Immunocytochemical characterization of 10 pancreatic tumours, associated with the glucagonoma syndrome, using antibodies to separate regions of the pro-glucagon molecule and other neuroendocrine markers

Histological diagnosis of neuroendocrine tumours can be hampered by their lack of peptide or amine immunoreactivity. In order to assess the usefulness of a range of specific and general markers of neuroendocrine differentiation, 10 pancreatic endocrine tumours, associated with high levels of circulating glucagon, were studied using histology, histochemistry, immunocytochemistry and electron microscopy. All cases showed immunoreactivity for one or other of the peptides derived from pro-glucagon, although only seven were found to contain immunoreactive pancreatic glucagon. The presence of secretory granules in eight of the tumours was demonstrated by electron microscopy, argyrophilia or chromogranin immunoreactivity. Not only was neuron specific enolase positively immunostained in all the tumours, thereby revealing their neuroendocrine nature, but also the intensity of the immunostain was higher in four of the five malignant ones than in the rest of the cases. Pancreatic polypeptide was present in non-glucagon cells in six out of 10 cases. Our results emphasize the importance of the use, not only of general histochemical and immunocytochemical tests but also antibodies to all possible derivatives of the precursor form of the active tumour product in the diagnosis of possible endocrine tumours. In this way, any abnormal molecular forms of the peptide synthesized by tumour cells with altered synthetic and secretory mechanisms may be detected.     

Immunohistochemical localization of acidic fibroblast growth factor in normal human enterochromaffin cells and related gastrointestinal tumours

Acidic fibroblast growth factor (aFGF) is a member of the structurally related heparin-binding growth factor family. The best studied members of this family are aFGF and basic FGF (bFGF), which are potent mitogens and differentiation factors for mesodermderived cells, including fibroblasts. This study was designed to verify the immunohistochemical expression of aFGF in normal human endocrine cells of the gut and in related endocrine tumours. We examined normal gastrointestinal mucosa from seven different subjects and 41 gut endocrine tumours from different sites, including stomach, duodenum, and small and large intestine, using an aFGF polyclonal antibody with no cross-reactivity for bFGF. We localized aFGF in a fraction of serotonin-producing enterochromaffin (EC) cells of the normal gut, while it was absent in gastrin (G), CCK, secretin (S), somatostatin (D) and glicentin (L) cells. aFGF immunoreactivity was also expressed in serotonin producing EC cell tumours, but not in other functional types of gut endocrine neoplasms investigated, including gastric ECL cell, duodenal somatostatin and gastrin cell, and rectal L cell tumours. A positive correlation was found between expression of aFGF and the amount of tumour fibrous stroma, suggesting that aFGF may be involved in proliferation and activity of stromal fibroblasts.     

Immunocytochemical demonstration of growth hormone-releasing factor in gastrointestinal and pancreatic endocrine tumors

Growth hormone-releasing factor (GRF), a linear peptide that exists in a number of different molecular forms (GRF-44, -40, -37, and-31) has been shown to be responsible for the acromegaly associated with certain endocrine tumors of the pancreas and other foregut-derived structures. With the use of two anti-sera (#1A850 and G59/901) directed against different segments of the GRF molecule, a series of 24 pancreatic and 35 gastrointestinal endocrine tumors, not associated with acromegaly, were surveyed systematically for immunocytochemical localization of GRF in the tumor cells. Strong immunoreactivity for GRF was encountered in 10 tumors (6 pancreatic and 4 gastrointestinal). While all ten tumors were immunoreactive against G59/901, which recognizes GRF-44, -40, and -37, two jejunal carcinoids showed additional immunostaining with 1A850 that is specific for GRF-44. Seven of these ten tumors were also immunoreactive for a variety of other regulatory peptides and neurotransmitters, including gastrin, insulin, glucagon, serotonin, substance P, somatostatin, pancreatic polypeptide, vasoactive intestinal peptide (VIP), and adrenocorticotropic hormone (ACTH). No consistent pattern of association between GRF and the other regulatory substances was evident. These findings indicate that, even in the absence of associated acromegaly, up to 17% of endocrine tumors of the gastro-entero-pancreatic (GEP) axis show immunoreactivity for GRF and that such reactivity is associated more frequently with pancreatic (25%) than with gastrointestinal (11%) endocrine tumors.     

