Mo1932 Fluorescent Endoscopy With SERS Nanoparticle for Detecting Colon Polyp in Azoxymethane-Induced Colon Cancer Mice

the EMT processes and stemness characteristics which fuel the fatal metastatic and drug resistant characteristics of advanced tumors. Mo1930

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ATM and P53 Expression Level Correlate With Sensitivity to Olaparib in Gastric Cancer Eiji Kubota, Chris T. Williamson, Susan P. Lees-Miller, Gwyn Bebb Background: Recent studies have demonstrated that small-molecule inhibitors of poly (ADPribose) polymerase (PARP) are promising anti-cancer drugs in the treatment of homologous recombination-defective tumors, such as BRCA1/2-deficient breast cancer. The ataxia telangiectasia mutated (ATM) plays a central role in the regulation of the cellular response to DNA damaging agents. We have previously shown that ATM-deficient Mantle Cell Lymphoma (MCL) cell lines are sensitive to the PARP inhibitor olaparib. Moreover, disruption of p53 in ATM-deficient MCL enhances sensitivity to olaparib. Up to 15% of gastric cancer (GC) cases are reported to have ATM deficiency, and ATM status is significantly associated with poor prognosis in GC. Here, we address whether the concept of PARP inhibitor synthetic lethality is applicable to GC with deficiency in ATM. Materials and Methods: 1. We determined relative ATM protein expression levels and ATM functionalities using western blot in a panel of GC cell lines (AGS, MKN1, MKN28, MKN45, KATO III, STKM-2, ISt-1, NUGC-4). After characterizing each GC cell line, we determined the sensitivity of GC cell lines to both ionizing radiation (IR) and olaparib by clonogenic survival assays. 2. We investigated the effect of olaparib in combination with the ATM inhibitor (KU-55933) on viability in ATMproficient GC cell lines, KATO III (p53del/del) and STKM-2 (p53wt/wt). We constructed ATM knockdown KATOIII and STKM-2 to investigate the role of ATM on sensitivity to olaparib. 3. To determine whether p53 deficiency enhanced olaparib sensitivity in the setting of ATM disruption, we constructed ATM/p53 double knockdown STKM-2. 4. To analyze the mechanism of cell death induced by olaparib, we performed apoptosis assay, and investigated the mRNA levels of p53-regulated genes by quantitative RT-PCR in the constructed cells exposed to olaparib. Results: 1. The ATM-deficient GC cell lines (NUGC-4, MKN45) were more sensitive to olaparib than the ATM-proficient counterparts (KATO III, STKM-2). ATM protein expression correlated with sensitivity to IR and olaparib, with correlation coefficients of 0.493 and 0.814, respectively. 2. Inhibition of ATM and ATM knockdown enhanced sensitivity to olaparib in KATO III (p53del/del), but not in STKM-2 (p53wt/wt). 3. ATM/p53 double knockdown significantly enhanced the sensitivity to olaparib in STKM-2. 4. Apoptosis was induced by combination of PARP and ATM inhibitor in KATO III. Apoptosis was also induced in ATM knockdown KATO III and ATM/p53 double knockdown STKM-2 when treated with olaparib. RT-PCR revealed that olaparib induced p53-independent apoptosis and regulation of cell cycle plays a crucial role in response to olaparib in GC. Conclusion: Our studies suggest that olaparib could have potential in the treatment of GC with low ATM protein expression levels, especially those with disruption of p53.

