PGP 9.5, a new marker for human neuroendocrine tumours

PGP 9.5 is a soluble protein isolated from brain and is a general marker for neuronal and neuroendocrine tissue. Its function is not known. Until now neurone specific enolase (NSE) has been the only general marker for the paracrine system and tumours derived from it. Seventy-four neuroendocrine tumours, 17 melanocytic naevi, 51 melanomas and four granular tumours were stained immunohistochemically for PGP 9.5 and NSE. A variety of pulmonary and non-neuroendocrine tumours were also stained. Two so-called goblet cell carcinoids of the appendix were included in the series. Using NSE 59/74 neuroendocrine tumours were positive and 58/74 stained for PGP 9.5. In combination 63/74 of these tumours were positive for either NSE or PGP 9.5 or both. Staining for PGP 9.5 was better for demonstration of nerves in routinely processed material than was staining for NSE. Twenty-one out of 43 primary melanomas stained for PGP 9.5 and 36 showed staining for NSE. Only two of eight metastatic melanomas melanocytic stained for PGP 9.5 while seven of these eight stained for NSE. Six of 17 melanocytic naevi stained for PGP 9.5 and five stained for NSE. All four granular cell tumours stained for PGP 9.5 and NSE. Both "goblet cell carcinoids' of the appendix were negative for NSE and PGP 9.5. Fifteen out of 32 pulmonary cancers showed staining for either marker and no non endocrine tumour showed any specific staining. Staining for PGP 9.5 is a valuable additional probe in the exploration of the paracrine system and the diagnosis of tumours arising from it.     

Neuroendocrine differentiation in adrenocortical carcinoma. New immunohistochemical findings supported by electron microscopy.

Ten adrenocortical carcinomas including two tumors with clinically detectable corticosteroid production, were immunohistochemically analyzed for their intermediate filament proteins, and for neuroendocrine markers. Keratins were present in 6 of 10, vimentin in all 10, and the 68 kilodalton kD neurofilament subunit protein in 6/10 tumors. Keratins numbers 8 and 18 were most prevalent, whereas only traces of keratins 19 and 7 were found. Eight tumors were positive for synaptophysin at least focally, and 3 showed extensive positivity in more than 30% of tumor cells. The tumors showed approximately similar levels of neuron-specific enolase (NSE) expression as judged by immunohistochemistry. Chromogranin was not detected, and there was no immunoreactivity for 3 neuropeptides (calcitonin, gastrin, somatostatin). In normal adrenal cortex, neuron-specific enolase, synaptophysin and neurofilaments were restricted to the nerves seen between the cortical cells. Electron microscopy revealed clusters of dense-core granules in 4 of 5 tumors, consistent with neuroendocrine granules. These results indicate that adrenocortical carcinomas may show signs of neuroendocrine differentiation and share some features with the adrenal medullary tumors.     

Immunohistochemical positivity for neuron-specific enolase and Leu-7 in malignant mesotheliomas.

The discovery of immunostaining for neuron-specific enolase (NSE) and Leu-7 in a small cell mesothelioma prompted us to study some putative immunohistochemical markers of neuroendocrine differentiation in malignant mesotheliomas and to examine any diagnostically important immunohistochemical distinctions or similarities between malignant mesothelioma and other histologically similar lung tumours. Most mesotheliomas were positive for NSE (96 per cent) and Leu-7 (70 per cent) and positivity for these two markers was also found in small cell carcinomas (NSE 25 per cent, Leu-7 81 per cent) and adenocarcinomas (NSE 28 per cent, Leu-7 28 per cent) but carcinosarcomas were positive for only NSE (44 per cent). Chromogranin A positivity was found only in occasional small cell carcinomas (6 per cent) and adenocarcinomas (6 per cent). No tumour was positive for bombesin. The high incidence of NSE and Leu-7 positivity in mesotheliomas is an important original observation because it guards against the unjustified exclusion of mesothelioma from a differential diagnosis on the basis of positivity for these two markers.     

