Endocrine-Related Cancer (2007) 14 853–863 Gastrointestinal stromal tumors regularly express synaptic vesicle proteins: evidence of a neuroendocrine phenotype Per Bu¨mming, Ola Nilsson1,Ha˚kan Ahlman, Anna Welbencer1, Mattias K Andersson 1, Katarina Sjo¨lund and Bengt Nilsson Lundberg Laboratory for Cancer Research, Departments of Surgery and 1Pathology, Sahlgrenska Academy at the University of Go¨teborg, Sahlgrenska University Hospital, SE-413 45 Go¨teborg, Sweden (Correspondence should be addressed to O Nilsson; Email: [email protected]) Abstract Gastrointestinal stromal tumors (GISTs) are thought to originate from the interstitial cells of Cajal, which share many properties with neurons of the gastrointestinal tract. Recently, we demonstrated expression of the hormone ghrelin in GIST. The aim of the present study was therefore to evaluate a possible neuroendocrine phenotype of GIST. Specimens from 41 GISTs were examined for the expression of 12 different synaptic vesicle proteins. Expression of synaptic-like microvesicle proteins, e.g., Synaptic vesicle protein 2 (SV2), synaptobrevin, synapsin 1, and amphiphysin was demonstrated in a majority of GISTs by immunohistochemistry, western blotting, and quantitative reversetranscriptase PCR. One-third of the tumors also expressed the large dense core vesicle protein vesicular monoamine transporter 1. Presence of microvesicles and dense core vesicles in GIST was confirmed by electron microscopy. The expression of synaptic-like microvesicle proteins in GIST was not related to risk profile or to KIT/platelet derived growth factor alpha (PDGFRA) mutational status. Thus, GISTs regularly express a subset of synaptic-like microvesicle proteins necessary for the regulated secretion of neurotransmitters and hormones. Expression of synaptic-like micro-vesicle proteins, ghrelin and peptide hormone receptors in GIST indicate a neuroendocrine phenotype and suggest novel possibilities to treat therapy-resistant GIST. Endocrine-Related Cancer (2007) 14 853–863 Introduction (Kindblom et al. 1998, Komuro 1999). GIST have dense core granules, or small empty vesicles, which resemble Gastrointestinal stromal tumors (GIST) are thought to synaptic vesicles (Erlandson et al. 1996). originate from stem cells that differentiate toward the Neuroendocrine function is defined by the presence interstitial cells of Cajal (ICC; Kindblom et al. 1998, of a regulated secretory pathway by which neuro- Sircar et al. 1999, Lee et al. 2001). The ICC form a transmitters and hormones are released upon stimu- complex cellular network that is positioned between lation. Two types of regulated secretory pathways have nerve endings and smooth muscle cells in the intestinal been characterized. One pathway utilizes large dense wall, and functions as a pacemaker system controlling core vesicles (LDCVs) containing e.g. chromogranins, motility and possibly also neurotransmission (Vannucchi vesicular monamine transporters (VMAT 1 and 2), and 1999). The tyrosine kinase receptor KIT is of vital synaptic vesicle protein 2 (SV2), and the other pathway importance for normal development of the intestinal uses synaptic-like microvesicles (SLMVs) containing pacemaker system (Huizinga et al. 1995, Hirota et al. e.g. synapsin 1, synaptophysin, synaptobrevin, and 1998). Both ICC and GISTs express KIT, and mutations SV2 (Rindi et al. 2004). Neurons predominantly in the KIT gene are regarded as an early pathogenetic contain SLMV, while neuroendocrine cells frequently event in the tumorigenesis of GIST. Ultrastructural contain both LDCV and SLMV. studies have demonstrated similarities between ICC and Chromogranin A (CgA), the major acidic protein of GISTs with presence of numerous cytoplasmic vesicles chromaffin granules, is present in most neuroendocrine Endocrine-Related Cancer (2007) 14 853–863 Downloaded from Bioscientifica.comDOI:10.1677/ERC-06-0014 at 09/25/2021 09:24:23AM 1351–0088/07/014–853 q 2007 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.orgvia free access PBu¨mming et al.: Neuroendocrine differentiation in GIST cell types, both normal and neoplastic (Lloyd & tissue (brain and gut) and endocrine tumors (ileal Wilson 1983). The expression of synaptophysin, carcinoid and adrenal pheochromocytoma) served as initially found in small vesicle membranes of neurons, positive controls for IHC. has also been demonstrated in normal and neoplastic neuroendocrine cells (Jahn et al. 1985). Expression of Confocal laser scanning microscopy CgA and/or synaptophysin in tumors has therefore been used as histopathologic criterion for neuroendo- Formalin-fixed paraffin embedded specimens from Z Z crine differentiation. normal stomach (n 2), normal ileum (n 2), normal Z Z Here, we report the expression profile of