Yuko Takehara

Tu1386 Systematic Genomic Characterization of Normal Mucosa, Adenomatous Polyps, and Cancer in Patients With Familial Adenomatous Polyposis

Introduction: Serum tryptase measurement is part of the routine diagnostic work-up of anaphylaxis or suspected mastocytosis. Levels >20 ng/ml would be consistent with recent anaphylaxis and are associated with an elevated risk for mastocytosis. The clinical relevance of a basal elevated tryptase between 11.5 and 20 remains unclear, and autosomal dominant inheritance of such elevations has recently been reported. In this study, families with inherited basal elevations in serum tryptase levels were evaluated at the NIH. The patients (pts) shared similar features, such as atopic symptoms, connective tissue abnormalities, autonomic dysfunctions, neuropsychiatric disorders, and chronic gastrointestinal (GI) symptoms. Here we describe the GI findings from this unique cohort of pts. Methods: Pts were referred to the NIH for evaluation, and the index ptss family was also invited to undergo evaluation. Pts were evaluated by a gastroenterologist and/or completed the Bowel Disease Questionnaire (BDQ). The ptss basic demographic information was noted as well as their GI clinical symptoms, and diagnostic and treatment histories. Laboratory data, including tryptase level, were taken, and endoscopic procedures were performed when clinically indicated. Results: A total of 28 pts with confirmed elevated tryptase levels were seen and evaluated at the NIH. 13 pts were evaluated by a gastroenterologist and completed the BDQ, 9 pts completed the BDQ only, and 6 pts were evaluated by a gastroenterologist only. These pts represented 16 different families. There were 15 males and 13 females, and the median age was 39.1 years. The average BMI was 25.6 and the median tryptase level was 17.9. Of the pts who completed the questionnaire, 13 complained of abdominal pain while 9 did not. Of the pts who had abdominal pain, 11 fulfilled Rome III criteria for irritable bowel syndrome (IBS). 6 had IBS-Mixed, 2 had IBS-Constipation, and 3 had IBS-Diarrhea. Of the pts without abdominal pain, 6 fulfilled Rome III criteria for chronic constipation (CC). Evaluation of upper tract symptoms revealed that 23 of the 28 pts had gastroesophageal reflux disease (GERD) based on history and/or BDQ, and GERD was common in pts with and without abdominal pain (14/16, 9/12, respectively). Conclusions: In this cohort of families with basal elevated tryptase levels, functional GI disorders such as IBS and CC, as well as GERD, were more frequently seen in this cohort than in the general population. An autosomal dominant pattern of inheritance has been discovered. The percentage of functional GI pts with this genetic variation remains unknown, however eliciting personal and familial histories of the comorbid symptoms seen in these families, and, potentially, serum tryptase levels, may be warranted in the workup of patients with functional GI disease.

Sa2043 Inherent Single Nucleotide Variants in the TBX21 Gene As Novel Prognostic Markers in Patients With Pancreatic Ductal Adenocarcinoma

G A A b st ra ct s metastatic PC patients. SOMAmer® reagents are a unique class of DNA aptamers that bind their protein targets with high selectivity and a median limit of detection of 40fM. We have reported that a plasma SOMApanel can detect resectable PC in a multi-center case-control study. Methods: Chart reviews were performed for the 100 PC patients from our site used in the prior study to determine their time to disease progression (PFS) through September 2014. Pre-treatment plasma samples collected between 2008 and 2010 were analyzed for CA 19-9 and a SOMAscanTM assay consisting of 1045 known proteins. Univariate Cox models were used for identifying biomarkers related to time to progression compared to patients who have not progressed. Short PFS was defined as those with documented progression less than one year from PC diagnosis or unrecognized metastatic disease found at surgery. Long PFS was defined as progression greater than one year. The top 10 proteins identified in this analysis were then compared to 42 Stage IV patients. Statistical p-values were calculated with log likelihood ratio test of Cox regression and corrected for multiple comparisons using q-values calculated with the Benjamin and Hochberg method. Results: Nine of 43 patients who had resection for pancreatic cancer were excluded due to noncancer cause of death in the first year or loss to follow up. Thirty-four patients were eligible for progression analysis: 14 with short PFS including 2 individuals with unrecognized metastatic disease and 7 with no disease progression with a minimum 4 years follow-up. As shown in the Figure, two of the 10 top performing prognostic proteins that had higher levels correlating with shorter PFS times were present at similar levels in baseline samples from subjects presenting with Stage IV PC. CA 19-9 was not prognostic in these samples. Conclusions: This study demonstrates the potential utility of identifying biomarkers that predict the presence of unrecognized metastatic disease defined as the detection of metastases at or the rapid recurrence following attempted curative resection of a PC. Future studies are planned to validate the results. If successful, this could lead to a test that identifies a subset of PC patients who can avoid the morbidity and health care costs associated with a major pancreatic resection.

225PEXTENSIVE METHYLATION OF EPIDERMAL GROWTH FACTOR-CONTAINING FIBULIN-LIKE EXTRACELLULAR MATRIX PROTEIN 1 (EFEMP1) PROMOTER COULD PREDICT MALIGNANT FORMATION IN INTRADUCTAL PAPILLARY MUCINOUS NEOPLASMS (IPMN)

