Noninvasive detection of aberrant DNA methylation could provide invaluable biomarkers for earlier detection of triple‐negative breast cancer (TNBC) which could help clinicians with easier and more efficient treatment options. We evaluated genome‐wide DNA methylation data derived from TNBC and normal breast tissues, peripheral blood of TNBC cases and controls and reference samples of sorted blood and mammary cells. Differentially methylated regions (DMRs) between TNBC and normal breast tissues were stringently selected, verified and externally validated. A machine‐learning algorithm was applied to select the top DMRs, which then were evaluated on plasma‐derived circulating cell‐free DNA (cfDNA) samples of TNBC patients and healthy controls. We identified 23 DMRs accounting for the methylation profile of blood cells and reference mammary cells and then selected six top DMRs for cfDNA analysis. We quantified un‐/methylated copies of these DMRs by droplet digital PCR analysis in a plasma test set from TNBC patients and healthy controls and confirmed our findings obtained on tissues. Differential cfDNA methylation was confirmed in an independent validation set of plasma samples. A methylation score combining signatures of the top three DMRs overlapping with the SPAG6, LINC10606 and TBCD/ZNF750 genes had the best capability to discriminate TNBC patients from controls (AUC = 0.78 in the test set and AUC = 0.74 in validation set). Our findings demonstrate the usefulness of cfDNA‐based methylation signatures as noninvasive liquid biopsy markers for the diagnosis of TNBC.

Gastric cancer (GC) is the fifth most common cancer and the third most frequent cause of cancer deaths worldwide. The high death rate associated with GC, and lack of appropriate biomarkers for diagnosis, prognosis, and treatment emphasize the need for identification of novel molecules. Given the emerging roles for long non-coding RNAs (lncRNAs) in cancer development, we studied novel lncRNA candidates involved in gastric carcinogenesis. LncRNA candidate discovery was performed using analyses of available datasets and literature. Validation was done using an internal sample set of GC/normal tissues, and external independent datasets. Network analysis and functional annotation of co-expressed protein coding genes were performed using the weighted gene correlation network analysis (WGCNA) and ingenuity pathway analysis. Two novel lncRNAs, PCAT18 and LINC01133, associated with GC development were identified by analysis of the discovery Gene Expression Omnibus (GEO) datasets. The down-regulation of these genes in GC tissues was successfully validated internally and externally. The results showed a tissue-specific down-regulation of PCAT18 and LINC01133 in gastrointestinal tissues. WGCNA and ingenuity pathway analyses revealed that the genes co-expressed with the two lncRNAs were mostly involved in metabolic pathways and networks of gastrointestinal disease and function. Our findings of a tissue-specific down-regulation of PCAT18 and LINC01133 in gastric and other gastrointestinal cancers imply that these lncRNAs may have a tumor suppressive function in the development of these tumor entities. The two lncRNA biomarkers may contribute to a better understanding of the complex mechanisms of gastric carcinogenesis.

Aberrant DNA methylation is often involved in carcinogenesis. Our initial goal was to identify DNA methylation biomarkers associated with pancreatic cancer. A genomewide methylation study was performed on DNA from pancreatic ductal adenocarcinoma (PDAC) and endocrine pancreas tumors. Validation of DNA methylation patterns and concomitant alterations in expression of gene candidates was performed on patient samples and pancreatic cancer cell lines. Furthermore, validation was done on independent data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). Finally, droplet digital PCR was employed to detect DNA methylation marks in cell‐free (cf) DNA isolated from plasma samples of PDAC patients and cancer‐free blood donors. Hypermethylation of the SST gene (encoding somatostatin) and concomitant downregulation of its expression were discovered in PDAC and endocrine tumor tissues while not being present in chronic pancreatitis (inflamed) tissues and normal pancreas. Fittingly, treatment with a somatostatin agonist (octreotide) reduced cell proliferation and migration of pancreatic cancer cells. Diagnostic performance of SST methylation in a receiver operating characteristic curve analysis was 100% and 89% for tissue and plasma samples, respectively. A large body of TCGA and GEO data confirmed SST hypermethylation and downregulation in PDAC and showed a similar effect in a broad spectrum of other tumor entities. SST promoter methylation represents a sensitive and promising molecular, pan‐cancer biomarker detectable in tumor tissue, and liquid biopsy samples.

