2 | 1.10 (0.61,1.97) | 0.75 | 1.15 (0.66,2.02) | 0.62 | ||
| TSER Functional Status | Arm 1 | Med | Low | 0.96 (0.67,1.38) | 0.83 | 0.92 (0.64,1.32) | 0.65 |
| High | Low | 0.34 (0.11,1.09) | 0.07 | 0.32 (0.10,1.03) | 0.06 | ||
| Arm 2 | Med | Low | 0.99 (0.65,1.49) | 0.95 | 0.97 (0.65,1.44) | 0.87 | |
| High | Low | 1.50 (0.71,3.15) | 0.28 | 1.46 (0.72,2.95) | 0.29 | ||
| Arm 3 | Med | Low | 1.02 (0.69,1.50) | 0.93 | 0.97 (0.67,1.41) | 0.88 | |
| High | Low | 1.11 (0.55,2.24) | 0.77 | 1.14 (0.58,2.22) | 0.70 |
Arm 1-Bolus 5-FU + XRT
Arm 2-PVI 5-FU + XRT
Arm 3-Bolus 5-FU + LV + LEV + XRT
Adjusted for age, gender, and treatment arm
We also explored analyses separately by treatment arm (Table 3). These analyses revealed few differences compared to the overall analyses, with the exception that patients with the MTHFR C677T TT genotype compared to those with homozygous wildtype at C677T locus had worse OS (HR=1.76; 95% CI: 1.06-2.93; p=0.03) and DFS (HR=1.84; 95% CI: 1.12-3.03; p=0.02) in treatment arm 2 (PVI-only arm - PVI before and after XRT). On the other hand, variants of the TS 1494del genotype had better OS and DFS than the wildtype in treatment arm 3 (bolus-only arm - with bolus FU + leucovorin (LV) before and after XRT). The HRs for OS comparing those with TS 1494del−/− and +/− genotypes were 0.61 (95%CI 0.39-0.98, p-value=0.04) and 0.65 (95%CI 0.42-0.99, p-value=0.04), respectively.
Toxicity Analysis
Because some of the toxicities, especially hematologic, in the overall trial differed by treatment arm 1, we performed stratified analyses for this endpoint. There was a trend towards increased gastrointestinal toxicity among variants of MTHFR C677T genotype in treatment arm 1 (HRs comparing TT and CT genotypes to CC were 2.97; 95%CI 1.13–7.86 and 1.29; 95%CI 0.72–2.30 respectively, p-trend=0.09). There were also trends towards reduced hematological toxicity among variants of SLC19A1 G80A in treatment arm 1 (HRs comparing AA and GA genotypes to GG genotype were 0.47; 95%CI 0.22– 1.00 and 0.83 95% CI 0.45– 1.56, p-trend=0.08) and reduced esophagitis/stomatitis among variants of TSER in treatment arm 3 (p-trend=0.06). However, no consistent patterns were observed across treatment arms.
Discussion
In this randomized controlled trial, we observed no statistically significant associations between functional polymorphisms in MTHFR, DHFR, SLC19A1, SHMT1, TS 1494del and TSER genes and clinical outcomes among stage II or III rectal cancer patients. Nevertheless, we observed worse OS and DFS among variants of MTHFR C677T and better OS and DFS among variants of TS 1494del compared to the wildtypes in analyses stratified by treatment arm.
Although few studies have investigated the associations of MTHFR genotypes with outcomes among rectal cancer patients, our findings are, in general, similar to what has been reported among colorectal cancer patients where studies, often small-sized, have observed no significant associations between MTHFR genotypes and OS or DFS [13, 14, 6, 19, 21, 23]. Likewise in a previous study among rectal cancer patients, MTHFR genotypes were not associated with OS and DFS among patients with rectal cancer, although they were associated with 5-FU toxicity 28.
Nevertheless, our findings suggest that whatever impact these variants may have on OS and DFS appear limited to only patients in a particular treatment arm (treatment arm 2 for MTHFR C677T TT and treatment arm 3 for TS 1494del genotype), which could be useful in targeted intervention. The MTHFR 677T allele has also been associated with lower response rates and worse tumor regression grade in rectal cancer patients receiving 5-FU-based chemotherapy 26, 29, 30. In a small study with 125 patients, the MTHFR 677T-1298A haplotype was associated with poor response to treatment 29. Rectal cancer patients without the MTHFR 677T-1298A haplotype had a 71% better response rate than patients with the MTHFR 677T-1298A haplotype 29. MTHFR is an essential regulatory enzyme in folate metabolism that diverts folate metabolites from pyrimidine synthesis towards methionine synthesis 8. The C to T mutation, which occurs commonly among Caucasians 31, 32, results in a reduction in MTHFR activity leading to lower levels of 5-methylTHF, accumulation of 5,10-methyleneTHF and elevated circulating homocysteine concentrations in people with borderline folate status 31-34.
