ZNF671 DNA methylation as a molecular predictor for the early recurrence of serous ovarian cancer

Serous ovarian cancer is the most frequent type of epithelial ovarian cancer. Despite the use of surgery and platinum‐based chemotherapy, many patients suffer from recurrence within 6 months, termed platinum resistance. Currently, the lack of relevant molecular biomarkers for the prediction of the early recurrence of serous ovarian cancers is linked to the poor prognosis. To identify an effective biomarker for early recurrence, we analyzed the genome‐wide DNA methylation status characteristic of early recurrence after treatment. The patients in The Cancer Genome Atlas (TCGA) dataset who showed a complete response after the first therapy were categorized into 2 groups: early recurrence serous ovarian cancer (ERS, recurrence ≤12 months, n = 51) and late recurrence serous ovarian cancer (LRS, recurrence >12 months, n = 158). Among the 12 differently methylated probes identified between the 2 groups, we found that ZNF671 was the most significantly methylated gene in the early recurrence group. A validation cohort of 78 serous ovarian cancers showed that patients with ZNF671 DNA methylation had a worse prognosis (P < .05). The multivariate analysis revealed that the methylation status of ZNF671 was an independent factor for predicting the recurrence of serous ovarian cancer patients both in the TCGA dataset and our cohort (P = .049 and P = .021, respectively). Functional analysis revealed that the depletion of ZNF671 expression conferred a more migratory and invasive phenotype to the ovarian cancer cells. Our data indicate that ZNF671 functions as a tumor suppressor in ovarian cancer and that the DNA methylation status of ZNF671 might be an effective biomarker for the recurrence of serous ovarian cancer after platinum‐based adjuvant chemotherapy.     

Genome‐wide DNA methylation analysis identifies MEGF10 as a novel epigenetically repressed candidate tumor suppressor gene in neuroblastoma

Neuroblastoma is a childhood cancer in which many children still have poor outcomes, emphasising the need to better understand its pathogenesis. Despite recent genome‐wide mutation analyses, many primary neuroblastomas do not contain recognizable driver mutations, implicating alternate molecular pathologies such as epigenetic alterations. To discover genes that become epigenetically deregulated during neuroblastoma tumorigenesis, we took the novel approach of comparing neuroblastomas to neural crest precursor cells, using genome‐wide DNA methylation analysis. We identified 93 genes that were significantly differentially methylated of which 26 (28%) were hypermethylated and 67 (72%) were hypomethylated. Concentrating on hypermethylated genes to identify candidate tumor suppressor loci, we found the cell engulfment and adhesion factor gene MEGF10 to be epigenetically repressed by DNA hypermethylation or by H3K27/K9 methylation in neuroblastoma cell lines. MEGF10 showed significantly down‐regulated expression in neuroblastoma tumor samples; furthermore patients with the lowest‐expressing tumors had reduced relapse‐free survival. Our functional studies showed that knock‐down of MEGF10 expression in neuroblastoma cell lines promoted cell growth, consistent with MEGF10 acting as a clinically relevant, epigenetically deregulated neuroblastoma tumor suppressor gene. © 2016 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc.     

Combination of RERG and ZNF671 methylation rates in circulating cell‐free DNA: A novel biomarker for screening of nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a prevalent malignancy in Southeast Asia, hence, identifying easily detectable biomarkers for NPC screening is essential for better diagnosis and prognosis. Using genome‐wide and targeted analyses based on next‐generation sequencing approaches, we previously showed that gene promoters are hypermethylated in NPC tissues. To confirm whether DNA methylation rates of genes could be used as biomarkers for NPC screening, 79 histologically diagnosed NPC patients and 29 noncancer patients were recruited. A convenient quantitative analysis of DNA methylation using real‐time PCR (qAMP) was carried out, involving pretreatment of tissue DNA, and circulating cell‐free DNA (ccfDNA) from nonhemolytic plasma, with methylation‐sensitive and/or methylation‐dependent restriction enzymes. The qAMP analyses revealed that methylation rates of RERG, ZNF671, ITGA4, and SHISA3 were significantly higher in NPC primary tumor tissues compared to noncancerous tissues, with sufficient diagnostic accuracy of the area under receiver operating characteristic curves (AUC). Interestingly, higher methylation rates of RERG in ccfDNA were statistically significant and yielded a very good AUC; however, those of ZNF671, ITGA4, and SHISA3 were not significant. Furthermore, the combination of methylation rates of RERG and ZNF671 in ccfDNA showed higher diagnostic accuracy than either of them individually. In conclusion, the methylation rates of specific genes in ccfDNA can serve as novel biomarkers for early detection and screening of NPC.     

