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Lundberg Laboratory for Cancer Research, Departments of Surgery and
1 Pathology, Sahlgrenska Academy at the University of Göteborg, Sahlgrenska University Hospital, SE-413 45 Göteborg, Sweden
(Correspondence should be addressed to O Nilsson; Email: ola.nilsson{at}llcr.med.gu.se)
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Abstract |
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Introduction |
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Neuroendocrine function is defined by the presence of a regulated secretory pathway by which neuro-transmitters and hormones are released upon stimulation. Two types of regulated secretory pathways have been characterized. One pathway utilizes large dense core vesicles (LDCVs) containing e.g. chromogranins, vesicular monamine transporters (VMAT 1 and 2), and synaptic vesicle protein 2 (SV2), and the other pathway uses synaptic-like microvesicles (SLMVs) containing e.g. synapsin 1, synaptophysin, synaptobrevin, and SV2 (Rindi et al. 2004). Neurons predominantly contain SLMV, while neuroendocrine cells frequently contain both LDCV and SLMV.
Chromogranin A (CgA), the major acidic protein of chromaffin granules, is present in most neuroendocrine cell types, both normal and neoplastic (Lloyd & Wilson 1983). The expression of synaptophysin, initially found in small vesicle membranes of neurons, has also been demonstrated in normal and neoplastic neuroendocrine cells (Jahn et al. 1985). Expression of CgA and/or synaptophysin in tumors has therefore been used as histopathologic criterion for neuroendocrine differentiation.
Here, we report the expression profile of synaptic vesicle proteins, demonstrating regular presence of SLMV, but not of LDCV, in GIST.
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Materials and methods |
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Criteria for the diagnosis of GIST were: (1) primary tumor site in the gastrointestinal tract and (2) characteristic histopathology including immunoreactivity toward KIT (CD 117). Based on tumor size and mitotic rate, the tumors were classified as very low-risk, low-risk, intermediate-risk, or high-risk GIST (Fletcher et al. 2002). To these four risk groups, we added a fifth, overtly malignant, which included all tumors with proven metastases at the time of diagnosis (Nilsson et al. 2005). In the present study, we separated the tumors into low-risk profile (very low-, low-, and intermediate-risk GIST, n = 29) and high-risk profile (high-risk and overtly malignant GIST, n = 12).
Mutation analysis was performed on all 41 tumors as previously described (Andersson 2005). The study was approved by the Ethical Trial Committee of Göteborg University and performed in accordance with the Helsinki Declaration of 1975.
Immunohistochemistry
Forty-one tumors (low-risk profile, n = 29; high-risk profile, n = 12) were analyzed by immunohistochemistry (IHC). Staining was performed on paraffin-embedded specimens. In patients with low-risk profile GIST, 27 primaries, one local recurrence and one metastasis were used for analysis. In patients with high-risk profile GIST, seven primaries, one local recurrence, and four metastases were used for analysis. Sections (4 µm) were subjected to heat-induced antigen retrieval using microwave treatment (Tris–EDTA, pH 9). Details of primary antibodies are given in Table 1
. Bound antibodies were visualized using EnVision+System-HRP labeled polymer (DakoCytomation, Dako, Glostrup, Denmark). Immunolabeling was graded as follows: 0, < 1% positive tumor cells; 1+, 1–24% positive tumor cells; 2+, 25–75% positive tumor cells; 3+, > 75% positive tumor cells. Normal tissue (brain and gut) and endocrine tumors (ileal carcinoid and adrenal pheochromocytoma) served as positive controls for IHC.
