Identification of DNA methylation markers for early detection of CRC indicates a role for nervous system-related genes in CRC.

PURPOSE: Colonoscopy and the fecal immunochemical test (FIT) are currently the most widely used screening modalities for colorectal cancer (CRC), however, both with their own limitations. Here we aim to identify and validate stool-based DNA methylation markers for the early detection of CRC and investigate the biological pathways prone to DNA methylation. METHODS: DNA methylation marker discovery was performed using The Cancer Genome Atlas (TCGA) colon adenocarcinoma data set consisting of normal and primary colon adenocarcinoma tissue. The performance of the five best candidate markers and a previously identified marker, NDRG4, was evaluated on tissues and whole stool samples of healthy subjects and CRC patients using quantitative MSP assays. The results were compared and combined with FIT data. Finally, pathway and gene ontology enrichment analyses were performed using ToppFun, GOrilla and clusterProfiler. RESULTS: GDNF, HAND2, SLC35F3, SNAP91 and SORCS1 were ranked as the best performing markers. Gene combinations of all five markers, NDRG4 and FIT were evaluated to establish the biomarker panel with the highest diagnostic potential, resulting in the identification of GDNF/SNAP91/NDRG4/FIT as the best performing marker panel. Pathway and gene ontology enrichment analyses revealed that genes associated with the nervous system were enriched in the set of best performing CRC-specific biomarkers. CONCLUSION: In silico discovery analysis using TCGA-derived data yielded a novel DNA-methylation-based assay for the early detection of CRC, potentially improving current screening modalities. Additionally, nervous system-related pathways were enriched in the identified genes, indicating an epigenetic regulation of neuronal genes in CRC.

Author Correction: Analysis of DNA methylation in cancer: location revisited.

The originally published article contained an error in the acknowledgements section in which the Universiteitsfonds Limburg/SWOL is incorrectly presented as awarding an SU2C- DCS International Translational Cancer Research Dream Team Grant (Stand Up To Cancer (SU2C)-AACR- DT1415, MEDOCC). This has been corrected in the HTML and PDF versions of the manuscript to reflect that Universiteitsfonds Limburg/SWOL is not the funding body that awarded this grant.

Analysis of DNA methylation in cancer: location revisited.

Changes in DNA methylation in cancer have been heralded as promising targets for the development of powerful diagnostic, prognostic, and predictive biomarkers. Despite the existence of more than 14,000 scientific publications describing DNA methylation-based biomarkers and their clinical associations in cancer, only 14 of these biomarkers have been translated into a commercially available clinical test. Methodological and experimental obstacles are both major causes of this disparity, but the genomic location of a DNA methylation-based biomarker is an intrinsic and essential property that also has an important and often overlooked role. Here, we examine the importance of the location of DNA methylation for the development of cancer biomarkers, and take a detailed look at the genomic location and other relevant characteristics of the various biomarkers with commercially available tests. We also emphasize the value of publicly available databases for the development of DNA methylation-based biomarkers and the importance of accurate reporting of the full methodological details of research findings.

Prognostic DNA methylation markers for sporadic colorectal cancer: a systematic review.

Background: Biomarkers that can predict the prognosis of colorectal cancer (CRC) patients and that can stratify high-risk early stage patients from low-risk early stage patients are urgently needed for better management of CRC. During the last decades, a large variety of prognostic DNA methylation markers has been published in the literature. However, to date, none of these markers are used in clinical practice. Methods: To obtain an overview of the number of published prognostic methylation markers for CRC, the number of markers that was validated independently, and the current level of evidence (LoE), we conducted a systematic review of PubMed, EMBASE, and MEDLINE. In addition, we scored studies based on the REMARK guidelines that were established in order to attain more transparency and complete reporting of prognostic biomarker studies. Eighty-three studies reporting on 123 methylation markers fulfilled the study entry criteria and were scored according to REMARK. Results: Sixty-three studies investigated single methylation markers, whereas 20 studies reported combinations of methylation markers. We observed substantial variation regarding the reporting of sample sizes and patient characteristics, statistical analyses, and methodology. The median (range) REMARK score for the studies was 10.7 points (4.5 to 17.5) out of a maximum of 20 possible points. The median REMARK score was lower in studies, which reported a p value below 0.05 versus those, which did not (p = 0.005). A borderline statistically significant association was observed between the reported p value of the survival analysis and the size of the study population (p = 0.051). Only 23 out of 123 markers (17%) were investigated in two or more study series. For 12 markers, and two multimarker panels, consistent results were reported in two or more study series. For four markers, the current LoE is level II, for all other markers, the LoE is lower. Conclusion: This systematic review reflects that adequate reporting according to REMARK and validation of prognostic methylation markers is absent in the majority of CRC methylation marker studies. However, this systematic review provides a comprehensive overview of published prognostic methylation markers for CRC and highlights the most promising markers that have been published in the last two decades.

Analysis of RET promoter CpG island methylation using methylation-specific PCR (MSP), pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM): impact on stage II colon cancer patient outcome.

BACKGROUND: Already since the 1990s, promoter CpG island methylation markers have been considered promising diagnostic, prognostic, and predictive cancer biomarkers. However, so far, only a limited number of DNA methylation markers have been introduced into clinical practice. One reason why the vast majority of methylation markers do not translate into clinical applications is lack of independent validation of methylation markers, often caused by differences in methylation analysis techniques. We recently described RET promoter CpG island methylation as a potential prognostic marker in stage II colorectal cancer (CRC) patients of two independent series. METHODS: In the current study, we analyzed the RET promoter CpG island methylation of 241 stage II colon cancer patients by direct methylation-specific PCR (MSP), nested-MSP, pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM). All primers were designed as close as possible to the same genomic region. In order to investigate the effect of different DNA methylation assays on patient outcome, we assessed the clinical sensitivity and specificity as well as the association of RET methylation with overall survival for three and five years of follow-up. RESULTS: Using direct-MSP and nested-MSP, 12.0 % (25/209) and 29.6 % (71/240) of the patients showed RET promoter CpG island methylation. Methylation frequencies detected by pyrosequencing were related to the threshold for positivity that defined RET methylation. Methylation frequencies obtained by pyrosequencing (threshold for positivity at 20 %) and MS-HRM were 13.3 % (32/240) and 13.8 % (33/239), respectively. The pyrosequencing threshold for positivity of 20 % showed the best correlation with MS-HRM and direct-MSP results. Nested-MSP detected RET promoter CpG island methylation in deceased patients with a higher sensitivity (33.1 %) compared to direct-MSP (10.7 %), pyrosequencing (14.4 %), and MS-HRM (15.4 %). While RET methylation frequencies detected by nested-MSP, pyrosequencing, and MS-HRM varied, the prognostic effect seemed similar (HR 1.74, 95 % CI 0.97-3.15; HR 1.85, 95 % CI 0.93-3.86; HR 1.83, 95 % CI 0.92-3.65, respectively). CONCLUSIONS: Our results show that upon optimizing and aligning four RET methylation assays with regard to primer location and sensitivity, differences in methylation frequencies and clinical sensitivities are observed; however, the effect on the marker's prognostic outcome is minimal.

CHFR promoter methylation indicates poor prognosis in stage II microsatellite stable colorectal cancer.