B. HCC of Sunitinib treated woodchuck showing partially preserved neoplastic cells (lower left) and expensive necrosis in the rest of the section. Mo1928 Chemotherapy With Bevacizumab for Advanced or Metastatic Colorectal Cancer Masaru Hirata [Background] We compared intention to treat analysis, patient survival and complication between FOLFOX+BEV, XELOX+BEV, and FOLFIRI+BEV among advanced or metastatic colorectal cancer patients. [Patients] Patients consisted of 56 cases (41 men and 15 women, 22 rectal cancer and 34 colon cancer, 28 advanced and 28 metastatic, 63.5 years old) who underwent chemotherapy with bevacizumab from July 2007 to June 2013. The location of metastasis were liver (32 cases), lung (13 cases), distant lymphnodes (11 cases), retroperitoneum (7 cases), peritoneum (4 cases), bone (2 cases), and ovary (1 case). [Method] All patients underwent complete resection of the primary carcinoma before chemotherapy. Time to progression analysis and patients survival, which were calculated by Kaplan-Meier method, and complication, were compared between FOLFOX+BEV, XELOX+BEV, and FOLFIRI+BEV group. [Results] 39 cases (69.6 %) out of 56 patients had k-ras wild type cancer and 17 (30.4 %) had k-ras mutant type. There was no significant difference about TTP of bevacizumab rgimen between k-ras wild group and k-ras mutant group, althouth k-ras wild group revealed significantly better patients survival. Bevacizumab based chemotherapy was performed as the first line in 41 cases (73.2 %), the second line in 8 cases (14.3 %), and the third line in 7 cases (12.5 %). TTP analysis revealed no significant difference between the first line group and other patients (second or third regimen group). Disease control rate (CR+PR+SD) was 83.3 % in FOLFOX+BEV group, 88.2 % in XELOX+BEV group, and 66.7 % in FOLFIRI+BEV group. Median TTP of FOLFOX+BEV group (225 days) or that of XELOX+BEV group (365 days) was significantly longer than that of FOLFIRI+BEV group (117 days). The rate of grade 3 or 4 adverse events was 3.6 % (1 gastrointestinal perforation and 1 fistula formation). [Discussion] FOLFOX + BEV or XELOX + BEV should be given to advanced or metastatic colorectal cancer at first.

Mo1931 Prognostic Significance of 18F-FDG PET/CT Defined Tumour Parameters in Patients With Esophageal Cancer Kieran Foley, Alex Karran, Charlotte E. Thomas, David S. Chan, Paul A. Blake, Patrick Fielding, Ashley Roberts, Wyn G. Lewis Purpose 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) combined with computed tomography (PET/CT) is now established as a routine staging investigation of esophageal cancer (EC). The aim of the study was to determine the prognostic significance of PET/CT derived tumour parameters including maximum standardized uptake value (SUVmax), tumour length (TL), metastatic length of disease (MLoD), metabolic tumour volume (MTV), total lesion glycolysis (TLG) and total local nodal metastasis count (PET/CT LNMC). Materials and Methods 103 pre-treatment EC patients (76 adenocarcinoma, 25 squamous cell carcinoma, 1 poorly differentiated and 1 neuroendocrine tumour) were staged using PET/CT. The prognostic value of the measured tumour parameters were tested using logrank analysis of the Kaplan-Meier method and Cox's proportional hazards method. Primary outcome measure was survival from diagnosis. Results Univariate analysis showed all parameters to have strong statistical significance in relation to survival. Multivariate analysis demonstrated three variables that were significantly and independently associated with survival; MLoD (HR 1.035, 95% CI 1.008 - 1.064, p=0.011), TLG (HR 1.002, 95% CI 1.000 - 1.003, p=0.018) and PET/CT LNMC (HR 1.261, 95% CI 1.105-1.439, p=0.015). Conclusion MLoD, TLG, and PET/CT LNMC are important prognostic indicators in EC. This is the first study to demonstrate an independent statistical association between TLG, MLoD and survival by multivariable analysis, and highlights the value of staging EC patients with PET/CT using functional tumour parameters.

