Increasing Dosage of Leucovorin Results in Pharmacokinetic and Gene Expression Differences When Administered as Two-Hour Infusion or Bolus Injection to Patients with Colon Cancer.

The combination of 5-fluorouracil (5-FU) and leucovorin (LV) forms the chemotherapy backbone for patients with colorectal cancer. However, the LV administration is often standardized and not based on robust scientific data. To address these issues, a randomized pharmacokinetics study was performed in patients with colon cancer. Thirty patients were enrolled, receiving 60, 200 or 500 mg/m2 LV as a single two-hour infusion. Blood, tumor, mucosa, and resection margin biopsies were collected. Folate concentrations were analyzed with LC-MS/MS and gene expression with qPCR. Data from a previous study where patients received LV as bolus injections were used as comparison. Saturation of methylenetetrahydrofolate (MeTHF) and tetrahydrofolate (THF) levels was seen after two-hour infusion and polyglutamated MeTHF + THF levels in tumors decreased with increasing LV dosage. The decrease was associated with decreased FPGS and increased GGH expression, which was not observed after LV bolus injection. In the bolus group, results indicate activation of a metabolic switch possibly promoting TYMS inhibition in response to 5-FU. Different metabolic mechanisms appear to be induced when LV is administered as infusion and bolus injection. Since maximal inhibition of TYMS by the 5-FU metabolite 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) requires excess polyglutamated MeTHF, the results point in favor of the bolus regimen.

Pharmacokinetics of preoperative intraperitoneal 5-FU in patients with pancreatic ductal adenocarcinoma.

PURPOSE: The aim was to investigate the pharmacokinetics of preoperatively administered intraperitoneal (IP) 5-FU in patients with resectable pancreatic ductal adenocarcinoma (PDAC) by analyzing levels of 5-FU and target metabolites in peritoneal fluid, plasma, liver, lymph nodes, pancreatic tumour, and pancreatic tissue. These results were correlated to expression of genes encoding enzymes of the 5-FU pathway and cell membrane transporters of 5-FU and FdUMP. METHODS: Twenty-two patients with PDAC were treated with IP 5-FU before surgery. The postoperative treatment followed a routine clinical protocol. 5-FU and its metabolites were analyzed by LC-MS/MS. The expression of genes encoding enzymes and transporters in the 5-FU pathway was analyzed by qPCR. RESULTS: After IP treatment, 5-FU could be detected in plasma, lymph nodes, liver, pancreatic tumour, and pancreatic tissue. The highest 5-FU concentration was found in the liver, also expressing high levels of the 5-FU transporter OAT2. 5-FU was converted to active FdUMP in all tissues and the highest concentration was measured in lymph nodes, liver and pancreatic tumour (18.5, 6.1 and 6.7 pmol/g, respectively). There was a correlation between the FdUMP and dUr levels in lymph nodes (r = 0.70, p = 0.0076). In tumours, there was an association between OAT2 expression and FdUMP concentration. CONCLUSION: The study shows uptake of IP 5-FU and drug metabolism to active FdUMP in pancreatic tumour, liver, and lymph nodes. Extended studies are warranted to evaluate the IP route for 5-FU administration in PDAC patients.

Plasma deoxyuridine as a surrogate marker for toxicity and early clinical response in patients with metastatic colorectal cancer after 5-FU-based therapy in combination with arfolitixorin.

