Genetic and epigenetic alteration as serum markers for cancer detection

Several studies have shown that epigenetic mechanisms, as microRNAs (miRs) expression and gene methylation are involved in cancer, by regulating cell proliferation, apoptosis and angiogenesis. Accordingly, epigenetic changes occur from early stages and accumulate during cancer progression, by contributing to cancer development and progression. Since tumour specific genetic and epigenetic alterations can be detected not only in cancer tissues but also in circulating serum or plasma cell-free DNA (cfDNA), this method is considered promising for improving non-invasive cancer detection and monitoring. The present work includes three papers presenting novel genetic and epigenetic changes that could contribute to the identification of new non-invasive cancer biomarkers. The aim of first work was to compare the status of KRAS mutation and SEPT9 methylation between the primary tumors and matched plasma samples in patients affected by colorectal cancer (CRC). KRAS mutations and SEPT9 promoter methylation were present in 34.1% (29/85) and in 95.3% (81/85) of the primary tumour tissue samples. Patients presented with both genetic and epigenetic alterations in tissue specimens (31.8%, 27/85) were considered for further analyses on cfDNA. In 4 primary tumours with KRAS mutations, identical mutations were not observed in the corresponding plasma samples. The median methylation rate in tumour tissues and plasma samples was 64.5% (12.2–99.8%) and 14.5% (0–45.5%), respectively. The median KRAS mutation load (for matched mutations) was 33.6% (1.2-86%) in tissues and 4% (0-17%) in plasma samples. A statistically significant correlation was found between tissue and plasma SEPT9 methylation rate (r=0.41, p=0.035), whereas no association was found between tissue and plasma KRAS mutation load (r=0.09, p=0.651). These data show a discrepancy in epigenetic versus genetic alterations detectable in cfDNA as markers for tumour detection. Many factors could affect the mutant cfDNA analysis including the sensitivity of the detection method and the presence of tumour clonal heterogeneity. The second line of research has focused on patients affected by epithelial ovarian cancer (EOC). Serum levels of miR-199a and miR-125b were found to be significantly higher in EOC patients compared to healthy controls (p=0.007 and p=0.002, respectively). A statistically significant correlation was found between miR-199a and miR-125b expression levels (r=0.38, p=0.03). The ROC curve analysis of the diagnostic performance on healthy controls and EOC patients revealed that HE4 had a significantly higher area under the curve (AUC: 0.90) when compared to CA125 (AUC: 0.85), miR-199a (AUC: 0.70) and miR-125b (AUC: 0.67). Despite the low specificity, mainly in pre-menopausal women, CA125 and HE4 seem to have better diagnostic performance compared to miRs investigated in our study. The third line of research has focused on investigate in human endometrial cancer (EC) the expression of miR-186, miR-222, miR-223, miR-204. Serum levels of miR-186, miR-222 and miR-223 resulted significantly up-regulated in patients compared to healthy controls (p=0.004, p=0.002 and p<0.0001). Contrarily, miR-204 resulted significantly down-regulated in EC patients compared to healthy controls (p<0.0001). A positive significant correlation was observed between miR-186 and both miR-222 (r=0.71, p<0.0001) and miR-223 (r=0.64, p<0.0001) as well also between miR-222 and miR-223 (r=0.57, p<0.0001). The AUCs for the selected miRs ranged from 0.70 to 0.87, significantly higher than for CA125 (0.59). Our results confirm that these miRs are implicated in EC and hold promise as a novel blood-based biomarker for the diagnosis. In conclusion, our results indicate that circulating nucleic acids are a potentially promising source of tumor-specific biomarkers in patients affected by different solid cancer. Accordingly, we have demonstrated that some circulating tumour-specific biomarkers can be detected at any time during the course of the disease and once detected indicate that a tumour is probably present. The biggest challenge remains to standardize the methodologies including sample storage and DNA or miRs extraction to translate the quantitation of circulating epigenetic biomarkers into a clinical routine for cancer diagnosis and prognosis prediction.