Immunohistochemical markers of small cell carcinoma and related neuroendocrine tumours of the lung

A selected group of 263 pulmonary neuroendocrine tumours comprised 156 small cell carcinomas, five combined cell carcinomas, nine atypical carcinoid/small cell carcinomas, 32 atypical carcinoids, ten large cell/small cell carcinomas, and 51 carcinoid tumours. These were compared with a group of 109 non-small cell carcinomas, using four markers of neuroendocrine differentiation to determine differences in reactivity between the two groups and among the variants of neuroendocrine tumour. The antibodies used were neuron-specific enolase (NSE), protein gene product (PGP) 9.5, human bombesin, and the C-terminal flanking peptide of human bombesin (CTP). Most small cell carcinomas, carcinoid tumours, and atypical carcinoid variants showed immunoreactivity for both NSE and PGP 9.5 but a significant number of non-small cell carcinomas, mainly squamous cell carcinomas, were also positive (11 and 35 per cent, respectively). Bombesin was specific for neuroendocrine tumours, being demonstrable in 35 per cent carcinoids and 24 per cent small cell carcinomas, but staining was focal and often confined to scattered cells. Diffuse strongly positive immunoreactivity for CTP was seen in the majority of malignant neuroendocrine tumours, but only 12 per cent of carcinoid tumours were positive and non-small cell carcinomas were negative. CTP is therefore of potential value as a specific marker of malignant neuroendocrine tumours, particularly if the amount of biopsy material is limited and the tumour is an unusual variant, such as atypical carcinoid or large cell-small cell carcinoma.     

Staining for factor VIII related antigen and Ulex europaeus agglutinin I (UEA-I) in 230 tumours. An assessment of their specificity for angiosarcoma and Kaposi''s sarcoma

In this study we examined the staining reactivity of commercially available antisera to factor VIII related antigen (F VIII RAg) and Ulex europaeus agglutinin I (UEA-I) on sections from 230 formalin fixed paraffin embedded tumours. These included 196 sarcomas, 20 carcinomas and 14 angiomas. All angiomas showed positive staining for F VIII RAg; all carcinomas showed negative staining; the vasoformative areas of all angiosarcomas stained positively but only four of six angiosarcomas showed positive staining of their solid areas; of seven Kaposi's sarcomas, all showed positive staining of vessels and six showed positive staining of the spindle cell component. In the remaining 181 non-vascular sarcomas there was a false positive result in four tumours (2.2%), three of which had a history of irradiation. Pre-radiotherapy biopsies of these three tumours stained negatively with anti-F VIII RAg. UEA-I was demonstrated in all the angiomas studied, in all angiosarcomas (including the solid components) and in well-formed vessels of all Kaposi's sarcomas, but only in the spindle cell component of 3/6. However, there was an unacceptably high rate of false positive staining amongst the carcinomas and non-vascular sarcomas. In conclusion, F VIII RAg is a specific but not a sensitive marker of angiosarcomas; UEA-I is a sensitive but not a specific marker of angiosarcomas.     

Immunocytochemical evidence for neuroendocrine differentiation in human breast carcinomas

Normal and neoplastic human breast tissues have been stained with antibodies recognizing neuroendrocrine differentiation. Fifteen out of 44 (34 per cent), breast carcinomas stained positively with monoclonal antibody LICRLON-E36, and 11 out of 44 (25 per cent) of these tumours stained with an antibody raised against neuron-specific enolase (NSE). Eight tumours stained positively with both antibodies. No correlation was observed between staining with these antibodies and the tumour histology, nor with the degree of cellular differentiation as indicated by staining with several cell surface directed monoclonal antibodies. Ultrastructural analysis of a series of breast tumours showed the presence of membrane-bound dense-core vesicles in almost all tumours, including E36 and NSE positive and negative tumours. The presence of these structures appears to be of little value in predicting neuroendocrine differentiation.     