PURPOSE: The aim was to explore the correlation between increasing doses of [6R]-5,10-methylenetetrahydrofolate (arfolitixorin) and plasma concentrations of deoxyuridine (dUr) in patients with metastatic colorectal cancer (mCRC), subjected to 5-fluorouracil (5-FU)-based chemotherapy. The aim was further to investigate the possibility to predict toxicity and clinical response during treatment using gender, age, and plasma dUr as explanatory variables. METHODS: Thirty-three patients from the ISO-CC-005 phase I/IIa study, which investigated safety and tolerability of arfolitixorin at four dose levels, were included. Toxicity and clinical response were evaluated after 4 cycles of chemotherapy. Plasma dUr was quantified before (0 h) and 24 h after 5-FU administration at the first (C1) and fourth (C4) cycle using LC-MS/MS. Fit modelling was used to predict toxicity and clinical response. RESULTS: The dUr levels increased with increasing arfolitixorin dose. Females had higher total and haematological toxicity scores (p = 0.0004 and 0.0089, respectively), and needed dose reduction more often than males (p = 0.012). Fit modeling showed that gender and the dUr levels at C1-0 h and C4-24 h predicted total toxicity (p = 0.0011), whereas dUr C4-0 h alone was associated with gastrointestinal toxicity (p = 0.026). Haematological toxicity was predicted by gender and age (p = 0.0071). The haematological toxicity score in combination with the dUr levels at C1-24 h and C4-24 h predicted early clinical response (p = 0.018). CONCLUSION: The dUr level before and during administration of 5-FU and arfolitixorin was predictive for toxicity and early clinical response and could be a potential surrogate marker for thymidylate synthase inhibition in patients with mCRC. TRIAL REGISTRATION: NCT02244632, first posted on ClinicalTrials.gov on September 19, 2014.

Folate pathway genes linked to mitochondrial biogenesis and respiration are associated with outcome of patients with stage III colorectal cancer.

5-fluorouracil in combination with the folate leucovorin is the cornerstone in treatment of colorectal cancer. Transport of leucovorin into cells, and subsequent metabolic action, require expression of several genes. The aim was to analyze if tumoral expression of genes putatively involved in leucovorin transport, polyglutamation, or metabolism was associated with outcome of patients with stage III colorectal cancer treated with adjuvant chemotherapy. A total of 363 stage III colorectal cancer patients who received adjuvant bolus 5-fluorouracil + leucovorin alone, or in combination with oxaliplatin according to Nordic bolus regimes were included. Expression of 11 folate pathway genes was determined in tumors using quantitative real-time polymerase chain reaction and related to disease-free survival. The median follow-up time was 5 years. During follow-up, 114 (31%) patients suffered from recurrent disease. A high tumoral expression of the genes SLC46A1/PCFT, SLC19A1/RFC-1, ABCC3/MRP3, GGH, and MTHFD1L, which are involved in folate transport, polyglutamation, or metabolism, was associated with longer disease-free survival of the patients. Each of these genes either encodes mitochondrial enzymes or is being regulated by mitochondrial transcription factors. Expression of the SLC46A1/PCFT gene was most strongly associated with disease-free survival, regardless of treatment regimen. In conclusion, the results show that expression of folate pathway genes are associated with outcome of colorectal cancer patients treated with adjuvant 5-fluorouracil in combination with leucovorin. A prospective study needs to be conducted to determine if expression of these genes can be used to predict response to leucovorin and other folates that are now being tested in clinical studies. Moreover, there seems to be a link between folate metabolism and mitochondrial biogenesis and respiration that deserves further exploration.

Relationship between folate concentration and expression of folate-associated genes in tissue and plasma after intraoperative administration of leucovorin in patients with colorectal cancer.

