Different Gene Expression Profiles in Metastasizing Midgut Carcinoid
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Endocrine-Related Cancer (2011) 18 479–489 Different gene expression profiles in metastasizing midgut carcinoid tumors Katarina Edfeldt, Peyman Bjo¨rklund, Go¨ran A˚ kerstro¨m, Gunnar Westin, Per Hellman and Peter Sta˚lberg Department of Surgical Sciences, Uppsala University, SE-751 85 Uppsala, Sweden (Correspondence should be addressed to P Sta˚lberg; Email: [email protected]) Abstract The genetic events leading the progression of midgut carcinoid tumors are largely unknown. The disease course varies from patient to patient, and there is a lack of reliable prognostic markers. In order to identify genes involved in tumor progression, gene expression profiling was performed on tumor specimens. Samples comprised 18 primary tumors, 17 lymph node (LN) metastases, and seven liver metastases from a total of 19 patients. Patients were grouped according to clinical data and histopathology into indolent or progressive course. RNA was subjected to a spotted oligo microarray and B-statistics were performed. Differentially expressed genes were verified using quantitative real-time PCR. Self-organizing maps demonstrated three clusters: 11 primary tumors separated in one cluster, five LN metastases in another cluster, whereas all seven liver metastases, seven primary, and 12 LN metastases formed a third cluster. There was no correlation between indolent and progressive behavior. The primary tumors with Ki67 O5%, with low frequency of the carcinoid syndrome, and a tendency toward shorter survival grouped together. Primary tumors differed in expression profile from their associated LN metastases; thus, there is evidence for genetic changes from primary tumors to metastases. ACTG2, GREM2, REG3A, TUSC2, RUNX1, TPH1, TGFBR2, and CDH6 were differentially expressed between clusters and subgroups of tumors. The expression profile that assembles tumors as being genetically similar on the RNA expression level may not be concordant with the clinical disease course. This study reveals differences in gene expression profiles and novel genes that may be of importance in midgut carcinoid tumor progression. Endocrine-Related Cancer (2011) 18 479–489 Introduction a lethal outcome; overall five-year survival is w60% Intestinal carcinoid tumors originate from the neuro- (Modlin et al.2003). The annual incidence is about two endocrine enterochromaffin (EC) cells of the gut. The cases per 1 00 000 persons, but the rate is increasing, WHO classification from 2000 divides the tumors into probably due to increased sensitivity and awareness in three groups: well-differentiated tumors, well-differ- the diagnostic phase. However, there is still often a entiated endocrine carcinomas, and poorly differen- malignant spread to regional lymph nodes (LNs) and tiated carcinomas (Kloppel et al. 2004). The older and the liver at the time of diagnosis, because the available previously more commonly used classification divides biomarkers are of low specificity and the clinical the tumors according to their embryologic origin into course often diffuses (Schnirer et al. 2003). foregut, midgut, and hindgut tumors, which also The tumors are neoplasms of peptide- and amine- reflects its localization in the gastrointestinal tract producing cells. Some patients will develop the carcinoid (Arnold 2005). syndrome due to excessive secretion of serotonin and Midgut carcinoid tumors are the most common type other metabolites, which includes flushing, diarrhea, of carcinoid tumors in the gastrointestinal tract and abdominal pain, bronchospasm, and heart disease and arise in the lower jejunum, ileum, appendix, and other symptoms resulting from fibrosis (Soga et al. cecum. This malignant neoplasm grows slowly (Ki67 1999). Symptoms and progression rate can greatly differ proliferating index is often !2%) but nevertheless has between individual patients and is almost impossible Endocrine-Related Cancer (2011) 18 479–489 Downloaded from Bioscientifica.comDOI: 10.1530/ERC-10-0256 at 09/27/2021 05:42:19AM 1351–0088/11/018–479 q 2011 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.orgvia free access K Edfeldt et al.: Gene expression profiles in carcinoid tumors to predict due to lack of reliable prognostic indicators. EC cells and/or bowel mucosa (Kidd et al. 2006, Leja The biomarkers used correlate to total tumor burden, et al. 2009), and in one microarray, the gene expression such as chromogranin A in serum and 5-hydroxy- was compared with a cell line (GOT1; Arvidsson et al. indoleacetic acid (5-HIAA) in urine. Several analyses 2008). It is difficult to extract normal EC cells from the have stated that factors leading to a bad prognosis bowel mucosa, because these cells are scattered are advanced