Sa1943 Feasibility Study Evaluating Whole Exome Sequencing of Endoscopic Ultrasound Fine Needle Aspiration (EUS FNA) Cytology Specimens

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AGA Abstracts

microRNA Markers for the Diagnosis of Pancreatic and Bile Duct Cancers Motohiro Kojima, Hiroko Sudo, Junpei Kawauchi, Satoko Takizawa, Satoshi Kondou, Hitoshi Nobumasa, Atsushi Ochiai It is recently reported that microRNAs (miRNAs) are stably present in serum and potentially useful in the diagnosis of cancer. Using highly sensitive microarray, 3D-Gene®, we examined comprehensive miRNA expression profiles in serum obtained from 66 pancreatic cancer patients, 66 bile duct cancer patients and 100 matched healthy volunteer as the study set, and found that 122 miRNA markers for pancreatic cancer and 125 miRNA markers for bile duct cancer differentiated pancreatic / bile duct cancer patients from healthy control with a statistical significance. We then validated the diagnostic performance of these markers using serum obtained from 33 pancreatic cancer patients, 33 bile duct cancer patients and 50 healthy controls. One of the single miRNA markers for pancreatic cancer yielded more than 90% diagnostic sensitivity and specificity, and combined use of two markers perfectly differentiated cancer and healthy samples. We also found that over 100 miRNA markers of pancreatic cancer overlapped with miRNA markers of bile duct cancer. Our results suggest that these miRNA markers have clinical values to screen out pancreatic and bile duct cancer patients. Sa1941 The Morphological Features of Colorectal Tumors That Are Associated With the Appearance of Methylated DNA in Plasma Dileep Mangira, Erin L. Symonds, Graeme P. Young, Stephen R. Cole, Libby Bambacas, David Murray, Rohan T. Baker, Lawrence C. LaPointe, Susanne K. Pedersen Background: Aberrant methylation of genes plays an important role in tumor development, with recent studies showing that tumor-derived hypermethylated DNA is detectable in free circulating nucleic acids extracted from the plasma of patients with colorectal cancer (CRC). Blood based CRC screening tests detecting methylated DNA could provide an additional avenue for improving screening participation rates. However, the biology/mechanisms resulting in appearance of tumor DNA into blood are not well understood since these markers are more frequently detected in tumour tissue than in blood. Aim: To determine whether the presence of methylated BCAT1 and IKZF1 in plasma correlates with morphological features of the colorectal cancer. Methods: Blood was collected from volunteers undergoing colonoscopy for any indication, or known to have CRC but prior to surgery (n = 1777). The level of methylated BCAT1 and/or IKZF1 in bisulphite converted DNA isolated from 4mL plasma was measured using a multiplexed PCR assay. Any sample containing detectable levels of either methylated gene was considered positive. Colorectal tumor pathology was assessed for size, location, depth of invasion (T stage), degree of differentiation and perivascular invasion. Lymph node involvement and presence of distant metastasis were also determined. Univariate (Chi2 test) and multivariate logistic regression analysis were performed to determine the factors associated with test positivity. A Spearman Rank correlation was performed to assess the correlation of tumor features to the levels of methylated BCAT1 and IKZF1 in plasma. P<0.05 was considered statistically significant. Results: A total of 127 cases of invasive CRC were identified (median age 68y, 62% male). Stage distribution (AJCC) was: I n = 33; II n = 36; III n = 35; IV n = 22; with one unstaged. Univariate analysis revealed that advanced stage, presence of vascular invasion, and presence of nodal or distant metastases were significantly associated with detection of the methylated genes in the blood (p<0.05, Table). Following adjustment for the biological features, multivariate logistic regression analysis revealed depth of invasion (T3: OR 69.84, 95% CI 5.65-863.12; T4: OR 187.17 95% CI 9.94-3525.43) and distal location (OR 5.99, 95% CI 1.42-25.32) were significantly associated with detectable marker (p<0.001). Concentration of BCAT1 and IKZF1 in plasma was strongly associated with depth of invasion (r=0.461, r = 0.481, respectively, p < 0.0001) and overall tumor size (r=0.348, r = 0.463, respectively, p < 0.0001). Conclusion: Plasma concentrations of methylated IKZF1 and BCAT1 correlate positively with depth of cancer invasion, advanced stage and presence of nodal or distant metastases. The appearance of tumor-associated DNA in the circulation appears to be dependent on depth of invasion and extent of spread. Table- Significant biological variables on univariate analysis

