Guido Rindi · Donatella Paolotti · Roberto Fiocca Bertram Wiedenmann . Jean-Pierre Henry Enrico Solcia
Vesicular monoamine transporter 2 as a marker of gastric enterochromaffin-like cell tumors
Received: 28 June 1999 / Accepted: 20 October 1999
Abstract The vesicular monoamine transporter 2 (VMAT2) facilitates the ATP-dependent accumulation of biogenic amine inside the secretory granules of endo- crine cells and neurons and was demonstrated in the his- tamine-producing enterochromaffin-like (ECL) cells of the stomach. In the present investigation, VMAT2 immu- nohistochemistry was tested in 85 endocrine tumors, of which 60 were well differentiated gastrointestinal and pancreatic growths, 5 poorly differentiated (neuro)endo- crine carcinomas (PDEC) and 1 mixed PDEC/ECL cell carcinoma of the stomach, 12 pheochromocytomas/para- gangliomas, 3 adrenocortical lesions, 2 parathyroid and 2 lung neuroendocrine tumors. Extensive and intense VMAT2 immunoreactivity was observed in 16 of 16 gas- tric ECL cell tumors, 6 of 6 adrenal pheochromocyto- mas, 2 of 2 chromaffin paragangliomas and in 3 of the 4 carotid body paragangliomas investigated. Rare VMAT2-positive cells were observed in 12 of 21 intesti- nal enterochromaffin (EC) cell tumors, in 9 of 11 pancre- atic neuroendocrine tumors, and in the mixed PDEC/ ECL cell carcinoma of the stomach (differentiated cells only). No VMAT2 immunoreactivity was observed in five gastrin, four somatostatin and three enterogluca-
G. Rindi X Department of Pathology, University of Brescia, Anatomia Patologica 2, Piazza Spedali Civili, 1, I-25124 Brescia, Italy
e-mail: rindi@master.cci.unibs.it
Tel .: +39-30-3995830, Fax: +39-30-3995053
D. Paolotti · E. Solcia
Department of Pathology, University of Pavia, Italy
R. Fiocca Department of Pathology, University of Genova, Italy
B. Wiedenmann Medizinische Klinik mit Schwerpunkt Hepatologie und Gastroenterologie, Universitätsklinikum Charité, Humbolt University, Berlin, Germany
J .- P. Henry Institut de Biologie Physico-Chimique, Paris, France
E. Solcia Research Direction, IRCCS Policlinico San Matteo, Pavia, Italy
gon/peptide YY tumors of the gastrointestinal tract, in six gastric PDECs, in three adrenocortical growths, and two parathyroid and two lung neuroendocrine tumors. These data support VMAT2 immunohistochemistry as being a useful tool for the diagnosis of gastric ECL cell tumors, separating them from all other endocrine tumors arising in the gastroduodenal area i.e., gastrin, somatostatin, EC cell and PDEC tumors, all of which proved essentially negative.
Key words VMAT2 . Stomach . ECL cell . Tumors . Immunohistochemistry
Introduction
At present, the specific identification of human entero- chromaffin-like (ECL) cells and related tumors may be achieved only using electron microscopy and histamine or histidine-decarboxylase histochemistry [5, 16, 29]. Unfortunately, electron microscopy has several disad- vantages, including the need of appropriately preserved and processed tissue, the limited sampling of tumor mass, and the relatively high costs. However, histamine immunohistochemistry is severely impaired by the low histamine content of human ECL cells and its rapid dis- solution in ordinary liquid fixatives. Moreover, to our knowledge no antibody for human histidine-decarboxyl- ase histochemistry is available that works in routine for- malin-fixed, paraffin sections. Thus, only a battery of light-microscopy methods, including positive chromo- granin A immunostaining and Grimelius or Sevier- Munger silver, coupled with negative tests for peptide hormones such as gastrin or somatostatin, may provide data supporting a reliable diagnosis of ECL cell tumor (neuroendocrine tumor) in routine histopathologic mate- rial [3, 20, 27].