PCDHB15 as a potential tumor suppressor and epigenetic biomarker for breast cancer

Breast cancer is among the most frequently diagnosed cancer types and the leading cause of cancer-related death in women. The mortality rate of patients with breast cancer is currently increasing, perhaps due to a lack of early screening tools. In the present study, using The Cancer Genome Atlas (TCGA) breast cancer dataset (n=883), it was determined that methylation of the protocadherin β15 (PCDHB15) promoter was higher in breast cancer samples than that in normal tissues. A negative association between promoter methylation and expression of PCDHB15 was observed in the TCGA dataset and breast cancer cell lines. In TCGA cohort, lower PCDHB15 expression was associated with shorter relapse-free survival times. Treatment with the DNA methyltransferase inhibitor restored PCDHB15 expression in a breast cancer cell line; however, overexpression of PCDHB15 was shown to suppress colony formation. PCDHB15 methylation detected in circulating cell-free DNA (cfDNA) isolated from serum samples was higher in patients with breast cancer (40.8%) compared with that in patients with benign tumors (22.4%). PCDHB15 methylation was not correlated with any clinical parameters. Taken together, PCDHB15 is a potential tumor suppressor in cases of breast cancer, which can be epigenetically silenced via promoter methylation. PCDHB15 methylation using cfDNA is a novel minimally invasive epigenetic biomarker for the diagnosis and prognosis of breast cancer.     

Methylomics analysis identifies epigenetically silenced genes and implies an activation of β‐catenin signaling in cervical cancer

Using DNA methylation biomarkers in cancer detection is a potential direction in clinical testing. Some methylated genes have been proposed for cervical cancer detection; however, more reliable methylation markers are needed. To identify new hypermethylated genes in the discovery phase, we compared the methylome between a pool of DNA from normal cervical epithelium (n = 19) and a pool of DNA from cervical cancer tissues (n = 38) using a methylation bead array. We integrated the differentially methylated genes with public gene expression databases, which resulted in 91 candidate genes. Based on gene expression after demethylation treatment in cell lines, we confirmed 61 genes for further validation. In the validation phase, quantitative MSP and bisulfite pyrosequencing were used to examine their methylation level in an independent set of clinical samples. Fourteen genes, including ADRA1D, AJAP1, COL6A2, EDN3, EPO, HS3ST2, MAGI2, POU4F3, PTGDR, SOX8, SOX17, ST6GAL2, SYT9, and ZNF614, were significantly hypermethylated in CIN3+ lesions. The sensitivity, specificity, and accuracy of POU4F3 for detecting CIN3+ lesions were 0.88, 0.82, and 0.85, respectively. A bioinformatics function analysis revealed that AJAP1, EDN3, EPO, MAGI2, and SOX17 were potentially implicated in β‐catenin signaling, suggesting the epigenetic dysregulation of this signaling pathway during cervical cancer development. The concurrent methylation of multiple genes in cancers and in subsets of precancerous lesions suggests the presence of a driver of methylation phenotype in cervical carcinogenesis. Further validation of these new genes as biomarkers for cervical cancer screening in a larger population‐based study is warranted.     

Urine and bladder washing cytology for detection of urothelial carcinoma: standard test with new possibilities

Background

Light microscopic evaluation of cell morphology in preparations from urine or bladder washing containing exfoliated cells is a standard and primary method for the detection of bladder cancer and also malignancy from other parts of the urinary tract. The cytopathologic examination is a valuable method to detect an early recurrence of malignancy or new primary carcinoma during the follow-up of patients after the treatment of bladder cancer.

Conclusions

Characteristic cellular and nuclear signs of malignancy indicate invasive or in situ urothelial carcinoma or high-grade papillary urothelial carcinoma. However, low sensitivity of the method reflects the unreliable cytopathologic diagnosis of low-grade urothelial neoplasms as cellular and nuclear signs of malignancy in these neoplasms are poorly manifested. Many different markers were developed to improve the diagnosis of bladder carcinoma on urinary samples. UroVysion™ test is among the newest and most promising tests. By the method of in situ hybridization one can detect specific cytogenetic changes of urothelial carcinoma.     