Mo1929 Small-Molecule Parkinson's Disease Kinase Inhibitor LRRK2-in-1 Demonstrates Potent Anti-Cancer Activity Through Inhibition of DCLK1 Nathaniel Weygant, Dongfeng Qu, William L. Berry, Randal May, Parthasarathy Chandrakesan, Daniel Owen, Naushad Ali, Ralf Janknecht, Sripathi M. Sureban, Courtney W. Houchen Background: Small-molecule kinase inhibitors hold significant promise in extending lifespan and improving outcomes for cancer patients. Doublecortin-like kinase 1 (DCLK1) is emerging as a tumor specific stem cell marker in colorectal and pancreatic cancer. Previous studies have demonstrated the therapeutic effects of inhibiting DCLK1 with siRNA as well as genetically targeting the DCLK1+ cell for deletion. However, the effects of inhibiting DCLK1 kinase activity have not been studied directly. Aim: To assess the effects of inhibiting DCLK1 kinase activity in gastrointestinal cancers. Methods: The recently invented small-molecule kinase inhibitor LRRK2-IN-1 demonstrates significant and relatively selective affinity for DCLK1 (Kd=5 nM). This compound was used to assess the effect of inhibiting DCLK1 kinase activity. Standard in vitro functional and molecular assays as well as cell cycle analyses were performed in AsPC-1 pancreatic and HCT116 colon cancer cell lines in order to assess LRRK2-IN-1's anticancer potential. DCLK1 was mutagenized to a kinase dead form (K419R) and overexpressed in order to provide a platform to assess DCLK1 kinase's specific contribution to LRRK2-IN-1's anticancer activity. Results: We found that LRRK2-IN-1 targets DCLK1 kinase activity (IC50=2.6 nM), inhibits phosphorylation of DCLK1, and alters downstream DCLK1 effectors including EMT and pluripotency factors and Notch signaling pathway genes. LRRK2-IN-1 demonstrated potent anti-cancer activity against AsPC-1 and HCT116 cell lines by inhibiting proliferation (IC50=~1.7 μM in both cell lines), migration, and invasion (>70% reduction) as well as inducing apoptosis and cell cycle arrest. Additionally, we found that LRRK2-IN-1 downregulates DCLK1 protein and mRNA expression and that this effect is likely mediated by inhibition of MAPK7(ERK5). Overexpression of DCLK1 rescued cells from LRRK2-IN-1 anticancer activity in proliferation and clonogenic assays relative to vector control and kinase-dead control cells. Additionally in a xenograft study, LRRK2-IN-1 proved to be effective in reducing tumor volume as a function of time (p<0.005) demonstrating its in vivo efficacy. Conclusion: The overall specificity of LRRK2-IN-1 for DCLK1 and the results of our in vitro and in vivo studies support the development of DCLK1 kinase inhibitors against cancer. Given the recalcitrance of advanced gastrointestinal cancers in general, this therapeutic concept may have the potential to overcome limitations of current therapies which only target proliferating cells in the primary tumor, by repressing

AGA Abstracts

Mo1932 Fluorescent Endoscopy With SERS Nanoparticle for Detecting Colon Polyp in Azoxymethane-Induced Colon Cancer Mice Wan-Sik Lee, Jesse Jokerst, Cristina Zavaleta, Steven Sensarn, Sanjiv S Gambhir Background : Detecting colon cancer at its early stage is important to prevent disease progression and improve survival. Endoscopic screening has limitations in discriminating benign from malignant lesion and often miss some proportion of polyps. Raman spectroscopy is an optical technique that has strong sensitivity and specificity in analyzing cell population and tumor tissue. SERS(surface enhanced raman scattering) nanoparticle(NP) is engineered to have fluorescent moiety and can be used for multimodality imaging of both fluorescent endoscopy and Raman spectroscopy. We tried to validate the possibility of EGFR(epithelial growth factor receptor) targeted SERS in detecting polyp by fluorescent endoscope in vivo and Raman spectroscopy ex vivo. Material : A/J mice were injected with 1mg/kg azoxymethane weekly for 6 weeks to develop colon polyp after 20 weeks. SERS NPs are first coated with a fluorophore(5-Rox) and remaining thiols react with a bis-maleimido PEG cross-linker to

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bind affibodies specific to EGFR. Custom made Spyglass fiberoptic endoscopy system was used to visualize mouse colon with fluorescent. Mice distal colon was sprayed with 200ul of 50pM NPs and allowed to incubate for 10 minutes. Then distal colon was washed with water and imaged with spyglass. Then mice were sacrificed and colon was extracted for Raman spectroscopy. Result : We Successful labeled S440 SERS nanoparticles with 5-ROX fluorophore and an EGFR-affibody. SpyGlass system is able to detect nanoparticles bound to tumors when applied at 50-pM concentration. SpyGlass video and Raman scan suggest polyp specificity of SERS NP, and qPCR results show EGFR up-regulation in tumor compared to normal. Conclusion : Fluorescent Endoscopy with SERS nanoparticle labled with EGFR showed specific binding to mice colon polyp. Raman spectroscopy gave additional corresponding result. These multi-modality imaging with targeted nanoparticle spraying concept could give rise to new diagnostic tool in the near future.

molecular profile directed therapy. However, KRAS mutations were not associated with disease progression or PFS.

