Virchows Arch (2005) 446: 604–612 DOI 10.1007/s00428-005-1222-6
ORIGINA L ARTI CLE
Guida Maria Portela-Gomes . Lars Grimelius . Mats Stridsberg . Enrica Bresaola . Giuseppe Viale . Giuseppe Pelosi
Expression of amino acid sequences of the chromogranin A molecule and synaptic vesicle protein 2 in neuroendocrine tumors of the lung Received: 22 December 2004 / Accepted: 7 February 2005 / Published online: 20 May 2005 # Springer-Verlag 2005
Abstract Chromogranin A (CgA) and its valuable complement synaptic vesicle protein 2 (SV2) are neuroendocrine (NE) markers. Post-translational processing of CgA has been reported to vary in different NE cell types and tumors, but little is known regarding the expression of various CgA epitopes and SV2 in NE pulmonary tumors. We studied the immunoreactivity to six CgA epitopes and SV2 in ten typical (TC) and ten atypical (ACT) carcinoids, five large-cell NE carcinomas (LCNEC) and five small-cell carcinomas (SCLC), also comparing the results with clinicopathological characteristics of tumors. The sequences CgA 17–38 (vasostatin), 176–195 (chromacin), 375–384 (parastatin) and 411–424 (C-terminal parastatin) and SV2 were relevant markers for the CT/ATC group, whereas the G. M. Portela-Gomes . L. Grimelius Unit of Pathology, Department of Genetics and Pathology, University Hospital, 751 85 Uppsala, Sweden G. M. Portela-Gomes Centre of Nutrition and Metabolism, Lisbon University, Lisbon, Portugal M. Stridsberg Department of Medical Sciences, Clinical Chemistry, University Hospital, 75185 Uppsala, Sweden E. Bresaola . G. Viale . G. Pelosi Division of Pathology and Laboratory Medicine, European Institute of Oncology and University of Milan School of Medicine, Milan, Italy G. Pelosi (*) Divisione di Anatomia Patologica e Medicina di Laboratorio, Istituto Europeo di Oncologia, Via G. Ripamonti 435, 20141 Milan, Italy e-mail:
[email protected] Tel.: +39-2-57489414 Fax: +39-2-57489417
antibody to CgA 176–195 was a better marker for the LCNEC/SCLC group. An inverse correlation was found between proliferative activity and granule-related markers in the CT/ACT group, and a direct correlation in poorly differentiated tumors. The expression of granule-related markers did not correlate with hormone content or clinical characteristics of NE tumors. The expression of CgA epitopes and SV2 occurs in all NE tumors, differing between better differentiated and poorly differentiated tumors but not within the respective groups. Keywords Chromogranin A . SV2 . Neuroendocrine . Lung . Tumor . Fragment . Immunohistochemistry
Introduction Neuroendocrine (NE) tumors of the lung are currently classified as typical carcinoid (TC), atypical carcinoid (ATC), large-cell NE carcinoma (LCNEC) and small-cell lung carcinoma (SCLC) [27]. Typical carcinoids are low-grade malignant tumors, ATC intermediate grade, and the two latter tumor types are highly malignant. The carcinoids account for 2% or less of all lung tumors, while LCNEC and SCLC represent together about 20% [7]. Different hormonal products have been immunohistochemically demonstrated in pulmonary NE tumors, some of which have given rise to endocrine-related syndromes, such as inappropriate secretion of antidiuretic hormone, Cushing syndrome, acromegaly or carcinoid syndrome [2]. Pulmonary