Recently, two integral membrane glycoproteins, ve- sicular monoamine transporters 1 and 2 (VMAT1 and 2), have been shown to facilitate the ATP-dependent accu- mulation of biogenic amine precursors into neuroendo-
crine secretory granules, where they may undergo decar- boxylation to corresponding monoamines [13, 30]. Inter- estingly, the two VMAT isoforms show broad selectivity for different amines and are differently distributed in various cell types. A combination of selectivity and re- dundancy is probably necessary to impart selective, ge- netically regulated, functional properties to different monoaminergic cell types [30]. VMAT1 was found to be expressed in serotonin-producing gastrointestinal entero- chromaffin (EC) tumors and the small intensely fluores- cent (SIF) cells of sympathetic ganglia, whereas VMAT2 was detected in histamine-producing gastric ECL cells, in addition to central and peripheral neurons [10, 12, 13, 32]. Only chromaffin cells of the adrenal medulla ex- pressed both VMAT1 and VMAT2. On this basis, VMAT isoforms have been tested in a case of a histamine-pro- ducing gastric endocrine tumor showing ultrastructural morphology of an ECL cell tumor. Only VMAT2 immu- noreactivity was found, thus raising the possibility that this may represent a useful marker for the diagnosis of ECL cell tumors [18].
In this study, VMAT2 immunohistochemistry has been applied to gastric endocrine tumors, most of which had been diagnosed as ECL cell tumors based on previ- ous morphological and clinico-pathological investiga-
Table 1 Vesicular monoamine transporter 2 (VMAT2) immunore- activity in 85 neuroendocrine tumors. PDEC poorly differentiated (neuro)endocrine carcinoma; No. number of investigated tumors; n number of positive tumors; intensity mean value of intensity of immunoreaction graded as +=weak; ++=moderate; +++=intense;
tions [20]. For comparison, endocrine tumors from the pancreas, intestine, adrenals, parathyroids, retroperitone- um, carotid body, and lung were also tested.
Materials and methods
A total of 85 neuroendocrine tumors from the files of the Depart- ments of Pathology of Pavia and Brescia were investigated. Of 23 gastric endocrine tumors, 21 of which were previously character- ized on histopathological, histochemical, ultrastructural and clin- ico-pathological grounds, 16 were well differentiated gastric ECL cell tumors, 1 was a pyloric gastrinoma, 5 were poorly differenti- ated gastric (neuro)endocrine carcinomas (PDECs) and 1 was a mixed PDEC/ECL cell carcinoma (for diagnostic criteria see [20] and [21]). Eight of the ECL cell tumors were of type I, associated with diffuse corpus-fundus chronic atrophic gastritis (A-CAG), 4 were of type II associated with Zollinger-Ellison syndrome (ZES) and type-1 multiple endocrine neoplasia (MEN-1), and 4 were of type III, sporadic. Of 62 non gastric tumors, 32 were tu- mors of the gut, 11 of the pancreas, 2 adenomas and 1 carcinoma of the adrenal cortex, 6 adrenal pheochromocytomas, 2 parathy- roid tumors, 2 retroperitoneal sympathetic paragangliomas, 4 ca- rotid body tumors, and 2 bronchial neuroendocrine tumors. Of 32 gut tumors, 4 were diagnosed as duodenal gastrin-producing G-cell tumors, one of which with MEN-1; 4 as duodenal, somato- statin-producing D-cell tumors; 21 as serotonin-producing EC cell neuroendocrine tumors (1 from duodenum, 18 from the ileum and 2 from the appendix) and 3 as enteroglucagon/peptide YY (peptide
| Site | Tumor type | Main cell type | No. | VMAT2 immunoreactivity | ||
|---|---|---|---|---|---|---|
| n | Intensity | % | ||||
| Stomach | Argyrophil carcinoid | ECL | 16 | 16 | ++ | 90a |
| Gastrinoma | G | 1 | 0 | – | – | |
| PDEC | Protoendocrine | 5 | 0 | – | – | |
| PDEC/carcinoid | Protoendocrine/ECL | 1 | 1 | ++ | 40b | |
| Duodenum | Gastrin cell | G | 4 | 0 | – | – |
| Somatostatin cell | D | 4 | 0 | – | – | |
| Argentaffin carcinoid | EC | 1 | 0 | – | – | |
| Ileum | Argentaffin carcinoid | EC | 18 | 12 | +/++ | 1-10c |
| Appendix | Argentaffin carcinoid | EC | 2 | 0 | – | – |
| Colon-rectum | Trabecular carcinoid | L | 3 | 0 | – | – |
