KI-67 Proliferation Index is a Better Prognostic Marker Than Mitotic Count in Neuroendocrine Tumors

intestinal inflammation by promoting the expansion of the mucosal CD11b+F4/80+ population and specifically inducing IL-17A-producing macrophages in experimental IBD.

642 Ultrastructral Evidence for a Mechanism of ECL Cell Transformation to Gastric Carcinoid in Mastomys Involving Impairment of Autophagy and Loss of Gastrin Control Reidar Alexander Vigen, Mark Kidd, Irvin Modlin, Duan Chen, Chun-Mei Zhao

AGA Abstracts

640 Morbidity and Mortality Associated With Desmoplasia in Midgut (Carcinoid) Neuroendocrine Tumours Fatima El-Khouly, Mohid S. Khan, Christos Toumpanakis, Olagunju A. Ogunbiyi, Marc C. Winslet, Martyn Caplin

Background and Aim: Development of gastric ECL cell hyperplasia, multiple micronodules and carcinoids (ECLoma) has been observed after long-term administration of proton pump inhibitor omeprazole in rats (after 1 year) or H2 receptor antagonist loxtidine in Mastomys (after 2 months). The aim of the present study was to evaluate whether the mechanism of gastric carcinoid genesis represented involved impairment of autophagy. Methods: Induction of hypergastrinemia and ECLoma in Mastomys was generated by oral loxtidine administration for 8 or 27 weeks. Four additional groups were included for comparison: a) control Mastomys without treatment; b) rats treated with omeprazole for 10 weeks; c) rats subjected to antrectomy for 10 weeks; and d) mice deficient in CCK2 receptor. Gastric tissue was collected after euthanasia, and ECL cells were analyzed by electron microscopy. Ultrastructural markers for cell transformation included numbers of granules, secretory vesicles and microvesicles, and the markers for autophagic pathway were the presence of vacuoles, lipofuscin bodies, and autophagysomes. Results: In comparison to untreated Mastomys, the ECL cell profile area, including nuclear and cytoplasmic areas, was not increased after loxtidine treatment for 8 or 27 weeks. Secretory vesicles, a hallmark feature of well-differentiated ECL cells, were unchanged after 8 weeks but markedly reduced after 27 weeks in both number and volume density. Granules were reduced in both number and volume density after both 8 and 27 weeks. Microvesicles were unchanged regardless of treatment. Vacuoles and lipofuscin bodies were found occasionally in the ECL cells in untreated or 8-week loxitidine-treated Mastomys but not in those after 27 weeks. The ultrastructural appearance of ECL cells in Mastomys treated with loxtidine for 8 or 27 weeks looked different from the ECL cells in rats treated with omeprazole for 10 weeks, which exhibited numerous large vacuoles and lipofuscin bodies. They were also different from the ECL cells in antrectomized rats, which displayed relatively numerous granules but few secretory vesicles and the appearance of autophagysomes. However, the ECL cells in 27-week, but not 8-week, loxtidine-treated Mastomys looked similar with the ECL cells in CCK2 receptor knockout mice. Conclusion: It seems likely that a combination of genetic predisposition and long-term hypergastrinemia leads to the initiation of gastric carcinoids and then impaired autophagy and loss of gastrin control advance the tumorigenesis of these lesions.