PURPOSE: The aim of study was to investigate the relationship between folate concentration and expression of folate-associated genes in tumour, mucosa and plasma of patients with colorectal cancer, after intraoperative administration of bolus leucovorin (LV). METHODS: Eighty patients were randomized into four groups to receive 0, 60, 200, or 500 mg/m2 LV, respectively. Tissue and plasma folate concentrations were assessed by LC-MS/MS. Gene expression of ABCC3/MRP3, FPGS, GGH, MTHFD1L, SLC46A1/PCFT, and SLC19A1/RFC-1 was determined using quantitative PCR. RESULTS: The folate concentration in tumour increased with increasing dosage of LV. Half of the patients treated with 60 mg/m2 did not reach a level above the levels of untreated patients. A significant correlation between folate concentration in tumour and mucosa was found in untreated patients, and in the group treated with 60 mg/m2 LV. The 5-MTHF/LV ratio correlated negatively with folate concentration in mucosa, whereas a positive correlation was found in tumour of patients who received 200 or 500 mg/m2 LV. A positive correlation was found between folate concentration and expression of all genes, except MTHFD1L, in patients who received LV. There was a negative correlation between 5-MTHF concentration in plasma of untreated patients and expression of GGH and SLC46A1/PCFT in tumour. CONCLUSIONS: The results indicate the possibility of using the individual plasma 5-MTHF/LV ratio after LV injection as a surrogate marker for tissue folate concentration. Expression of several folate-associated genes is associated with folate concentration in tissue and plasma and may become useful when predicting response to LV treatment.

Folates as adjuvants to anticancer agents: Chemical rationale and mechanism of action.

Folates have been used with cytotoxic agents for decades and today they are used in hundreds of thousands of patients annually. Folate metabolism is complex. In the treatment of cancer with 5-fluorouracil, the administration of folates mechanistically leads to the formation of [6R]-5,10-methylene-tetrahydrofolate, and the increased concentration of this molecule leads to stabilization of the ternary complex comprising thymidylate synthase, 2'-deoxy-uridine-5'-monophosphate, and [6R]-5,10-methylene-tetrahydrofolate. The latter is the only natural folate that can bind directly in the ternary complex, with other folates requiring metabolic activation. Modulation of thymidylate synthase activity became central in the study of folate/cytotoxic combinations and, despite wide use, research into the folate component was neglected, leaving important questions unanswered. This article revisits the mechanisms of action of folates and evaluates commercially available folate derivatives in the light of current research. Better genomic insight and availability of new analytical techniques and stable folate compounds may open new avenues of research and therapy, ultimately bringing increased clinical benefit to patients.

Reduced folate and serum vitamin metabolites in patients with rectal carcinoma: an open-label feasibility study of pemetrexed with folic acid and vitamin B12 supplementation.

The objectives of this single-center, open-label, phase II study were to evaluate (a) the feasibility and safety of neoadjuvant administration of pemetrexed with oral folic acid and vitamin B12 (FA/B12) in newly diagnosed patients with resectable rectal cancer and (b) intracellular and systemic vitamin metabolism. Patients were treated with three cycles of pemetrexed (500 mg/m, every 3 weeks) and FA/B12 before surgery. The reduced folates tetrahydrofolate, 5-methyltetrahydrofolate, and 5,10-methylenetetrahydrofolate were evaluated from biopsies in tumor tissue and in adjacent mucosa. Serum levels of homocysteine, cystathionine, and methylmalonic acid were also measured. All 37 patients received three cycles of pemetrexed; 89.2% completed their planned dosage within a 9-week feasibility time frame. Neither dose reductions nor study drug-related serious adverse events were reported. Reduced folate levels were significantly higher in tumor tissue compared with adjacent mucosa at baseline. After FA/B12 administration, tissue levels of reduced folates increased significantly and remained high during treatment in both tumor and mucosa until surgery. Serum levels of cystathionine increased significantly compared with baseline after FA/B12 administration, but then decreased, fluctuating cyclically during pemetrexed therapy. Homocysteine and methylmalonic acid levels decreased significantly after FA/B12 administration, and remained below baseline levels during the study. These results indicate that administration of three neoadjuvant cycles of single-agent pemetrexed, every 3 weeks, with FA/B12 in patients with resectable rectal cancer is feasible and tolerable. Tissue and serum vitamin metabolism results demonstrate the influence of pemetrexed and FA/B12 on vitamin metabolism and warrant further study.

Phase 1 dose de-escalation trial of the endogenous folate [6R]-5,10-methylene tetrahydrofolate in combination with fixed-dose pemetrexed as neoadjuvant therapy in patients with resectable rectal cancer.

