Merkel Cell Virus (MCV) is a newly discovered polyomavirus, recently found in a rare skin cancer, Merkel cell carcinoma (MCC). However, MCV has also been detected in some normal tissue samples. We tested and compared the relative quantity of the MCV in a set of diverse human tissue samples with the MCC samples. The levels of MCV in MCCs were over 60 times higher than the highest values in all other tissues. Low quantities of MCV were detected in diverse tissue samples independently of malignant or benign histologic status. Higher levels of the virus were found in the upper aerodigestive tract, digestive system, and saliva compared to the lung and genitourinary system samples. These results confirm that MCV is widespread in the human body and suggest a possible fecal‐oral transmission route similar to the Hepatitis A virus. Despite widespread presence of the virus, it appears that only neuroendocrine skin cells are susceptible to transformation by MCV.

Purpose: To establish a novel panel of cancer-specific methylated genes for cancer detection and prognostic stratification of early-stage non–small cell lung cancer (NSCLC). Experimental Design: Identification of differentially methylated regions (DMR) was performed with bumphunter on “The Cancer Genome Atlas (TCGA)” dataset, and clinical utility was assessed using quantitative methylation-specific PCR assay in multiple sets of primary NSCLC and body fluids that included serum, pleural effusion, and ascites samples. Results: A methylation panel of 6 genes (CDO1, HOXA9, AJAP1, PTGDR, UNCX, and MARCH11) was selected from TCGA dataset. Promoter methylation of the gene panel was detected in 92.2% (83/90) of the training cohort with a specificity of 72.0% (18/25) and in 93.0% (40/43) of an independent cohort of stage IA primary NSCLC. In serum samples from the later 43 stage IA subjects and population-matched 42 control subjects, the gene panel yielded a sensitivity of 72.1% (31/41) and specificity of 71.4% (30/42). Similar diagnostic accuracy was observed in pleural effusion and ascites samples. A prognostic risk category based on the methylation status of CDO1, HOXA9, PTGDR, and AJAP1 refined the risk stratification for outcomes as an independent prognostic factor for an early-stage disease. Moreover, the paralog group for HOXA9, predominantly overexpressed in subjects with HOXA9 methylation, showed poor outcomes. Conclusions: Promoter methylation of a panel of 6 genes has potential for use as a biomarker for early cancer detection and to predict prognosis at the time of diagnosis. Clin Cancer Res; 23(22); 7141–52. ©2017 AACR.

Purpose: To evaluate aberrant promoter hypermethylation of candidate tumor suppressor genes as a means to detect epigenetic alterations specific to solid tumors, including head and neck squamous cell carcinoma (HNSCC). Experimental Design: Using promoter regions identified via a candidate gene and discovery approach, we evaluated the ability of an expanded panel of CpG-rich promoters known to be differentially hypermethylated in HNSCC in detection of promoter hypermethylation in serum and salivary rinses associated with HNSCC. We did preliminary evaluation via quantitative methylation-specific PCR (Q-MSP) using a panel of 21 genes in a limited cohort of patients with HNSCC and normal controls. Using sensitivity and specificity for individual markers as criteria, we selected panels of eight and six genes, respectively, for use in salivary rinse and serum detection and tested these in an expanded cohort including up to 211 patients with HNSCC and 527 normal controls. Results: Marker panels in salivary rinses showed improved detection when compared with single markers, including a panel with 35% sensitivity and 90% specificity and a panel with 85% sensitivity and 30% specificity. A similar pattern was noted in serum panels, including a panel with 84.5% specificity with 50.0% sensitivity and a panel with sensitivity of 81.0% with specificity of 43.5%. We also noted that serum and salivary rinse compartments showed a differential pattern of methylation in normal subjects that influenced the utility of individual markers. Conclusions: Q-MSP detection of HNSCC in serum and salivary rinses using multiple targets offers improved performance when compared with single markers. Compartment-specific methylation in normal subjects affects the utility of Q-MSP detection strategies.

The human cysteine dioxygenase 1 (CDO1) gene is a non-heme structured, iron-containing metalloenzyme involved in the conversion of cysteine to cysteine sulfinate, and plays a key role in taurine biosynthesis. In our search for novel methylated gene promoters, we have analyzed differential RNA expression profiles of colorectal cancer (CRC) cell lines with or without treatment of 5-aza-2′-deoxycytidine. Among the genes identified, the CDO1 promoter was found to be differentially methylated in primary CRC tissues with high frequency compared to normal colon tissues. In addition, a statistically significant difference in the frequency of CDO1 promoter methylation was observed between primary normal and tumor tissues derived from breast, esophagus, lung, bladder and stomach. Downregulation of CDO1 mRNA and protein levels were observed in cancer cell lines and tumors derived from these tissue types. Expression of CDO1 was tightly controlled by promoter methylation, suggesting that promoter methylation and silencing of CDO1 may be a common event in human carcinogenesis. Moreover, forced expression of full-length CDO1 in human cancer cells markedly decreased the tumor cell growth in an in vitro cell culture and/or an in vivo mouse model, whereas knockdown of CDO1 increased cell growth in culture. Our data implicate CDO1 as a novel tumor suppressor gene and a potentially valuable molecular marker for human cancer.

Lungs resected for adenocarcinomas often harbour minute discrete foci of cytologically atypical pneumocyte proliferations designated as atypical adenomatous hyperplasia (AAH). Evidence suggests that AAH represents an initial step in the progression to adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) and fully invasive adenocarcinoma. Despite efforts to identify predictive markers of malignant transformation, alterations driving this progression are poorly understood. Here we perform targeted next-generation sequencing on multifocal AAHs and different zones of histologic progression within AISs and MIAs. Multiregion sequencing demonstrated different genetic drivers within the same tumour and reveal that clonal expansion is an early event of tumorigenesis. We find that KRAS, TP53 and EGFR mutations are indicators of malignant transition. Utilizing droplet digital PCR, we find alterations associated with early neoplasms in paired circulating DNA. This study provides insight into the heterogeneity of clonal events in the progression of early lung neoplasia and demonstrates that these events can be detected even before neoplasms have invaded and acquired malignant potential.