INTRODUCTION Midgut neuroendocrine tumours (NETs) form part of a heterogenous group characterized by their ability to secrete hormones and other mediators which may cause carcinoid syndrome. They may also present with complex problems related to mesenteric fibrosis/desmoplasia. The cause of desmoplasia is unknown, however it may be associated with peptides secreted by NETs. It has been previously reported that 30% of patients with midgut NETs are found to have desmoplasia on CT at presentation and this increases as disease progresses. Desmoplasia may cause symptoms secondary to mesenteric artery/vein occlusion, as well as bowel obstruction. AIM To assess morbidity and mortality associated with desmoplasia in midgut NETs. METHODS A retrospective review of our NET database for patients with cofirmed midgut NET and desmoplasia was performed. Information available included: age, sex, diagnosis, previous treatments, current management and outcomes. 107 midgut NET patients with desmoplasia were identified. Full data was available for 99 patients (51 males and 48 females). Desmoplasia was identified at surgery or at CT imaging. Overall survival (OS) was calculated from time of diagnosis. RESULTS 61 (62%) patients were symptomatic at diagnosis or during the course of their disease, from either obstructive or ischaemic cause. The remaining 38 (38%) patients were asymptomatic and desmoplasia was diagnosed incidentally on CT. 68 (69%) patients had undergone surgical procedures, including resection of the primary, re-resection, stoma formation and bypass. Of the 48 patients who had undergone surgical resection of the primary tumour, 31 (65%) had presented with obstructive symptoms. 6 (6%) patients developed symptoms from superior mesenteric vein occlusion, needing long-term anticoagulation. 2 patients had short bowel syndrome and 5 (5%) patients required long-term total parenteral nutrition. 36 patients died. Total median OS was 96 months. The median OS survival in the symptomatic group was 81 months and not reached in the asymptomatic group (log-rank p=0.059). The median OS for those who underwent primary resection was 144 months, whilst the median OS for those not resected was 75 months (log-rank p=0.128). CONCLUSIONS The majority of patients with midgut NETs and desmoplasia are symptomatic requiring surgical intervention. Patients may present acutely with bowel obsturction due to tumour or the effects of desmoplasia. Desmoplasia may progress despite medical treatments. Patients with mesenteric artery/vein occlusion may be managed with anticoagulation. The median OS in symptomatic patients was less than the total median OS. These patients have complex symptomatology which can only be dealt with by experienced multidisciplinary approach. Further research is required into the pathogenesis of desmoplasia to enable better treatment.

643 The Role of Serotonin and the 5HT7 Receptor in IGF-I Synthesis and Secretion in the “Carcinoid” Tumor: Hepatocyte Microenvironment Bernhard Svejda, Mark Kidd, Ben J. Lawrence, Daniele Alaimo, Simon Schimmack, Irvin Modlin Background/Aim: In the liver, physiological levels (~1nm) of serotonin (5-HT) regulate hepatocyte proliferation via 5-HT2A,B receptors. Little is known about the effect at pathological levels (0.1-1um) of serotonin on hepatocyte function when 5-HT secreting neuroendocrine tumors (NETs - or carcinoids) metastasize to the liver. We hypothesized that 5-HT secreted by tumors induced growth factor secretion from hepatocytes which regulated growth in this hepatic tumor microenvironment. Methods: The 5-HT receptor profile was evaluated on isolated pure preparations (>99%) of rat hepatocytes by PCR and confirmed with sequencing. The effects of 5-HT (physiological-1nM; and pathological-1um) and the highly selective 5-HT7 receptor blocker, SB269970HCl, on primary cultured rat hepatocytes were assessed using WST1 cell proliferation assays. ELISA and western blots were performed to delineate 5-HT-mediated signaling pathways (PKA, cAMP, pCREB, AKT, ERK). Growth factor synthesis and secretion from hepatocytes was evaluated using real-time PCR and ELISA. Results: The 5-HT7 receptor was confirmed to be expressed in rat hepatocytes by PCR and sequencing. Cell viability (WST1) was significantly increased at physiological (121%) and pathological (126%) 5-HT levels after 1 and 4 hrs of treatment (p<0.05). cAMP levels and PKA activity were significantly increased at physiological (132-164%) and pathological (10-6M, 159204%) levels. pCREB was elevated in a time-dependent fashion, between 2-4 hrs (180%) by physiological 5-HT and within 1 hr (195%) by pathological 5-HT concentrations. Additionally, a PKA-independent increase of AKT but not ERK was noted after 5-HT treatment. IGF-I - a critical regulator of carcinoid proliferation - was elevated at transcript and secretion levels by pathological 5-HT levels (138-280%, p<0.05) as was HGF (164-252%), a known hepatocyte mitogen. These effects were reversible by SB269970HCl. Conclusions: Hepatocytes express the cAMP-coupled 5-HT7 receptor which signals via CREB and AKT and is linked to IGF-I synthesis, secretion and growth. Physiological 5-HT levels regulate growth via PKA/CREB (PKA-dependent) as well as AKT (PKA-independent), but not ERK signaling. Pathological 5-HT concentrations induce growth factor production and secretion. As the latter are regulators of tumor proliferation, metastatic carcinoids regulate their growth phenotype via IGF-I from hepatocytes. Targeting the latter with 5-HT7 receptor antagonists may inhibit the hepatic tumor microenvironment limiting metastases proliferation.

