Lung cancer epigenetics: Emerging biomarkers

Lung cancer is the leading cause of cancer-related deaths worldwide, and the 5-year survival rate is still very poor due to the scarcity of effective tools for early detection. The discovery of highly sensitive and specific biomarkers highlighting pathological changes early enough to allow clinical intervention is therefore of great importance. In the last decade, epigenetics and particularly research on DNA methylation have provided important information towards a better understanding of lung cancer pathogenesis. Novel and promising molecular biomarkers for diagnosis and prognosis of lung cancer are continuously emerging in this area, requiring further evaluation. This process includes extensive validation in prospective clinical trials before they can be routinely used in a clinical setting. This review summarizes the evidence on epigenetic biomarkers for lung cancer, focusing on DNA methylation. © 2013 Future Medicine Ltd

KMT2C promoter methylation in plasma-circulating tumor DNA is a prognostic biomarker in non-small cell lung cancer

MLL3 histone methyltransferase, encoded by the KMT2C gene, is a tumor suppressor that has an essential role in cell-type-specific gene expression. We evaluated the prognostic significance of KMT2C promoter methylation as a circulating epigenetic biomarker in plasma cell-free DNA (cfDNA) in non-small cell lung cancer (NSCLC). We examined the methylation status of KMT2C promoter using a novel highly specific and sensitive real-time methylation-specific PCR (MSP) assay in (a) operable NSCLC: 48 fresh-frozen NSCLC tissues, their corresponding adjacent non-neoplastic tissues, and 48 matched plasma samples; (b) metastatic NSCLC: 91 plasma samples; and (c) 60 plasma samples from healthy donors (HD). KMT2C promoter methylation in plasma cfDNA was detected in 7/48 (14.6%) patients with operable and in 18/91 (19.8%) patients with advanced NSCLC but in none (0/60, 0%) of the plasma samples from HD. In operable NSCLC, in corresponding adjacent non-neoplastic tissue samples, KMT2C promoter methylation was detected in 3/48 (6.3%) cases. Moreover, in operable NSCLC, KMT2C promoter methylation in plasma cfDNA was related to reduced disease-free survival (ΗR = 0.239; P = 0.001) and worse overall survival (OS; HR = 0.342, P = 0.023). In metastatic NSCLC, KMT2C promoter methylation in plasma cfDNA was related to worse progression-free survival (PFS; HR = 0.431; P = 0.005) and worse OS (HR = 0.306; P < 0.001). Our data strongly suggest that the detection of KMT2C promoter methylation in plasma cfDNA predicts poor prognosis in patients with both operable and metastatic NSCLCs. KMT2C promoter methylation in plasma cfDNA therefore merits further evaluation and validation as a noninvasive circulating epigenetic biomarker. © 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd

SOX17 promoter methylation in plasma circulating tumor DNA of patients with non-small cell lung cancer

SOX17 belongs to the high-mobility groupbox transcription factor superfamily and down-regulates the Wnt pathway. The aim of our study was to evaluate the prognostic significance of SOX17 promoter methylation in circulating tumor DNA (ctDNA) in plasma of non-small cell lung cancer (NSCLC) patients. Methods: We examined the methylation status of SOX17 promoter in 57 operable NSCLC primary tumors and paired adjacent non-cancerous tissues and in ctDNA isolated from 48 corresponding plasma samples as well as in plasma from 74 patients with advanced NSCLC and 49 healthy individuals. SOX17 promoter methylation was examined by Methylation Specific PCR (MSP). Results: In operable NSCLC, SOX17 promoter was fully methylated in primary tumors (57/57, 100%), and in corresponding ctDNA (27/48, 56.2%) while it was detected in only 1/49 (2.0%) healthy individuals. In advanced NSCLC, SOX17 promoter was methylated in ctDNA in 27/74 (36.4%) patients and OS was significantly different in favor of patients with non-methylated SOX17 promoter (p = 0.012). Multivariate analysis revealed that SOX17 promoter methylation in ctDNA was an independent prognostic factor associated with OS in patients with advanced but not operable NSCLC. Conclusions: Our results show that SOX17 promoter is highly methylated in primary tumors and in corresponding plasma samples both in operable and advanced NSCLC. In the advanced setting, SOX17 promoter methylation in plasma ctDNA has a statistical significant influence on NSCLC patient's survival time. Detection of SOX17 promoter methylation in plasma provides prognostic information and merits to be further evaluated as a circulating tumor biomarker in patients with operable and advanced NSCLC. © 2016 by De Gruyter

