Sa1898 Next Generation Sequencing (NGS)-Based Transcriptomic Approaches for the Identification of Novel Colorectal Cancer (CRC)-Specific miRNA Biomarkers

AA with CRC provides insight into identification of novel somatic mutations in APC from African Americans with CRC. Our data suggest an association between specific mutations in the WNT signaling pathway and risk of CRC. The analysis of the pathogenicity of these novel variants might help understand the aggressive nature of CRC in African Americans and may lead to the development of more effective ways to reduce cancer morbidity and mortality in this population.

Sa1898

Background: CRC is the second leading cause of cancer-related deaths worldwide. Aberrant expression of several microRNAs (miRNAs) has been reported in several cancers, including CRC. Although many previous studies have conducted miRNA profiling using conventional hybridization-based microarray platforms, these approaches have several limitations including a lack of sensitivity, narrow dynamic range, and non-specific hybridization. In comparison to microarray platforms, NGS-based sequencing technologies enable direct determination of the identity and abundance of whole transcripts, which facilitates the identification of comprehensive, novel, and cancer-specific miRNAs. Aim: This study aimed to perform a comprehensive discovery of CRC-specific miRNAs using NGS-based whole transcriptome profiling. Materials and Methods: CRC-specific miRNA profiles were generated by NGSbased whole transcriptome analysis in matched pairs of CRC and adjacent normal mucosa (NM). Quantitative RT-PCR validation of the newly discovered miRNA signature was performed in 32 CRC and matched 16 NM tissue specimens. The clinical significance of the miRNA signature was evaluated in a second, independent cohort of 175 CRC tissues. Results: NGS analysis identified 15 CRC-specific miRNAs compared to matched NM tissues (P<0.05 and Min count>100). Among 15 miRNAs, 3 miRNAs (miR-31, miR-182, and miR-224) were quantitatively validated (P<0.0001). All 3 miRNAs were elevated in more advanced CRC, and their expression was significantly associated with poor patient survival. While high miR-224 (HR= 2.1, P=0.04) expression in CRC tissues was an independent prognostic factor, elevated levels of miR-182 expression independently predicted distant metastasis (OR=3.6, P=0.01). Conclusion: We discovered novel CRC-specific miRNA signatures using NGS-based whole transcriptome profiling. MiR-224 and miR-182 expression have the potential to serve as novel biomarkers for predicting prognosis and metastasis in CRC patients.

Sa1896 Nanomaterial-Based Sensor Technology Can Detect Gastric Cancer and Peptic Ulcer Disease With a High Accuracy From an Exhaled Air Sample Haitham Amal, Marcis Leja, Konrads Funka, Roberts Skapars, Inta Liepniece-Karele, Ilze Kikuste, Aigars Vanags, Ivars Tolmanis, Hossam Haick BACKGROUND. Early and non-invasive detection of gastric cancer (GC) is remaining a challenge in many high-risk areas. OBJECTIVES. To investigate the feasibility of gastric cancer and peptic ulcer disease detection by measuring volatile organic compounds (VOCs) in the exhaled breath by a nanomaterial-based sensor technology. METHODS. Alveolar exhaled breath samples were collected from 99 GC patients, 53 peptic ulcer disease (PUD) patients and 342 controls in Latvia (Caucasian population) having been investigated by upper endoscopy. Nanomaterial-based sensors - Gold nano particles (GNP) and single-wall carbon nanotube (SWCNT) were used to discriminate the GC and the PUD groups from the healthy controls by loading discriminant factor analysis (DFA) pattern recognition. Classification success was calculated by (i) building an algorithm for 70% of the samples as a training set and (ii) randomly blinding 30% of the samples as a validation set. RESULTS. The blind DFA models showed: (i) An excellent discrimination between the GC patients and controls (91% accuracy); (ii) An excellent discrimination between the GC patients and PUD patients (86% accuracy); (iii) Good discrimination between the PUD patients and controls (80% accuracy). CONCLUSIONS: The obtained results are demonstrating the good potential of VOC detection in exhaled breath by nanomaterial-based sensor technology in diagnosing GC and PUD. Performance characteristics of nanomaterial-based sensor technology to detect gastric cancer and peptic ulcer disease