BACKGROUND: Modufolin® ([6R]-5,10-methylene tetrahydrofolate; [6R]-MTHF) is an endogenous biomodulator that is being developed as an alternative to leucovorin, a folate prodrug used in the treatment of colorectal cancer. The objective of this phase 1 dose de-escalation trial was to estimate the minimum tolerated dose of [6R]-MTHF to be used in combination with pemetrexed 500 mg/m(2) in the neoadjuvant treatment of patients with rectal cancer. METHODS: Adult patients (≥18 years) with resectable rectal adenocarcinoma were allocated to [6R]-MTHF doses of 500, 100, 50, and 10 mg/m(2) in combination with pemetrexed 500 mg/m(2). [6R]-MTHF was administered as an intravenous (i.v.) bolus injection 1 week prior to the first dose of pemetrexed and then once weekly for 9 weeks; pemetrexed was administered by i.v. infusion once every 21 days for three cycles. RESULTS: Twenty-four patients (mean [SD] age, 63.1 [12.9] years) were enrolled in the study. A total of 72 treatment-related adverse events (AEs) were reported, of which the most common were fatigue (n = 17; 23.6 %), nausea (n = 10; 13.9 %), and diarrhea (n = 5; 6.9 %). The incidence of treatment-related AEs by [6R]-MTHF dose level (500, 100, 50, 10 mg/m(2)) was 11.1 % (n = 8), 13.9 % (n = 10), 45.8 % (n = 33), and 29.2 % (n = 21), respectively. There were no dose-limiting toxicities, and only two (2.8 %) treatment-related AEs were grade 3 in severity. Of the 11 serious AEs reported, none were considered to be related to [6R]-MTHF treatment. CONCLUSIONS: The results of this phase 1 study indicate that the estimated minimum tolerated dose of [6R]-MTHF was 100 mg/m(2) once weekly in combination with pemetrexed 500 mg/m(2). The low toxicity profile of [6R]-MTHF supports its further evaluation as a component of systemic chemotherapy in the management of colon and rectal cancer.

Expression of Folate Pathway Genes in Stage III Colorectal Cancer Correlates with Recurrence Status Following Adjuvant Bolus 5-FU-Based Chemotherapy.

Colorectal cancer is commonly treated with 5-fluorouracil and 5-formyltetrahydrofolate (leucovorin). Metabolic action of leucovorin requires several enzymatic steps that are dependent on expression of corresponding coding genes. To identify folate pathway genes with possible impact on leucovorin metabolism, a retrospective study was performed on 193 patients with stage III colorectal cancer. Relative expression of 22 genes putatively involved in leucovorin transport, polyglutamation and metabolism was determined in tumor and mucosa samples using quantitative real-time polymerase chain reaction. After surgery, patients received adjuvant 5-fluorouracil-based bolus chemotherapy with leucovorin during six months, and were followed for 3 to 5 years. Cox regression analysis showed that high tumoral expression of the genes SLC46A1/PCFT (proton-coupled folate transporter) and SLC19A1/RFC-1 (reduced folate carrier 1) correlated significantly (p < 0.001 and p < 0.01, respectively) with a decreased risk of recurrent disease, measured as disease-free survival (DFS). These two genes are involved in the transport of folates into the cells and each functions optimally at a different pH. We conclude that SLC46A1/PCFT and SLC19A1/RFC-1 are associated with DFS of patients with colorectal cancer and hypothesize that poor response to 5-fluorouracil plus leucovorin therapy in some patients may be linked to low expression of these genes. Such patients might need a more intensified therapeutic approach than those with high gene expression. Future prospective studies will determine if the expression of any of these genes can be used to predict response to leucovorin.

A pharmacokinetic and pharmacodynamic investigation of Modufolin® compared to Isovorin® after single dose intravenous administration to patients with colon cancer: a randomized study.