carcinoids but not the high-grade NE tumors may also be associated with neoplastic or hyperplastic lesions of the pituitary and parathyroid glands and of endocrine pancreas, mainly as a part of MEN-1 syndrome [7]. Several studies have been carried out in order to identify histopathological characteristics useful in assisting the differential diagnosis of these tumor types and/or in predicting their biological behaviour [3, 13, 14, 27, 28]. The most specific and suitable markers so far for highlighting NE differentiation in lung tumors are chromogranin A (CgA) and synaptophysin (Syn), but more recently synaptic ve-
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sicle protein 2 (SV2) has also been proposed as a useful marker for NE differentiation [22]. CgA is a glycoprotein localized in the secretory granules [29], whereas Syn is an integral membrane glycoprotein detectable in small vesicles [17]. SV2 has been demonstrated in vesicular structures of the mammalian nervous system [4] and in various NE cell types [22]. Recently, interest has been focused on the expression of various epitopes of the CgA molecule in different NE cell types of the gastroenteropancreatic system and derived tumors [23– 25]. CgA has ten potential cleavage sites, which can give rise to post-translational processing of the CgA molecule. However, data on the prevalence and clinicopathological implications of the immunoreactivity to both different CgA epitopes and SV2 in NE tumors of the lung are lacking. An essential factor for tumor growth is neoangiogenesis [9], which can be inferred by microvessel density (MVD) in tumor sections after selective immunostaining of endothelial cells [31]. In carcinoids of the gastroenteropancreatic system and in NE-differentiated adenocarcinomas of the prostate, the hypervascular status of the neoplasm has been linked to the presence of CgA-immunoreactive NE tumor cells, which produce a vascular endothelial growth factor [6, 16, 26]. Moreover, it has been shown that CgA is able to prevent the endothelial cell cytoskeleton rearrangement and vascular leakage mediated by cytokines [10], and that vasostatins may inhibit vascular contractions [1]. The aim of the present study was to analyse the expression of epitopes of the CgA molecule and SV2 in various types of NE lung tumors and to evaluate if these results can give additional information to other tumor characteristics, such as histological typing, tumor cell proliferation, neoangiogenesis and peptide hormone pattern.
Materials and methods Patients and tumors Tissue specimens from surgically resected pulmonary NE tumors from 30 patients (10 CT, 10 ACT, 5 LCNEC and 5 SCLC) were included in the study. The tissue specimens were fixed in buffered formalin and routinely processed to paraffin. These tumors were selected among 117 consecutive NE lung tumors of various histologic types, diagnosed at the Division of Pathology and Laboratory Medicine of the European Institute of Oncology, Milan, Italy, surgically removed between 1996 and 2002. To ensure an accurate staging, all tumors were removed by radical surgery, with extensive mediastinal lymph node dissection. All ten CT were arbitrarily chosen with no regional lymph node metastases at the time of diagnosis, whereas all the