鹅胃肠内分泌细胞的免疫组织化学定位

应用６种胃肠激素抗血清，对鹅胃肠各段的内分泌细胞分布进行了免疫组织化学定位。腺胃显示有较多促胃泌素释放肽和生长抑素细胞。肌胃有较多促胃泌素释放肽、生长抑素和胃泌素细胞，少量胰多肽细胞。幽门部有大量的生长抑素细胞，密集的胃泌素细胞，偶见胰多肽细胞。小肠内有胃泌素、胰多肽和生长抑素细胞，细胞类型和数量由前段向后段逐渐减少。未检出胃动素和抑胃肽细胞。     

Differential expression of vesicular monoamine transporter (VMAT) 1 and 2 in gastrointestinal endocrine tumours.

Neuroendocrine tumours are characterized by their capacity to produce hormones, which are stored in vesicles and secretory granules. Demonstration of granule/vesicle proteins in tumours is taken as evidence of neuroendocrine differentiation. Vesicular monoamine transporters (VMAT1 and VMAT2) mediate the transport of amines into vesicles of neurons and endocrine cells. The expression of VMAT1 and VMAT2 and the usefulness of VMAT1 and VMAT2 in the histopathological diagnosis of gastrointestinal endocrine tumours have not been fully explored. This study therefore investigated the expression of VMAT1 and VMAT2 in 211 human gastrointestinal tumours by immunocytochemistry and western blotting. VMAT1 and/or VMAT2 were demonstrated in the majority of amine-producing endocrine tumours of gastric, ileal, and appendiceal origin. Serotonin-producing endocrine tumours (ileal and appendiceal carcinoids) expressed predominantly VMAT1, while histamine-producing endocrine tumours (gastric carcinoids) expressed VMAT2 almost exclusively. In peptide-producing endocrine tumours such as rectal carcinoids and endocrine pancreatic tumours, only a small number of immunopositive tumour cells were observed. No labelling was found in non-endocrine tumours, including gastric, colorectal and pancreatic adenocarcinomas and gastrointestinal stromal tumours. In conclusion, VMAT1 and VMAT2 are differentially expressed by gastrointestinal endocrine tumours, with a pattern specific for each tumour type, reflecting their neuroendocrine differentiation and origin. VMAT1 and VMAT2 may therefore become valuable markers in the classification of neuroendocrine tumours and may also indicate patients suitable for radioisotope treatment operating via these transporter systems.     

Immunohistochemical localization of factor X-like antigen in pancreatic islets and their tumours

Summary The authors have investigated by immunohistochemistry the distribution of factor X-like antigen in normal pancreatic islets and in a series of 46 pancreatic endocrine tumours. It was found that both glucagon-producing (A) cells and pancreatic polypeptide-producing (PP) cells are immunoreactive for the antigen. Benign glucagonomas and PP-omas presented the highest concentrations of immunoreactive material whose intracellular distribution was consistent with localization within cell secretory granules. Some benign insulinomas also presented factor X immunostaining in spite of the absence of the antigen in normal insulin-producing B cells. Although malignant tumours usually exhibited very low or no immunostaining, two of three malignant glucagonomas showed scattered, intensely immunoreactive cells. The factor X-like antigen identified in this study was found to differ from chromogranin A and B. The possible implications of the present findings for coagulative disorders associated with glucagonomas or diabetes are discussed.     

Pancreatic endoproteases and pancreatic secretory trypsin inhibitor immunoreactivity in human Paneth cells.

Normal and metaplastic gastrointestinal mucosa obtained at surgical resection were studied by light microscopy, using the unlabelled antibody enzyme method for immunohistochemical staining of lysozyme, pancreatic endoproteases, and pancreatic secretory trypsin inhibitor (PSTI). Paneth cells in the mucosa of normal small intestine, gastric mucosa with intestinal metaplasia, and colonic metaplastic mucosa were found to contain anionic trypsin, cationic trypsin, lysozyme, and PSTI immunoreactivity, but not chymotrypsin and elastase immunoreactivity. Normal gastric and colonic mucosa and some goblet cells in the small intestine showed positive PSTI immunoreactivity but no endoprotease immunoreactivity. The presence of immunoreactive trypsin and immunoreactive PSTI in the Paneth cells, which are of secretory type, probably indicates an important extrapancreatic source of these proteins rather than a storage of endocytosed material.     