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Formalin-fixed paraffin embedded specimens from normal stomach (n = 2), normal ileum (n = 2), normal colon (n = 2), and GIST (gastric; low-risk profile, n = 1 and high-risk profile, n = 2) were analyzed by confocal laser scanning microscopy. Sections (4 µm) were subjected to heat-induced antigen retrieval using microwave treatment (Tris–EDTA, pH 9) followed by incubation with a mixture of primary antibodies (anti-CD117 1:20 and anti-SV2 1:100), rinsing in PBS and incubated with a mixture of secondary antibodies conjugated with Alexa Fluor 594 (goat anti-rabbit, 1:2000; A11037 [GenBank] , Invitrogen) and Alexa Fluor 488 (goat anti-mouse, 1:2000; A11029 [GenBank] , Invitrogen, Carlsbad, CA, USA). Coverslips were mounted with ProLong Gold antifade reagent with DAPI (P36931, Molecular Probes, Eugene, OR, USA). The fluorescent cells were analyzed by confocal microscopy using a Zeiss LSM 510 META system with the LSM–FCS software supplied by the manufacturer (Carl Zeiss, Jena, Germany). Emission spectra were recorded with the META multichannel detector using the following settings: Hoechst 33258, Diode laser 405 nm excitation, 422–476 nm emission detection; Alexa Fluor 488, Argon laser 488 nm excitation, 497–550 nm emission detection and Alexa Fluor 594, DPSS-laser 561 nm excitation, 583–668 nm emission detection. Images were recorded with an optical section of 0.8 µm using a Plan-FLUAR 100X/1.45 N.A. oil immersion objective.
Electron microscopy
Specimens from GIST (gastric; low-risk profile, n = 1 and high-risk profile, n = 1) were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 1–2 days, followed by postfixation in 1% osmium tetroxide for 1 h. Specimens were dehydrated and embedded in epoxy resin (TAAB 812). Ultrathin sections were placed on copper grids, contrasted with uranyl acetate and lead citrate before examination and photography in a Philips CM12 electron microscope.
Western blot
Biopsies from ten patients with GIST were analyzed by western blotting (low-risk profile, n = 4; high-risk profile, n = 6). In patients with low-risk profile GIST, only primary tumors were analyzed. In patients with high-risk profile GIST, one primary, one local recurrence and four metastases were used for analysis. Neuroendocrine tumors served as positive controls (sporadic adrenal pheochromocytoma, well-differentiated endocrine carcinoma of the ileum–midgut carcinoid, and sporadic endocrine pancreatic carcinoma–malignant insulinoma). Western blot was performed as previously described (Jakobsen et al. 2002). Briefly, tumor tissues (200 mg/sample) were homogenized and assayed for protein content according to Bradford. Aliquots of proteins (40 µg) were separated by electrophoresis on precast polyacrylamide gels (10% NuPAGE Bis–Tris gels; Invitrogen) and transferred to polyvinyl difluoride membranes using a NOVEX blotting system (Invitrogen). The membranes were incubated with primary antibodies (Table 1
) at +4 °C overnight, followed by incubation with alkaline phosphatase-conjugated secondary antibodies and then with CDP-Star (Tropix, Bedford, MA, USA) as substrate. The membranes were exposed to hyper-film ECL (Amersham Pharmacia Biotech, Buckinghamshire, UK) at room temperature for 10–300 s. Molecular weight markers (See-Blue Plus 2 and MagicMark; Invitrogen) were used to calculate the apparent size of immunoreactive proteins.
Quantitative reverse transcriptase PCR (Q-PCR)
RNA extraction
Biopsies from ten patients with GIST were analyzed by Q-PCR (low-risk profile, n = 5; high-risk profile, n = 5). In patients with low-risk profile GIST, four primaries, and one local recurrence were used for analysis. In patients with high-risk profile GIST, one primary, one local recurrence, and three metastases were used for analysis. A human midgut carcinoid cell line (GOT1; Kölby et al. 2001) and a human colorectal adenocarcinoma cell line (Colo 205-CCL-222; ATCC, Manassas, VA, USA) were used as calibrators for Q-PCR assay. Colorectal adenocarcinoma (primaries, n = 2; lymph node metastasis, n = 1) and well-differentiated endocrine carcinoma of the ileum (midgut carcinoid; primary, n = 1; liver metastases, n = 2) served as control tumors in the Q-PCR analysis. Total RNA was prepared by homogenizing tumor biopsies in Trizol Reagent (Invitrogen; cat. no. 15596-026) using a Tissuelyser (QIAGEN, Solna, Sweden; cat. no. 85220) followed by RNA purification using FastRNA Kit, Green (QBIOgene, Irvine, CA, USA; cat. no. 6040-600). Total RNA from cultured cell lines was prepared using Trizol and the RNeasy Mini Kit (QIAGEN, cat. no. 74104). The quality of extracted RNA was examined by gel electrophoresis (Agilent 2100 Bioanalyser, Agilent Technologies, Santa Clara, CA, USA; cat. no. 62938-90030). Before cDNA synthesis, RNA samples were subjected to DNase treatment using DNA-Free (Ambion Inc., Austin, TX, USA; cat. no. 1906).