other tumours were metastatically spread to regional lymoh nodes. Lymph node metastases were at the pN1 (8 ACT, 4 LCNEC and 1 SCLC) or pN2 (5 ACT, 1 LCNEC, and 4 SCLC) level according to the last international tumor-node-metastasis (TNM) staging system (6th edn, 2003) [30], involving the hilar/peribronchial nodes or mediastinal nodes, respectively. One LCNEC and four SCLC patients underwent neo-adjuvant combination chemotherapy by using CAVE protocol (cyclophosphamide, doxorubicin, vincristine and etoposide). For each case, all paraffin blocks were retrieved and archival hematoxylin-eosin sections reviewed. Tumor recurrences were seen in 12 (40%) patients (1 CT, 2 ACT, 4 LCNEC and 5 SCLC), with localization in ipsilateral lung (1 CT, 1 ACT, 4 LCNEC and 1 SCLC), liver (3 LCNEC and 2 SCLC), ovary (1 ACT), bone (3 SCLC), or brain (2
Table 1 Antibodies used in the current study Antibodies
m/P Clone
Specificity
Dilution Source
CgA
m
LK2H10
1:1000
Synthetic human CgA (CgA2)
p
–
CgA 250–301 N-terminal pancreastatin CgA 17–38 vasostatin
Synthetic human CgA (CgA4) Synthetic human CgA (CgA10) Synthetic human CgA (CgA11) Synthetic human CgA (CgA8) Synthetic human CgA (CgA9) Synaptic vesicle protein 2
p p p p p m
– – – – – SV2
CgA 116–130 chromostatin CgA 176–195 chromacin CgA 238–247 CgA 375–384 GE25 (mid-parastatin) CgA 411–424 C-terminal parastatin Synaptic vesicle protein 2
1:800 1:1600 1:1600 1:400 1:800 1:50
Synaptophysin ACTH Calcitonin CD34 GRP/Bombesin Serotonin alpha-HCG Ki-67
m m p m p m p m
SY 38 02A3 – QBEnd/10 – 5HT-H209 – MIB-1
Synaptophysin Human ACTH 24–39 Human calcitonin 1–32 110 kD glycoprotein Human GRP 14–27 5-HT Alpha-chain Ki-67 antigen
1:20 1:100 1:20 1:400 1:400 1:10 1:200 1:400
CgA chromogranin A; m monoclonal; p polyclonal
1:800
Boehringer-Mannheim, Mannheim, Germany M. Stridsberg, Dept. Clin.Chem., Uppsala, Sweden M. Stridsberg M. Stridsberg M. Stridsberg M. Stridsberg M. Stridsberg RB Kelly, Dept. Biochem. Biophys., UCSF, CA DakoCytomation, Glostrup, Denmark DakoCytomation, Glostrup, Denmark DakoCytomation, Glostrup, Denmark DakoCytomation, Glostrup, Denmark DakoCytomation, Glostrup, Denmark DakoCytomation, Glostrup, Denmark Signet Laboratories, Dedham, MA Immunotech, Marseille, France
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SCLC). The mean age of the patients at the time of surgery was 54±15 years (median: 59, range 26–75) for CT and ACT group, and 60±11 years (median: 59, range 45–74) for LCNEC and SCLC (NS). The gender distribution was 12 males (7 CT/ACT and 5 LCNEC/SCLC) and 18 females (13 CT/ACT and 5 LCNEC/SCLC). Complete clinical follow-up was available for all patients with a mean duration of 29±19 months (median 24; range 9–85); during this period eight (26.7%) died of disease (2 ACT, 3 LCNEC and 3 SCLC). No patients presented with MEN-1 or any other endocrine-related syndromes. The diagnosis of NE tumor was based on previously refined morphological and immunohistochemical criteria, including immunoreactivity for pan-endocrine markers CgA, Syn, and for a variety of respiratory tract-related hormones, including GRP, calcitonin, ACTH, serotonin and alphaHCG) [20, 21]. Tumor size ranged from 0.7 to 4.0 cm in