Immunostaining of neuron-specific enolase is a valuable aid to the cytological diagnosis of neuroendocrine tumours of the lung

The neuroendocrine marker neuron-specific enolase (NSE) has been reported to be detectable by immunohistochemistry in paraffin sections of neuroendocrine neoplasms of the lung. There is no study of immunodetection of NSE in cytological smear preparations from these tumours. We have examined 10 cases of small cell carcinoma of the lung, using cells obtained from serous fluids or bronchial biopsies, and found all but one had NSE-like immunoreactivity. No such immunoreactivity was found in 26 serous fluids and 19 biopsies from non-small-cell carcinomas. It is suggested, therefore, that immunostaining for NSE is a valuable aid to the cytological diagnosis of small cell carcinoma of the lung.     

Expression of erythropoietin and neuroendocrine markers in clear cell renal cell carcinoma

The aim of the study was to investigate the expression of erythropoietin and neuroendocrine markers in clear cell renal cell carcinoma (CCRCC). We retrospectively reviewed the medical records and re‐evaluated histopathological specimens of 33 patients with CCRCC and compared with those of 11 cases of non‐CCRCC. All patients were treated with a partial or radical nephrectomy at St. Olavs Hospital, Trondheim University Hospital, between 2010 and 2016. Thirty‐three patients who were diagnosed with CCRCC had a total of 35 tumours, where 34 of the tumours were CCRCC and one was papillary adenoma. Thirty‐three (97%) of 34 CCRCCs were positive for erythropoietin, and the same 33 (97%) tumours demonstrated strong expression for neuron‐specific enolase (NSE). Two (6%) of 34 CCRCCs had a positive reaction for synaptophysin, and three (9%) of 34 were positive for CD56. Erythropoietin and NSE were negative in non‐CCRCCs, and chromogranin A was negative in all tumours. The above findings suggest that there is a strong association between CCRCC and the expression of erythropoietin and NSE.     

IMMUNOREACTIVE NEURON-SPECIFIC ENOLASE (NSE) IS EXPRESSED IN TESTICULAR CARCINOMA-IN-SITU

Neuron-specific enolase (NSE) is a well-known marker of tumours that have neuroendocrine origin. High levels of NSE have also been described in various types of testicular germ cell neoplasms, particularly in seminomas. To evaluate the presence of NSE in testicular carcinoma- in situ (CIS), a preinvasive stage of testicular germ cell tumours, a panel of CIS tissue specimens was examined. Fifteen of 18 (83 per cent) CIS samples showed immunohistochemical staining with anti-NSE monoclonal antibody. Immunoreactivity has also been found in overt testicular germ cell tumours, including seminomas, non-seminomas, and a mixed germ cell tumour. As the co-existence of high NSE production and gene amplification of N- myc has been reported in some tumours, including germ cell tumours, the expression of the protein product of N- myc was also examined in this study, but only sporadic cases showed N- myc staining. These results are evidence against a relationship between NSE and N- myc in testicular germ cell tumours. The high expression of NSE in CIS and overt germ cell tumours may be due to the increased gene dosage effect associated with the overrepresentation of isochromosome 12p.     

Immunoexpression of the α subunit of a guanine nucleotide-binding protein (Go) in pulmonary neuroendocrine cells and neoplasms

The α subunit of a GTP-blndlng protein, Go, was investigated in pulmonary neuroendocrine neoplasms and fetal tissues of the lung by an immunohistochemlcal method. Positive immunostaining for the α subunit of Go (Goα) was found predominantly on the cell membrane and found occasionally in the cytoplasm. Typical carcinoids were all positively stained (9/9), and small cell carcinoma showed weaker and less frequent staining (5 positive cases in 10). Atypical carcinoids were variously stained (3/4). The tendency for obvious neuroendocrine differentiation to be immunohistochemically determined in typical carcinoids and not in small cell carcinoma is also true of staining for neuron specific enolase (NSE), chromogranin A (CG-A) and synaptophysin. In the lung, Goα-immunostaining was positive not only in nerve tissues but also in the airway epithelium. In the fetal lung, serial sections immunostained for NSE, CG-A and Goα confirmed that Goα-immunoreactive cells belong to the neuroendocrine cell population. The biological significance of Goα is unclear in normal and neoplastic lung tissues, but Goα is a useful marker of neuroendocrine cells and neoplasms of the lung.     