PURPOSE: Leucovorin is commonly used as folate supplement in 5-fluorouracil-based chemotherapy, but needs to be converted to active 5,10-methylenetetrahydrofolate (methyleneTHF) intracellularly. This provides for interindividual differences. MethyleneTHF has recently been developed into the stable, distributable drug, Modufolin®. The aim was to compare the concentration of folate metabolites in tumor, mucosa, and plasma of patients with colon cancer after administration of Modufolin® or Isovorin® (levo-leucovorin). METHODS: Thirty-two patients scheduled for colon resection were randomized to receive Modufolin® or Isovorin® at dosage of 60 or 200 mg/m². The study drug was given as one i.v. bolus injection after anesthesia. Plasma was collected for pharmacokinetic (PK) analysis before, during, and after surgery. Tissue biopsies were collected at surgery. Folate metabolites were analyzed by LC-MS/MS. RESULTS: MethyleneTHF and THF concentrations were significantly higher in mucosa (p < 0.01, both dosages) and tumors (p < 0.01, 200 mg/m²) after Modufolin® as compared to Isovorin® administration. The results correlated with PK observations. The Modufolin® to Isovorin® C(max) ratio for methyleneTHF was 113 at 200 mg/m² and 52 at 60 mg/m²; the AUC(last) ratios were 17 and 9, respectively. The THF plasma concentrations were also higher after Modufolin® administration (C(max) ratio 23, AUC(last) ratio 13 at 200 mg/m²; C(max) ratio 15, AUC(last) ratio 11 at 60 mg/m²). CONCLUSION: Modufolin® administration resulted in significantly higher methyleneTHF levels than Isovorin® and may potentially increase the efficacy of 5-fluorouracil-based chemotherapy. The results encourage further evaluation of Modufolin® as a substitute to Isovorin® including the potential clinical benefits.

Folate levels measured by LC-MS/MS in patients with colorectal cancer treated with different leucovorin dosages.

PURPOSE: Calcium folinate (leucovorin), which is converted in vivo into biologically active folate, enhances the potency of 5-fluorouracil (5-FU)-based chemotherapy in colorectal cancer. A common dosage of leucovorin in adjuvant and palliative settings is 60 mg/m(2). The aim was to determine the levels of tetrahydrofolate (THF), 5,10-methylenetetrahydrofolate (methyleneTHF), and 5-methyltetrahydrofolate (methylTHF) in tumour and mucosa of colorectal cancer patients who received different dosages of leucovorin intravenously at time of surgery. METHODS: Eighty patients scheduled for colorectal resection with indication of colorectal cancer were randomised into four groups to receive leucovorin at 0, 60, 200, or 500 mg/m(2), respectively. Blood samples were taken 10 and 30 min after leucovorin administration. Biopsy samples from tumour and mucosa were collected and snap-frozen at surgery. The levels of THF, methyleneTHF, and methylTHF in tumour and mucosa were assessed by liquid chromatography electrospray ionisation tandem mass spectrometry (LC-MS/MS) and the results were related to clinical diagnosis and therapeutic regimens. RESULTS: The folate levels in tissue revealed extensive inter-individual variability. The mean methyleneTHF value for the four treatment groups were 880, 1,769, 3,024 and 3,723 pmol/gww. Only half of the patients who received 60 mg/m(2) leucovorin had higher levels of methyleneTHF in tumour than patients who received 0 mg/m(2) leucovorin. Rectal cancer patients had significantly lower levels of methyleneTHF compared with colon cancer patients. CONCLUSIONS: There was a large inter-patient variability of tissue folate levels in colorectal cancer patients after supplementation with leucovorin at standardised dosage. High leucovorin doses were needed to exceed baseline methyleneTHF values, especially in rectal cancer patients. The results indicate that the standardised leucovorin dose may be insufficient to attain the full antitumour effect of 5-FU. Further studies are needed to establish whether higher dosage yields a better treatment response.