cDNA synthesis
cDNA was synthesized from extracted RNA using TaqMan reverse transcription reagents according to the manufacturer’s protocol (Applied Biosystems, Foster City, CA, USA; cat. no. N808-0234). For every cDNA synthesis reaction, a control reaction containing the same components was run, but without reverse transcriptase. The tubes were placed in a GeneAmp PCR System 2400 (Applied Biosystems) and a one-cycle PCR was performed using the following parameters: 25 °C for 10 min, 48 °C for 30 min, 95 °C for 5 min, followed by incubation at 4 °C. The cDNA was kept at –20 °C until assay.
Primers and probes
Primers and probes were purchased from Applied Biosystems. Hs00249911_m1 (vesicle-associated membrane protein 1: VAMP1; synaptobrevin 1), Hs00360269_m1 (vesicle-associated membrane protein 2: VAMP2; synaptobrevin 2), Hs00155723_m1 (amphiphysin: AMPH), Hs00199577_m1 (synapsin 1: SYN1), Hs00372069_m1 (synaptic vesicle glycoprotein 2A: SV2A), Hs00174029_m1 (v-kit: KIT), Hs00154441_ml (CgA: CHGA), Hs00300531_m1 (synaptophysin: SYP), Hs00161839_ml (vesicular monoamine, member 1: SLC18A1 solute carrier family 18: VMAT1), Hs00161858_ml (vesicular monoamine, member 2: SLC18A2 solute carrier family 18: VMAT2). ß-Actin primers and probe were used as a control (TaqMan ß-actin Control Reagent, cat. no. 401846).
Real-time PCR assay
The PCR assays were performed in 96-well optical plates with closed optical caps, using an ABI Prism 7700. A master mix was prepared for the PCR reactions, using 2 x Assay Mix containing primers, probes and TaqMan Universal PCR Master Mix (part. no 4304437).
Each well received 5 µl cDNA and 40 µl master mix. The samples were subjected to an initial heating for 2 min at 50 °C, followed by heating for 10 min. at 90 °C. The reaction consisted of 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The samples were analyzed in triplicate.
The cycle threshold (Ct) for target genes and ß-actin was determined for each sample. Values were expressed as –
Ct (target gene: ß-actin), or number of target copies per 1000 copies of ß-actin.
Statistical analysis
Continuous data from the different groups were compared using the non-parametric Mann–Whitney U test. Categorical data from different groups were compared using Fisher’s exact test. All P-values were two-tailed. P < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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, Fig. 1). Results of western blot showed that, in GISTs, KIT migrated as a major band with an apparent weight of 108 kDa, and with a minor band of ~104 kDa (Fig. 2). Q-PCR demonstrated high expression of KIT in all GISTs (Table 5, Fig. 3). Activating KIT mutations were found in 27 (66%) out of 41 GISTs. There were no differences in overall KIT/PDGFRA mutation frequency between low- and high-risk profile GISTs. Details of KIT/platelet derived growth factor alpha (PDGFRA) mutational analysis are given in Table 2.
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SLMV proteins were shown to be expressed in GIST, as determined by IHC, western blot, and Q-PCR. Positive staining for SV2 was observed in the majority of GISTs (Table 3). In positive tumors, moderate to strong staining was observed in the cytoplasm of tumor cells, with a granular pattern (Fig. 1). Western blot showed that SV2 migrates as a broad band of ~75–100 kDa (Table 4, Fig. 2). Q-PCR showed expression of SV2A in all GISTs, with median values similar to those observed in well differentiated endocrine carcinomas (WDEC) of the ileum/carcinoids, and two orders of magnitude higher than those observed in colorectal adenocarcinomas (Table 5 and Fig. 3). The localization of SV2 in GIST and normal ICC was further studied by confocal laser scanning microscopy (Fig. 4). In GIST, a granular SV2 staining was observed in the cytoplasm of KIT positive tumor cells. In normal intestine, no SV2 staining could be detected in KIT positive ICC. However, a close spatial relationship between SV2 positive nerve terminals and KIT positive ICC was demonstrated.