Endocrine differentiation of extra-pulmonary small cell carcinoma demonstrated by immunohistochemistry using antibodies to PGP 9.5, neuron-specific enolase and the C-flanking peptide of human pro-bombesin

Several recent studies have confirmed the endocrine nature of small cell carcinoma of the lung. In extra-pulmonary sites, small cell 'undifferentiated' carcinomas have classical morphological features similar to their pulmonary counterpart. We therefore investigated, using immunocytochemistry, the possibility that the non-pulmonary neoplasms may also be endocrine in nature. Sections of 29 small cell carcinomas from oesophagus, stomach, larynx, colon and urinary bladder were immunostained using antisera to protein gene product 9.5 (PGP 9.5), neuron-specific enolase (NSE), cytokeratin, leucocyte common antigen and peptides including bombesin, the C-flanking peptide of human probombesin, adrenocorticotrophic hormone, neurotensin, calcitonin and pancreatic polypeptide. All the tumours showed immunoreactivity for at least one of the two general endocrine markers PGP 9.5 and NSE. Twenty-three of the 29 cases were immunoreactive for PGP 9.5, 27 for NSE. All were positive for cytokeratin and negative for leucocyte common antigen. Of the regulatory peptides, immunoreactivity was obtained with antisera to bombesin (one case), the C-flanking peptide of human pro-bombesin (14 cases), adrenocorticotrophic hormone (one case) and calcitonin (three cases). No PGP 9.5-, NSE- or peptide-like immunoreactivity was detected in 25 control tumours from similar sites, including lymphomas and poorly differentiated tumours. These results suggest that non-pulmonary small cell carcinoma has an endocrine character.     

Clusterin is highly expressed in pancreatic endocrine tumours but not in solid pseudopapillary tumours

AIMS: Clusterin is a sulphated glycoprotein, implicated in many processes, including tumorigenesis. Several studies have reported its overexpression in many human neoplasms, including prostatic and pancreatic adenocarcinoma, but its expression has not been described previously in other pancreatic tumours. Our aim was to investigate the expression of clusterin by immunohistochemistry in 30 endocrine pancreatic tumours (ENTs) and 22 solid pseudopapillary tumours (SPPTs) to document its potential in differential diagnosis, and the possible correlation between this expression and clinicopathological parameters. METHODS AND RESULTS: Cytoplasmic positivity was scored qualitatively (weak, moderate or strong immunoreactivity) and quantitatively on a four-tiered scale. The pattern of immunoreactivity (cytoplasmic, secretory or Golgi pattern) was also assessed. Except for scattered tumour cells in five cases, all SPPTs were negative, while all ENTs showed strong immunoreactivity in a variable proportion of tumour cells. Neither the reactivity score nor the pattern of immunoreactivity was correlated with tumour size, vascular permeation, perineural invasion or lymph node metastasis. DISCUSSION: The expression of clusterin in all ENTs is of interest and could be an additional useful marker in the differential diagnosis with SPPTs. However, the lack of correlation between clusterin expression and clinicopathological parameters rules out a role as a predictive marker for endocrine tumour aggressiveness.     

Synaptic vesicle protein 2, A new neuroendocrine cell marker

Synaptic vesicle protein 2 (SV2) is a glycoprotein identified in the nervous system of several species, including man, but its occurrence in the human neuroendocrine (NE) cell system has not been investigated. By using a monoclonal antibody to SV2, immunoreactivities were demonstrated in NE cell types in human gastrointestinal tract, pancreas, anterior pituitary gland, thyroid, parathyroid, and adrenal medulla, and also in chief cells of gastric oxyntic mucosa. Immunoelectron microscopy of pancreatic islets revealed SV2 immunoreactivity in secretory granules. Comparison of SV2, synaptophysin, and chromogranin A immunoreactivity showed more SV2- and synaptophysin- than chromogranin A-immunoreactive cells in the antrum and pancreas. In the other gastrointestinal regions and in the other endocrine organs more SV2- than synaptophysin-immunoreactive cells were seen. More chromogranin A- than SV2-immunoreactive cells were observed in duodenum, colon, and parathyroid. Various NE tumors were examined and all contained SV2-immunoreactive cells. The staining patterns with the three markers agreed well, except in hindgut carcinoids, which showed strong SV2 immunoreactivity, weak synaptophysin but no chromogranin A immunostaining. In pituitary adenomas more cells were immunoreactive to SV2 than to the other two antibodies. In conclusion, SV2 is recognized as a further broad marker for NE cells and widens the arsenal of diagnostic tools for NE tumors. It is of special importance for identifying hindgut carcinoids.     