age, LN involvement, presence of more throughout the gastrointestinal mucosa, and isolating than five liver metastases, lack of symptoms at the time these cells will result in contamination from surround- of diagnosis, high levels of 5-HIAA, high levels of ing tissue. Owing to these difficulties, this study has a plasma chromogranin A or neuropeptide K, and the new approach. To clarify the gene expression pattern in carcinoid syndrome (Janson et al.1997, Soreide et al. midgut carcinoid tumors, with emphasis on genes 2000, Hellman et al.2002, Kolby et al.2004, Ahmed involved in progression and aggressiveness, an et al.2009). expression microarray assay was performed comparing Today, surgery is the only potential cure. To achieve primary midgut carcinoid tumors with metastases and prolonged survival and symptom relief, the patients are comparing groups of tumors with different clinical treated with cytotoxic agents, biological therapies, and features. tumor-targeted radionucleotides (Schnirer et al. 2003). Liver metastases can be treated with surgery, radio- frequency ablation, or liver embolization (Eriksson Materials and methods et al. 2008). Nevertheless many tumors are resistant to therapy and there is an urgent need to develop Tumor material and RNA preparation new therapies. Primary and metastatic midgut carcinoid tumors were Besides the lack of diagnostic and therapeutic tools, snap frozen in liquid nitrogen and kept at K70 8C. The a greater understanding of molecular arrangements in tumors came from a cohort of patients who were midgut carcinoid tumors is required. Loss of chromo- diagnosed with midgut carcinoid tumor and operated at some 18q has been found to be the most frequent Uppsala University Hospital between 1989 and 2007. genetic aberration (Zhao et al. 2000, Kytola et al. 2001, Patient records were evaluated, and totally 42 tumors Lollgen et al. 2001, Tonnies et al. 2001, Wang et al. from 19 patients were selected for further analysis. 2005, Kulke et al. 2008). No alterations in the tumor Clinical features are shown in Table 1; two patients had suppressor genes DPC4/SMAD4 and DCC, located on been treated with interferon a prior to surgery and ten chromosome 18, are found indicating that other, with somatostatin analogues. According to the WHO currently unknown genes are important for patho- classification, all of the tumors were well-differentiated genesis (van Eeden & Offerhaus, 2006). Moreover, neuroendocrine carcinomas. Endocrine tumors have previous research has treated neuroendocrine neo- often been treated as a homogenous group in the plasms as a homogenous group of tumors. This is most literature, but resent research has stated their hetero- likely insufficient, because recent studies have demon- geneity; thus, pancreatic neuroendocrine tumors are strated the heterogeneous nature and different gene foregut and not midgut carcinoid tumors. In 11 patients expression profiles among subgroups of tumors (Zhao (no. 1–5, 7–10, 17, and 19), RNA was extracted from et al. 2000, Kytola et al. 2001, Tonnies et al. 2001, both primary tumors and LN metastases, and from five Wang et al. 2005). patients (no. 11–15), RNA was extracted from the To our knowledge, only three expression microarray primary tumor, LN, and liver metastases. From one assays, which include midgut carcinoid tumors, have patient (no. 6), tumor material came from only the been performed until today. The gene expression primary tumor, and from another patient (no. 16), the profiles have only been analyzed in a limited number tumor material came from the primary tumor and liver of tumors; in total, three primary tumors, 16 liver metastasis, and from one patient (no. 18) RNA was metastases, and none LN metastases. There are some extracted from LN and liver metastasis. Total RNA suggestions of novel genes (PNMA2, SPOCK1, was extracted using TriZol Reagent (Invitrogen) SERPINA10, GRIA2, GRP112, OR51E1, CXCL14, according to the manufacturer’s instructions. Sections NKX23, NAP1L1, MAGE-D2, MTA-1, and APLP1) from all the tumors were stained with Mayer’s that may be of importance for tumorigenesis, but their hematoxylin and histopathologically evaluated, and role in the development of midgut carcinoid tumors is the tissues used contained at least 80%, and in most still unknown (Kidd et al. 2006, Arvidsson et al. 2008, cases more than 90%, of neoplastic cells. When Leja et al. 2009). In two of the microarrays, the gene needed, the tumor was macroscopically resected from expression in tumor tissues was compared with normal excessive stromal tissue, blood vessels, bowel mucosa, Downloaded from Bioscientifica.com at 09/27/2021 05:42:19AM via free access 480 www.endocrinology-journals.org www.endocrinology-journals.org Table 1 Patient characteristics PCA PCA Follow-up Follow-up Tumor no: