Elisabeth Odin

A Study of the MTHFR Gene Polymorphism C677T in Colorectal Cancer

PURPOSE The aim of this study was to examine the clinical significance of the methylenetetrahydrofolate reductase (MTHFR) gene polymorphism C677T in colorectal cancer (CRC). The hypothesis was that the genotype could affect the risk of cancer development and the results of cancer treatment. PATIENTS AND METHODS Genotyping was made for a random 30% (n = 544) of all patients treated for CRC at our unit from 1999 to 2006 (n = 1812). Basic clinical and pathologic factors were analyzed by genotype group and also compared with those of the entire cohort. Tolerability of chemotherapy and possible side effects were analyzed by genotype. Survival was analyzed by genotype for all stages for patients treated between 1999 and 2003. The genotype prevalence was also compared with a control material of healthy blood donors. RESULTS No genotype was associated with an increased risk of CRC or higher cancer stage. The patients with CT/TT genotype had significantly greater risk of suffering side effects from fluoropyrimidine (5-fluorouracil) treatment (P < .05). In stage III colon cancer, the patients with CT/TT genotype had a poorer prognosis than those with the CC genotype. The difference was significant in univariate (P < .003) and multivariate (P < .040) analysis. Though the genotype-associated side effect risks remained in stage IV, the effect on survival was not significant (P < .1). CONCLUSION The MTHFR polymorphism C677T does, in our material, not affect the risk of CRC; however, it can affect the sensitivity to chemotherapy and the risk of side-effects and therefore survival in stage III and possibly stage IV colon cancer. It could be a future predictive factor in the choice of a treatment regimen.

Comparison of bacterial quantities in left and right colon biopsies and faeces

AIM To compare quantities of predominant and pathogenic bacteria in mucosal and faecal samples. METHODS Twenty patients undergoing diagnostic colonoscopy with endoscopically and histologically normal mucosa were recruited to the study, 14 subjects of which also supplied faecal (F) samples between 15 d to 105 d post colonoscopy. Mucosal biopsies were taken from each subject from the midportion of the ascending colon (right side samples, RM) and the sigmoid (left side samples, LM). Predominant intestinal and mucosal bacteria including clostridial 16S rRNA gene clusters IV and XIVab, Bacteroidetes, Enterobacteriaceae, Bifidobacterium spp., Akkermansia muciniphila (A. muciniphila), Veillonella spp., Collinsella spp., Faecalibacterium prausnitzii (F. prausnitzii) and putative pathogens such as Escherichia coli (E. coli), Clostridium difficile (C. difficile), Helicobacter pylori (H. pylori) and Staphylococcus aureus (S. aureus) were analysed by quantitative polymerase chain reaction (qPCR). Host DNA was quantified from the mucosal samples with human glyceraldehyde 3-phosphate dehydrogenase gene targeting qPCR. Paired t tests and the Pearson correlation were applied for statistical analysis. RESULTS The most prominent bacterial groups were clostridial groups IV and XIVa+b and Bacteroidetes and bacterial species F. prausnitzii in both sample types. H. pylori and S. aureus were not detected and C. difficile was detected in only one mucosal sample and three faecal samples. E. coli was detected in less than half of the mucosal samples at both sites, but was present in all faecal samples. All detected bacteria, except Enterobacteriaceae, were present at higher levels in the faeces than in the mucosa, but the different locations in the colon presented comparable quantities (RM, LM and F followed by P(1) for RM vs F, P(2) for LM vs F and P(3) for RM vs LM: 4.17 ± 0.60 log(10)/g, 4.16 ± 0.56 log(10)/g, 5.88 ± 1.92 log(10)/g, P(1) = 0.011, P(2) = 0.0069, P(3) = 0.9778 for A. muciniphila; 6.25 ± 1.3 log(10)/g, 6.09 ± 0.81 log(10)/g, 8.84 ± 1.38 log(10)/g, P(1) < 0.0001, P(2) = 0.0002, P(3) = 0.6893 for Bacteroidetes; 5.27 ± 1.68 log(10)/g, 5.38 ± 2.06 log(10)/g, 8.20 ± 1.14 log(10)/g, P(1) < 0.0001, P(2) ≤ 0.0001, P(3) = 0.7535 for Bifidobacterium spp.; 6.44 ± 1.15 log(10)/g, 6.07 ±1.45 log(10)/g, 9.74 ±1.13 log(10)/g, P(1) < 0.0001, P(2) ≤ 0.0001, P(3) = 0.637 for Clostridium cluster IV; 6.65 ± 1.23 log(10)/g, 6.57 ± 1.52 log(10)/g, 9.13 ± 0.96 log(10)/g, P(1) < 0.0001, P(2) ≤ 0.0001, P(3) = 0.9317 for Clostridium cluster XIVa; 4.57 ± 1.44 log(10)/g, 4.63 ± 1.34 log(10)/g, 7.05 ± 2.48 log(10)/g, P(1) = 0.012, P(2) = 0.0357, P(3) = 0.7973 for Collinsella spp.; 7.66 ± 1.50 log(10)/g, 7.60 ± 1.05 log(10)/g, 10.02 ± 2.02 log(10)/g, P(1) ≤ 0.0001, P(2) = 0.0013, P(3) = 0.9919 for F. prausnitzsii; 6.17 ± 1.3 log(10)/g, 5.85 ± 0.93 log(10)/g, 7.25 ± 1.01 log(10)/g, P(1) = 0.0243, P(2) = 0.0319, P(3) = 0.6982 for Veillonella spp.; 4.68 ± 1.21 log(10)/g, 4.71 ± 0.83 log(10)/g, 5.70 ± 2.00 log(10)/g, P(1) = 0.1927, P(2) = 0.0605, P(3) = 0.6476 for Enterobacteriaceae). The Bifidobacterium spp. counts correlated significantly between mucosal sites and mucosal and faecal samples (Pearson correlation coefficients 0.62, P = 0.040 and 0.81, P = 0.005 between the right mucosal sample and faeces and the left mucosal sample and faeces, respectively). CONCLUSION Non-invasive faecal samples do not reflect bacterial counts on the mucosa at the individual level, except for bifidobacteria often analysed in probiotic intervention studies.

Molecular determinants of efficacy for 5‐FU‐based treatments in advanced colorectal cancer: mRNA expression for 18 chemotherapy‐related genes

5‐Fluorouracil (5‐FU)‐based regimens remain a cornerstone in the treatment of colorectal cancer (CRC). However, the attendant toxicity prevents these regimens from reaching maximum therapeutic potential. In this retrospective analysis, we examined the pretreatment expression of 18 genes in archival tumor bank samples from patients with advanced CRC to determine if one or more of the selected genes showed promise as either a prognostic or predictive marker of 5‐FU‐based treatment outcomes. One hundred and forty‐four CRC patient samples (collected from 1983 to 2004) were analyzed via real‐time PCR for gene expression. Univariate analyses were used to correlate gene expression with efficacy and time‐to‐event variables. Low thymidine phosphorylase (TP), dihydrofolate reductase, dihydropyrimidine dehydrogenase (DPD), excision repair cross‐complementing 1 (ERCC1) and thymidylate synthase gene expression were associated with better time‐to‐progression in the entire population. Low TP, DPD and ERCC1 expression were independently associated with improved overall survival. Low TP gene expression was also predictive of response. This study suggests that TP gene expression in particular is a predictive as well as a prognostic biomarker for advanced CRC patients. Gene panels assessing pretreatment TP, DPD, ERCC1, dihydrofolate reductase and thymidylate synthase gene expression may help improve the therapeutic potential of 5‐FU‐ or other novel antifolate‐based regimens. Further analysis of the prognostic or predictive value of these genes in prospective trials in CRC patients seems warranted.

p16INK4a gene promoter hypermethylation in mucosa as a prognostic factor for patients with colorectal cancer.

