Elevated EVL Methylation Level in the Normal Colon Mucosa Is a Potential Risk Biomarker for Developing Recurrent Adenomas.

BACKGROUND: Individuals with adenomatous colorectal polyps undergo repeated colonoscopy surveillance to identify and remove metachronous adenomas. However, many patients with adenomas do not develop recurrent adenomas. Better methods to evaluate who benefits from increased surveillance are needed. We evaluated the use of altered EVL methylation as a potential biomarker for risk of recurrent adenomas. METHODS: Patients with ≥1 colonoscopy had EVL methylation (mEVL) measured with an ultra-accurate methylation-specific droplet digital PCR assay on normal colon mucosa. The association between EVL methylation levels and adenoma or colorectal cancer was evaluated using three case/control definitions in three models: unadjusted (model 1), adjusting for baseline characteristics (model 2), and an adjusted model excluding patients with colorectal cancer at baseline (model 3). RESULTS: Between 2001 and 2020, 136 patients were included; 74 healthy patients and 62 patients with a history of colorectal cancer. Older age, never smoking, and baseline colorectal cancer were associated with higher levels of mEVL (P ≤ 0.05). Each log base 10 difference in mEVL was associated with an increased risk of adenoma(s) or cancer at/after baseline for model 1 [OR, 2.64; 95% confidence interval (CI), 1.09-6.36], and adenoma(s) or cancer after baseline for models 1 (OR, 2.01; 95% CI, 1.04-3.90) and model 2 (OR, 3.17; 95% CI, 1.30-7.72). CONCLUSIONS: Our results suggest that EVL methylation level detected in the normal colon mucosa has the potential to be a biomarker for monitoring the risk for recurrent adenomas. IMPACT: These findings support the potential utility of EVL methylation for improving the accuracy for assigning risk for recurrent colorectal adenomas and cancer.

Cohort profile: Biomarkers related to folate-dependent one-carbon metabolism in colorectal cancer recurrence and survival - the FOCUS Consortium.

PURPOSE: The overarching goal of the FOCUS (biomarkers related to folate-dependent one-carbon metabolism in colorectal cancer (CRC) recurrence and survival) Consortium is to unravel the effect of folate and folate-mediated one-carbon metabolism (FOCM) biomarkers on CRC prognosis to provide clinically relevant advice on folate intake to cancer patients and define future tertiary prevention strategies. PARTICIPANTS: The FOCUS Consortium is an international, prospective cohort of 2401 women and men above 18 years of age who were diagnosed with a primary invasive non-metastatic (stages I-III) CRC. The consortium comprises patients from Austria, two sites from the Netherlands, Germany and two sites from the USA. Patients are recruited after CRC diagnosis and followed at 6 and 12 months after enrolment. At each time point, sociodemographic data, data on health behaviour and clinical data are collected, blood samples are drawn. FINDINGS TO DATE: An increased risk of cancer recurrences was observed among patients with higher compared with lower circulating folic acid concentrations. Furthermore, specific folate species within the FOCM pathway were associated with both inflammation and angiogenesis pathways among patients with CRC. In addition, higher vitamin B6 status was associated with better quality of life at 6 months post-treatment. FUTURE PLANS: Better insights into the research on associations between folate and FOCM biomarkers and clinical outcomes in patients with CRC will facilitate the development of guidelines regarding folate intake in order to provide clinically relevant advice to patients with cancer, health professionals involved in patient care, and ultimately further tertiary prevention strategies in the future. The FOCUS Consortium offers an excellent infrastructure for short-term and long-term research projects and for combining additional biomarkers and data resulting from the individual cohorts within the next years, for example, microbiome data, omics and multiomics data or CT-quantified body composition data.

Higher vitamin B6 status is associated with improved survival among patients with stage I-III colorectal cancer.