Pretherapeutic uracil and dihydrouracil levels in saliva of colorectal cancer patients are associated with toxicity during adjuvant 5-fluorouracil-based chemotherapy.

PURPOSE: 5-fluorouracil (5-FU) competes with uracil (Ura) as a substrate for dihydropyrimidine dehydrogenase (DPD). Low DPD activity impairs breakdown of Ura to dihydrouracil (UH2) and is associated with toxicity during 5-FU-based chemotherapy. Calculation of the 5-FU dose is based on body surface area, and new tools are needed to individualize treatment. The aim of study was to measure Ura and UH2 in saliva of patients with colorectal cancer and relate levels to treatment-induced toxicity. METHODS: Saliva was collected from 73 patients with stage III colorectal cancer prior to adjuvant 5-FU-based treatment. Ura and UH2 were analyzed by a column-switching HPLC method. Toxicity was evaluated before each treatment cycle and the highest grade was noted at end of treatment. RESULTS: Toxicity was more common and severe among women compared with men. The Ura and UH2 concentrations in saliva were 5.0 ± 6.8 and 5.0 ± 4.0 nmol/ml, respectively. The UH2/Ura ratio was lower in women compared with men (1.2 ± 1.0 and 2.2 ± 2.5, respectively, p = 0.0026). Patients who needed to reduce the drug dose during treatment (or terminate treatment) due to toxicity had a lower ratio (1.3 ± 0.85) compared to patients who completed treatment without dose reduction (4.1 ± 4.3, p < 0.0001). CONCLUSION: Sampling of saliva is a quick, noninvasive, safe and painless process that gives information about patients Ura and UH2 levels prior to chemotherapeutical treatment. This information may be useful in order to predict and prevent occurrence of treatment-related toxicities which otherwise may limit drug administration.

Folate Levels and Polymorphisms in the Genes MTHFR, MTR, and TS in Colorectal Cancer.

AIM: The aim of the study was to explore and describe the effect of polymorphisms in folate-associated genes regarding the levels of different folate forms and their distribution in tumors and mucosa in patients with colorectal cancer. MATERIALS AND METHODS: Tumor and mucosa tissues from 53 patients with colorectal cancer were analyzed. The concentrations of tetrahydrofolate (THF), 5-methylTHF, and 5,10-methyleneTHF were measured by liquid chromatography-mass spectrometry. Genotyping of polymorphisms in the folate-associated genes methylenetetrahydrofolate reductase (MTHFR, C677T), methionine synthase (MTR, A2756G), and thymidylate synthase (TS, 5'-TSER 28 bp tandem repeat and 3'-TSUTR 6 bp deletion/insertion), were done by real-time polymerase chain reaction. Folate levels and distributions were determined in the total patient cohort and after subgrouping by genotypes. RESULTS: The total folate level, as well as the THF and 5,10-methyleneTHF levels, were significantly higher in the tumor compared with mucosa tissue (P = 0.030, 0.031, and 0.015, respectively). The individual variation in folate levels in both tumor and mucosa were larger than the variation found when the patients were subgrouped by the gene polymorphisms. No significant differences in the mean concentration of any folate in the mucosa or tumor tissue were found in relation to the analyzed polymorphisms. The percentage level of 5,10-methyleneTHF in tumors was highest in patients with the MTHFR 677 CC genotype, and lowest in patients with the TT genotype (P = 0.033). A significantly lower percentage level of the 5,10-methyleneTHF level was found in tumors of patients with the 5'-TSER 3R/3R genotype (P = 0.0031). CONCLUSION: A significant difference was found between the percentage level of 5,10-methyleneTHF in tumor tissues in relation to the MTHFR C677T and 5'-TSER 28 bp repeat polymorphisms. However, no differences were found in the actual tissue folate levels, or in their distribution, in relation to the polymorphisms in the MTHFR, MTR, or TS genes. These findings could be of importance for further research in the field by explaining some of the difficulties of obtaining reproducible and uniform results when using a few selected polymorphisms as predictive markers.