Immunocytochemical localization of neuron specific enolase in small cell carcinomas and carcinoid tumours of the lung

Carcinoid tumours and small cell carcinomas of the lung share many characteristics with normal neuroendocrine cells. While carcinoid tumours contain many dense-cored neurosecretory granules and are frequently argyrophil, small cell carcinomas are poorly granulated and rarely argyrophil, which casts doubt on their neuroendocrine nature. Immunostaining of the enzyme neuron specific enolase (NSE) was recently used to demonstrate the neuroendocrine components of the lung including nerves and neuroendocrine cells. We therefore used NSE immunostaining to investigate neuroendocrine differentiation in 79 lung tumours, including 18 bronchial carcinoids and 31 small cell carcinomas, and compared these results with those obtained with silver stains. Thirteen of the 18 carcinoids were reactive to silver, all other types being negative. NSE-immunoreactivity occurred in 16 carcinoids and 18 small cell carcinomas. None of the squamous cell carcinomas, large cell anaplastic carcinomas and adenocarcinomas examined showed NSE-immunoreactivity. Radioimmunoassay of extractable NSE from 10 fresh lung tumours correlated well with the immunostaining results, demonstrating large amounts in two small cell carcinomas (334 and 517 ng/mg protein) and three carcinoids (152, 908, and 1143 ng/mg protein). Values were much lower for four squamous cell carcinomas (31-44 ng/mg protein) and one large cell anaplastic carcinoma (30 ng/mg protein) and were accounted for by the presence of NSE-positive nerves and neuroendocrine cells in the surrounding lung. NSE immunostaining is a useful marker of neuroendocrine differentiation in lung tumours and should prove particularly valuable in the diagnosis of small cell anaplastic tumours and their metastases.     

Carcinoids of the gastrointestinal tract: importance of determining differentiation and proliferation markers

The group of 35 carcinoid tumours obtained from 34 patients was reviewed according to recent histopathological criteria. Consequently, evaluation of the Grimelius staining and immunohistochemical detection of chromogranin A (CgA), Leu-7 (CD-57), synaptophysin, neuron-specific enolase (NSE), (beta-III tubulin, Ki-67 and proliferating cell nuclear antigen (PCNA) was performed. The majority of tumours (29, i.e. 83%) were classified as typical carcinoids composed predominantly of mixed solid and trabecular or solid and tubular growth patterns. Six tumours (17%) revealed more prominent cytological abnormalities corresponding with the diagnosis of atypical carcinoid. The majority of tumours (31, i.e. 93.9%) showed granular cytoplasmic positivity in Grimelius staining and diffuse cytoplasmic positivity of NSE (34, i.e. 97.1%). All of the 32 stained tumour samples showed positive immunoreactivity for synaptophysin. A high percentage of tumours (32, i.e. 91.4%) revealed also a positive reaction with antibody TU-20 detecting (beta-III tubulin, a marker of an early stage of neuronal differentiation. Thirty-four tumours (97.1%) showed granular cytoplasmic positivity for both markers of neuroendocrine granules (CgA and Leu-7). One tumour (2.9%) was positive only for Leu-7. Tumour cells revealed predominantly low proliferative activity evaluated by PCNA and Ki-67 immunodetection. Higher degree of proliferation was observed especially in atypical carcinoids.     

Expression of c-erbB-2 protein, neuron-specific enolase and DNA flow cytometry in locally advanced transitional cell carcinoma of the urinary bladder

Expression of c-erbB-2 protein, neuron-specific enolase (NSE) and DNA ploidy was studied by immunohistochemistry and flow cytometry in formalin-fixed and paraffin-embedded specimens from 104 patients with locally advanced transitional cell carcinoma of the bladder. Positive membrane bound c-erbB-2 staining was found in 15% of the tumours, and 38% of the tumours were positive for NSE. Only one tumour stained positively for both NSE and c-erbB-2. Expression of c-erbB-2 protein and NSE was neither correlated to tumour stage nor to histopathological grade. The frequency of non-diploid tumours was 78% in 49 c-erbB-2/NSE negative tumours, 98% in 40 NSE positive tumours, and 100% in 16 c-erbB-2 positive tumours ( P =0.004). Whether the c-erbB-2 expression is a useful prognostic marker in addition to other conventional parameters, remains to be shown.     