BACKGROUND: Folate-mediated 1-carbon metabolism requires several nutrients, including vitamin B6. Circulating biomarker concentrations indicating high vitamin B6 status are associated with a reduced risk of colorectal cancer (CRC). However, little is known about the effect of B6 status in relation to clinical outcomes in CRC patients. OBJECTIVES: We investigated survival outcomes in relation to vitamin B6 status in prospectively followed CRC patients. METHODS: A total of 2031 patients with stage I-III CRC participated in 6 prospective patient cohorts in the international FOCUS (folate-dependent 1-carbon metabolism in colorectal cancer recurrence and survival) Consortium. Preoperative blood samples were used to measure vitamin B6 status by the direct marker pyridoxal 5'-phosphate (PLP), as well as the functional marker HK-ratio (HKr)[3'-hydroxykynurenine: (kynurenic acid + xanthurenic acid + 3'-hydroxy anthranilic acid + anthranilic acid)]. Using Cox proportional hazards regression, we examined associations of vitamin B6 status with overall survival (OS), disease-free survival (DFS), and risk of recurrence, adjusted for patient age, sex, circulating creatinine concentrations, tumor site, stage, and cohort. RESULTS: After a median follow-up of 3.2 y for OS, higher preoperative vitamin B6 status as assessed by PLP and the functional marker HKr was associated with 16-32% higher all-cause and disease-free survival, although there was no significant association with disease recurrence (doubling in PLP concentration: HROS, 0.68; 95% CI: 0.59, 0.79; HRDFS, 0.84; 95% CI: 0.75, 0.94; HRRecurrence, 0.96; 95% CI: 0.84, 1.09; HKr: HROS, 2.04; 95% CI: 1.67, 2.49; HRDFS, 1.56; 95% CI: 1.31, 1.85; HRRecurrence, 1.21; 95% CI: 0.96,1. 52). The association of PLP with improved OS was consistent across colorectal tumor site (right-sided colon: HROS, 0.75; 95% CI: 0.59, 0.96; left-sided colon: HROS, 0.71; 95% CI: 0.55, 0.92; rectosigmoid junction and rectum: HROS, 0.61; 95% CI: 0.47, 0.78). CONCLUSION: Higher preoperative vitamin B6 status is associated with improved OS among stage I-III CRC patients.

Circulating B-vitamin biomarkers and B-vitamin supplement use in relation to quality of life in patients with colorectal cancer: results from the FOCUS consortium.

BACKGROUND: B vitamins have been associated with the risk and progression of colorectal cancer (CRC), given their central roles in nucleotide synthesis and methylation, yet their association with quality of life in established CRC is unclear. OBJECTIVES: To investigate whether quality of life 6 months postdiagnosis is associated with: 1) circulating concentrations of B vitamins and related biomarkers 6 months postdiagnosis; 2) changes in these concentrations between diagnosis and 6 months postdiagnosis; 3) B-vitamin supplement use 6 months postdiagnosis; and 4) changes in B-vitamin supplement use between diagnosis and 6 months postdiagnosis. METHODS: We included 1676 newly diagnosed stage I-III CRC patients from 3 prospective European cohorts. Circulating concentrations of 9 biomarkers related to the B vitamins folate, riboflavin, vitamin B6, and cobalamin were measured at diagnosis and 6 months postdiagnosis. Information on dietary supplement use was collected at both time points. Health-related quality of life (global quality of life, functioning scales, and fatigue) was assessed by the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire 6 months postdiagnosis. Confounder-adjusted linear regression analyses were performed, adjusted for multiple testing. RESULTS: Higher pyridoxal 5'-phosphate (PLP) was cross-sectionally associated with better physical, role, and social functioning, as well as reduced fatigue, 6 months postdiagnosis. Associations were observed for a doubling in the hydroxykynurenine ratio [3-hydroxykynurenine: (kynurenic acid + xanthurenic acid + 3-hydroxyanthranilic acid + anthranilic acid); an inverse marker of vitamin B6] and both reduced global quality of life (ß = -3.62; 95% CI: -5.88, -1.36) and worse physical functioning (ß = -5.01; 95% CI: -7.09, -2.94). Dose-response relations were observed for PLP and quality of life. No associations were observed for changes in biomarker concentrations between diagnosis and 6 months. Participants who stopped using B-vitamin supplements after diagnosis reported higher fatigue than nonusers. CONCLUSIONS: Higher vitamin B6 status was associated with better quality of life, yet limited associations were observed for the use of B-vitamin supplements. Vitamin B6 needs further study to clarify its role in relation to quality of life.

Circulating Folate and Folic Acid Concentrations: Associations With Colorectal Cancer Recurrence and Survival.

BACKGROUND: Folates, including folic acid, may play a dual role in colorectal cancer development. Folate is suggested to be protective in early carcinogenesis but could accelerate growth of premalignant lesions or micrometastases. Whether circulating concentrations of folate and folic acid, measured around time of diagnosis, are associated with recurrence and survival in colorectal cancer patients is largely unknown. METHODS: Circulating concentrations of folate, folic acid, and folate catabolites p-aminobenzoylglutamate and p-acetamidobenzoylglutamate were measured by liquid chromatography-tandem mass spectrometry at diagnosis in 2024 stage I-III colorectal cancer patients from European and US patient cohort studies. Multivariable-adjusted Cox proportional hazard models were used to assess associations between folate, folic acid, and folate catabolites concentrations with recurrence, overall survival, and disease-free survival. RESULTS: No statistically significant associations were observed between folate, p-aminobenzoylglutamate, and p-acetamidobenzoylglutamate concentrations and recurrence, overall survival, and disease-free survival, with hazard ratios ranging from 0.92 to 1.16. The detection of folic acid in the circulation (yes or no) was not associated with any outcome. However, among patients with detectable folic acid concentrations (n = 296), a higher risk of recurrence was observed for each twofold increase in folic acid (hazard ratio = 1.31, 95% confidence interval = 1.02 to 1.58). No statistically significant associations were found between folic acid concentrations and overall and disease-free survival. CONCLUSIONS: Circulating folate and folate catabolite concentrations at colorectal cancer diagnosis were not associated with recurrence and survival. However, caution is warranted for high blood concentrations of folic acid because they may increase the risk of colorectal cancer recurrence.