| Pancreas | Nonfunctioning | Various | 3 | 3 | ++ | 1-20 |
| Functioning | B | 3 | 2 | ++ | 1-10 | |
| A | 2 | 2 | ++ | 10-30 | ||
| G | 3 | 2 | ++ | 1-10 | ||
| Parathyroid | Adenoma | Chief | 2 | 0 | – | – |
| Lung | Argyrophil carcinoid | P | 2 | 0 | – | – |
| Adrenal | Cortical adenoma | Cortical | 2 | 0 | – | – |
| Cortical carcinoma | Cortical | 1 | 0 | – | – | |
| Pheochromocytoma | Chromaffin | 6 | 6 | +++ | 40-80 | |
| Retroperitoneum | Paraganglioma | Chromaffin | 2 | 2 | ++ | 15-40 |
| Carotid body | Paraganglioma | Non-chromaffin | 4 | 3 | +++ | 5-80 |
a One case 30%; b Restricted to well-differentiated cells only; c One case 30%
A
B
C
D
with C-terminus and N-terminus tyrosine)-producing L-cell tu- mors of the rectum. The 11 pancreatic tumors were all well differ- entiated with variable tumor cell populations at immunohisto- chemical analysis. According to the pertinent clinical settings, three were diagnosed as nonfunctioning and eight as functioning tumors (three insulinomas, three gastrinomas, and two glucagono- mas). For non-ECL cell tumors, the diagnosis was based on con- ventional histopathological criteria, positive silver stain and im- munoreactivity for the general neuroendocrine markers chromo- granin-A or synaptophysin, coupled with immunoreactivity for the cell-specific endocrine peptides or amines and electron microsco- py [4, 6, 7, 14, 28]. Samples of non-tumor gastrointestinal muco- sa, pancreas and adrenals from tumor-bearing and tumor-free cases were also investigated.
Formalin- or Bouin-fixed, 4-um paraffin sections were brought to water and immunostained with anti-human VMAT2 affinity-pu- rified serum (see below for details) after microwave antigen retri- eval [9], and with antibodies specific for the gastrointestinal hor-
A
B
C
D
E
F
mones serotonin, gastrin, somatostatin, glycentin, and peptide YY using the avidin-biotin-peroxidase technique [17], as detailed elsewhere [5, 7, 14, 20]. For double localization studies, serial or reverse face 2-um paraffin sections were used. Controls consisted of omission of the first layer and use of tissue with or without the pertinent antigen.
The anti VMAT2 serum was generated by immunization of rabbits with a fragment encoding the amino acids 42-133 of bo- vine VMAT2, corresponding to the large intravesicular loop [22]. Subsequently the serum was first filtered through glutathione- S-transferase (GST) immobilized on glutathione-sepharose, to eliminate antiGST antibodies, and then the anti VMAT2 antibod- ies were purified by adsorbing the flowthrough on the VMAT fusion protein immobilized on glutathione-sepharose.
Results
The results are summarized in Table 1. Specific VMAT2 immunoreactivity was found in 49 of the 85 endocrine tumors investigated, including all the 16 ECL cell tu- mors (irrespective of clinicopathological subtypes), 1 mixed PDEC/ECL cell tumor, 12 of 21 EC cell tumors, 9 of 11 pancreatic tumors, the 6 adrenal pheochromo- cytomas, the 2 extra-adrenal sympathetic paraganglioma, and 3 of 4 carotid body paragangliomas (Fig. 1 and Fig. 2). Of digestive tract tumors, VMAT2 immunoreactivity was restricted to gastric ECL cell, intestinal EC cell, and pancreatic growths. VMAT-2 immunoreactivity was cy- toplasmic and granular, and varied in intensity in differ- ent tumor cell types. Of 16 well-differentiated gastric ECL cell tumors, 15 were intensely positive in the large majority of tumor cells (Fig. 1A, B). One type-III ECL cell tumor showed only about 30% of tumor cells posi- tive for VMAT2. This tumor previously proved to be moderately differentiated (G2) on histological and histo- chemical grounds [21]. VMAT2-positive EC cells ranged from 1% to 10% in tumors (Fig. 2A), one case display- ing up to 30% of positive cells. In pancreatic tumors, VMAT2-positive cells ranged from 1% to 30% and ap- peared to be mainly concentrated in areas rich in pancre- atic polypeptide-producing (PP) or glucagon-producing