Cyclin-dependent kinase (cdk6) and p16 in pancreatic endocrine neoplasms

AIMS: p16 and p27, the inhibitors of cyclin-dependent kinases, have been reportedly decreased in certain human tumours, including a few endocrine tumours. The current study used immunocytochemical staining to compare the staining intensity of cdk6 and its inhibitor, p16, in pancreatic endocrine neoplasms with normal pancreatic islets. METHODS: Twenty-four primary pancreatic endocrine neoplasms, consisting of 12 insulinomas, one glucagomoma, three pancreatic polypeptide (PP)-omas, five gastgrinomas and three non-fuctioning tumours, were immunocytochemically studied for cdk6 and p16 compared with the adjacent non-neoplastic islets. RESULTS: In the normal islets, cdk6 staining was strongly positive for islet cell nuclei and cytoplasms, whereas p16 was strongly positively stained for islet cell cytoplasms. Insulinomas, glucagonoma, PP-omas and non-functioning tumours were weakly stained for cdk6 and p16. Among five gastrinomas, three tumours were moderately stained and two tumours were more weakly stained for cdk6 and p16. Thus, tumour cells were weaker stained for cdk6 and p16 compared with the strong staining of normal islet cells. No distinct immunostaining difference was observed among five kinds of pancreatic endocrine neoplasms. CONCLUSIONS: The decreased immunocytochemical staining for cdk6 and p16 is consistently observed in five kinds of pancreatic endocrine neoplasms. This decreased cdk6 and p16 in pancreatic endocrine neoplasms may be a part of the cell cycle event in tumour transformation and progression, and the same process may involve other endocrine tumours.     

Islet amyloid polypeptide in proliferating pancreatic B cells during development, hyperplasia, and neoplasia in humans and mice.

The occurrence of islet amyloid polypeptide (IAPP) immunoreactivity was investigated in fetal pancreas, islet cell hyperplasia, and tumors in humans and mice. Transgenic mice heritably developing endocrine tumors of the pancreas (AVP/SV40, Rip 1 Tag2/Rip2PyST1 and Glu2-Tag strains) were used as murine models of islet cells proliferative disease. In the mouse, IAPP immunoreactivity was found in B cells at embryonic day 12 (E12), paralleling the onset of insulin immunoreactivity. In hyperplastic/dysplastic islets and in B-cell tumors of transgenic mice (n = 16), IAPP immunoreactivity was localized consistently to insulin-immunoreactive cells. Ultrastructural single- and double-immunogold labeling of transgenic mice B-cell tumors (n = 3) showed insulin and IAPP to be colocalized in beta granules. In human fetuses, IAPP immunoreactivity was found in insulin-immunoreactive B cells, but at a later gestational age than the onset of insulin immunoreactivity. In pancreatic specimens of infantile/neonatal persistent hyperinsulinemic hypoglycemia (11 cases) and in pancreatic endocrine tumors (21 cases, 10 of which were functioning insulinomas), IAPP immunoreactivity was found consistently in insulin-immunoreactive B cells. Congo-red-positive amyloid deposits present in tumors also were IAPP immunoreactive. Ultrastructural single and double immunogold labeling of infantile/neonatal persistent hyperinsulinemic hypoglycemia cases (n = 3) and functioning insulinomas (n = 2) showed IAPP and insulin to be colocalized in beta granules. In addition, IAPP immunoreactivity was observed in amyloidlike fibrils. These findings indicate that IAPP is a constitutive component of B cells. Possible relationships between IAPP and insulin expression and interspecies differences are suggested and discussed.     

Development of colonic and pancreatic endocrine tumours in mice expressing a glucagon-SV40 T antigen transgene

We report the histological, immunohistochemical and ultrastructural changes in mice containing a chimeric glucagon-simian virus 40 T antigen (SV40Tag) gene. Transgene expression was detected in endocrine cells of pancreas, small and large intestine. Hyperplasia of glucagon-containing cells developed in pancreas and large bowel by gestational day 19. In large bowel, hyperplastic cells increased in number postnatally and invasive carcinomas were identified at 4 weeks; several animals had lymph node metastases. In contrast, no pathology was detected in the small bowel in any of the transgenic mice. Colonic tumours expressed SV40Tag, proglucagon-derived peptides and peptide YY (PYY); scattered cells contained cholecystokinin or glycoprotein hormone -subunit. Somatostatin or serotonin was also detected in some tumours. By electron microscopy, the colonic tumours retained features of endocrine differentiation, but secretory granules were smaller than those of non-tumorous intestinal glucagon-producing L cells. In postnatal pancreas, atypical cells containing SV40Tag and glucagon were initially clustered at the periphery of islets; this atypical hyperplasia progressed to neoplasia by 11–12 weeks. Some neoplastic pancreatic cells contained glucagon, PYY or vasoactive intestinal peptide immunopositivity, but most were negative for all peptides; they contained immunoreactivity for tyrosine hydroxylase and by electron microscopy, pancreatic tumour cells had neuronal features. Pancreatic polypeptide was not detected in the non-tumorous islets of transgenic animals. This line of transgenic mice provides a model for the analysis of endocrine tumour progression in the gut and pancreas.     