Sa1899 Fecal Recovery of Ingested Cellular DNA: Implications for Noninvasive Detection of Upper Gastrointestinal Neoplasms Benjamin B. Strauss, Tracy C. Yab, Helen M. O, William R. Taylor, Douglas W. Mahoney, Julie A. Simonson, John D. Christensen, Suresh T. Chari, David A. Ahlquist Background: Stool DNA (sDNA) testing, shown to effectively detect colorectal neoplasms, represents a potential noninvasive approach to the screen-detection of upper gastrointestinal (UGI) neoplasms as well. However, luminal digestion by DNAases from saliva, pancreatic excretion, and colonic microflora represents a potential impediment; and little is known about the fecal recovery efficiency of DNA exfoliated from UGI tumors. Aims: To establish a safe human ingestion model that approximates the quantity of cellular DNA shed daily from a size range of UGI neoplasms and to estimate fecal DNA marker recovery rates. Methods: Healthy volunteers (n=10, as per power estimate) were asked to ingest two different doses of raw salmon, 0.3g and 30g, simulating the estimated mass exfoliated daily from 1 cm and 4.5 cm lesions. To approach a steady-state, each dose was ingested over 3 consecutive days along with a regimented diet. Dose order was randomized in a 2x2 crossover design with a minimum 5-day interval washout period. Following defecation of an indicator dye that was ingested with test meals, all stools were collected over the next 48 hours, stored at -20C, and then batch assayed in blinded fashion. Hybrid bead capture of ingested salmon DNA was performed on fecal supernatants using probes targeting short interspersed nuclear sequences (SlmII) that are pathognomic for the Salmonidae family (i.e., no homology with human or bacterial DNA). Captured DNA was washed and quantified with real-time PCR using primers to generate 178, 138, 88 and 55 base pair (bp) amplicons. Results: SlmII sequences were successfully recovered in stool from all volunteers after both doses of ingested salmon, and the recovered quantity was proportional to the amount ingested. However, fecal recovery efficiencies of the ingested salmon marker were very low at <0.1%. In contrast, recoveries of fragmented Slmll spiked directly into stool averaged 15.7% (range 14.8-16.7%) across volunteers. Absolute fecal recovery rates of ingested cellular DNA varied inversely and exponentially with targeted amplicon size (Figure); mean recovery rates were 0.49, 0.91, 3.63, and 7.31 copies per 100,000 copies of ingested SlmII for 178, 134, 88, and 55 bp amplicons, respectively, p=0.0001. Neither SlmII dose nor oro-anal transit time affected recovery efficiencies. Conclusions: While recovery rates are low, ingested cellular DNA simulating daily amounts shed from UGI tumors can readily be detected in stool. Assay of shorter-fragment analyte increases recovery. This ingestion model has potential value in studying the effects of various perturbations relevant to the fecal recovery of DNA exfoliated from UGI tumors.

GC-gastric cancer PUD- peptic ulcer disease Sa1897 Novel Evidence for Interleukin-8 (IL-8) As a Predictive Biomarker of Response to 5fluorouracil Based Chemotherapy in Colorectal Cancer Lucía Pérez-Carbonell, Cecilia M. Egoavil, Estefanía Rojas, Cristina Alenda, Maria Rodriguez-Soler, Carla Guarinos, Miriam Juarez-Quesada, Montserrat Andreu, Xavier Llor, Antoni Castells, Rodrigo Jover, C. Richard Boland, Ajay Goel Background: Recently evidence suggests that the expression and secretion of IL-8 by colon cancer cells not only promotes proliferation, invasion and stem cell-like activity, but also increases chemoresistance to oxaliplatin chemotherapy. However, no previous studies have evaluated the role of IL-8 expression in 5Fluorouracil (5FU)-based chemotherapy, which remains the standard of care for treating patients with colorectal cancer (CRC). The objective of the present study was to interrogate whether IL-8 is involved in determining the lack of response to 5FU-based chemotherapy, using a systematic approach that involves its functional characterization in colon cancer cell lines and subsequent validation of this phenomenon in clinical specimens from a population-based cohort of CRC patients. Methods: We established isogenic pairs of HCT116 colon cancer cells that were rendered chemoresistant to 5FU (5FU-R), in which we assessed the effect of IL-8 inhibition by siRNA transfection and by treating them with the IL-8 receptor antagonist, SCH-527123. For clinical validation, we analyzed tissues that were obtained from 415 CRC patients enrolled in a population-based, prospective, multicenter, cohort (EPICOLON II), who were uniformly treated with a 5FUbased chemotherapeutic regimen. We interrogated IL-8 expression in the cell lines and clinical specimens using qRT-PCR and immunohistochemistry. Results: IL-8 expression was elevated in several pairs of 5FU-R colon cancer cell lines compared to the parental ones. Inhibition of IL-8 by either siRNA transfection or treating with the CXCR2 antagonist decreased cell viability, proliferation, colony formation, and increased apoptosis and cell arrest in 5FU-R HCT116 cells. For clinical validation, among stage II-III CRC patients, 26.3% (45/171) had elevated expression of IL-8 levels in their tumor tissues. In these patients, IL-8 up-regulation emerged as an independent predictor of poor survival (HR= 2.17, 95%CI: 1.1-4.3, p=0.027). Interestingly, high expression of IL-8 was an independent risk factor for poor disease-free survival in patients treated with 5-FU based adjuvant chemotherapy (HR=3.8, 95%CI: 1.66-8.7, p=0.001), while only lymph node metastasis independently predicted survival in non-treated patients. Conclusions: Our data reveal that IL-8 is frequently over-expressed in CRC cell lines and tissues, and direct inhibition of IL8 or indirect inhibition by a CXCR2 antagonist decreased tumorigenic behaviors and 5FU resistance in the 5FU-R HCT116 cells. We also identified that elevated IL-8 expression serves as an independent prognostic and predictive biomarker in stage II and III CRC patients, suggesting that it is a potential target for personalizing treatment in patients with this malignancy.

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AGA Abstracts

AGA Abstracts

Next Generation Sequencing (NGS)-Based Transcriptomic Approaches for the Identification of Novel Colorectal Cancer (CRC)-Specific miRNA Biomarkers Keun Hur, Guorong Xu, Yoshinaga Okugawa, Yuji Toiyama, Aaron N. Chang, C. Richard Boland, Ajay Goel