CT (mean±SD: 2.4±0.9; median 2.45), from 1.2 to 6.2 cm in ATC (mean±SD: 2.7±1.5; median 2.25), from 3.1 to 8.0 cm in LCNEC (mean±SD: 4.9±1.8; median 4.5) and from 2.5 to 9.0 cm in SCLC (mean±SD: 4.5±2.8; median 3.0) (CT versus ACT, NS; LCNEC versus SCLC, P=0.060; CT/ACT versus LCNEC/SCLC, P=0.037). Immunohistochemistry Sections 5 μm thick were cut from representative tissue blocks and immunostained with polyclonal antibodies raised to six specific regions of the CgA molecule [amino acid sequences CgA 17–38 (vasostatin), 116–130 (chromostatin), 176–195 (chromacin), 238–247, 375–384 9 (mid-portion of parastatin) and 411–424 (C-terminal parastatin)], all previously characterized by Portela-Gomes and Stridsberg
Table 2 Distribution of chromogranin A fragments nd SV2 immunoreactivity according to tumor typing Tumor type
17–38 vasostatin
116–130 chromostatin
176–195 chromacins
238–247
375–384 mid-parastatin (GE-25)
411–424 C-terminal parastatin
mCgA 250–301 N-terminal pancreastatin
SV-2
CT #11 CT #12 CT #13 CT #14 CT #15 CT #16 CT #17 CT #18 CT #19 CT #20 ACT #1 ACT #2 ACT #3 ACT #4 ACT #5 ACT #6 ACT #7 ACT #8 ACT #9 ACT #10 LCNEC #21 LCNEC #22 LCNEC #23 LCNEC #24 LCNEC #25 SCLC #26 SCLC #27 SCLC #28 SCLC #29 SCLC #30
2/*(**) 4/**(*) 3/*(**) 4/*** 3/*** 4/*** 3/** 4/*** 4/**(*) 4/*** 4/*** 4/*** 3/*(**) 4/*** 4/*** 4/*** 4/*** 4/*** 4/*(**) 4/*** 2(3)/**(*) 2/*(*) 3/* 2/*(**) 1/** 4/*** 2/* – 3/*(**) 3/*(**)
1/*(*) 4/*** 2/* 2/*(*) 2/* 2/*(*) 3/* 3/*(*) 1/* 4/** 3/* 3/* 3/** 2/* 3/*(*) 1/** 4/**(*) 4/**(*) 4/*(**) 3/*(*) 2/**(*) 2/* 3/* 3/*(**) 1/** – 2/* 4/* 3/*(*) 3/*
3(4)/** 4/*** 4/** 4/*** 4/*** 4/*** 4/*** 4/*** 4/*** 4/*** 4/*** 4/*** 4/*** 3(4)/*** 4/*** 4/*** 4/*** 4/*** 4/*** 4/*** 4/**(*) 3/**(*) 4/**(*) 3/*(**) 4/** 4/*** 2/**(*) 4/** 4/*(**) 4/***
1/* 3/* 1/* 4/** 3/** 4/*(*) 3/*(*) 4/** 1/* 4/** 4/** 3/*(*) 4/*(*) 2(3)/* 4/** 4/*(*) 4/* 3/* 3/* 3/(*) 3/** 2/* 3/* 3/(*) – 2/* 2/* 4/* 2/* 2/*
3/* 4/**(*) 3/*(**) 3/** 3/** 4/*** 4/*** 4/** 4/*(*) 4/**(*) 4/*** 4/*** 4/*** 3/** 4/*** 4/*** 4/**(*) 3/**(*) 3/**(*) 4/*(*) 2/** 3/*(**) 3/* 2/*(**) 3/* 2/* 2/* 3/* 3/* 2/*
4/** 3/* 4/* 4/*(*) 3/*(*) 4/* 3/**(*) 3/* – 4/*(*) 4/*** 3/*(**) 4/* 3/* 4/**(*) 4/** 4/** 3/** 4/**(*) 4/** 3/** 4/** 3(4)/* 3/* 3/* 3/* 2/* 4/*(*) 3/*(*) 3/*(*)
3(4)/*(*) 4/*** 4/*** 4/*** 4/*** 4/*** 4/*** 4/*** 4/*** 4/*** 4/*** 4/*** 4/*** 3(4)/*** 4/*(*) 4/*** 4/*** 4/**(*) 4/*** 4/*** 3/**(*) 3/**(*) 3/**(*) 3/*(**) 1/*** 4/*** 1/** – 4/*(**) 4/***
4/** 4/*** 4/**(*) 4/** 3/* 2/* 3/** 4/*(*) 4/*(*) 4/*(*) 4/**(*) 4/**(*) 4/** 3/*(*) 4/**(*) 4/** 4/**(*) 4/**(*) 3/*(*) 4/** 1/* – 3/* 2/(*) – 4/*** – 1/* 3/* 4/**
mCgA monoclonal antibody to chromogranin A; CT typical carcinoid; ACT atypical carcinoid; LCNEC large-cell neuroendocrine carcinoma; SCLC small-cell lung carcinoma Percentages of tumor cells displaying immunoreactivity: −, negative; 1, 90% positive Intensity of immunoreaction: ***, strong; **, moderate; *, weak; (*), very weak