Rare loss of function variants in candidate genes and risk of colorectal cancer.

Although ~ 25% of colorectal cancer or polyp (CRC/P) cases show familial aggregation, current germline genetic testing identifies a causal genotype in the 16 major genes associated with high penetrance CRC/P in only 20% of these cases. As there are likely other genes underlying heritable CRC/P, we evaluated the association of variation at novel loci with CRC/P. We evaluated 158 a priori selected candidate genes by comparing the number of rare potentially disruptive variants (PDVs) found in 84 CRC/P cases without an identified CRC/P risk-associated variant and 2440 controls. We repeated this analysis using an additional 73 CRC/P cases. We also compared the frequency of PDVs in select genes among CRC/P cases with two publicly available data sets. We found a significant enrichment of PDVs in cases vs. controls: 20% of cases vs. 11.5% of controls with ≥ 1 PDV (OR = 1.9, p = 0.01) in the original set of cases. Among the second cohort of CRC/P cases, 18% had a PDV, significantly different from 11.5% (p = 0.02). Logistic regression, adjusting for ancestry and multiple testing, indicated association between CRC/P and PDVs in NTHL1 (p = 0.0001), BRCA2 (p = 0.01) and BRIP1 (p = 0.04). However, there was no significant difference in the frequency of PDVs at each of these genes between all 157 CRC/P cases and two publicly available data sets. These results suggest an increased presence of PDVs in CRC/P cases and support further investigation of the association of NTHL1, BRCA2 and BRIP1 variation with CRC/P.

Dynamic plasma microRNAs are biomarkers for prognosis and early detection of recurrence in colorectal cancer.

BACKGROUND: Plasma microRNAs (miRNAs) are promising non-invasive biomarkers for colorectal cancer (CRC) prognosis. However, the published studies to date have yielded conflicting and inconsistent results for specific plasma miRNAs. METHODS: We have conducted a study using robust assays to assess a panel of nine miRNAs for CRC prognosis and early detection of recurrence. Plasma samples from 144 patients in a prospective CRC cohort study were collected at diagnosis, 6, 12, and 24 months after diagnosis. miRNAs were assayed by Taqman qRT-PCR to generate miRNA normalised copy numbers. RESULTS: Preoperative high plasma miRNA levels were associated with increased recurrence risk for miR-200b (HR [95% CI]=2.04 [1.00, 4.16], P=0.05), miR-203 (HR=4.2 [1.48, 11.93], P=0.007), miR-29a (HR=2.61 [1.34,5.07], P=0.005), and miR-31 (HR=4.03 [1.76, 9.24], P=0.001). Both plasma miR-31 (AUC: 0.717) and miR-29a (AUC: 0.703) could discriminate recurrence from these patients without recurrence. In addition, high levels of miR-31 during surveillance was associated with a three-fold increased risk of recurrence across all time points. Dynamic postoperative plasma miR-141 and 16 levels correlated with recurrence in the surveillance samples. CONCLUSIONS: Pre-operative plasma miR-29a, 200b, 203, and 31 are potential CRC prognosis biomarkers. In addition, dynamic postoperative miR-31, 141 and 16 levels are potential biomarkers for the early detection of recurrence during CRC surveillance.

MethyLight droplet digital PCR for detection and absolute quantification of infrequently methylated alleles.

Aberrant DNA methylation is a common epigenetic alteration found in colorectal adenomas and cancers and plays a role in cancer initiation and progression. Aberrantly methylated DNA loci can also be found infrequently present in normal colon tissue, where they seem to have potential to be used as colorectal cancer (CRC) risk biomarkers. However, detection and precise quantification of the infrequent methylation events seen in normal colon is likely beyond the capability of commonly used PCR technologies. To determine the potential for methylated DNA loci as CRC risk biomarkers, we developed MethyLight droplet digital PCR (ddPCR) assays and compared their performance to the widely used conventional MethyLight PCR. Our analyses demonstrated the capacity of MethyLight ddPCR to detect a single methylated NTRK3 allele from among more than 3125 unmethylated alleles, 25-fold more sensitive than conventional MethyLight PCR. The MethyLight ddPCR assay detected as little as 19 and 38 haploid genome equivalents of methylated EVL and methylated NTRK3, respectively, which far exceeded conventional MethyLight PCR (379 haploid genome equivalents for both genes). When assessing methylated EVL levels in CRC tissue samples, MethyLight ddPCR reduced coefficients of variation (CV) to 6-65% of CVs seen with conventional MethyLight PCR. Importantly, we showed the ability of MethyLight ddPCR to detect infrequently methylated EVL alleles in normal colon mucosa samples that could not be detected by conventional MethyLight PCR. This study suggests that the sensitivity and precision of methylation detection by MethyLight ddPCR enhances the potential of methylated alleles for use as CRC risk biomarkers.

Plasma 25-hydroxyvitamin D3, folate and vitamin B12 biomarkers among international colorectal cancer patients: a pilot study.

Vitamin D and folate are associated with decreased colorectal cancer risk and their association with colorectal cancer prognosis is under investigation. We assessed the levels of plasma 25-hydroxyvitamin D3 (25(OH)D3), folate and vitamin B12 in an international pilot study in order to determine variability of these biomarkers based on geographical location. Plasma 25(OH)D3, folate and vitamin B12 concentrations were measured in 149 invasive, newly diagnosed colorectal cancer cases from Heidelberg (Germany), Seattle (WA, USA), and Tampa (FL, USA) and in ninety-one age- and sex-matched controls. Their associations with potential predictors were assessed using multivariate linear regression analyses. Plasma 25(OH)D3, folate and vitamin B12 concentrations differed by location. Other predictors were season for 25(OH)D3 and tumour stage (vitamin B12). Season-corrected average 25(OH)D3 concentrations were higher in Heidelberg (31·7 ng/ml; range 11·0-83·0 ng/ml) than in Seattle (23·3 ng/ml; range 4·0-80·0 ng/ml) and Tampa (21·1 ng/ml; range 4·6-51·6 ng/ml). In Heidelberg, a strong seasonal variation was observed. Folate (11·1 ng/ml) and vitamin B12 (395 pg/ml) concentrations in Heidelberg were lower than those in Seattle (25·3 ng/ml and 740 pg/ml, respectively) and Tampa (23·8 ng/ml and 522 pg/ml, respectively). Differences in plasma 25(OH)D3 and folate concentrations between Heidelberg and the US sites were observed, probably reflecting variation in outdoor activities and sun-avoidance behaviour during summer as well as in folic acid fortification and supplement use. Intra-site differences at each study location were greater than between-location variability, suggesting that individual health behaviours play a significant role. Nevertheless, the intra-site differences we observed may be due to chance because of the limited sample size. Our pilot study illustrates the value of an international cohort in studying colorectal cancer prognosis to discern geographical differences in a broad range of exposures.

Maternal microchimerism in healthy adults in lymphocytes, monocyte/macrophages and NK cells.

During pregnancy some maternal cells reach the fetal circulation. Microchimerism (Mc) refers to low levels of genetically disparate cells or DNA. Maternal Mc has recently been found in the peripheral blood of healthy adults. We asked whether healthy women have maternal Mc in T and B lymphocytes, monocyte/macrophages and NK cells and, if so, at what levels. Cellular subsets were isolated after fluorescence activated cell sorting. A panel of HLA-specific real-time quantitative PCR assays was employed targeting maternal-specific HLA sequences. Maternal Mc was expressed as the genome equivalent (gEq) number of microchimeric cells per 100,000 proband cells. Thirty-one healthy adult women probands were studied. Overall 39% (12/31) of probands had maternal Mc in at least one cellular subset. Maternal Mc was found in T lymphocytes in 25% (7/28) and B lymphocytes in 14% (3/21) of probands. Maternal Mc levels ranged from 0.9 to 25.6 and 0.9 to 25.3 gEq/100,000 in T and B lymphocytes, respectively. Monocyte/macrophages had maternal Mc in 16% (4/25) and NK cells in 28% (5/18) of probands with levels from 0.3 to 36 and 1.8 to 3.2 gEq/100,000, respectively. When compared to fetal Mc, as assessed by quantification of male DNA in women with sons, maternal Mc was substantially less prevalent in all cellular subsets; fetal Mc prevalence in T and B lymphocytes, monocyte/macrophages and NK cells was 58, 75, 50 and 62% (P=0.01, P=0.005, P=0.04, P=0.05) respectively. In summary, maternal Mc was identified among lymphoid and myeloid compartments of peripheral blood in healthy adult women. Maternal Mc was less frequent than fetal Mc in all cellular subsets tested. Studies are needed to investigate the immunological effects and function of maternal Mc and to explore whether maternal Mc in cellular subsets has biological effects on her progeny.