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). In tumors, strong staining was seen predominantly in tumor cell neurite- and bouton-like structures (Fig. 1). Q-PCR showed expression of synapsin 1 in all GISTs, with median values two orders of magnitude lower than those observed in WDEC of the ileum/carcinoids, but one order of magnitude higher than those observed in colorectal adenocarcinomas (Table 5 and Fig. 3).
Positive staining for amphiphysin was observed in the majority of GISTs (Table 3). Moderate staining was seen in the majority of tumor cells, with a granular cytoplasmic pattern and frequently with perinuclear localization (Fig. 1). In GISTs, amphiphysin migrated as multiple bands, with a major band of ~85 kDa, and minor bands of 110, 120 and 155 kDa (Table 4 and Fig. 2). Q-PCR showed expression of amphiphysin in all GISTs with median values similar to those observed in WDEC of the ileum/carcinoids and one order of magnitude higher than those observed in colorectal adenocarcinomas (Table 5 and Fig. 3).
Positive staining for synaptobrevin was observed in the majority of GISTs (Table 3). A moderate number of tumor cells were stained. Moderate staining with a granular pattern was seen in the tumor cell cytoplasm (Fig. 1). In GISTs, synaptobrevin migrated as a major band of ~15 kDa (Table 4 and Fig. 2). Q-PCR showed expression of synaptobrevin 1 and 2 in all GISTs. The expression values for synaptobrevin 2 were one order of magnitude higher than those for synaptobrevin 1. Expression values for synaptobrevin 1 and 2 in GISTs were similar to those in WDEC of the ileum/carcinoids (Table 5 and Fig. 3).
Synaptophysin, synapsin 2A, syntaxin, SNAP-25, and GAP-43 could not be demonstrated by IHC, but expression of synaptophysin was detected by Q-PCR in all GISTs, although with median values two orders of magnitude lower than those observed in WDEC of the ileum/carcinoids and similar to those observed in colorectal adenocarcinomas.
LDCV proteins are not regularly expressed in GIST
LDCV proteins were demonstrated in a minority of GISTs (Table 3). CgA was undetectable in all GISTs by IHC (Table 3). Q-PCR showed expression of CgA in five GISTs, with median values five orders of magnitude lower than those in WDEC of the ileum/carcinoids and similar to those observed in colorectal adenocarcinomas. VMAT1 was negative in the majority of GISTs, but a small number of tumor cells were stained in one-third of the tumors (Table 3). Moderate staining was observed in the cytoplasm of tumor cells, predominantly localized to the perinuclear area (Fig. 1). VMAT2 was undetectable in all GISTs by IHC. Western blot analysis was negative for both VMAT1 and VMAT2 (Table 4). Q-PCR detected expression of VMAT1 in only two GISTs. The expression levels of VMAT 1, in these two tumors were four orders of magnitude lower than those observed in WDEC of the ileum/carcinoids, and similar to those observed in colorectal adenocarcinomas. Expression of VMAT 2 was detected in all but one GIST, with median values three orders of magnitude lower than those observed in WDEC of the ileum/carcinoids, and one order of magnitude lower than those observed in colorectal adenocarcinomas (Table 5 and Fig. 3).
Ultrastructural evidence for SLMV in GIST
Analysis of GIST by electron microscopy demonstrated presence of numerous vesicles in tumor cells. The majority of the vesicles was of clear type with an approximate diameter of 40–80 nm and located predominantly in perinuclear and Golgi areas. The ultrastructural appearance was consistent with that of SLMV. In some tumor cells, vesicles with an approximate diameter of 80–120 nm and an electron dense core were also demonstrated (Fig. 5).