Sa1942 Targeted Cancer Gene Sequencing Identifies Potential Causative Novel Candidate Mutations in African Americans Colon Carcinogenesis Hassan Ashktorab, Michael L. Nickerson, Edward L. Lee, Babk Shokrani, Ali Afsari, Adeyinka O. Laiyemo, Sudhir Varma, Sara E. Bass, Joseph Boland, Wen-Yi Huang, Meredith Yeager, Lee E. Moore, Tahmineh Haidary, Hassan Brim Background: Much of the underlying genetic ‘cancer driver' mutations in sporadic colorectal cancer (CRC) have not been characterized by race. Aim: Here, we report the identification of distinct novel variants from CRC patients in mismatch repair (MMR) genes MHS3 and MSH6, and APC. Method: We developed a panel of 20 frequently altered colon cancer genes comprised of ACVR2A, APC, ARID1A, BRAF, FAM123B, FBXW7, KRAS, MSH2, MSH3, MSH6, NRAS, PIK3CA, POLE, PTEN, SMAD2, SMAD4, SOX9, TCF7L2, TGFBR2, and TP53 for targeted sequencing in 138 colon tissues comprised of 26 normal tissue sample, 22 adenomas, 33 advanced adenoma and 57 colorectal tumors. Multiplex PCR and Ion Torrent sequencing was used to examine 98.8% of the targeted exons and splice junctions at a depth of sequencing that allowed for high confidence variant calling (most bases were covered by ≥ 500 reads). After alignment and variant calling, we annotated the variants with information from the 1000 Genomes Project, COSMIC, Polyphen2, and PFAM domain and transcription factor motifs. Results: Excluding synonymous SNVs, 212 deleterious variants in adenoma, 760 in advanced adenoma, and 2624 variants in tumors were detected. Three were known pathogenic variants (MSH3 V250M, MSH6 T627I and APC R1432X). Novel variants (1591 and 1363) were found in MMR genes (MSH6 and MSH3) and APC gene, respectively. Most of the MMR (449) and APC (563) variants are deleterious mutations, respectively, suggesting the value of a broad cancer gene panel. Spearman correlation between number of deleterious mutations and progression of disease (normal-adenoma-cancer) for MSH3, MSH6 and APC was 0.27, 0.28 and 0.36 respectively. The occurrence of potentially deleterious MMR and APC gene variants suggests possible misclassification of MMR status. Notably, among the 57 CRC cases, [(22/57=39%) for MSH3, MSH6; and (36/57=63% for APC)] carried likely deleterious MMR and APC mutations, respectively, suggesting the value of a broad cancer gene panel. Conclusion: These findings further highlight the relevance of APC gene in CRC onset but also the potential underestimation of the MSI-H in sporadic CRC as many of the novel mutations in MMR genes detected here were of a deleterious nature. Sa1943 Feasibility Study Evaluating Whole Exome Sequencing of Endoscopic Ultrasound Fine Needle Aspiration (EUS FNA) Cytology Specimens Ferga C. Gleeson, Sarah E. Kerr, Benjamin R. Kipp, Eric W. Klee, Konstantinos Lazaridis, Eric Wieben, Michael J. Levy Background and Objective: Whole exome sequencing (WES) is a highly complex molecular evaluation to identify DNA mutations in the protein coding region of the cancer genome. It typically requires greater DNA quality and quantity than formalin fixed, paraffin embedded tumor tissue can provide. As cytology specimens are less invasive and more cost effective to obtain than snap frozen surgical excision specimens, our objective was to determine if high-quality exome sequencing data could be generated from a variety of EUS FNA tumor cytology samples to potentially determine somatic alterations that may be relevant to understanding tumor biology or targeted treatment for individualized medicine. Methods: We analyzed 12 DNA samples extracted from clinically obtained alcohol fixed direct cytology smears (n = 9) or cell pellets (n = 3) yielding 0.4 - 3.3 ug of DNA (mean 1.4 ug), as measured by fluorometry. Samples were derived from fine needle aspiration/biopsy representing a.) adrenal gland metastasis from lung cancer, b.) gastric gastrointestinal stromal tumors, c.) malignant lymph nodes from rectal cancer and, d.) primary pancreatic cancer, with 3 samples multiplexed per lane, and 3 samples per cancer type. Bioinformatic analysis included standard secondary analysis using Genome GPS and tertiary review of results to verify high or low