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[23, 25]. The SV2 antibody was instead monoclonal, and was previously characterized by Buckley and Kelly [4]. Dewaxed sections were initially treated with 5% hydrogen peroxide for 12 min for blocking endogenous peroxidase activity and reacted with the panel of reagents listed in Table 1 using either a Ventana NEXES automatic immunostainer (Ventana, Tucson, Ariz., USA) for commercial CgA (clone LK2H10), SV2 and CgA region-specific antibodies, or a Dako Autostainer (DakoCytomation, Glostrup, Denmark) for all the other reagents under investigation, both of which were used according to the manufacturers’ suggestions. Whenever appropriate, before immunostaining the sections were pretreated in a microwave oven with either citrate buffer at pH 6.0 or EDTA buffer at pH8 as retrieval solution. Peroxidase activity was then developed with 3-3′-diaminobenzidine-copper sulphate (Sigma Chemical Co., St Louis, Mo., USA) to obtain a bright brownishblack end product. The control stainings for all reactions entailed (i) omission of the primary antibodies or (ii) replacement of the first layer of antibody by nonimmune serum diluted 1:10, (iii) replacement of the CgA regionspecific antibodies by the diluent alone and preincubation (24 h) of primary antibodies with the relevant antigen (10 nmol per ml diluted antibody solution, respectively) before application to the sections. Appropriate internal positive controls represented by the scattered NE cells of the normal bronchial mucosa were checked in every staining run. n The density of immunoreactive cells was semiquantitatively calculated on a five-tier scale, as being either negative or positive in 90% tumor cells, respectively. For CgA and SV2, the immunostaining intensity was also evaluated subjectively by using a three-tier scale (strong, moderate, weak/very weak).
Tumor cell proliferation was evaluated by Ki-67 index and tumor neoangiogenesis was inferred by MVD after CD34 immunostaining of endothelial cells as previously detailed [19]. Statistical analysis Qualitative data were compared using the Fisher exact t-test or chi-square test. Differences in continuous data (i.e. CgA fragments, SV2, MVD, Ki-67 index, tumour size) were contrasted employing the Mann–Witney test or the Kruskal–Wallis test if medians were compared between two or more groups, respectively. All correlation tests were calculated using Spearman’s rank test (r). All P-values were based on two-sided testing.
Results Immunostaining of scattered normal NE cells of adjacent bronchi and bronchioli with the various CgA regionspecific antibodies and SV2 showed that immunoreactive cells to CgA 176–195, the monoclonal CgA antibody and SV2 were the most commonly observed, whereas only occasional or no immunoreactive cells were observed to CgA 116–130, 238–247 and 411–424. The findings of immunostaining for both NE markers (commercial CgA and SV2) and region-specific epitopes of the CgA molecule are presented in Tables 2 and 3. Representative features of CgA molecule fragment and SV2 immunoreactivity are depicted in Figs. 1 and 2, respectively. For the commercial CgA antibody and SV2, the frequency of immunoreactive cells was significantly higher in
Table 3 Chromogranin (Cg) A and SV2 immunoreactive cells in 30 pulmonary neuroendocrine tumours Antibody
CgA 17–38 (CgA2) CgA 116–130 (CgA4) CgA 176–195 (CgA10) CgA 238–247 (CgA11) CgA 375–384 (CgA8) CgA411–424 (CgA9) mabCgA SV2
Tumour category
CT/ACT LCNEC/SCLC CT/ACT LCNEC/SCLC CT/ACT LCNEC/SCLC CT/ACT LCNEC/SCLC CT/ACT LCNEC/SCLC CT/ACT LCNEC/SCLC CT/ACT LCNEC/SCLC CT/ACT LCNEC/SCLC
No. of tumors with different percentage intervals of immunoreactive cells (%) neg