641 KI-67 Proliferation Index is a Better Prognostic Marker Than Mitotic Count in Neuroendocrine Tumors Mohid S. Khan, Fatima El-Khouly, Christos Toumpanakis, Tim Meyer, Martyn Caplin, Tu Vinh Luong Introduction: Gastroenteropancreatic (GEP) neuroendocrine tumors (NETs) are uncommon tumors but with an increasing prevalence. They are generally slow-growing but vary in their degree of proliferation. The recent classification system of NETs proposed by the European Neuroendocrine Tumor Society (ENETS) utilises Ki-67 proliferation index and/or mitotic count to assign grade (low, intermediate, high). It has been adopted into routine practice but there is limited data on the relationship between these indices and whether they have similar prognostic value. We have previously demonstrated a discrepancy in grade assignment depending on whether Ki-67 or mitotic count is used. Aims: To explore the prognostic value of grading according to Ki-67 and mitotic count in GEP NETs. Methods: 285 NET patients, 144 midgut and 141 pancreatic, had their cases reviewed. Ki-67 index, evaluated by staining tumour sections with MIB-1 antibody, was determined by assessing the percentage of positively staining tumour cell nuclei in 2000 tumour cells. Mitotic counts (per 10 high power fields) were evaluated in at least 40 fields in areas of highest mitotic activity. Grades (1 to 3) were assigned according to the new TNM classification using both indices. Slides were assessed by an experienced pathologist with expertise in NETs. Progression-free survival (PFS) and overall survival (OS) from date of diagnosis were estimated using Kaplan-Meier methodology and multivariate analysis performed with Cox Regression. Results: Complete data was available in 131/144 midgut and 136/141 pancreatic NETs. As previously reported, there was disagreement in grade assignment depending on whether Ki-67 or mitotic count was used (midgut: 38% discordance, pancreatic: 51%). Median follow up was 42 months in midgut and 46 months in pancreatic NETs. 5-year PFS and OS for pancreatic NETs were 29% and 53% respectively. 5-year PFS and OS for midgut NETs were 37% and 61%. On univariate analysis, grade according to either Ki-67 or mitotic count was prognostic of PFS and OS in both midgut and pancreatic NETs. However, on multivariate analysis, only grade according to Ki-67 was an independent prognostic indicator of PFS and OS (p<0.001). Grade according to mitotic count was not (p>0.16). Conclusion: Based on the current classification thresholds, grade according to Ki-67 offers better prognostic value than grade according to mitotic count. Further studies evaluating grading thresholds of mitotic counts and investigation of other mitotic markers are required.

AGA Abstracts

644 Targeting PI3K/AKT Pathway is an Effective Strategy for the Treatment of Neuroendocrine Tumors Harboring PIK3CA Mutations Jun Song, Jing Li, Ji Tae Kim, Heidi Weiss, Courtney M. Townsend, B. M. Evers The PI3K/AKT and RAS/RAF/MEK/ERK pathways are vital signal transduction cascades that regulate growth and survival. These pathways are often altered in a number of human malignancies. Oncogenic activating mutations in the PIK3CA (encodes p110α catalytic subunit of PI3K) and RAS genes are frequent in human cancers. However, the importance of these pathways has not been extensively explored in neuroendocrine tumors (NETs). Recently, combination treatment, targeting both pathways, has emerged as a more effective strategy for treating cancers, particularly those with both PI3K and RAS mutations. Here, we determined: i) mutations in hot-spot regions of PIK3CA in NET cells, and ii) the roles of PI3K/AKT and Ras/Raf/MEK/ERK signaling pathways in the regulation of NET cell

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