Colorectal cancer (CRC), the second leading cause of cancer mortality, constitutes a significant global health burden. An accurate, noninvasive detection method for cRc as complement to colonoscopy could improve the effectiveness of treatment. In the present study, SureSelectXT Methyl-Seq was performed on cancerous and normal colon tissues and CLDN1, INHBA and SLC30A10 were found as candidate methylated genes. MethyLight assay was run on formalin-fixed paraffin-embedded (FFPE) and fresh case and control tissues to validate the methylation of the selected gene. the methylation was significantly different (p-values < 2.2e-16) with a sensitivity of 87.17%; at a specificity cut-off of 100% in FFPE tissues. Methylation studies on fresh tissues, indicated a sensitivity of 82.14% and a specificity cut-off of 92% (p-values = 1.163e-07). The biomarker performance was robust since, normal tissues indicated a significant 22.1-fold over-expression of the selected gene as compared to the corresponding CRC tissues (p-value < 2.2e-16) in the FFPE expression assay. In our plasma pilot study, evaluation of the tissue methylation marker in the circulating cell-free DNA, demonstrated that 9 out of 22 CRC samples and 20 out of 20 normal samples were identified correctly. In summary, there is a clinical feasibility that the offered methylated gene could serve as a candidate biomarker for CRC diagnostic purpose, although further exploration of our candidate gene is warranted.Colorectal cancer (CRC) constitutes a significant global health burden, leading to over 862,000 deaths globally in 2018. It is the second main cause of cancer mortality in the world and currently stands as the third most common cancer, with a yearly incidence of over one million and eight thousand cases worldwide 1 . Its leading cause of death is due to liver metastasis with a median survival rate of approximately 30 months. Generally, half of the patients with CRC develop tumor recurrences 2 . For early-diagnosed CRC patients, the 5-year survival rates are approximately 90% but this lowers to less than 10% in patients with extensive metastases. Thus, the most effective approach to reduce CRC incidence and its mortality is early detection of colonic lesions 3 . Fortunately, because of implementation and growth of wide spread cancer screening assays, such as colonoscopy as well as increasingly effective therapies, the mortality rate of CRC is lowering in many countries 2 .

Detecting cfDNA in plasma or serum could serve as a 'liquid biopsy', for circulating tumor DNA with aberrant methylation patterns offer a possible method for early detection of several cancers which could avoid the need for tumor tissue biopsies. Bone Morphogenetic Protein 3 (BMP3) was identified as a candidate tumor suppressor gene putatively down-regulated in colorectal cancer (CRC). In this study, we aimed to assess the potential role of BMP3 promoter methylation changes in plasma DNA for detection of colorectal cancerous and precancerous lesions. Plasma DNA samples were extracted from 50 patients with histologically diagnosed polyps or tumor and 50 patients reported negative for polyps or tumors. The procedure consists of bisulfite conversion of the extracted DNA, purification of bis-DNA, and BMP3 methylation status analysis by using the bisulfite specific high resolution melting analysis. This study demonstrated that there was a significantly higher frequency of BMP3 methylated DNA in plasma in patients with polyps versus healthy controls with a sensitivity and specificity of 40 and 94%, respectively. In conclusion, our results demonstrated that BMP3 DNA methylation in plasma had not have sufficient sensitivity and it should be used in combination with other biomarkers for the detection of CRC.

A large number of tumor-related methylated genes have been suggested to be of diagnostic and prognostic values for CRC when analyzed in patients' stool samples; however, reported sensitivities and specificities have been inconsistent and widely varied. This meta-analysis was conducted to assess the detection accuracy of stool DNA methylation assay in CRC, early stages of CRC (advanced adenoma, non-advanced adenomas) and hyperplastic polyps, separately. We searched MEDLINE, Web of Science, Scopus and Google Scholar databases until May 1, 2016. From 469 publications obtained in the initial literature search, 38 studies were included in the final analysis involving 4867 individuals. The true positive, false positive, true negative and false negative of a stool-based DNA methylation biomarker using all single-gene tests considering a certain gene; regardless of a specific gene were pooled and studied in different categories. The sensitivity of different genes in detecting different stages of CRC ranged from 0% to 100% and the specificities ranged from 73% to 100%. Our results elucidated that SFRP1 and SFRP2 methylation possessed promising accuracy for detection of not only CRC (DOR: 31.67; 95%CI, 12.31–81.49 and DOR: 35.36; 95%CI, 18.71–66.84, respectively) but also the early stages of cancer, adenoma (DOR: 19.72; 95%CI, 6.68–58.25 and DOR: 13.20; 95%CI, 6.01–28.00, respectively). Besides, NDRG4 could be also considered as a significant diagnostic marker gene in CRC (DOR: 24.37; 95%CI, 10.11–58.73) and VIM in adenoma (DOR: 15.21; 95%CI, 2.72–85.10). In conclusion, stool DNA hypermethylation assay based on the candidate genes SFRP1, SFRP2, NDRG4 and VIM could offer potential diagnostic value for CRC based on the findings of this meta-analysis.

DNA methylation is an important epigenetic modification involved in many biological processes and diseases. Computational analysis of differentially methylated regions (DMRs) could explore the underlying reasons of methylation. DMRFusion is presented as a useful tool for comprehensive DNA methylation analysis of DMRs on methylation sequencing data. This tool is designed base on the integration of several ranking methods; Information gain, Between versus within Class scatter ratio, Fisher ratio, Z-score and Welch's t-test. In this study, DMRFusion on reduced representation bisulfite sequencing (RRBS) data in chronic lymphocytic leukemia cancer displayed 30 nominated regions and CpG sites with a maximum methylation difference detected in the hypermethylation DMRs. We realized that DMRFusion is able to process methylation sequencing data in an efficient and accurate manner and to provide annotation and visualization for DMRs with high fold difference score (p-value and FDR<0.05 and type I error: 0.04).

ObjectiveIn the present study, we investigated different simple and cost effective methods to evaluate and validate cell free DNA (cfDNA) isolation. The ability of the QIAamp DNA Blood Mini Kit method to extract cfDNA was assessed by several approaches, including purification of endogenous cfDNA and exogenous spike-in control material, prior to plasma extraction, and followed by quantitative-PCR.ResultsUsing QIAamp DNA Blood Mini kit, nearly 27% (380 bp) to 35% (173 bp) cfDNA was recovered with a higher recovery of smaller size cfDNA (173 bp) in comparison to larger ones (380 bp). These simple laboratory methods can be used to assess the efficiency of any cfDNA isolation method.Electronic supplementary materialThe online version of this article (10.1186/s13104-018-3866-8) contains supplementary material, which is available to authorized users.