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.

Thymidine Phosphorylase Gene Expression in Stage III Colorectal Cancer.

BACKGROUND: The thymidine phosphorylase (TP) enzyme has several tumor-promoting functions. The aim of this study was to explore TP gene expression in relation to clinical and histopathological data obtained from patients with stage III colorectal cancer. METHODS AND RESULTS: TP gene expression was analyzed by real-time quantitative PCR in tumor and mucosa samples from 254 patients. TP gene expression in tumors correlated with lymph node staging, with higher expression relating to a higher number of positive nodes and a worse N-stage. Higher TP expression was also associated with a worse histological tumor grade. Patients with rectal cancer had significantly higher TP expression in mucosa and tumors compared with patients having colon cancer. CONCLUSION: Higher intratumoral TP expression appears to be related to a worse N stage, and thus, with a worse prognosis. TP gene expression measured in a preoperative biopsy could be of interest in preoperative staging.

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.

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

BACKGROUND: 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 (UH(2)) seem promising for clinical use. METHODS: Urine was collected from 56 untreated volunteers and 143 patients with CRC before adjuvant treatment. Ura and UH(2) were analyzed using a column-switching high-performance liquid chromatography method that incorporates reversed-phase and cation-exchange columns. Ura, UH(2), and UH(2)/Ura levels were related to toxicity. RESULTS: Ura and UH(2) in patients were not different from controls. UH(2) was significantly higher in women compared with men. The UH(2)/Ura ratio, however, did not differ according to sex. Low UH(2) and UH(2)/Ura levels were associated with diarrhea in men. Women experiencing thrombocytopenia had significantly higher Ura compared with women with no thrombocytopenia. The UH(2)/Ura ratio correlated negatively with total toxicity score in men (r = -0.39, P = .020). CONCLUSION: Pretherapeutic Ura and UH(2) levels per se may be related to risk of side effects during adjuvant 5-FU-based treatment, whereas the UH(2)/Ura ratio may not always reveal such a risk. Sex is a strong risk factor for toxicity, showing the importance of evaluating male and female patients separately.

Gene polymorphisms MTHFRC677T and MTRA2756G as predictive factors in adjuvant chemotherapy for stage III colorectal cancer.

BACKGROUND: The aim of this study was to explore the effect in stage III colorectal cancer of functional gene polymorphisms methylenetetrahydrofolate reductase (MTHFR C677T) and methionine synthase (A2756G), in the folate metabolism on outcome and risk of toxicity for adjuvant chemotherapy. A secondary aim was to investigate any possible interdependency between the two genes. PATIENTS AND METHODS: one hundred and fifty randomly chosen patients with stage III colorectal cancer, treated with adjuvant chemotherapy, were genotyped by real-time PCR. Patient and treatment data were retrieved and assessed for demography, pathology, chemotherapy tolerability and survival after adjuvant therapy. The polymorphisms were studied separately and in combination to discover possible interactions. RESULTS: Patients with MTHFR 677 CC genotype carried lower risks of suffering from nausea (p=0.027), parasthesia (p=0.0042) and need for dose reduction (p=0.025). The CC genotype was also associated with better survival (p<0.034). There was interdependency with MTR A2756G. Patients with MTR AG/GG in combination with MTHFR CT/TT genotypes carried the highest risk of side-effects. CONCLUSION: Functional polymorphisms of MTHFR C677T and MTR A2756G can affect outcome and risk of toxicity during adjuvant chemotherapy in stage III colorectal cancer. Their possible interdependence brings attention to the function of folate metabolism overall regarding its association with 5-fluoruracil related toxicity. Our results could explain some of the difficulties of obtaining reproducible and uniform results when using single polymorphisms as predictive markers.

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.

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.