Circulating cell-free DNA in breast cancer: size profiling, levels, and methylation patterns lead to prognostic and predictive classifiers

Blood circulating cell-free DNA (ccfDNA) is a suggested biosource of valuable clinical information for cancer, meeting the need for a minimally-invasive advancement in the route of precision medicine. In this paper, we evaluated the prognostic and predictive potential of ccfDNA parameters in early and advanced breast cancer. Groups consisted of 150 and 16 breast cancer patients under adjuvant and neoadjuvant therapy respectively, 34 patients with metastatic disease and 35 healthy volunteers. Direct quantification of ccfDNA in plasma revealed elevated concentrations correlated to the incidence of death, shorter PFS, and non-response to pharmacotherapy in the metastatic but not in the other groups. The methylation status of a panel of cancer-related genes chosen based on previous expression and epigenetic data (KLK10, SOX17, WNT5A, MSH2, GATA3) was assessed by quantitative methylation-specific PCR. All but the GATA3 gene was more frequently methylated in all the patient groups than in healthy individuals (all p < 0.05). The methylation of WNT5A was statistically significantly correlated to greater tumor size and poor prognosis characteristics and in advanced stage disease with shorter OS. In the metastatic group, also SOX17 methylation was significantly correlated to the incidence of death, shorter PFS, and OS. KLK10 methylation was significantly correlated to unfavorable clinicopathological characteristics and relapse, whereas in the adjuvant group to shorter DFI. Methylation of at least 3 or 4 genes was significantly correlated to shorter OS and no pharmacotherapy response, respectively. Classification analysis by a fully automated, machine learning software produced a single-parametric linear model using ccfDNA plasma concentration values, with great discriminating power to predict response to chemotherapy (AUC 0.803, 95% CI [0.606, 1.000]) in the metastatic group. Two more multi-parametric signatures were produced for the metastatic group, predicting survival and disease outcome. Finally, a multiple logistic regression model was constructed, discriminating between patient groups and healthy individuals. Overall, ccfDNA emerged as a highly potent predictive classifier in metastatic breast cancer. Upon prospective clinical evaluation, all the signatures produced could aid accurate prognosis. © 2019, Springer Nature Limited

Breast cancer metastasis suppressor-1 promoter methylation in cell-free DNA provides prognostic information in non-small cell lung cancer