We observed no overall statistically significant associations between TS genotypes and OS and DFS. Among patients in treatment arm 3, however, variants in the TS 1494del gene were associated with better OS and DFS compared with the wildtype which has an allele frequency of 0.29 35. There are conflicting reports on the associations of TS genotypes with clinical outcomes among rectal cancer patients treated with 5-FU. Some studies have reported associations of TS genotypes with DFS and tumor regression while others have not 36-39. However, most of these studies had small number of patients and were conducted in the neoadjuvant setting.
We observed no significant associations between DHFR 19 bp deletion, SHMT1 C1420T, SLC19A1 G80A polymorphisms and OS and DFS. The DHFR genotypes affect serum folate levels and thus could potentially mediate the effect of 5-FU treatment in cancer patients 40-42. Homozygosity for the 19 bp deletion polymorphism in intron 1 of DHFR is related to an increase in DHFR mRNA 43 and a decrease in plasma homocysteine 41. Although the heterozygous genotype for the 19 bp deletion polymorphism in DHFR gene was marginally associated with OS in our study, the homozygous genotype was not. The result needs to be interpreted with caution since the trend analysis was not statistically significant.
We observed no statistically significant associations between these genotypes and toxicity, although for some genotypes, our analyses suggest that the effect on toxicity might depend on type of treatment. In a previous study among rectal cancer patients, the MTHFR haplotype (677C–1298C) was shown to be protective against severe diarrhea and mucositis among patients treated with 5-FU but not among those treated with combined 5-FU/irinotecan 28. Furthermore, patients with the 1298A/A genotype were reported to have a higher risk of developing grade 3–4 diarrhea or mucositis compared to patients with the A/C or C/C 28, we did not observe such differences in our analyses of combined MTHFR genotypes.
The therapeutic effectiveness and cytotoxicity of 5-FU is due to the impact of folate on DNA replication 3, 4. Cancer cells as well as normal but rapidly replicating cells of the gastrointestinal and hematopoietic systems are especially susceptible to the effects of 5-FU, hence, the major sites of 5-FU toxicity are the gastrointestinal and hematopoietic cells 4. MTHFR and TS genotypes have been reported to modify the severity of oral mucositis following hematopoietic stem cell transplantation 44.
Strengths
Our study has several strengths. It is a large study ancillary to a completed clinical trial with a well-defined clinical database. The 5-FU chemotherapy and radiation therapy were administered according to a well-defined study protocol, and toxicities were coded according to established criteria and guidelines.
Limitations
Although the number of participants was large, because of limited DNA availability, it represented only 40% of patients registered for the clinical trial. Moreover, due to differing adverse event profiles in the three study arms, the sample sizes to assess the associations between genotypes and adverse events required separate analyses within treatment arms, limiting the power to detect associations. Due to the poor quality of DNA isolated from FFPE -LCM tissue, we were unable to obtain genotypes for all 792 of the patients for whom we had specimens, despite up to three attempts. We also recognize that the stratification resulted in a number of statistical comparisons; results thus require further replication in comparable settings.
Conclusions
Genetic variability in folate-metabolizing enzymes was only to a limited degree associated with clinical outcomes among rectal cancer patients treated with 5-FU. Because of the inter-individual variability in response to 5-FU therapy and toxicity, identification of other genotypes that may predict therapeutic response and toxicity still remains a priority.
Precis: There were no statistically significant associations between eight functionally significant polymorphisms in six genes (MTHFR (C667T, A1298C), SLC191A (G80A), SHMT1 (C1420T), DHFR (Del19bp)), TS 1494del, TSER) involved in folate metabolism and overall survival (OS), disease free survival (DFS) and toxicity in the overall analyses. Nevertheless, there was a trend towards worse DFS, reduced hematological and gastrointestinal toxicities among patents with variant alleles in MTHFR C677T, SLC19A1 G80A, TSER, respectively.
Table 2.