Gastric Stimulator, A Novel Approach in Treating Malignancy Associated Gastroparesis Hamza Shah, Archana Kedar, Lindsay McElmurray, Christopher J. Lahr, Brian Beauerle, Thomas L. Abell Background: Malignancy Associated Gastroparesis (MAG) has been increasingly recognized in the last decade, but therapies for this condition are lacking. One novel treatment for MAG is the use of a gastric electrical stimulation (GES), to alleviate symptoms when medical therapy is ineffective. We describe 6 patients with a variety of cancer related conditions who met criteria for MAG and underwent temporary Gastric Electrical Stimulation (GES) followed by permanent implant. Methods: 6 patients (3 male, 3 female) mean age of 57.7 years, were retrospectively reviewed for baseline physiologic measures and symptom outcomes after treatment. Of the six patients, 2 were diagnosed with esophageal cancer, 2 had a history of Hodgkin's lymphoma, 1 with gastric cancer, and 1 with breast cancer. Patients with esophageal and gastric cancer were in remission after esophagectomy and gastrectomy, respectively. Symptoms were scored by three variables: nausea (N), vomiting (V), and GI total symptom score (TSS). Baseline physiologic profiles were measured by gastric emptying scan (4 hr solid phase) and cutaneous electrogastrogram (EGG) and mucosal electrogram (EG) frequency. GI symptoms were measured over 6 days after temporary endoscopic GES placement and 1 yr after subsequent permanent gastric stimulator placement. Patients were implanted under a Humanitarian Use Device (HUD) protocol. Temporary stimulation was performed as previously described (CTRN 00432835; GIE 2011;74:496-503) followed by permanent GES implant. Results: Baseline gastric emptying studies in all patients except 1 (near total gastrectomy) displayed delayed gastric emptying (mean 29.9% retention at 4 hours; normal <10%). EGG (mean frequency 5.29 cpm) and EG (mean frequency 6.59) showed evidence of neuromuscular dysfunction (normal 2.7-3.3 cpm). Symptom scores in N, V, and GI TSS showed reduction after gastric stimulator placement (Table 1). Patients as a group improved with both temporary and permanent GES. The most significant improvement was seen by day 3 with the temporary stimulator placement and 1 year with the permanent stimulator placement (Figure 1). Statistical significance was noted between baseline vomiting and TSS when compared to 3 days post temporary stimulator and 1 year permanent stimulator placement. Conclusions: In a small sample of patients who met criteria for MAG, GES appears to offer relief of symptoms. After placement of GES, symptom scores in Nausea, Vomiting, and TSS all displayed reduction from baseline. This retrospective study suggests that GES is a potential option for patients who display MAG. Formal prospective studies of MAG using temporary and or permanent GES may be warranted. Baseline, Temporary and Permanent Measures for GES in MAG