Diagnostic potential of TERT promoter and FGFR3 mutations in urinary cell‐free DNA in upper tract urothelial carcinoma

Most upper tract urothelial carcinomas (UTUC) are muscle invasive at the time of diagnosis. Current standard methods for the diagnosis of UTUC are invasive. Urine cytology is the only non‐invasive test for detecting UTUC, but its sensitivity is low. A novel non‐invasive assay for UTUC detection would improve patient outcome. This study aimed to investigate the mutation of cell‐free DNA (cfDNA) in urine supernatant to develop a reliable diagnostic biomarker for UTUC patients. We studied urinary cfDNA from 153 individuals, including 56 patients with localized UTUC, and carried out droplet digital PCR assay for TERT promoter and FGFR3 hotspot mutations. We could detect mutations of TERT C228T in 22/56 (39.3%), TERT C250T in 4/56 (7.1%), and FGFR3 S249C in 9/56 (16.1%) patients. FGFR3 mutation was detected only in ≤pT1 tumors (positive predictive value: 100.0%). In combination with cytology results, the sensitivity was 78.6%, and the specificity was 96.0%. Although these data need to be validated in a larger‐scale cohort, mutation analysis of TERT promoter and FGFR3 in urinary cfDNA has the potential to be a non‐invasive diagnostic marker and reliable factor for tumor staging.     

Epigenetic inactivation of the candidate tumor suppressor gene ASC/TMS1 in human renal cell carcinoma and its role as a potential therapeutic target

This study investigated the epigenetic alteration and biological function of the pro-apoptotic gene ASC/TMS1 in renal cell carcinoma. ASC/TMS1 was downregulated in five out of six RCC cell lines. A significant downregulation was also detected in sixty-seven paired renal tumors compared with adjacent non-cancerous tissues. The downregulation of ASC/TMS1 was correlated with promoter hypermethylation and could be restored with demethylation treatment. Re-expression of ASC/TMS1 in silenced RCC cell lines inhibited cell viability, colony formation, arrested cell cycle, induced apoptosis, suppressed cell invasion and repressed tumorigenicity in SCID mice. The antitumorigenic function of ASC/TMS1 in renal cancer was partially regulated by activation of p53 and p21 signaling. In addition, restoration of ASC/TMS1 sensitizes RCC cells to DNA damaging agents. Knockdown of ASC/TMS1 reduced DNA damaging agents-induced p53 activation and cell apoptosis. Moreover, ASC/TMS1 hypermethylation was further detected in 41.1% (83/202) of RCC tumors, but only 12% in adjacent non-cancerous tissues. ASC/TMS1 methylation was significantly correlated with higher tumor nuclear grade. In conclusion, ASC/TMS1 is a novel functional tumor suppressor in renal carcinogenesis. ASC/TMS1 tumor specific methylation may be a useful biomarker for designing improved diagnostic and therapeutic strategies for RCC.     

TMEM196 acts as a novel functional tumour suppressor inactivated by DNA methylation and is a potential prognostic biomarker in lung cancer

Epigenetic silencing of tumour suppressors contributes to the development and progression of lung cancer. We recently found that TMEM196 was hypermethylated in lung cancer. This study aimed to clarify its epigenetic regulation, possible roles and clinical significance. TMEM196 methylation correlated with loss of protein expression in chemical-induced rat lung pathologic lesions and human lung cancer tissues and cell lines. 5-aza-2′-deoxycytidine restored TMEM196 expression. Moreover, TMEM196 hypermethylation was detected in 61.2% of primary lung tumours and found to be associated with poor differentiation and pathological stage of lung cancer. Functional studies showed that ectopic re-expression of TMEM196 in lung cancer cells inhibited cell proliferation, clonogenicity, cell motility and tumour formation. However, TMEM196 knockdown increased cell proliferation and inhibited apoptosis and cell-cycle arrest. These effects were associated with upregulation of p21 and Bax, and downregulation of cyclin D1, c-myc, CD44 and β-catenin. Kaplan–Meier survival curves showed that TMEM196 downregulation was significantly associated with shortened survival in lung cancer patients. Multivariate analysis showed that patients with TMEM196 expression had a better overall survival. Our results revealed for the first time that TMEM196 acts as a novel functional tumour suppressor inactivated by DNA methylation and is an independent prognostic factor of lung cancer.     

A novel and non-invasive approach utilising nasal washings for the detection of nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is an epithelial cancer of the nasopharynx which is highly associated with Epstein–Barr virus (EBV). Worldwide, most of the top 20 countries with the highest incidence and mortality rates of NPC are low- and middle-income countries. Many studies had demonstrated that EBV could be detected in the tissue, serum and plasma of NPC patients. In this study, we explored the potential of assays based on non-invasive nasal washings (NW) as a diagnostic and prognostic tool for NPC. A total of 128 patients were evaluated for NW EBV DNA loads and a subset of these samples were also tested for 27 EBV and human miRNAs shortlisted from literature. EBV DNA and seven miRNAs showed area under the receiver operating characteristic curve (AUC) values of more than 0.7, suggestive of their potential utility to detect NPC. Logistic regression analyses suggested that combination of two NW assays that test for EBNA-1 and hsa-miR-21 had the best performance in detecting NPC. The trend of NW EBV DNA load matched with clinical outcome of 71.4% (10 out of 14) NPC patients being followed-up. In summary, the non-invasive NW testing panel may be particularly useful for NPC screening in remote areas where healthcare facilities and otolaryngologists are lacking, and may encourage frequent testing of individuals in the high risk groups who are reluctant to have their blood tested. However, further validation in an independent cohort is required to strengthen the utility of this testing panel as a non-invasive detection tool for NPC.     