Polymorphisms in folate metabolism in relation to survival among rectal cancer patients (SWOG9304)
| Gene | Polymorphism | Genotype | n/N (%) | Overall Survival* | Disease -free Survival* | ||
|---|---|---|---|---|---|---|---|
| HR (95% CI) | *p-value | HR (95% CI) | *p-value | ||||
| MTHFR | A1298C | AA | 318/693 (46%) | 1.00 (reference) | 1.00 (reference) | ||
| AC | 320/693 (46%) | 0.94 (0.75,1.17) | 0.59 | 0.89 (0.72,1.11) | 0.31 | ||
| CC | 55/693 (8%) | 0.74 (0.48,1.15) | 0.19 | 0.75 (0.49,1.14) | 0.18 | ||
| ptrend =0.21 | ptrend =0.13 | ||||||
| C677T | CC | 301/643 (47%) | 1.00 (reference) | 1.00 (reference) | |||
| CT | 253/643 (39%) | 1.18 (0.93,1.50) | 0.17 | 1.18 (0.94,1.49) | 0.16 | ||
| TT | 89/643 (14%) | 1.29 (0.94,1.78) | 0.12 | 1.33 (0.97,1.81) | 0.08 | ||
| ptrend =0.08 | ptrend =0.05 | ||||||
| A1298C,C677T | 0 variant | 94/609 (15%) | 1.00 (reference) | 1.00 (reference) | |||
| 1 variant | 250/609 (41%) | 1.11 (0.79,1,58) | 0.54 | 1.11 (0.80,1.56) | 0.53 | ||
| >1 variant | 265/609 (44%) | 1.19 (0.85,1.68) | 0.32 | 1.19 (0.85,1.66) | 0.31 | ||
| ptrend =0.85 | ptrend =0.79 | ||||||
| SLC19A1 | G80A | GG | 211/669 (32%) | 1.00 (reference) | 1.00 (reference) | ||
| GA | 300/669 (45%) | 1.03 (0.80,1.34) | 0.80 | 0.97 (0.76,1.25) | 0.83 | ||
| AA | 158/669 (24%) | 1.17 (0.87,1.57) | 0.30 | 1.06 (0.80,1.41) | 0.68 | ||
| ptrend =0.32 | ptrend =0.74 | ||||||
| DHFR | Del19bp | wt/wt | 185/648 (29%) | 1.00 (reference) | 1.00 (reference) | ||
| wt/del | 268/648 (41%) | 0.76 (0.59,0.99) | 0.04 | 0.85 (0.66,1.10) | 0.22 | ||
| del/del | 195/648 (30%) | 0.88 (0.67,1.16) | 0.36 | 0.92 (0.70,1.20) | 0.53 | ||
| ptrend =0.37 | ptrend =0.54 | ||||||
| SHMT1 | C1420T | CC | 332/666 (50%) | 1.00 (reference) | 1.00 (reference) | ||
| CT | 240/666 (36%) | 0.96 (0.76,1.22) | 0.77 | 1.00 (0.79,1.26) | 0.99 | ||
| TT | 94/666 (14%) | 1.07 (0.78,1.48) | 0.68 | 1.18 (0.87,1.61) | 0.29 | ||
| ptrend =0.83 | ptrend =0.40 | ||||||
| TS 1494del | 6bp+/6bp+ | 236/719 | 1.00 (reference) | 1.00 (reference) | |||
| 6bp+/6bp− | 287/719 | 0.93 (0.73,1.19) | 0.58 | 0.89 (0.70,1.13) | 0.35 | ||
| 6bp−/6bp− | 196/719 | 0.81 (0.61,1.06) | 0.13 | 0.79 (0.61,1.03) | 0.09 | ||
| ptrend =0.14 | ptrend =0.09 | ||||||
| TSER | 2R/2R | 134/716 | 1.00 (reference) | 1.00 (reference) | |||
| 2R/3R | 391/716 | 0.93 (0.71,1.23) | 0.61 | 0.89 (0.68,1.17) | 0.41 | ||
| 3R/3R | 191/716 | 0.85 (0.62,1.17) | 0.32 | 0.82 (0.60,1.11) | 0.20 | ||
| ptrend =0.33 | ptrend =0.21 | ||||||
| TSER Functional Status | Low | 375/692 | 1.00 (reference) | 1.00 (reference) | |||
| Intermediate | 274/692 | 0.99 (0.79,1.24) | 0.94 | 0.95 (0.77,1.18) | 0.66 | ||
| High | 43/692 | 0.90 (0.57,1.43) | 0.66 | 0.90 (0.58,1.40) | 0.65 | ||
| ptrend =0.74 | ptrend =0.56 |
HR- Hazard Ratio, 95% CI- 95% Confidence Interval, *p-value from Wald Chi-Square Test in Cox Regression
0variant: homozygous for common alleles for both polymorphisms
1variant: heterozygous for one of the polymorphisms
>1 variant: heterozygous for both polymorphisms or homozygous for the rare allele
Adjusted for type of surgery, nodal involvement, primary tumor stage, and time from surgery to registration
Acknowledgments
Financial support: This work was supported by the National Institute of Health under RO1 CA105145 and also in part by the following PHS Cooperative Agreement grant numbers awarded by the National Cancer Institute, DHHS: U10 CA32102, CA38926, CA46113 and CA58882.
Footnotes
Conflict of interest statement:
The authors declare no conflicts of interest.
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