Mart DeLaCruz

Dysregulation of microRNAs in colonic field carcinogenesis: implications for screening.

Colorectal cancer (CRC) screening tests often have a trade-off between efficacy and patient acceptability/cost. Fecal tests (occult blood, methylation) engender excellent patient compliance but lack requisite performance underscoring the need for better population screening tests. We assessed the utility of microRNAs (miRNAs) as markers of field carcinogenesis and their potential role for CRC screening using the azoxymethane (AOM)-treated rat model. We found that 63 miRNAs were upregulated and miR-122, miR-296-5p and miR-503# were downregulated in the uninvolved colonic mucosa of AOM rats. We monitored the expression of selected miRNAs in colonic biopsies of AOM rats at 16 weeks and correlated it with tumor development. We noted that the tumor bearing rats had significantly greater miRNA modulation compared to those without tumors. The miRNAs showed good diagnostic performance with an area under the receiver operator curve (AUROC) of >0.7. We also noted that the miRNA induction in the colonic mucosa was mirrorred in the mucus layer fecal colonocytes isolated from AOM rat stool and the degree of miRNA induction was greater in the tumor bearing rats compared to those without tumors. Lastly, we also noted significant miRNA modulation in the Pirc rats- the genetic model of colon carcinogenesis, both in the uninvolved colonic mucosa and the fecal colonocytes. We thus demonstrate that miRNAs are excellent markers of field carcinogenesis and could accurately predict future neoplasia. Based on our results, we propose an accurate, inexpensive, non-invasive miRNA test for CRC risk stratification based on rectal brushings or from abraded fecal colonocytes.

In vivo measurement of the shape of the tissue-refractive-index correlation function and its applicationto detection of colorectal field carcinogenesis

Polarization-gated spectroscopy is an established method to depth-selectively interrogate the structural properties of biological tissue. We employ this method in vivo in the azoxymethane (AOM)-treated rat model to monitor the morphological changes that occur in the field of a tumor during early carcinogenesis. The results demonstrate a statistically significant change in the shape of the refractive-index correlation function for AOM-treated rats versus saline-treated controls. Since refractive index is linearly proportional to mass density, these refractive-index changes can be directly linked to alterations in the spatial distribution patterns of macromolecular density. Furthermore, we found that alterations in the shape of the refractive-index correlation function shape were an indicator of both present and future risk of tumor development. These results suggest that noninvasive measurement of the shape of the refractive-index correlation function could be a promising marker of early cancer development.

Nano-Architectural Alterations in Mucus Layer Fecal Colonocytes in Field Carcinogenesis: Potential for Screening

Current fecal tests (occult blood, methylation, DNA mutations) target minute amounts of tumor products among a large amount of fecal material and thus have suboptimal performance. Our group has focused on exploiting field carcinogenesis as a modality to amplify the neoplastic signal. Specifically, we have shown that endoscopically normal rectal brushings have striking nano-architectural alterations which are detectable using a novel optical technique, partial wave spectroscopic microscopy (PWS). We therefore wished to translate this approach to a fecal assay. We examined mucus layer fecal colonocytes (MLFC) at preneoplastic and neoplastic time points (confirmed with rat colonoscopy) in the azoxymethane (AOM)-treated rat model and conducted PWS analysis to derive the nano-architectural parameter, disorder strength (Ld). We confirmed these results with studies in a genetic model (the Pirc rat). We showed that MLFC appeared microscopically normal, consistent with field carcinogenesis. Ld was elevated at an early time point (5 weeks post-AOM injection, effect size = 0.40, P = 0.024) and plateaued before adenoma formation (10 weeks post-AOM, effect size = 0.66, P = 0.001), with no dramatic increase once tumors developed. We replicated these data in the preneoplastic Pirc rat with an effect size in the MLFC that replicated the rectal brushings (increase vs. age-matched controls of 62% vs. 74%, respectively). We provide the first demonstration of a biophotonics approach to fecal assay. Furthermore, targeting the nano-architectural changes of field carcinogenesis rather than the detection of tumor products may provide a novel paradigm for colorectal cancer screening. Cancer Prev Res; 6(10); 1111–9. ©2013 AACR.