Background: Breast-cancer metastasis suppressor 1 (BRMS1) gene encodes for a predominantly nuclear protein that differentially regulates the expression of multiple genes, leading to suppression of metastasis without blocking orthotropic tumour growth. The aim of the present study was to evaluate for the first time the prognostic significance of BRMS1 promoter methylation in cell-free DNA (cfDNA) circulating in plasma of non-small cell lung cancer (NSCLC) patients. Towards this goal, we examined the methylation status of BRMS1 promoter in NSCLC tissues, matched adjacent non-cancerous tissues and corresponding cfDNA as well as in an independent cohort of patients with advanced NSCLC and healthy individuals. Methods: Methylation of BRMS1 promoter was examined in 57 NSCLC tumours and adjacent non-cancerous tissues, in cfDNA isolated from 48 corresponding plasma samples, in cfDNA isolated from plasma of 74 patients with advanced NSCLC and 24 healthy individuals. Results: The BRMS1 promoter was highly methylated both in operable NSCLC primary tissues (59.6%) and in corresponding cfDNA (47.9%) but not in cfDNA from healthy individuals (0%), while it was also highly methylated in cfDNA from advanced NSCLC patients (63.5%). In operable NSCLC, Kaplan-Meier estimates were significantly different in favour of patients with non-methylated BRMS1 promoter in cfDNA, concerning both disease-free interval (DFI) (P=0.048) and overall survival (OS) (P=0.007). In advanced NSCLC, OS was significantly different in favour of patients with non-methylated BRMS1 promoter in their cfDNA (P=0.003). Multivariate analysis confirmed that BRMS1 promoter methylation has a statistical significant influence both on operable NSCLC patients’ DFI time and OS and on advanced NSCLC patients’ PFS and OS. Conclusions: Methylation of BRMS1 promoter in cfDNA isolated from plasma of NSCLC patients provides important prognostic information and merits to be further evaluated as a circulating tumour biomarker

Methylation status of the APC and RASSF1A promoter in cell-free circulating DNA and its prognostic role in patients with colorectal cancer

DNA methylation is the most frequent epigenetic alteration. Using methylation-specific polymerase chain reaction (MSP), the methylation status of the adenomatous polyposis coli (APC) and Ras association domain family 1 isoform A (RASSF1A) genes was examined in cell-free circulating DNA from 155 plasma samples obtained from patients with early and advanced colorectal cancer (CRC). APC and RASSF1A hypermethylation was frequently observed in both early and advanced disease, and was significantly associated with a poorer disease outcome. The methylation status of the APC and RASSF1A promoters was investigated in cell-free DNA of patients with CRC. Using MSP, the promoter methylation status of APC and RASSF1A was examined in 155 blood samples obtained from patients with CRC, 88 of whom had operable CRC (oCRC) and 67 had metastatic CRC (mCRC). The frequency of APC methylation in patients with oCRC was 33%. Methylated APC promoter was significantly associated with older age (P=0.012), higher stage (P=0.014) and methylated RASSF1A status (P=0.050). The frequency of APC methylation in patients with mCRC was 53.7%. In these patients, APC methylation was significantly associated with methylated RASSF1A status (P=0.016). The frequency of RASSF1A methylation in patients with oCRC was 25%. Methylated RASSF1A in oCRC was significantly associated with higher stage (P=0.021). The frequency of RASSF1A methylation in mCRC was 44.8%. Methylated RASSF1A in mCRC was associated with moderate differentiation (P=0.012), high levels of carcinoembryonic antigen (P=0.023) and methylated APC status (P=0.016). Patients with an unmethylated APC gene had better survival in both early (81±5 vs. 27±4 months, P<0.001) and advanced disease (37±7 vs. 15±3 months, P<0.001), compared with patients with methylated APC. Patients with an unmethylated RASSF1A gene had better survival in both early (71±6 vs. 46±8 months, P<0.001) and advanced disease (28±4 vs. 16±3 months, P<0.001) than patients with methylated RASSF1A. The observed significant correlations between APC and RASSF1A promoter methylation status and survival may be indicative of a prognostic role for these genes in CRC, which requires additional testing in larger studies. © 2016, Spandidos Publications. All rights reserved

Assessment of SOX17 DNA methylation in cell free DNA from patients with operable gastric cancer. Association with prognostic variables and survival