Low gene expression of folylpolyglutamate synthase (FPGS) in colorectal mucosa correlates with low folate levels and poor survival of colorectal cancer (CRC) patients. Because gene-specific hypermethylation is affected by the folate level, the hypermethylation status in mucosa may also be linked to clinical outcome of CRC patients. The tumor suppressor gene p16INK4a (p16) regulates the cell cycle and angiogenic switch. In human neoplastic tissues, the main mechanism of p16 inactivation is promoter methylation. The aim of the study was to determine whether hypermethylation of the p16 promoter could be detected in mucosa of CRC patients (n = 181) and to analyze if hypermethylation was related to survival. The relation between p16 hypermethylation and expression of FPGS and two other folate-associated genes, reduced folate carrier 1 (RFC-1), and thymidylate synthase (TS), was analyzed (n = 63). The results showed that p16 was hypermethylated in 65 (36%) of the mucosa samples and that hypermethylation was age-related (P = 0.029). After adjustment for known risk factors, Cox regression analysis showed that Dukes’ A–C patients with p16 hypermethylation in mucosa had an increased risk of cancer-related death (hazard ratio = 2.9, P = 0.007) and shorter disease-free survival (hazard ratio = 2.5, P = 0.015) compared with patients with no p16 hypermethylation. RFC-1 and FPGS gene expression levels were significantly correlated in patients lacking p16 hypermethylation in mucosa (P = 0.0003), but not at all correlated in patients having hypermethylation in mucosa (P = 1.0). In conclusion, p16 hypermethylation in mucosa of CRC patients was identified as an independent prognostic parameter for cancer-specific survival as well as an independent predictor of DFS. The results suggest that there might be a connection between folate-associated gene expression and p16 methylation status.

MTHFR, MTR, and MTRR polymorphisms in relation to p16INK4A hypermethylation in mucosa of patients with colorectal cancer.

We recently analyzed the hypermethylation status of the p16INK4a (p16) gene promoter in normal-appearing mucosa obtained from patients with colorectal cancer. Hypermethylation of p16 was associated with reduced survival of these patients. In the present study, germ line polymorphisms in the folate- and methyl-associated genes, methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR) and methionine synthase reductase (MTRR), were analyzed in the same patient cohort to find a possible link between these genetic variants and p16 hypermethylation. Genomic DNA was extracted from blood of patients (n = 181) and controls (n = 300). Genotype analyses were run on an ABI PRISM® 7900HT sequence-detection system (Applied Biosystems), using real-time polymerase chain reaction and TaqMan chemistry. The results showed that the genotype distributions of the patient and control groups were similar. No significant differences in cancer-specific or disease-free survival of stage I–III patients according to polymorphic variants were detected, nor were any differences in cancer-specific or disease-free survival detected when patients were subgrouped according to the MTHFR or MTR genotype groups and dichotomized by p16 hypermethylation status in mucosa. However, patients with the MTRR 66 AA/AG genotypes were found to have a significantly worse cancer-specific survival when the mucosa were positive, compared with negative, for p16 hypermethylation (hazard ratio 2.7; 95% confidence interval 1.2–6.4; P = 0.023). In contrast, there was no difference in survival among patients with the MTRR 66 GG genotype stratified by p16 hypermethylation status. These results indicate a relationship between genetic germ-line variants of the MTRR gene and p16 hypermethylation in mucosa, which may affect the clinical outcome of patients with colorectal cancer.

Pretherapeutic uracil and dihydrouracil levels of colorectal cancer patients are associated with sex and toxic side effects during adjuvant 5-fluorouracil–based chemotherapy†

In Nordic countries, the standard treatment of colorectal cancer (CRC) in the adjuvant setting is bolus 5‐fluorouracil (5‐FU) plus leucovorin alone or in combination with oxaliplatin. 5‐FU competes with the natural occurring pyrimidine uracil (Ura) as a substrate for dihydropyrimidine dehydrogenase (DPD; enzyme commission number 1.3.1.2). Low DPD activity is associated with toxicity during treatment. Pretherapeutic detection of DPD deficiency could prevent severe toxicity otherwise limiting drug administration. Assays showing that DPD deficiency impairs breakdown of Ura to dihydrouracil (UH2) seem promising for clinical use.