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Sa1947 RNA Sequencing Identifies Unique and Commonly Expressed Genes in Sessile Serrated and Hyperplastic Colon Polyps and Demonstrates Molecular Overlap Between Syndromic and Sporadic Sessile Serrated Polyps Priyanka Kanth, Mary P. Bronner, Deborah Neklason, Randall W. Burt, Curt Hagedorn, Don Delker Purpose: Colorectal cancer (CRC) is the 2nd leading cause of cancer related deaths and 3rd most common cancer in the United States. Serrated colon polyps are found in 12-36% of patients undergoing routine screening colonoscopy. Sessile serrated polyps (SSA/P) are a subtype of serrated polyps that are now considered pre-malignant and "Serrated Polyp Pathway" has been described suggesting SSA/P may progress and contribute to 20-30% of CRC. The Serrated Polyposis Syndrome (SPS) is an extreme phenotype, with multiple serrated polyps, known to carry a high risk of colon cancer however the underlying pathway is poorly understood. Differentiating SSA/P from traditional hyperplastic polyps by colonoscopy or histopathology remains difficult. This makes it necessary to identify better molecular markers for accurate detection of SSA/P and for adding insight into understanding the "Serrated Polyp pathway' in greater detail. Methods: The aim of the study was to identify novel molecular markers of SSA/P using RNA sequencing gene expression analyses. This approach detects differentially expressed genes with greater sensitivity and specificity, does not require prior knowledge of their sequence and includes novel and non-protein coding regulatory RNAs. We analyzed SSA/Ps from patients with SPS (n=12), sporadic SSA/P (n= 10), traditional hyperplastic polyps (n=10), colon cancer (n=4) and normal colon tissues (n=20). Colon samples were prospectively collected from subjects undergoing screening or surveillance colonoscopy. Cancer samples were obtained from the tissue bank at the University hospital. Results: 1918 ≥ 1.5 fold highly differentially expressed genes were found in SSA/P when compared to hyperplastic polyps (Fig 1). An overlap of gene expression was noted in SSA/P's from patients with SPS and with sporadic SSA/P's. 343 genes that were ≥ 2 fold changed from controls were common to SSA/Ps and colon cancer as compared to only 71 genes overlapping between hyperplastic polyps and cancer (Fig 2). Conclusion: This is the first study comparing differentially expressed genes in SSA/Ps in both syndromic and sporadic patient cohorts using comprehensive RNA sequencing analysis. RNA sequencing identified 1918 uniquely expressed genes in SSA/P's compared to HPs. The molecular overlap between sporadic and syndromic SSA/P's indicates a likely common underlying pathway in development of colon cancer from SSA/P's. A number of common genes found in SSA/P's and colon cancer tissues suggest a possible role of these genes in the development of colon cancer from SSA/P's.