4 Fig. 2 A Isolated vesicular monoamine transporter 2 (VMAT2) immunoreactive tumor cells in an argentaffin enterochromaffin (EC) cells tumor of the small intestine. B Absence of VMAT2 im- munoreactivity in a poorly differentiated (neuro)endocrine carci- noma of the stomach. Note the structure made by solid aggregates with central necrosis. C VMAT2 (top) and glucagon (below) im- munoreactivities in consecutive sections of a nonfunctioning tu- mor of the pancreas rich in glucagon-producing A and PP cells. Note the diffuse and even distribution of glucagon immunoreactiv- ity - only a fraction of glucagon-positive cells appear to colocalize VMAT2. D One single, VMAT2-immunoreactive pancreatic duct cell (arrowhead, top left) colocalizes PP-immunoreactivity (ar- rowhead, below left) in consecutive sections. Note that some PP- immunoreactive cells are negative for VMAT2; VMAT2 positive cells (top, right) colocalizing insulin-immunoreactivity (below, right) in consecutive sections: the arrowheads indicate one area rich in cells colocalizing both antigens. E, F Strong VMAT2 im- munoreactivity in non-chromaffin cells (non-CC) of a carotid body paraganglioma (E) and in chromaffin cells (CC) of a pheo- chromocytoma (F). A-D Immunoperoxidase, ABC method
(A) cells in consecutive sections, although usually corre- sponding to a fraction only of the latter cells (Fig. 2C and Table 1). Weak immunostaining of some cells in in- sulinomas was also observed. Adrenal and extra-adrenal paragangliomas showed intense VMAT2 immunoreactiv- ity accounting for 40-80% of tumor cells (Fig. 2E, F). No VMAT2 immunoreactivity was detected in gastrin-, somatostatin- (Fig. 1D) or enteroglucagon/peptide YY- producing gut endocrine tumors, in non-argentaffin bronchial neuroendocrine tumors, in gastric PDECs (Fig. 2B), in parathyroid and adrenocortical tumors.
Scattered VMAT2-immunoreactive cells resembling ECL cells in morphology and distribution were found in the acidopeptic, non-tumor mucosa of several gastric samples. Intense VMAT2 reactivity was also observed in the hyperplastic and/or precarcinoid (dysplastic) ECL cell changes associated with ECL cell tumors in samples from hypergastrinemic patients with diffuse A-CAG or ZES-MEN-1 syndrome (Fig. 1C). No VMAT2-positive cells were found in the pyloric or intestinal mucosa, apart from rare, weakly reactive cells observed in occa- sional specimens of the small intestine (not shown). In serial sections of non-tumor pancreas, VMAT2 immuno- reactivity was colocalized in PP cells, either in islets, es- pecially of the posterior part of the head (PP-rich islets), or scattered in the exocrine parenchyma (Fig. 2D), in glucagon cells (not shown), and in a subpopulation of in- sulin-producing (B) cells (Fig. 2D). No significant colo- calization was appreciated in somatostatin-producing D cells.
VMAT2-immunoreactive neurons and/or nerves were observed in the gut wall and in the adrenal capsule. Some VMAT2-reactive mononuclear cells scattered in tumor and non-tumor connective tissue were interpreted as mast cells. VMAT2 immunoreactivity was consider- ably influenced by histology procedures. In general, Bouin-fixed tissues gave more intense and reproducible reactions than formalin-fixed ones. Microwave treatment was necessary in most cases, especially with Bouin or prolonged formalin fixation; however, poor staining was observed in samples after short formalin fixation.
Discussion
The above findings outline the specificity of the hista- mine transporter VMAT2 as a marker of gastric ECL cell tumors and related hyperplastic or dysplastic lesions. Ex- tensive and intense VMAT2 immunoreactivity was de- tected in all clinicopathological subtypes of well-differ- entiated ECL cell tumors. These include sporadic type- III cases, usually not associated with hypergastrinemia and sometimes showing clinical evidence of histamine production [1, 18], as well as those associated with hy- pergastrinemia, either due to diffuse chronic atrophic gastritis of the corpus-fundus mucosa (type-I cases) or to combined MEN/ZES (type-II cases), usually lacking signs of histamine hypersecretion and the histamine syn- drome [2, 3, 8, 20, 27]. In addition, most cells forming
the hyperplastic-dysplastic growths associated with type- I and type-II ECL cell tumors were intensely VMAT2 positive.