Rapid method for relative gene expression determination in human tissues using automated capillary gel electrophoresis and multicolor detection.

The aim of this study was to evaluate a direct and automated post-polymerase chain reaction (PCR) detection system to simultaneously determine the relative gene expression levels of nine cancer-related human genes. Total RNA was prepared from flash-frozen biopsies derived from human colorectal tumors or normal mucosa and reverse-transcribed to cDNA which was PCR-amplified using primer pairs corresponding to the studied genes. In each reaction, the forward primer was labeled with a fluorescent dye. The PCR products were pooled and an internal size standard with a uniquely colored fluorescent dye was added. The samples were then subjected to automated capillary gel electrophoresis. Fragment analysis software was used to calculate the relative gene expression using beta-actin as the reference gene. We found that automated capillary gel electrophoresis with multicolor detection is a rapid, accurate and highly reproducible method for separation and quantification of PCR-amplified cDNA.

Chemical stability and human plasma pharmacokinetics of reduced folates

The in vitro stability and plasma pharmacokinetics of 5,10-methylenetetrahydrofolic acid (CH2FH4), tetrahydrofolic acid (FH4), 5-methyltetrahydrofolic acid (CH3FH4), and 5-formyltetrahydrofolic acid (5-CHOFH4) were studied in view of their potential usefulness in cancer chemotherapy. Analysis of reduced folates was done on a high-performance liquid chromatography (HPLC) system. The high sensitivity of FH4 and CH2FH4 to oxidation can be circumvented by use of high concentrations of the folates, addition of ascorbate, and by thorough exclusion of atmospheric O2. Intravenous injection of 200 mg FH4 or CH2FH4 resulted in average peak concentrations of 69.2 +/- 3.2 nmol/ml and 46.3 +/- 2.6 nmol/ml, respectively. The plasma concentration curves support the concept that these highly oxygen-sensitive reduced folates can be reliably administered as pharmaceuticals to cancer patients through the use of a suitable air-occlusive system for their preparation and administration.

Determination of reduced folates in tumor and adjacent mucosa of colorectal cancer patients using LC-MS/MS.

A liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS) method has been developed for the determination of 5,10-methylenetetrahydrofolate (methyleneTHF), tetrahydrofolate (THF) and 5-methyltetrahydrofolate (methylTHF) in colorectal mucosa and tumor tissues. The folate extraction method includes homogenization, heat and folate conjugase treatment to hydrolyze polyglutamyl folate to monoglutamyl folate. Before analysis on LC-MS/MS, simple and fast sample purification with ultrafiltration (molecular weight cut-off membrane, 10 kDa) was performed. Folates were detected and quantified using positive electrospray. The method described in the present paper was successfully applied to determine the level of three folate monoglutamates in mucosa and tumor samples from 77 colorectal cancer patients, starting from a limited amount of tissue. The results showed that the LC-MS/MS method has a great advantage over other previously used methods because of its high sensitivity and selectivity. Significantly higher levels of methyleneTHF and THF were found in tumor compared with matched mucosa tissues. Folate levels in adjacent mucosa were associated with tumor location, age and gender. The correlation between folate levels and tumor site further strengthens the fact that development of right- and left-sided tumors follows different pathways.

Rapid Quantitative PCR Determination of Relative Gene Expression in Tumor Specimens Using High-Pressure Liquid Chromatography

A reverse transcriptase polymerase chain reaction (rt-PCR) for quantification of gene expression has been optimized for analysis of folylpolyglutamate synthase (FPGS) and thymidylate synthase (TS), using β-actin as an internal standard (house-keeping gene). Total RNA was isolated from tumor tissue, reversely transcribed to cDNA and PCR amplified with primers specific for TS, FPGS and β-actin in separate vials. PCR products were separated and quantified by high-pressure liquid chromatography (HPLC) without addition of radioactive or fluorescent markers, which minimizes labor and occupational hazards. The day-to-day variation in the HPLC analysis was 2.7% and the within sample variations for rt-PCR/HPLC analysis of TS and FPGS were 18.5% for both assays. This method provides a tool for convenient gene expression analysis in clinical biopsies.