Background: DNA methylation represents one of the most common epigenetic changes in human cancer providing important information regarding carcinogenesis. A possible role as a prognostic indicator has also been proposed. The aim of our study was to evaluate the prognostic significance of SOX17 promoter methylation status in patients with operable gastric cancer. Methods: Using methylation-specific PCR (MSP) we examined the incidence and prognostic significance of SOX17 methylation status in cell free circulating DNA in the serum of 73 patients with operable gastric cancer. Fifty-one patients were male (69.9%), their median age was 65 years, 43 patients (58.9%) had regional lymph node involvement and all had a Performance Status (WHO) of 0-1. Results: SOX17 promoter was found to be methylated in 43 out of 73 gastric cancer serum samples examined (58.9%). All 20 control serum samples from healthy individuals were negative. Overall survival (OS) was found to be significantly associated with SOX17 methylation (p=0.049). A significant correlation between methylation status and differentiation (p=0.031) was also observed. No other significant associations between different tumor parameters examined and SOX17 methylation status were observed. Conclusions: SOX17 promoter methylation in cell free DNA of patients with operable gastric cancer is a frequent event and may provide important information regarding prognosis in this group of patients. © 2013 by Walter de Gruyter Berlin Boston 2013

Prognostic Role of RASSF1A, SOX17 and Wif-1 Promoter Methylation Status in Cell-Free DNA of Advanced Gastric Cancer Patients

Epigenetic modification of several genes is a key component in the development of gastric cancer. The methylation status of RASSF1A, SOX17 and Wif-1 genes was evaluated in the cell free circulating DNA of 70 patients with advanced gastric cancer, using methylation-specific PCR. Patients with higher cell-free DNA concentration seem to have lower PFS, than patients with lower cell-free DNA concentration (p = 0.001). RASSF1A was the tumor suppressor gene, most frequently methylated in metastatic gastric cancer patients, followed by SOX17 and Wif-1 (74.3%, 60.0% and 47.1%, respectively). Patients having the SOX17 promoter methylated, had lower progression free survival and overall survival, than unmethylated ones (p < 0.001). Patients having the Wif-1 promoter methylated, had lower progression free survival and overall survival, than unmethylated ones (p = 0.001). Patients having the RASSF1A promoter methylated, had lower progression free survival and overall survival, than unmethylated ones (p = 0.004). Promoter methylation of the examined genes was significantly associated with a decrease in progression free survival and overall survival, comparing to that of patients without methylation. Simultaneous methylation of the above genes was associated with even worse progression free survival and overall survival. The methylation of RASSF1A, SOX-17 and Wif-1 and genes, is a frequent epigenetic event in patients with advanced gastric cancer

Prognostic role of APC and RASSF1A promoter methylation status in cell free circulating DNA of operable gastric cancer patients

Gastric carcinogenesis is a multistep process including not only genetic mutations but also epigenetic alterations. The best known and more frequent epigenetic alteration is DNA methylation affecting tumor suppressor genes that may be involved in various carcinogenetic pathways. The aim of the present study was to investigate the methylation status of APC promoter 1A and RASSF1A promoter in cell free DNA of operable gastric cancer patients.Using methylation specific PCR, we examined the methylation status of APC promoter 1A and RASSF1A promoter in 73 blood samples obtained from patients with gastric cancer.APC and RASSF1A promoters were found to be methylated in 61 (83.6%) and 50 (68.5%) of the 73 gastric cancer samples examined, but in none of the healthy control samples ( p<. 0.001). A significant association between methylated RASSF1A promoter status and lymph node positivity was observed ( p= 0.005). Additionally, a significant correlation between a methylated APC promoter and elevated CEA ( p= 0.033) as well as CA-19.9 ( p= 0.032) levels, was noticed. The Kaplan-Meier estimates of survival, significantly favored patients with a non-methylated APC promoter status ( p= 0.008). No other significant correlations between APC and RASSF1A methylation status and different tumor variables examined was observed.Serum RASSF1A and APC promoter hypermethylation is a frequent epigenetic event in patients with early operable gastric cancer. The observed correlations between APC promoter methylation status and survival as well as between a hypermethylated RASSF1A promoter and nodal positivity may be indicative of a prognostic role for those genes in early operable gastric cancer. Additional studies, in a larger cohort of patients are required to further explore whether these findings could serve as potential molecular biomarkers of survival and/or response to specific treatments. © 2015 Elsevier B.V