MiR-4500 is epigenetically downregulated in colorectal cancer and functions as a novel tumor suppressor by regulating HMGA2

This study aimed to understand the exact function and potential mechanism of miR-4500 in colorectal cancer (CRC). In this study, the expression of miR-4500 was decreased in both CRC cells and tissues, and downregulated miR-4500 indicated advanced tumor stage and poor survival. By bisulfite sequencing analysis, we found that the CpG island in the promoter region of miR-4500 was hypermethylated in CRC cells and tissues compared with normal control cells and non-tumor tissues, respectively. Functionally, gain- and loss-of-function analyses indicated the tumor suppressor role of miR-4500: it suppressed cell proliferation, cell cycle progression, migration, and invasion. Predictive algorithms and experimental analyses identified HMGA2 as a direct target of miR-4500. Reintroducing HMGA2 impaired the inhibitory effects of miR-4500 on cell growth and motility. Clinically, higher HMGA2 protein expression in CRC tissues was associated with advanced tumor stage and poor survival. An inverse correlation was found between miR-4500 levels and HMGA2 protein expression. Taken together, this study provides the first evidence that miR-4500 functions as a novel tumor suppressor in the miR-4500/HMGA2 axis in colorectal carcinogenesis, and restoring miR-4500 expression might represent a promising therapeutic strategy for CRC.     

Urinary laminin‐γ2 is a novel biomarker of non‐muscle invasive urothelial carcinoma

Lack of appropriate biomarkers has hampered early detection of urothelial cancer (UC), therefore, development of biomarkers for its diagnosis at earlier stages is of importance. Laminin‐332 (Ln‐332, formerly Ln‐5), a component of basement membranes, consists of Ln‐α3, Ln‐β3, and Ln‐γ2 polypeptides. However, monomeric Ln‐γ2 alone is frequently expressed in malignant neoplasms. If Ln‐γ2 is also expressed in UC and secreted into the urine, its detection could be useful for UC diagnosis. Here, we evaluated Ln‐γ2 levels from 60 patients with urinary diseases (including UC) by Western blotting, and detected it in approximately 53% of UC cases. Using immunohistochemistry, we confirmed Ln‐γ2 expression in UC tissues that were positive for Ln‐γ2, whereas Ln‐α3 expression was absent. We next developed a sandwich enzyme‐linked immunosorbent assay and applied it for screening 39 patients with non‐muscle invasive UC and 61 patients with benign urologic diseases. The Ln‐γ2 levels were higher in UC patients than in those with benign urologic diseases. Ln‐γ2 was detected even in patients with earlier stages of UC, such as Ta, T1, or carcinoma in situ. The sensitivity of Ln‐γ2 testing for UC was 97.4%, and the specificity was 45.9%, using a cut‐off of 0.5 μg/g∙crn. Ln‐γ2 had greater diagnostic value for detecting non‐muscle invasive UC compared to conventional urine cytology and available biomarkers for UC, and may be useful as a urine biomarker for the diagnosis and monitoring of UC.     

Aberrant methylation of the PTPRO gene in peripheral blood as a potential biomarker in esophageal squamous cell carcinoma patients

Epigenetic inactivation of protein tyrosine phosphatase receptor-type O (PTPRO), a new member of the PTP family, has been described in several forms of cancer. We evaluated PTPRO promoter hypermethylation as a potential biomarker in esophageal squamous cell carcinoma (ESCC). This alteration was observed in 27 (75%) of 36 primary tumors and correlated significantly with depth of invasion (T-stage, P = 0.013). Among matched peripheral blood samples from ESCC patients, 13 (36.1%) of 36 exhibited detectable methylated PTPRO in plasma, while 15 (41.7%) of 36 had this abnormality in buffy coat. No methylated PTPRO was observed in normal peripheral blood samples from 10 healthy individuals. In addition, demethylation by 5-aza-dC treatment led to gene reactivation in PTPRO-methylated and -silenced ESCC cell lines. To our knowledge, this is the first report of methylated PTPRO as a noninvasive tumor biomarker in peripheral blood. These findings suggest that hypermethylated PTPRO occurs frequently in ESCC. Further, detection in peripheral blood of ESCC patients suggests potential clinical application for noninvasive diagnosis and disease monitoring.