Sa1944 Immunohistochemical Localisation of Kallikrein-Related Protease 14 (KLK14) in Colon Tissues During Cancer Development: Expression and Clinical Evaluation Maroulio Talieri, Dimitrios Kypraios, Marina Devetzi, Georgia Papachristopoulou, Apostolos Malachias, Stefanos P. Bassioukas, Stavros Stavrinides, Dimitrios Xinopoulos, Loukas Theodoropoulos INTRODUCTION: Human kallikrein-related peptidases (KLKs) constitute a family of 15 serine proteases with diverse roles in both physiological and pathological processes, including carcinogenesis. KLK14 is a steroid hormone-regulated gene encoding for a trypsin-like protease, expressed mainly in the central nervous system and endocrine tissues. KLK14 has been reported to have a potential value as tumor biomarker for various types of cancer, including prostate, breast, ovarian, non-small cell lung and colon carcinomas. AIM: To investigate the localisation of KLK14 in colon tissues at different stages of colon cancer development, as well as to determine its expression on these tissues and evaluate it clinically. MATERIALS AND METHODS: In this study, we assessed the KLK14 antigen expression of 75 colon cancer tissues, using immunohistochemistry. RESULTS: KLK14 is intensely expressed on the epithelium of colonic carcinoma and adenoma, but not on the normal colonic mucosa. KLK14 staining is mainly observed on epithelial cells and less on the stroma. KLK14 staining is cytoplasmic, but is also seen on the colon lumen and mucus. Neoplastic cells show cytoplasmic staining on the lumen surface. Colon cancer shows granular cytoplasmic staining, whereas colon adenomas show a cytoplasmic homogeneous staining and is present only on the lumen surface. On the adenomas' columnar epithelium cytoplasmic expression is observed around the nucleus, present on the lumen and mucus, picture corresponding to protease. Poorly differentiated tumors of more advanced stage showed higher KLK14 immunostaining. Follow-up analysis revealed that KLK14 was significantly associated with shorter Overall Survival (OS) (p=0.02) and Disease-Free Survival (DFS) (p=0.01). CONCLUSION: Present data suggests that KLK14 protein is intensely expressed on the epithelium of colon cancer and adenoma tissues, whereas not on the normal colonic epithelium. Also, KLK14 is up-regulated in colon cancer and its expression predicts poor prognosis for colon cancer patients.

Fig 1: Genes differentially expressed ≥ 1.5 fold and FDR < 0.05 in sessile serrated polyps (syndromic and sporadic) and hyperplastic polyps by RNA sequencing.

Sa1946 Low-Expression of the Tumor Suppressor Opcml Is an Independent Prognostic Factor in Gastric Cancer Progression Xiangbin Xing Gastric cancer is the second leading cause of cancer-related death worldwide while its prognostic markers and potential treatment targets remain under investigation. OPCML falls into the IgLON family of Ig domain-containing GPI-anchored cell adhesion molecules, and was recently found to play a crucial role in the development of human cancer. We found that OPCML expression was significantly diminished or lost in gastric cancer tissues as compared to matched adjacent non-cancerous gastric tissues and was also markedly reduced in gastric cancer cell lines. The reduced expression of OPCML was associated with promoter methylation. Ectopic expression of OPCML in gastric cancer cells significantly inhibited cell viability (p<0.01) and anchorage-dependent (p<0.001) and -independent (p<0.001) colony formation in vitro, and suppressed tumor growth in vivo. Moreover, OPCML inhibited growth of gastric cancer cells via arresting cell cycle in G0/G1 phase and inducing cell apoptosis. In addition, reduced OPCML expression had significant association with more advanced tumor stages (p=0.007) and poorer tumor differentiation (p<0.001) in gastric cancer. Interestingly, Cox multivariate analysis revealed that OPCML was an independent prognostic factor for survival of gastric cancer patients (HR=2.34; p=0.002). Our data reveal that OPMCL functions as a tumor suppressor in gastric cancer and its low-expression is an independent predictive factor for poor survival of gastric cancer patients.

Fig 2: Overlapping genes between SSA/P and Colon cancer. Genes differentially expressed ≥ 2 fold and FDR < 0.01 in sessile serrated polyps (syndromic and sporadic), hyperplastic polyps and colon cancer by RNA sequencing.

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quality sequencing outcomes. In addition, results were stratified by tumor type, if there was variability in the sequence quality, reads generated, or basic coverage metrics. To assess the fidelity of variant calls in the EUS FNA exome data, results were compared to mutations previously detected by a 50 gene cancer panel. Results: The sequencing study was successful with a sufficient/expected number of reads per sample generated (Table 1). The mean percent duplicate reads per sample was 12.9% (range 4.8-38.6%). Exome coverage varied between samples; 50-85% of the exome was covered at 50x, and 20-50% of the exome was covered at 100x. The 50 gene panel comparator set comprised of 87 mutations across 12 samples. Comparison revealed that 86 of 87 mutations reported by the panel were also detected by exome sequencing. Conclusion: This is the first report to demonstrate the success of EUS FNA exome-sequencing. There was sufficient coverage in all samples and the variant identification between the 50 gene panel and the exome sequencing was highly concordant. The findings reveal the potential clinical utility of this method when applied to routine clinical cytology specimens to identify potential therapeutic targets which could be readily applied in a clinical research trial and eventually direct clinical care. Table 1