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There were no significant differences in the staining pattern, molecular size or expression levels of SLMV proteins when low- and high-risk profile GISTs were compared. Similarly, there were no significant differences in SLMV protein expression between GISTs with wild-type KIT/PDGFRA and mutated KIT/PDGFRA.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The diagnosis and classification of neuroendocrine tumors requires a combination of morphological analysis and demonstration of one or several of the synaptic vesicle protein known to be involved in the regulated secretory pathway. Traditionally, the presence of CgA and/or synaptophysin has been a prerequisite for the histopathological diagnosis of neuroendocrine tumors.
In recent years, multiple synaptic vesicle protein besides CgA and synaptophysin have been demonstrated in neuroendocrine tumors, e.g., SV2 (Portela-Gomes et al. 2000, Jakobsen et al. 2002), VMAT 1 and 2 (Jakobsen et al. 2001), and amphiphysin (Zanner et al. 2004). The expression of several SLMV proteins in GIST therefore suggests that these tumors have a neural or neuroendocrine differentiation. This is further supported by the observation of numerous clear vesicles in GIST having an ultrastructural appearance of SLMV.
Quantitation of synaptic vesicle protein by Q-PCR revealed specific expression patterns in GIST, WDEC/carcinoids and colorectal adenocarcinomas. In WDEC/carcinoids, both LDCV and SLMV proteins were highly expressed, while in GIST only SLMV proteins were shown to be highly expressed. Colorectal adenocarcinomas showed low expression of both LDCV and SLMV proteins. Q-PCR analysis readily distinguished the three tumor types in this study and the technique may prove to be a useful tool to complement IHC in the molecular characterization of tumors (Kidd et al. 2006).
Neuroendocrine differentiation, i.e., positive CgA and/or synaptophysin staining has been documented in some non-endocrine tumors, e.g., prostatic carcinoma (Hirano et al. 2005), breast carcinoma (van Krimpen et al. 2004), and gastrointestinal carcinoma (Grabowski et al. 2002). However, in non-endocrine tumors, neuroendocrine differentiation is usually demonstrated in a small subset of tumor cells. Neuroendocrine differentiation in some types of non-endocrine tumors has been associated with poor prognosis (Di Sant’Agnese 2001). In this series, we could not detect any correlation between tumor site, risk profile, or KIT/PDGFRA mutational status and expression of synaptic vesicle protein. The significance of SLMV expression in GIST is currently unknown and must be explored further. Confocal laser scanning microscopy failed to demonstrate the synaptic vesicle protein SV2 in ICC, while this technique readily demonstrated SV2 in GIST. This finding suggests that SLMV protein expression in ICC is low or absent, and that expression of SLMV proteins is up-regulated during transformation and development of GIST.
The regulated secretion in neuroendocrine cells and tumors is controlled by G protein-coupled membrane receptors. High expression of G protein-coupled receptors, e.g., somatostatin receptors, has been demonstrated in neuroendocrine tumors (Barnett 2003) and GIST (Montella et al. 2005). Extremely high densities of bombesin subtype 2 (gastrin-releasing peptide-GRP)-receptors, vasoactive intestinal peptide subtype 2-receptors, and cholecystokinin subtype 2-receptors have been demonstrated in the majority of GISTs (Reubi et al. 2004), further supporting the presence of a regulated secretion in GIST. In the future, radiolabeled peptides/analogs may be used for both localization and targeted treatment of GIST, especially in patients who are not responsive to treatment with tyrosine kinase inhibitors.
In conclusion, our study shows that GISTs regularly express SLMV proteins, indicating a neuroendocrine phenotype. This suggests that novel strategies of treatment of these tumors may soon be a reality.
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Acknowledgements |
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Supported by the Swedish Cancer Society, Swedish Research Council (5220), Sahlgrenska Academy (the government ALF agreement), the Inga-Britt and Arne Lundberg Research Foundation, the Johan Jansson Foundation for Cancer Research, the Assar Gabrielsson Research Foundation, Göteborg Medical Society, the King Gustav V Jubilee Clinical Cancer Research Foundation, and Sahlgrenska University Hospital Research Foundation. The authors do not have any financial or personal relationships between themselves that might bias this work.
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References |
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