Mo1933 Development of Esophageal and Anal Cancer Patient Derived Tumor Xenograft (PDTX) Models Using a Novel Implantation Technique Matthew Read, Maria-Pia Bernardi, David Liu, Christina M. Fennell, Robert G. Ramsay, Alexander Heriot, Cuong Duong, Wayne A. Phillips, Nicholas J. Clemons Background: Esophageal cancer, a highly fatal disease, is the 6th most common malignancy worldwide and the incidence of one sub-type, esophageal adenocarcinoma (EAC) has risen dramatically in recent decades. Although a relatively rare tumor, a similar rapid increase in incidence has been observed for anal squamous cell carcinoma (SCC). Treatments for esophageal cancer and anal SCC have essentially stayed the same over the past few decades, and in both cases there are no second line therapies for patients who relapse or patients with metastatic disease. Therefore, there is a critical need to develop new therapeutic options for patients with these cancers. However, progress has been hindered by a lack of preclinical models for both EC and ASCC. Thus, our aim was to develop robust, validated patient derived tumor xenograft (PDTX) models of EC and ASCC, to provide tools for the pre-clinical testing of new therapies in these cancers. Methods: Matrigel coated tumor biopsies were implanted subcutaneously (s.c.) on the flank, or intramuscularly (i.m.) in the dorsal muscle of immunocompromised mice. Histological (H&E stained) sections, immunohistochemistry for known esophageal cancer and anal SCC markers, and targeted gene sequencing were used to compare multiple generations of PDTX tumors with the corresponding original patient tumor. Cohorts of mice carrying PDTX tumors were treated with the same chemotherapy regimen as the corresponding patient to compare PDTX tumor response with patient tumor response. Results: EC (both EAC and esophageal SCC), and anal SCC PDTXs were successfully established from both fresh and cryopreserved tumor biopsies. The i.m. implantation technique had a superior engraftment rate compared with s.c. implantation, with a per tumor success rate of 95% vs. 25% and a per biopsy success rate of 48% vs. 7%. Furthermore, i.m. tumors grew more quickly than tumors that grew s.c. PDTX tumor differentiation status, protein expression, mutation profile, and response to chemotherapy were consistent with original patient tumors. In addition, HER2+ EAC PDTX tumors were successfully treated with trastuzumab, demonstrating the potential for using the model to test molecular targeted therapies. Furthermore, PDTX tumors established from the primary tumor of patients who developed distant metastases also metastasized in mice. Conclusions: PDTX models for esophageal cancer and anal SCC were successfully established using an i.m. technique, which was more robust than the commonly used s.c. approach. PDTX tumors were phenotypically comparable to original patient tumors and demonstrated similar biological behaviour, consistent with the notion that PDTX models are representative of patient tumors. We propose that our models will be important tools for the development of much needed new drug treatments for esophageal cancer and anal SCC. Mo1934 Treatment Naive Locally Advanced Rectal Cancer Lymph Node KRAS Mutation Profile As Revealed by EUS FNA Cytology Next-Generation Sequencing: An Additional Piece to the Puzzle Ferga C. Gleeson, Benjamin R. Kipp, Jesse S. Voss, Michael B. Campion, Michael Henry, Andrew P. Sciallis, Rondell Graham, Konstantinos Lazaridis, George Vasmatzis, Michael J. Levy Background: Molecular cancer diagnostics is rapidly emerging from a solely investigational tool to one used in clinical practice. Next-generation sequencing (NGS) is arguably one of the most significant technological advances in the biological sciences of the last 30 years. The RAS (HRAS, NRAS and KRAS) oncogene family was the first human tumor oncogene discovered. Acquired mutations in KRAS are an early step in carcinogenesis, identified in 40 % of colorectal cancers. An FDA approved genetic test kit is designed to detect the presence of 7 KRAS mutations in codons 12 and 13 to predict lack of response to targeted monoclonal antibodies in colorectal cancer. Aims: Firstly, to assess the frequency of RAS mutations and the presence of incrementally identified mutations not currently identified by FDA approved KRAS genetic test kits. Secondly, to assess if the RAS oncogene family lymph node (LN) mutation profile is associated with disease progression or progression free survival (PFS) in patients with locally advanced rectal cancer. Methods: The Ion AmpliSeq™ V2 Cancer Hotspot NGS Cancer Panel and MiSeq sequencers were used to simultaneously sequence and assess for ≥ 2,500 possible mutations in 50 oncogene and tumor suppressor genes from 102 archived (2002-2010) EUS FNA malignant LN cytology specimens. The cancer panel can detect 63 COSMIC mutations in the KRAS gene. Results: KRAS, NRAS and HRAS mutations were present in 31%, 6%, and 1% of LNs, respectively. The RAS oncogene family was associated with FBXW7 mutations rather than any other panel tested mutation (p=0.0063). The most common KRAS mutations (n=32) occurred at codons 12 (84%) and 13 (9%) of exon 2, respectively. We also identified 2 (6.3%) patients without mutations in codons 12-13, presenting with a rare mutation in codon 19 and 146, c.57G>T, p.Leu19Phe and c.436G>A, p.Ala146Thr, respectively. Disease progression or PFS was not associated with any KRAS mutation or KRAS mutation at codon 12 or 13, respectively. Conclusion: When compared to the Ion AmpliSeq™ V2 Cancer Hotspot NGS Cancer Panel, FDA approved assays may miss alterations in the KRAS gene that are used to guide tumor

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AGA Abstracts

AGA Abstracts

Mo1935