MicroRNAs as Novel Targets for NSAID Chemoprevention of Colon Carcinogenesis

Background & Objective: In the United States, colorectal cancer (CRC) is the third most prevalent and deadly malignancy. Development of novel CRC-specific abnormal DNA biomarkers may advance non-invasive, cost-efficient population-based CRC screening and ultimately reduces CRC death. DNA hypermethylation is a common epigenetic abnormality in CRCs and represents a promising class of cancer biomarkers. The objective of the current study was to develop optimal DNA hypermethylation-based biomarkers for use in fecesor serum-based average-risk CRC screening. Design: We first applied DNAmethylationmicroarray analysis in order to identify novel loci demonstrating neoplasia-specific methylation in the colon. This array analysis allowed us to investigate 55% of CpG islands within the genome and was applied to 17 primary CRCs relative to 8 non-neoplastic colonic tissues (NCs) from neoplasia-free subjects. The detected CRC-associated hypermethylation events were then individually measured in 113 colonic tissues comprising 51 CRCs, 9 adenomas, 19 NCs from CRC patients (CRC-NCs), and 34 NCs from neoplasia-free subjects (control NCs) using highly sensitive and quantitative real-time quantitative methylation-specific PCR (qMSP) assays. Receiver-operator characteristics (ROC) curve analysis was applied to the qMSP data in order to assess each individual event as well as combination of events for their ability to discriminate neoplastic from non-neoplastic cases. Results: Microarray-based global methylation profiles discriminated CRCs from NCs. A bioinformatic filtering of the microarray data identified 169 candidate CRC-associated hypermethylation events, including one previously validated hypermethylation marker for fecal DNA-based CRC detection, SFRP2. Fourteen of these 169 loci were evaluated using qMSP assays. Ten of these 14 methylation events significantly distinguished CRCs from control NCs (p<.01). Of these ten events, methylation of VSX2 achieved the highest discriminative accuracy (83.3% sensitivity and 92.3% specificity; Area under ROC curve, or AUC, 0.93, p<1E-6). Similarly, CRC-NCs were significantly discriminated from control NCs by methylation of ALX3 (AUC 0.78, p<1E-4). The discrimination of CRC-NCs from control NCs was improved by a multi-locus methylation panel (AUC 0.83, p<1E-6) relative to ALX3. Although the sample numbers were small, two methylation events significantly distinguished adenomas from control NCs (p<.01). Conclusions: Systematic methylome analysis has identified 11 novel methylation events in neoplastic and non-neoplastic colonic mucosa from CRC patients that accurately discriminate CRC patients from controls. These markers merit further evaluation as candidate biomarkers for stool and circulating DNA-based CRC detection.

Sa1909 Fatty Acid Synthase Modulates NSAID Sensitivity via 15-Hydroxyprostaglandin Dehydrogenase (15-PGDH): Implications for Personalizing Chemopreventive Strategies

G A A b st ra ct s for intractable anastomosis stenosis resistant to repeated EBD.(Purpose)The utility of mucosal incision with steroid injection for intractable anastomotic stenosis after gastrectomy is considered.(Material and Methods)The data of a total of 520 consecutive patients who underwent gastrectomy between 2009 and 2014were analyzed retrospectively with a view to determining the incidence of anastomotic stenosis. The period after operation when symptoms of stenosis appeared, methods of gastrectomy and reconstruction of surgery, complications, methods of treatment of stenosis, times of EBD, and the period until symptoms were disappeared were analyzed. We performed EBD only for cases with severe anastomosis stenosis. Mucosal incision was performed by needle knife for thickest part of stricture scar. As steroid injection, 80mg triamcinolone was performed for lack of mucosa after mucosal incision. (Results)Seven patients (1.3%) were developed severe stenosis which needed to be treated by EBD. The median interval between the surgery and detection of stenosis was 1.5 months. The methods of operation and reconstruction after gastrectomy are proximal gastrectomy with esophagogastric anastomosis for 6 patients and with esophago-jejuno anastomosis for 1 patient. 3 patients were added mucosal incision and steroid injection. No serious complication was happened . The median time of EBD until the lumen was kept was 7 times in all patients. The median period after first EBD until the lumen was kept was 10 months in patients conducted only balloon dilatation without mucosal incision, while the median period after mucosal incision was 1 month in patients with mucosal incision(P<0.001).(Conclusion) Mucosal incision with steroid injection was shown to be safety and useful method for severe anastomotic stenosis after gastrectomy. The adaptation of this method is thought to be cases with severe stricture of anastomoses resistant to EBD dilatation, which shows thick scar and restenosis by mucosal defect by EBD. Steroid mucosal injection is thought to be very useful for maintaining the lumen without stricture. Mucosal incision with steroid injection showed to have possibility to improve stenosis for severe stricture resistant of EBD in a short period.