Glucagon-, glicentin-, and pancreatic polypeptide-like immunoreativities in rectal carcinoids and related colorectal cells.

Three nonargentaffin rectal carcinoids have been investigated immunohistochemically. In one case most tumor cells reacted with antiglucagon sera as well as with antiglicentin, antibovine pancreatic polypeptide (BPP), and antihuman pancreatic polypeptide (HPP) sera; they were identified ultrastructurally as L cells. Another case showed glucagon-, glicentin-, and BPP-immunoreactive cells but lacked HPP immunoreactivity. In the third case glucagon- and glicentin-immunoreactive cells were well represented, while PP immunoreactivities were scarce. Parallel investigations of human rectal and sigmoid mucosa showed numerous cells reacting with glucagon, glicentin, and BPP antisera, most of which lacked HPP immunoreactivity. Cells reacting with glucagon and glicentin antisera, while lacking PP immunoreactivities, were also found. Thus, both tumor and nontumor cells produce glucagonlike immunoreactive (GLI) peptides--one of which may be glicentin or a related molecule--as well as PP-related sequences, although differing histochemically and ultrastructurally from glucagon or PP cells of the human pancreas. It is concluded that nonargentaffin rectal carcinoids are histogenetically linked to nonargentaffin endocrine cells of the human rectum.     

The occurrence and clinicopathological significance of serotonin immunoreactive cells in large bowel carcinoma

We studied the incidence, clinicopathological relevance and prognostic significance of serotonin immunoreactive cells in a clinically well-documented series of 300 large bowel cancer patients. Serotonin immunoreactive cells were detected in 8 per cent of the carcinomas, occurring either as focal clusters (4.7 per cent) or as occasional single cells (3.3 per cent). Both types frequently displayed presence of mucin and/or immunoreactivity for secretory component as well. In respect of localization, stage or tumour extension and histological grade tumours with serotonin immunoreactivity showed no significant differences in comparison with carcinomas lacking this feature. In terms of survival, however, the tumours with serotonin immunoreactivity demonstrated a more aggressive clinical course in comparison with tumours without these cells. This phenomenon reached borderline statistical significance. It is therefore concluded that the study of serotonin immunoreactivity in large bowel carcinomas enables the identification of a subpopulation of colorectal carcinomas with a relatively poor prognosis.     

Immunocytochemical studies on the islet and the gut of the arctic lamprey, Lampetra japonica

The endocrine cells and nerves in the islet and the gut of the arctic lamprey Lampetra japonica were examined immunocytochemically by using antisera against brain-gut peptides and amine. The cellular composition of the islets as reported by previous researchers in European species of the lamprey was confirmed in the present study. The islet consisted exclusively of insulin immunoreactive cells in the larvae (ammocoetes), whereas in the adult somatostatin immunoreactive cells were added to the insulin immunoreactive cells; the gut epithelium in the adult was now devoid of somatostatin cells. In the gut of the lamprey, the endocrine cells--which were flask-shaped with a cytoplasmic process extended to the lumen--were classified into three types in the larvae, but were represented by a single type in the adult. In the larval lamprey, the first type was immunoreactive for somatostatin, the second one for gastrin/cholecystokinin (CCK) and the third cell type was immunoreactive for glucagon, pancreatic polypeptide and FMRFamide, simultaneously. In the gut of the adult lamprey, the single type of endocrine cell reacted simultaneously to C-terminal specific anti-glucagon serum, N-terminal specific anti-glucagon serum, anti-bovine PP serum, anti-neuropeptide Y serum and anti-FMRFamide serum. These cells occurred most frequently in the upper intestine, their distribution decreasing from the middle to the lower intestine. Two types of peptide containing nerves were identified in the islet and the gut of the larval and adult lamprey. The first type of neurons (perikarya and fibers) was immunoreactive for serotonin and calcitonin gene-related peptide (CGRP), and was located in the mucous and muscular layer of the intestine and in the islet. The second type of neurons contained both serotonin- and gastrin releasing peptide (GRP)-like immunoreactivities and was scattered exclusively in the muscular layer of the gut. In larval and adult lampreys, a few serotonin/CGRP immunoreactive nerve cell bodies and beaded fibers were found in the connective tissue around the islet cell cords. These nerve fibers were sometimes closely apposed to the blood capillaries and to the islet cells. These findings indicate that a neuroendocrine correlation comparable with that in mammals may have been established in the islet of this most primitive vertebrate.     