Aberrant DNA methylation has been investigated in carcinogenesis and as biomarker for the early detection of colorectal cancer (CRC). The present study aimed to define the methylation status in the regulatory elements of two proapoptotic genes, Fas cell surface death receptor (FAS) and BCL2-associated X protein (BAX). DNA methylation analysis was performed in tumor and adjacent normal tissue using HpaII/MspI restriction digestion and methylation-specific polymerase chain reaction (PCR). The results observed downregulation of the FAS and BAX genes in the CRC tissues compared with the adjacent normal samples. Furthermore, demethylation using 5-aza-2′-deoxycytidine treatment followed by reverse-transcription quantitative PCR were performed on the HT-29 cell line to measure BAX and FAS mRNA expression following demethylation. The 5-aza-2′-deoxycytidine treatment resulted in significant FAS gene upregulation in the HT-29 cell line, but no significant difference in BAX expression. Furthermore, analysis of CpG islands in the FAS gene promoter revealed that the FAS promoter was significantly hypermethylated in 53.3% of tumor tissues compared with adjacent normal samples. Taken together, the results indicate that decreased expression of the FAS gene due to hypermethylation of its promoter may lead to apoptotic resistance, and acts as an important step during colorectal carcinogenesis.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is by far the most common type of pancreatic cancer. It constitutes about 90% of tumors of the exocrine pancreas. The aggressive nature of PDAC along with its poor diagnostic markers contributes to a high lethality of this disease. Studies from other cancer entities implicate the role of viruses in tumor development. However, there have been no established reports about virus(es) associated with pancreatic cancer. The present study identified a virus by genomic and digital microRNAome subtraction between healthy and PDAC patients, suggesting that this virus may play a role in carcinogenesis. Experimental procedures: Representational difference analysis (RDA) was utilized to perform an experimental genome-wide search for DNA sequences that occur in PDAC but not in normal tissues. Ten healthy and ten PDAC tissue DNA samples were used for the generation of representations and subtractive hybridization. The difference products (DPs) so obtained were paired end sequenced on the Illumina MiSeq platform. The sequencing data were aligned against the viral RefSeq database. We also did in silico subtraction of microRNA content (miRNA-seq) of PDAC samples. Eight PDAC samples were analyzed for viral sequences using relevant RefSeq databases. The two approaches (RDA and miRNA-seq) were pursued in parallel. RT-qPCR analysis and the in vitro functional characterization of viral sequences in PDAC cell lines were respectively performed using hydrolysis probes and second generation microRNA mimic systems. Stable cell lines expressing viral microRNA and luciferase were produced using lentivirus for mice in vivo studies. Results and Summary: We found DNA sequences of a virus in the difference products of RDA whose signature was also implicated in the microRNA analysis. Further, using RT-qPCR, we identified that a particular microRNA from this virus is expressed at significantly higher levels in PDAC samples than in normal tissue. This observation was also verified by droplet digital PCR analysis. Non-metastatic PDAC cell lines overexpressing viral microRNA showed significant invasion against relevant controls, interestingly with no significant change in proliferation. We are currently analyzing the functional consequences of these viral sequences and their involvement in carcinogenesis. Conclusion: From two different robust approaches, these findings strongly indicate viral sequences associated with PDAC that could affect pathways relevant to tumor development. Citation Format: Mohanachary Amaravadi, Agnes Hotz-Wagenblatt, Sandeep Kumar Botla, Pouria Jandaghi, Mehdi Manoochehri, Nathalia Giese, Markus W. Büchler, Andrea S. Bauer, Jörg D. Hoheisel. Genomic and microRNAome subtraction identifies pathogenic viral sequences in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2718. doi:10.1158/1538-7445.AM2015-2718