Su2032 Differential Early Metabolic Changes in Colon Carcinogenesis for Health Disparities: Physiological and Molecular Evidence

Background: Epidemiologic data unequivocally demonstrate a 23% higher incidence in colonic neoplasia in blacks with concomitant 50% increase in colorectal (CRC) mortality compared to whites. However, the biological basis for this has been largely unexplored. The role of early metabolic alterations such as the Warburg effect is clearly appreciated in established cancers however it is not known in early neoplasia. Given that metabolic CRC risk factors (i.e. diabetes, obesity) disproportionately impact blacks, we used polarizationgated spectroscopy (PGS) to examine the microvascular blood flow as a surrogate of metabolic dysregulation in early neoplastic transformation. To explore potential molecular correlates behind PGS, we chose to investigate two master regulators of early CRC, Sirt 6 and Hif-1α, which couple metabolic dysregulation (Sebastian et al., Cell 2012). Methods: For this study we used a fiberoptic PGS probe to measure oxyhemoglobin (OHb) and deoxyhemoglobin (DHb) within rectal microvasculature in 80 prospectively recruited black patients. We compared results to a previous cohort of ~120 white patients in the superficial mucosa (~150μm). We assessed Sirt 6 and Hif-1α expression from rectal biopsies of 80 patients by RT-PCR. Field carcinogenesis was defined as presence of molecular changes in advanced adenomas (AAs) evident elsewhere in the colon. Results: In blacks, there was a striking induction in DHb (220% of control) with OHb being more muted (150% of control). Thus, from a diagnostic perspective, DHb appears to be a stronger marker than OHb. This was in contradistinction to whites, where only OHb was statistically significant with diagnostic potential. The molecular basis of this finding was supported by metabolic regulators, Sirt 6 and Hif-1α, in blacks. The reduced expression of Sirt6 mRNA (Figure 1) appears to give a marked induction of Hif-1α mRNA(Figure 2). Conclusions: We demonstrate herein, for the first time, that there differential metabolic changes in blacks than whites. This was supported physiologically with differential diagnostic effects of DHb versus OHb which implies altered oxygen extraction consonant with Warburg effect. The molecular underpinnings may be related to the key metabolic sensor, Sirt 6 through control of Hif-1α. This work provides potentially important insights into the pathogenesis of CRC disparities and demonstrates the power of optical spectroscopy provide physiological common denominator of the myriad of complex molecular alterations in early carcinogenesis.

548 Correlation of Spectral Signatures With Rectal Prostaglandin E2 Levels As Biomarkers for Aspirin Chemoprevention of Colon Carcinogenesis: Results From a Placebo-Controlled Double Blinded Phase 2B Trial

G A A b st ra ct s had ≥1 adenoma at repeat colonoscopy. Compared to no-therapy, patients exposed to metformin-only had a lower risk of adenoma recurrence irrespective of exam indication (screening, n= 622, HR=0.69 CI: 0.51-0.93; surveillance n= 1308, HR=0.88 CI: 0.71-1.08; diagnostic n=667, HR=0.58 CI: 0.41-0.84). However, the association was not statistically significant in patients who underwent surveillance exams. Conclusion: Metformin use was associated with a reduced risk of recurrent or new primary adenoma after polypectomy. If confirmed in further studies in other subgroups of patients and in randomized trials, metformin could have an important role in the secondary prevention of colorectal adenomas.

1022 Targeting Fatty Acid Synthase (FASN) As a Modality to Ameliorate Obesity Related Colorectal Cancer (CRC)

Background: Recent research demonstrates association between colonic polyps and insulin resistance(IR). However, it is unclear if this association correlates with polyp neoplastic potential(NP) and whether metabolic features such as non-alcoholic fatty liver disease(NAFLD) or components of the metabolic syndrome(MS) are also associated with colonic neoplasia. Aim: To evaluate the associations between various colonic polyps and metabolic alterations. Methods: Case-control study in consecutive subjects(40-70y), undergoing colonoscopy. Cases with colonic polyps and controls with no polyps were evaluated for metabolic and anthropometric parameters, including ultrasound for NAFLD and liver steatosis quantitation by Hepato-Renal-Index(HRI). We excluded subjects at high risk for colonic neoplasia and severe systemic/metabolic illness. T test, Chi-square test and logistic regression analysis were used. Results: Out of 538 participants (mean age 58.8±6.3, 53.5% men) 269 cases with colonic polyps were compared to 269 controls (Table 1). Dose response association was detected between polyp NP(hyperplastic, non advanced and advanced adenomas), HOMA IR score(P for trend=0.03) and HRI (P for trend=0.01). Polyp risk factors in multivariate analysis, adjusting for age and gender, were: MS(OR=1.92, 95%CI 1.34-2.75, P<0.001), low HDL (OR=1.67, 1.14-2.44, P<0.001), abdominal obesity(OR=1.60, 1.10-2.32, P=0.01), and NAFLD (OR=1.47, 1.04-2.08, P=0.02). In cases of advanced adenoma, number of polyps was positively correlated with fasting serum HOMA IR(r=0.258, P<0.01), waist circumference(r=0.206, P=0.03), HRI(r=0.202, P=0.04), and MS components(r=0.224, P= 0.02). Conclusions: Our initial results strongly suggest that IR, MS and NAFLD predict colonic polyps and their neoplastic potential. This data opens venue for prediction models to improve CRC prevention programs. Comparison of Cases with Colonic Polyps and Controls