The prevalence and clinical significance of chromogranin A and secretogranin II immunoreactivity in colorectal adenocarcinomas

Colorectal adenocarcinomas may display features of endocrine differentiation, shown by argyrophil stains and by the expression of endocrine markers such as chromogranin A. We investigated chromogranin A and secretogranin II immunoreactivity in a series of 208 carcinomas of the large bowel to assess the prevalence and clinical significance of endocrine differentiation. Tumours expressing endocrine markers were classified as low expressors (< than 1 immunoreactive tumour cell/mm²) and high expressors (> than 1 immunoreactive tumour cell/mm²). There were 33 (16%) carcinomas showing both chromogranin A and secretogranin II immunoreactivity: 11 tumours (5%) were high expressors. Endocrine differentiation was not related to the disease stage, tumour location, grade, DNA ploidy and p53 protein accumulation. In the entire series chromogranin A immunoreactivity did not provide prognostic information using univariate and multivariate analysis. A worse overall survival (P=0.048) was demonstrated for the stage III patients with high expressor tumours, but there were only five patients in this group. The results of our investigation suggest that chromogranin A immunoreactivity is not a useful variable in the prognostic assessment of colorectal adenocarcinomas.     

Insulin-like growth factor-II in endocrine pancreatic tumours. Immunohistochemical, biochemical and in situ hybridization findings

In earlier studies a high-molecular-weight (HMW) insulin-like growth factor-II (IGF-II) peptide was identified in adult human pancreas and localized to the insulin-producing B-cells. This peptide has now been investigated in neoplastic insulin cells. Forty endocrine pancreatic tumours and 17 pancreatic adenocarcinomas of ductal type were included in the study. All cases were investigated with immunohistochemical techniques using antibodies to IGF-II, insulin, pro-insulin, glucagon, somatostatin, pancreatic polypeptide, gastrin and vasoactive intestinal peptide (VIP). Frozen tissue from nine tumours and two normal pancreatic glands was extracted, gel separated, and quantified using radioimmunoassay. The tumours were also investigated by in situ hybridization. IGF-II-immunoreactive cells were found in nearly all the 18 insulin-producing tumours (16/18), in a minority of the other endocrine tumours, but not in pancreatic adenocarcinomas. All extracts from the endocrine tumours showed varying amounts of IGF-II and had different molecular-weight forms. The immunohistochemical and radioimmunoassay findings are both based on immunological binding and were further confirmed by Northern blot and in situ hybridization. These results show that IGF-II is expressed in insulin-producing tumours as well as in pancreatic tumours producing other peptides, in contrast to normal pancreatic islets where IGF-II is found exclusively in insulin-producing cells.     

Prevalence of CD99 protein expression in pancreatic endocrine tumours (PETs)

AIMS: To determine the prevalence of CD99 expression in pancreatic endocrine tumours (PETs). We evaluated CD99 expression and analysed Ki67 labelling by immunohistochemistry in PETs. METHODS AND RESULTS: Thirty-eight PETs from 33 patients were analysed. CD99 immunoreactivity was consistently observed in normal islets of the pancreas, regardless of the cell type. Tumours comprising more than 30% CD99+ cells were defined as positively immunoreactive for CD99. CD99 expression was observed in 20 of the 38 PETs examined, but not in any of the pancreatic tumours of other histological subtypes (10 ductal adenocarcinomas, five intraductal papillary-mucinous tumours, and two acinar cell tumours). Loss of CD99 expression was related to markers of worse prognosis for PET, including gross local invasion, metastasis to the lymph nodes or other organs, lymphatic or blood vessel invasion, and neuroendocrine carcinoma (NEC). Thus, CD99 expression may have an efficiency comparable to that of high Ki67 labelling index (5% or more) for prognostication. CONCLUSIONS: CD99 expression was observed frequently and exclusively in PETs, and loss of CD99 expression in PETs was found to be associated with ominous prognostic indicators.