A variable, usually minor population of moderately VMAT2-reactive cells was observed in 12 of 20 seroto- nin-producing, EC cell tumors of the distal small intes- tine and no reactivity in the single duodenal EC cell tumor investigated (Fig. 2A). This finding may be relat- ed to the property of occasional catecholamine produc- tion reported for EC cell tumors [15], in addition to their regular and extensive serotonin production, and may contribute to the relative heterogeneity of EC tumor cells previously appreciated on histochemical grounds [31]. The vast majority of EC tumor cells were, however, VMAT2 negative, in keeping with the lack of VMAT2 in normal human EC cells, known to express VMAT1 only [13]. In addition, no VMAT2 was observed in intestinal, well-differentiated, peptide hormone-producing tumors such as gastroduodenal gastrin and somatostatin cell tu- mors or colorectal enteroglucagon/peptideYY cell tu- mors. The extensive and intense VMAT2 expression ob- served in well-differentiated gastric ECL cell tumors rel- ative to the substantial lack of VMAT2 in other endo- crine gut tumors supports the specificity of VMAT2 ex- pression for ECL cell tumors. Considering that the endo- crine cell population of the human stomach is composed by a variety of cell types, including, besides ECL cells, G, D, EC, X (A-like) and P cells [23, 26], all of which may also occur in tumors [20, 25], our results support the usefulness of VMAT2 as a tool for the differential di- agnosis of gastric endocrine tumors. Indeed, none of the above cell types, apart from ECL cells, and none of the tumors arising in the gastroduodenal area, except for ECL cell tumors, have been found to react with VMAT2 antibodies.
A limited proportion of VMAT2 positive cells were observed in the pancreatic endocrine tumors investigat- ed, mainly concentrated in areas rich in PP or A cells in consecutive sections (Fig. 2C), although only a fraction of these cells were involved. Weak VMAT2 immunore- activity was also observed in limited subpopulations of insulinomas (Table 1). In addition, VMAT2 expression was localized in PP, A, and B islet cells of non-tumor pancreatic samples. These findings are in accordance with the previously reported presence of uncharacterized reactive cells in normal human pancreas [13] and indi- cate that some A, B, and PP islet cells may indeed ac- tively transport amines, as repeatedly shown in islet cells of mammals injected with amine precursors [24]. Wheth- er such cells in man do normally store monoamines as they do in other species and what the physiological sig- nificance of such a feature is remain to be elucidated.
In keeping with VMAT2 demonstration in all chro- maffin cells of human adrenal medulla and the extraction of VMAT2 mRNA from human pheochromocytomas [13], we detected widespread VMAT2 immunoreactivity in all cases of the adrenal pheochromocytomas investi- gated. In addition, we immunolocalized VMAT2 in ex- tra-adrenal paragangliomas, either of the retroperitoneal
chromaffin type or of the carotid body non-chromaffin type. Both kinds of tumors are known to produce cate- cholamines [11, 19]. Despite the widespread pattern of VMAT2 reactivity, these tumors should not interfere with the histopathological characterization of ECL cell tumors unless at metastatic sites.
No VMAT2 immunoreactivity was observed in the pure gastric PDECs investigated. At the ultrastructural level, gastric PDEC cells show evident small synaptic- like vesicles with rare, immature or abortive endocrine granules, and retain, at light microscopy, neuroendocrine antigens of the cytosol (e.g., NSE and PGP9.5) or synap- tic-like vesicles (synaptophysin), while lacking almost completely markers of the secretory granule, such as chromogranin-A and hormones [20, 21]. Therefore, the lack of VMAT2 immunoreactivity in PDECs reflects the poorly differentiated status and the poor secretory gran- ule content of their cells.
In conclusion, the data here presented support the usefulness of VMAT2 to separate gastric ECL cell tu- mors from other types of gastrointestinal endocrine tu- mors on routine histology specimens. In particular, VMAT2 immunohistochemistry adds a specific positive marker to the general endocrine markers such as chro- mogranin A, synaptophysin, or silver stains and to the specific but negative peptide hormone tests so far uti- lized. Although, as shown in previous studies, minor populations of gastrin-, somatostatin-, or pancreatic polypeptide-producing cells may be found in tumors mostly composed of ECL cells [3, 20], our study con- firms that the large majority of gastric endocrine tumors is made by ECL cells. The detection of an overwhelming population of VMAT2-positive cells allows us to easily ascertain the ECL cell nature of such tumors - even in the absence of electron microscopy - and to separate them from other gastrointestinal tumors such as the VMAT2-negative, gastrin, somatostatin, and enteroglu- cagon cell tumors or the poorly differentiated, highly malignant (neuro)endocrine carcinomas. Previous inves- tigations show absence of VMAT2 immunoreactivity in the rare gastric EC cell tumors [18]. Separation of gastric ECL cell tumors from EC cell tumors of the distal intes- tine and pancreatic endocrine tumors is facilitated, be- sides by their different topography and specific hormone contents, by the focal pattern of VMAT2 immunoreactiv- ity of the latter tumors relative to the widespread pattern of ECL cell tumors.
Acknowledgements This study was supported by grants from the Italian Ministry of Health to IRCCS Policlinico San Matteo, from MURST and University of Brescia and Pavia to GR and ES.
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