ABSTRACT Introduction: Intraductal papillary mucinous neoplasms (IPMN) of the pancreas are pancreatic cystic tumors. Genetic and epigenetic alterations are believed to be accumulated during tumor progression. Whereas the specific genetic mutations and DNA methylation for each type of lesion have been somewhat known, the fundamental understanding for the dynamics of methylation expansion across specific CpG dinucleotides in a gene promoter during carcinogenesis remains unclear. We sought to evaluate whether the expansion of methylation in the EFEMP1 promoter could serve as a predictive biomarker for malignant IPMNs. Methods: KRAS mutations and the methylation status of two discrete regions within the EFEMP1 promoter in 65 IPMN tissues, including 30 IPMNs with low- and intermediate-grade dysplasia, 12 IPMNs with high-grade dysplasia and 23 invasive IPMN, were determined using genomic sequencing and High Sensitive Assay for bisulfate modification DNA (Hi-SA) - a modified COBRA by which fluorescence labeled DNA fragments are detected using a genetic analyzer (Nagasaka T et al., JNCI 2009), respectively. Thereafter, correlation between mutant KRAS, methylation status and various clinic-pathological features were examined. Results: KRAS mutations were detected in 42.9% of low- and intermediate-grade IPMNs, 33.3% of high-grade IPMNs and 71.4% of invasive IPMNs. All mutations were confined to codon 12 of the KRAS gene, and these mutations did not correlate with a specific histological grade or type of lesions, or any of the other clinic-pathological features. On the other hand, while the methylation of the region 1 or region 2 within the EFEMP1 promoter was observed in more than 80% of non-invasive IPMNs and invasive IPMNs, simultaneous methylation of both regions occurred in 0% of noninvasive vs. 34.7% of invasive IPMNs (p = 0.00148). These results suggest that KRAS mutations and extensive methylation within the EFEMP1 promoter were one of the early and late events in cancer progression, respectively. Conclusions: The extensive methylation in EFEMP1 promoter could be a potential predictive marker for identification of invasive IPMNs, and provides an attractive rationale for developing this molecular signature as a substrate for the development of non-invasive screening for invasive IPMNs. Disclosure: All authors have declared no conflicts of interest.

220PCYTOKERATIN 19, A NOVEL PROGNOSTIC BIOMARKER FOR HEPATOCELLULAR CARCINOMA, IS REGULATED BY DNA METHYLATION

ABSTRACT Aim: The expression of CK19 protein has recently been proposed as a novel predictor for poor prognosis following curative resection in patients with hepatocellular carcinoma (HCC). While the mechanisms underlying regulation of CK19 expression in HCC remain unclear, the presence of a CpG island within its promoter region implicates DNA methylation as a potential epigenetic process in this malignancy. Methods: A panel of 564 surgically resected HCCs at Okayama University between 2000 and 2010 were analyzed for CK19 expression and its promoter methylation status. Among all HCCs, we excluded all tumors with recurrence, cancers with TNM stage ≥IIIB, lesions with preoperative therapy, transplantation, and combined hepatocellular-cholangiocarcinomas. CK19 expression was evaluated by immunohistochemistry in FFPE tissues. CK19 promoter methylation was evaluated for region 1 and region 2 by Hi-SA - a modified COBRA by which fluorescence labeled DNA fragments are detected using a genetic analyzer (Nagasaka T et al., JNCI 2009). Molecular results were correlated with clinicopathological features, followed by Kaplan-Meier survival analysis. Results: A total of 125 cases matched for the selection criteria. Among these, 29 HCCs demonstrated positive CK19 staining, and these patients corresponded with significantly poor survival following surgery (p = 0.025), and extra-hepatic metastasis-free survival (p = 0.017). Multivariate analysis revealed that CK19-positive was an independent prognostic factor for survival, following surgery (HR2.85, 95%CI 1.21-6.41, p = 0.018). Methylation analysis revealed that methylation rates at both regions of CK19 gene were statistically higher in HCCs with loss of CK19 expression compared to CK19-positive group (p = 0.0002, p Conclusions: Our data report that CK19 expression is an important prognostic biomarker for HCC. Furthermore, since CK19s expression was strongly associated with the methylation of its promoter region, our study provides novel mechanistic insights for the participation of this epigenetic alteration in the development of HCC. Disclosure: All authors have declared no conflicts of interest.

Su1995 Genetic and Epigenetic Alterations in the Netrin-1 Receptors, UNC5c and DCC, Constitutes a Previously Unrecognized Pathway in Gastric Cancer Progression

Background: Downregulated expression of the netrin-1 dependence receptors, UNC5C and DCC, is a frequent event in significant proportion of colorectal tumors. However, the tumor suppressive role of this important cancer-related pathway in other gastrointestinal cancers remains unclear. We hypothesized that similar to colorectal cancer, down-regulation of UNC5C and DCC may have an important growth-regulatory function in gastric tumorigenesis as well. Methods: We performed a series of genetic (loss of heterozygosity, LOH) and epigenetic analysis (aberrant DNA methylation) for UNC5C and DCC in a cohort of 105 sporadic gastric cancers and the corresponding normal gastric mucosa. Results: In this study, the distribution of gastric cancers as categorized by TNM staging system (UICC 7th) was as follows; 20, 24, 28, and 29 cases in stages I, II, III, and IV, respectively. Overall, 58% (48 out of 83 informative cases) and 76% (65 out of 85 informative cases) of gastric cancers demonstrated either a genetic or epigenetic alteration in the UNC5C and DCC genes, respectively. Interestingly, although epigenetic mechanisms were the primary cause for UNC5C inactivation, methylation-induced transcriptional silencing served also served as the second hit responsible for the DCC gene loss. Taken together, 52% (36 out of 69 gastric cancers with informative data) of gastric cancers showed cumulative defects in these two dependence receptors. The frequency of LOH and aberrant methylation of both dependence receptors gradually increased with progressive tumor stages. Conclusions: This is the first study that unravels the observation that majority of gastric cancers harbor defects in netrin1 receptors. Our observation that both genetic and epigenetic alterations are present early, and continue to escalate in a stepwise manner with the disease progression, highlights the fundamental importance of this growth regulatory pathway in gastric carcinogenesis.