Modern Pathology (2013) 26, 139–147 & 2013 USCAP, Inc. All rights reserved 0893-3952/13 $32.00 139 Heterogeneity in signaling pathways of gastroenteropancreatic neuroendocrine tumors: a critical look at notch signaling pathway He Wang1, Yili Chen1, Carlos Fernandez-Del Castillo2, Omer Yilmaz1 and Vikram Deshpande1 1Department of Pathology, Massachusetts General Hospital, Boston, MA, USA and 2Department of Surgery, Massachusetts General Hospital, Boston, MA, USA The molecular pathogenesis of gastroenteropancreatic neuroendocrine tumors is largely unknown. We hypothesize that gastroenteropancreatic neuroendocrine tumors are heterogeneous with regard to these signaling pathways and these differences could have a significant impact on the outcome of clinical trials. We selected 120 well-differentiated neuroendocrine tumors including tumors originating in pancreas (n ¼ 74), ileum (n ¼ 31), and rectum (n ¼ 15). Immunohistochemistry was performed on tissue microarrays using the following antibodies: NOTCH1, HES1, HEY1, pIGF1R, and FGF2. Gene profiling study was performed by using human genome U133A 2.0 array and data were analyzed. The gene profiling results were selectively confirmed by using quantitative reverse-transcription PCR. Initial immunohistochemical analysis showed NOTCH1 was uniformly expressed in rectal neuroendocrine tumors (100%), a subset of pancreatic neuroendocrine tumors (34%), and negative in ileal neuroendocrine tumors. Similarly, a downstream target of NOTCH1, HES1 was preferentially expressed in rectal neuroendocrine tumors (64%), a subset of pancreatic neuroendocrine tumors (10%), and uniformly negative in ileal neuroendocrine tumors. Messenger RNAs for NOTCH1, HES1, and HEY1 were 2.32-, 2.44-, and 2.39-folds, respectively, higher in rectal neuroendocrine tumors as compared with ileal neuroendo- crine tumors. Global gene expression profiling showed 95 genes were differentially expressed in small intestinal vs rectal neuroendocrine tumors, with changes as high as 50-fold. These genes were concentrated in several signal transduction pathways including cancer endocrine pathway and cell growth/proliferation pathway. The differential expression of selected genes including ISL LIM homeobox 1, cathepsin B, glucagon, and tryptophan hydroxylase 1 were confirmed by qPCR and immunohistochemistry. Our results confirm the heterogeneity in signaling pathways of gastroenteropancreatic neuroendocrine tumors. NOTCH1 inhibitors are unlikely to provide benefit in ileal neuroendocrine tumors; conversely, their efficacy in rectal neuroendocrine tumors needs further study. Further analysis of signaling pathways is critical for designing clinical trials in gastroenteropancreatic neuroendocrine tumors. Modern Pathology (2013) 26, 139–147; doi:10.1038/modpathol.2012.143; published online 24 August 2012 Keywords: carcinoid tumor; neuroendocrine tumor; notch Neuroendocrine tumors are a diverse group of ileum, appendix, and ascending colon), and hindgut neoplasms that have traditionally been divided acc- (inferior mesenteric artery distribution including ording to their embryonic origins into foregut (eso- descending colon and rectum).1,2 Similar to the phagus, stomach, pancreas, and duodenum), midgut epithelial neoplasms of the gastrointestinal tract, (superior mesenteric artery distribution including there is significant morphological and immuno- histochemical diversity across neuroendocrine tumors of the tubular gut. However, unlike epithelial Correspondence: Dr V Deshpande, MD, Department of Pathology, neoplasms of the gastrointestinal tract little is Massachusetts General Hospital, Warren 3, 55 Fruit Street, known about the molecular heterogeneity or genetic Boston, MA 02114, USA. E-mail: [email protected] underpinnings of neuroendocrine tumors of the Received 12 March 2012; revised 9 July 2012; accepted 9 July tubular gut. One well-documented genetic alteration 2012; published online 24 August 2012 in neuroendocrine tumors of the gastrointestinal tract www.modernpathology.org Notch signaling in carcinoid tumors 140 H Wang et al is inactivation of MEN1 gene. The genetic alterations Hospital between 2006 and 2009. All cases were are also location-specific events: MEN1 somatic routine diagnostic surgical specimens including mutations occur most frequently in foregut biopsies and resections. Tissues were fixed in 4% neuroendocrine tumors, 18q LOH almost exclusively buffered formalin, processed using standard proce- in midgut and hindgut neuroendocrine tumors.3 dures, and embedded in paraffin. All specimens Understanding the molecular pathology of neuro- were reviewed by an experienced gastrointestinal endocrine tumors is critical for developing medical pathologist, and reclassification (if necessary) was therapy. The