78 Reversal of Hypermethylation As a Novel Strategy for NSAID Chemoprevention of Colon Carcinogenesis: Potential Role of Dnmt1

BACKGROUND AND AIM: Endoscopy is the first level procedure in the diagnosis of gastrointestinal (GI) masses. However gastro-duodenal or colonic lesions which mainly involve submucosa or subserosa as well as small bowels lesions may be difficult to diagnose. The present study aimed to evaluate the role of ultrasound (US)-guided percutaneous biopsy in diagnosing GI tract lesions. PATIENTS AND METHODS: 114 patients (63 male 51 female patients, age range 55-80 years, median age 68 years) underwent US-guided biopsy of GI lesions from 2000 to 2012. In 36 patients the lesion was in the small bowel, thus endoscopically inaccessible; in 50 patients previous histology of endoscopic biopsies was repeatedly negative; in 18 patients, stenosis did not allow bioptic sampling and in 10 patients age, severe cardiological disease and sepsis prevented endoscopy. We used multi-frequency convex probes with side adapters and 18G cutting needle for histological sampling. After biopsy, patients were monitored by measuring blood pressure and pulse rate during the following 3-4 hours to evaluate possible complications. Histology was compared with postsurgery histological evaluation in 73 cases. In the remaining, histology of US-guided biopsy was the only clue to diagnosis. RESULTS: Biopsy specimens were taken from stomach in 38 cases, small bowel in 36 cases and colon-sigma in 40 cases. Final diagnosis was malignant lesions in all but three cases. One case was intestinal endometriosis, the second was bowel duplication, whereas in the third case it was a false-negative result, the correct diagnosis being gastric adenocarcinoma. Sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy of biopsy were respectively 99%, 100%, 100%, 66%, 99%.We observed no mortality and one complication: A case of melena due to bleeding from gastric gastrointestinal stromal tumor that was effectively controlled by positioning of metal clips. CONCLUSION: US-guided percutaneous biopsy of GI tract lesions may be an alternative, safe and effective procedure for diagnosis of GI tract lesions in those cases where conventional diagnostic approach is not feasible or successful.

Sa1835 Nanocytomics as a Novel Modality for Determining Transcriptional Activity

to the squamocolumnar junction (SCJ) were quantified and compared between Pre-CE-IM and Post-CE-IM groups. RESULTS: 3D-OCT provided 80x 100x larger field-of-view compared to biopsy and reliably (in 100% cases) sufficient imaging depth, into the muscularis mucosa, for detecting SSIM. In comparison, only 40% to 80% biopsies reached the lamina propria as reported in recent studies. SSIM was found in 72% (13/18) patients in the PreCE-IM group, and 63% (10/16) patients in the Post-CE-IM group. The number of SSIM per patient in the Post-CE-IM group [7.1 (9.3)] was significantly lower compared to the Pre-CE-IM group [34.4 (44.6); p = 0.02]. SSIM gland size (p = 0.69) and distribution (p = 0.54) were not significantly different before and after CE-IM. CONCLUSIONS: The prevalence of SSIM in patients who responded to treatment after RFA is higher than previously reported using four-quadrant biopsy. Compared to other advanced imaging modalities only imaging surface [narrow band imaging (NBI), chromoendoscopy] or small focal areas [confocal, elastic scattering spectroscopy (ESS), etc.], 3D-OCT provides both subsurface and broad area of view uniquely suited to potentially evaluate patients before and after ablation treatment. ACKNOWLEDGEMENT: NIH 5R01-CA075289-14, K99-EB010071-01A1, and AFOSR FA9550-10-1-0063 and FA9550-10-1-0551.