synaptic colon (n 2), and GIST (gastric; low-risk profile, n 1 Z vesicle proteins, demonstrating regular presence of and high-risk profile, n 2) were analyzed by confocal SLMV, but not of LDCV, in GIST. laser scanning microscopy. Sections (4 mm) were subjected to heat-induced antigen retrieval using microwave treatment (Tris–EDTA, pH 9) followed by Materials and methods incubation with a mixture of primary antibodies (anti- Criteria for GIST – diagnosis and classification CD117 1:20 and anti-SV2 1:100), rinsing in PBS and incubated with a mixture of secondary antibodies Criteria for the diagnosis of GIST were: (1) primary tumor conjugated with Alexa Fluor 594 (goat anti-rabbit, site in the gastrointestinal tract and (2) characteristic 1:2000; A11037, Invitrogen) and Alexa Fluor 488 (goat histopathology including immunoreactivity toward KIT anti-mouse, 1:2000; A11029, Invitrogen, Carlsbad, CA, (CD 117). Based on tumor size and mitotic rate, the USA). Coverslips were mounted with ProLong Gold tumors were classified as very low-risk, low-risk, antifade reagent with DAPI (P36931, Molecular Probes, intermediate-risk, or high-risk GIST (Fletcher et al. Eugene, OR, USA). The fluorescent cells were analyzed 2002). To these four risk groups, we added a fifth, overtly by confocal microscopy using a Zeiss LSM 510 META malignant, which included all tumors with proven system with the LSM–FCS software supplied by the metastases at the time of diagnosis (Nilsson et al. 2005). manufacturer (Carl Zeiss, Jena, Germany). Emission In the present study, we separated the tumors into low-risk spectra were recorded with the META multichannel profile (very low-, low-, and intermediate-risk GIST, detector using the following settings: Hoechst 33258, nZ29) and high-risk profile (high-risk and overtly Diode laser 405 nm excitation, 422–476 nm emission malignant GIST, nZ12). detection; Alexa Fluor 488, Argon laser 488 nm Mutation analysis was performed on all 41 tumors as excitation, 497–550 nm emission detection and Alexa previously described (Andersson 2005). The study was Fluor 594, DPSS-laser 561 nm excitation, 583–668 nm approved by the Ethical Trial Committee of Go¨teborg emission detection. Images were recorded with an optical University and performed in accordance with the section of 0.8 mm using a Plan-FLUAR 100X/1.45 N.A. Helsinki Declaration of 1975. oil immersion objective. Immunohistochemistry Electron microscopy Z Forty-one tumors (low-risk profile, n 29; high-risk Specimens from GIST (gastric; low-risk profile, nZ1 Z profile, n 12) were analyzed by immunohistochem- and high-risk profile, nZ1) were fixed in 2.5% istry (IHC). Staining was performed on paraffin- glutaraldehyde in 0.1 M cacodylate buffer for 1– embedded specimens. In patients with low-risk profile 2 days, followed by postfixation in 1% osmium GIST, 27 primaries, one local recurrence and one tetroxide for 1 h. Specimens were dehydrated and metastasis were used for analysis. In patients with embedded in epoxy resin (TAAB 812). Ultrathin high-risk profile GIST, seven primaries, one local sections were placed on copper grids, contrasted with recurrence, and four metastases were used for analysis. uranyl acetate and lead citrate before examination and Sections (4 mm) were subjected to heat-induced photography in a Philips CM12 electron microscope. antigen retrieval using microwave treatment (Tris– EDTA, pH 9). Details of primary antibodies are given in Table 1. Bound antibodies were visualized using Western blot EnVisionCSystem-HRP labeled polymer (DakoCyto- Biopsies from ten patients with GIST were analyzed by mation, Dako, Glostrup, Denmark). Immunolabeling western blotting (low-risk profile, nZ4; high-risk was graded as follows: 0, !1% positive tumor cells; profile, nZ6). In patients with low-risk profile GIST, 1C, 1–24% positive tumor cells; 2C, 25–75% positive only primary tumors were analyzed. In patients with tumor cells; 3C, O75% positive tumor cells. Normal high-risk profile GIST, one primary, one local Downloaded from Bioscientifica.com at 09/25/2021 09:24:23AM via free access 854 www.endocrinology-journals.org Endocrine-Related Cancer (2007) 14 853–863 Table 1 Antibodies used for immunohistochemistry and western blot Antibody Species Dilution (IHC) Dilution (WB) Clone Code no. Source Anti-amphiphysin Mouse 1:250 1:250 3 VAM-SV030 Nordic Biosite AB, Ta¨by, Sweden Anti-b-actin Mouse – 1:1000 mAbcam8226 AB8226 Abcam Ltd, Cambridge, UK Anti-CD117 Rabbit 1:100 1:500 – A4502 DakoCytomation Denmark A/S, Glostrup, Denmark Anti-CgA Mouse 1:1600 – LK2H10 MAB319 Chemicon International Inc., Temecula, CA, USA Anti-GAP43 Mouse 1:20 – 1G7 NCL-GAP43 Novocastra Labora- tories Ltd, Newcastle upon Tyne, UK Anti-SNAP25 Mouse 1:2000 – SP12 NB09 Oncogene Research