PURPOSE: Data on the prognostic significance of promoter CpG island methylation in colorectal cancer (CRC) are conflicting, possibly due to associations between methylation and other factors affecting survival such as genetic alterations and use of adjuvant therapy. Here, we examine the prognostic impact of promoter methylation in patients with CRC treated with surgery alone in the context of microsatellite instability (MSI), BRAF and KRAS mutations. EXPERIMENTAL METHODS: One hundred and seventy-three CRCs were analyzed for promoter methylation of 19 tumor suppressor and DNA repair genes, the CpG island methylator phenotype (CIMP), MSI, the exon 15 V600E BRAF mutation and KRAS codon 12 and 13 mutations. RESULTS: Unsupervised hierarchical clustering based on methylation status of 19 genes revealed three subgroups: cluster 1 [CL1, 57% (98/173) of CRCs], cluster 2 [CL2, 25% (43/173) of CRCs], and cluster 3 [CL3, 18% (32/173) of CRCs]. CL3 had the highest methylation index (0.25, 0.49, and 0.69, respectively, P = <0.01) and was strongly associated with CIMP (P < 0.01). Subgroup analysis for tumor stage, MSI, and BRAF status showed no statistically significant differences in survival between CL1, CL2, and CL3 nor between CIMP and non-CIMP CRCs. Analyzing genes separately revealed that CHFR promoter methylation was associated with a poor prognosis in stage II, microsatellite stability (MSS), BRAF wild-type (WT) CRCs: multivariate Cox proportional HR = 3.89 [95% confidence interval (CI), 1.58-9.60, P < 0.01; n = 66] and HR = 2.11 (95% CI, 0.95-4.69, P = 0.068, n = 136) in a second independent population-based study. CONCLUSIONS: CHFR promoter CpG island methylation, which is associated with MSI, also occurs frequently in MSS CRCs and is a promising prognostic marker in stage II, MSS, BRAF WT CRCs.

Promoter CpG island methylation of RET predicts poor prognosis in stage II colorectal cancer patients.

Improved prognostic stratification of patients with TNM stage II colorectal cancer (CRC) is desired, since 20-30% of high-risk stage II patients may die within five years of diagnosis. This study was conducted to investigate REarranged during Transfection (RET) gene promoter CpG island methylation as a possible prognostic marker for TNM stage II CRC patients. The utility of RET promoter CpG island methylation in tumors of stage II CRC patients as a prognostic biomarker for CRC related death was studied in three independent series (including 233, 231, and 294 TNM stage II patients, respectively) by using MSP and pyrosequencing. The prognostic value of RET promoter CpG island methylation was analyzed by using Cox regression analysis. In the first series, analyzed by MSP, CRC stage II patients (n = 233) with RET methylated tumors had a significantly worse overall survival as compared to those with unmethylated tumors (HRmultivariable = 2.51, 95%-CI: 1.42-4.43). Despite a significant prognostic effect of RET methylation in stage III patients of a second series, analyzed by MSP, the prognostic effect in stage II patients (n = 231) was not statistically significant (HRmultivariable = 1.16, 95%-CI 0.71-1.92). The third series (n = 294), analyzed by pyrosequencing, confirmed a statistically significant association between RET methylation and poor overall survival in stage II patients (HRmultivariable = 1.91, 95%-CI: 1.04-3.53). Our results show that RET promoter CpG island methylation, analyzed by two different techniques, is associated with a poor prognosis in stage II CRC in two independent series and a poor prognosis in stage III CRC in one series. RET methylation may serve as a useful and robust tool for clinical practice to identify high-risk stage II CRC patients with a poor prognosis. This merits further investigation.

Promoter CpG island methylation markers in colorectal cancer: the road ahead.

Despite increasing knowledge on the biology, detection and treatment of colorectal cancer (CRC), the disease is still a major health problem. Hypermethylation of promoter regions of genes has been studied extensively as a contributor in CRC carcinogenesis. In addition, it is the topic of many studies focusing on biomarkers for the early detection, prediction of prognosis and treatment outcome. Methylation markers may be preferred over current screening and test methods as they are stable and easy to detect. However, almost no methylation marker is currently being used in clinical practice, often due to a lack of sensitivity, specificity, or validation of the results. This review summarizes the current knowledge of hypermethylation biomarkers for CRC detection, progression and treatment outcome.