Assessment of three epigenotypes in colorectal cancer by combined bisulphite restriction analysis.

Background: Recent investigations have demonstrated the clear heterogeneity of sporadic colorectal cancer (CRC) with regard to CpG island methylation. Two unsupervised cluster analyses revealed that CRCs form three distinct DNA methylation subsets, which are referred to as the high-, intermediate- and low-methylation epigenotypes (HME, IME, and LME, respectively). A recent study by Yagi et al. found a fairly sensitive and specific identification of HME, IME and LME using two marker panels analysed by MALDI-TOF mass spectrometry (MassARRAY). However, the expensive equipment required for this method substantially increases the cost and complexity of the assay. Findings: In this article, we demonstrate the assessment of HME, IME and LME in a group of 233 sporadic CRCs using seven markers proposed by Yagi et al. The DNA methylation of each marker was quantified using combined bisulphite restriction analysis (COBRA) together with an analysis of various genetic factors associated with CRC (the BRAF and KRAS mutations and microsatellite instability (MSI)). The baseline methylation of each marker was generated from pooled DNA isolated from 50 normal colon tissues. Conclusions: We demonstrate that the correlation of HME, IME and LME epigenotyped by COBRA using different molecular classifiers is similar to that achieved by MassARRAY. Therefore, epigenotyping CRCs using COBRA is a simple, specific and cost-effective method that has the potential to be widely used in CRC research

Nomograms for predicting local recurrence, distant metastases, and overall survival for patients with locally advanced rectal cancer on the basis of European randomized clinical trials.

International audiencePURPOSE: The purpose of this study was to develop accurate models and nomograms to predict local recurrence, distant metastases, and survival for patients with locally advanced rectal cancer treated with long-course chemoradiotherapy (CRT) followed by surgery and to allow for a selection of patients who may benefit most from postoperative adjuvant chemotherapy and close follow-up. PATIENTS AND METHODS: All data (N = 2,795) from five major European clinical trials for rectal cancer were pooled and used to perform an extensive survival analysis and to develop multivariate nomograms based on Cox regression. Data from one trial was used as an external validation set. The variables used in the analysis were sex, age, clinical tumor stage stage, tumor location, radiotherapy dose, concurrent and adjuvant chemotherapy, surgery procedure, and pTNM stage. Model performance was evaluated by the concordance index (c-index). Risk group stratification was proposed for the nomograms. RESULTS: The nomograms are able to predict events with a c-index for external validation of local recurrence (LR; 0.68), distant metastases (DM; 0.73), and overall survival (OS; 0.70). Pathologic staging is essential for accurate prediction of long-term outcome. Both preoperative CRT and adjuvant chemotherapy have an added value when predicting LR, DM, and OS rates. The stratification in risk groups allows significant distinction between Kaplan-Meier curves for outcome. CONCLUSION: The easy-to-use nomograms can predict LR, DM, and OS over a 5-year period after surgery. They may be used as decision support tools in future trials by using the three defined risk groups to select patients for postoperative chemotherapy and close follow-up (http://www.predictcancer.org)

Protein tyrosine phosphatase receptor-like genes are frequently hypermethylated in sporadic colorectal cancer

Introduction: The activity of phosphatases could be influenced by genetic, as well as epigenetic alterations. In our study we have investigated the methylation status of four PTPRs: PTPRM, PTPRT, PTPRR and PTPRZ1, which were pre-selected using microarray techniques as being alternatively methylated in sporadic colorectal cancer. Materials and methods: The analyses were carried out on 131 surgical specimens obtained from sporadic colorectal cancer patients. The methylation status of the four genes was examined using MSP. Results: The analysis of promoter methylation using an Illumina 27K microarray revealed four protein tyrosine phosphatases PTPRM, PTPRT, PTPRR and PTPRZ1 as being hypermethylated with beta-value ≥ 0.2 and p≤0.05. Subsequent analysis using MSP confirmed these observations - the frequency of promoter methylation was significantly higher in tumor cells compared to matched normal tissue for each of the analyzed genes. There was no association observed between the methylation status of PTPRs and either CIMP, K-ras (codon 12) and BRAF (exon 15, V600E) mutations or tumor localization (proximal/distal). The results of our study show a statistically significant difference between promoter methylation in cancerous and healthy tissue. This result supports the hypothesis that the PTPR family plays an important role in the etiology of colorectal cancer