current medical therapies, including based on criteria suggested by the WHO.6,9 somatostatin analogs, interferon-a,andcytotoxic chemotherapy, have been somewhat effective in alleviating symptoms, but have done little in prevent- TMA Construction ing neuroendocrine tumor progression.1,4 There are In all, 120 consecutive selected specimens were more than 100 ongoing clinical trials on neuro- included in the study, comprising 31 cases of endocrine tumor, targeting phosphatidylinositol 30 neuroendocrine tumors from small intestine, 15 kinase, mitogen-activated protein kinase, mTOR, and cases from rectum, and 74 pancreatic neuroendo- VEGFA pathways.5 However, there has been no crine tumors. Histological slides for each specimen systematic effort to explore the signaling trans- included in the tissue microarray were reviewed, duction pathways in human neuroendocrine tumors, and the corresponding region on the paraffin block and even less is known about the variability in these identified. Cores of 2 mm diameter were chosen for signaling pathways across the gastrointestinal tract. this study. The mean number of cores examined per For example, in spite of the success of sunitinib in sample was 1.6 (range 1–3). patients with advanced neuroendocrine tumors, relatively little is known about the expression pattern and activation status of growth factor Human Paraffin-Embedded Neuroendocrine Tumor receptors such as VEGFA in human neuroendocrine mRNA Expression Profiling tumors. Understanding these signaling pathways is particularly significant given the potential RNA was extracted from formalin-fixed paraffin- heterogeneity of these neoplasms.6 embedded tissue blocks after macrodissection. Although immunohistochemical and PCR-based Briefly, ‘donor’ region of neuroendocrine tumors, approaches are sufficient to assess expression of composed at least 95% of tumor cells, were selected proteins/genes, genome-wide microarray identified under the microscope, and these were profiling provides a more comprehensive approach marked up on each glass slide and tissue block. to the understanding of tumor signal transduction. Tissue cores of 1 mm diameter were extracted from Fresh-frozen tissue is a common source of high each marked region by using tissue arrayer. quality messenger RNA for microarray studies. The Human Genome U133A 2.0 arrays (Affyme- Messenger RNA from formalin-fixed and paraffin- trix, Santa Clara, CA, USA) were used for microarray embedded tissue blocks was traditionally regarded as analysis. Human neuroendocrine tumors\RNA was unsuitable for gene microarray applications because purified using QIAzol lysis reagent and RNeasy mini of degradation and chemical modification. This kit (QIAGEN, Valencia, CA, USA). A total of 11 traditional view has recently been challenged, and samples were analyzed, including 5 samples from several groups have reported quantitative gene small intestine and 6 samples from rectum. Each expression and microarray profiling data from for- RNA sample was prepared individually and hybri- malin-fixed and paraffin-embedded tissue blocks.7,8 dized to a different microarray chip. Sample label- The goal of this study was to explore signal ing and processing were performed according to the transduction pathways and gene expression profile manufacture’s protocol. Double-stranded comple- in three common gastrointestinal neuroendocrine mentary DNA was synthesized, and biotinylated tumors: rectum, small intestine and pancreas, and cRNA was prepared and then hybridized to the assess the heterogeneity in the signaling pathways arrays. The arrays were then stained with streptavi- across these tumors. Identification of novel signal- din R-phycoerythrin (Molecular Probes, Eugene, ing pathway may uncover potential druggable OR, USA) using GeneChip Fluidics Station 400, targets. Identifying differences in signal transduc- and scanned with a GeneChip scanner 3000 (Affy- tion pathways among these neuroendocrine tumors metrix). The microarray data were analyzed using provides important baseline data for evidence-based Array Studio (Omics Soft, Morrisville, NC, USA). clinical trials for neuroendocrine tumors. The data was normalized with Robust Multiarray Averaging (RMA) method. Principal component analysis was used as quality control to detect Materials and methods outlier(s) among the samples. One small intestinal Tissue Samples sample was detected as an outlier and was elimi- nated from further analysis. The normalized data of All study cases were selected from the archives of the two tissues were then analyzed for differential the Department of Pathology, Massachusetts General gene expression using one-way ANOVA. Changes in Modern Pathology (2013) 26, 139–147 Notch signaling in carcinoid tumors H Wang et al 141