Lectin binding sites in developing mouse limb buds

Summary The binding sites of the following biotinylated lectins were demonstrated in serial paraffin sections of fore- and hindlimb buds from day-9 to day-16 mouse embryos with the Avidin-Biotin-Peroxidase Complex (ABC) procedure: Concanavalin A (Con A), Soybean Agglutinin (SBA), Wheat Germ Agglutinin (WGA), Peanut Agglutinin (PNA), Ricinus Communis Agglutinin I (RCA), Ulex Europaeus I Agglutinin (UEA), and Dolichos Biflorus Agglutinin (DBA). Alternating neighbouring sections were used to compare the distribution of PNA staining, PNA staining after neuraminidase treatment (N-PNA) and the autoradiographic sites of [³⁵S]-sulphate uptake. Unspecific binding sites common to all lectins tested were observed in periderm and chondrocytes. Several lectin affinities were seen in the undifferentiated mesoderm (Con A, WGA, RCA), blood vessels (WGA, PNA, N-PNA, RCA, UEA, DBA) and macrophages (Con A, WGA, N-PNA, RCA). A very selective and mainly extracellular affinity to N-PNA was demonstrated in the condensed preskeletal mesoderm, where it characterizes indistinct prospective chondrogenic, perichondral and pre-articular areas. Comparison with the distribution pattern of [³⁵S]-sulphate uptake and other previously published histochemical data suggests that N-PNA staining occurs at the late blastema stage, i.e. after the stage of cell condensation and before the earliest deposit of stainable matrix in chondrogenic areas. This property later disappears from the chondrifying rudiments, and is maintained in perichondral and pre-articular tissues. Surprisingly, only the pre-articular areas bind PNA without pretreatment with neuraminidase. A transient RCA binding probably related to terminal morphogenesis was detected in the undifferentiated distal part of the predigital columns of day-12 and day-13 limb buds. From the day-13 stage onwards, diverse new lectin affinities appeared in differentiating tissues, such as pretendinous rudiments, perichondrium and prospective periosteum, muscular connective tissue, myotubes, superficial fasciae and prospective dermis. A strong SBA and PNA staining was also detected in the extracellular matrix associated with the epithelial septa separating the roots of the digits in day-15 and day-16 limb buds.     

Immunohistochemistry of neurone specific enolase with gamma subunit specific anti-peptide monoclonal antibodies.

AIMS--To investigate the application in immunohistochemistry of gamma-subunit specific anti-peptide monoclonal antibodies to human neurone specific enolase (NSE); and to determine their reactivity with formalin fixed, wax embedded sections of normal tissue and neuroendocrine tumours. METHODS--Immunohistochemical staining was performed on sections of formalin fixed, wax embedded tissue with two monoclonal antibodies (NSE-P1 and NSE-P2) raised against different synthetic peptides specific for the gamma subunit of human enolase (neurone specific enolase). RESULTS--Both antibodies gave strong immunostaining in normal tissues and cells known to contain NSE. There was no immunoreactivity in tissues containing either the alpha alpha or beta beta isozymes of enolase. The reactivity of the antibodies with a range of neuroendocrine tumours was also studied and both antibodies gave strong immunostaining of tumour cells in the different tumours. CONCLUSIONS--The use of synthetic peptides from defined regions of a molecule as immunogenes provides antibodies of high specificity. These monoclonal antibodies to NSE are ideally suited for immunohistochemical studies and they should be particularly useful in histopathology as they react with epitopes which are resistant to formalin fixation and wax embedding.     

Gastrointestinal autonomic nerve tumours and their separation from other gastrointestinal stromal tumours: an ultrastructural and immunohistochemical study of seven cases

Gastrointestinal stromal tumours (GIST) represent a heterogeneous group whose classification frequently requires ultrastructural and immunohistochemical studies. In a retrospective study of the ultrastructural findings of 24 gastrointestinal stromal tumours, whose light microscopic study has yielded ambiguous results and in which accurate diagnosis had required ultrastructural support, seven were found to have the characteristics of gastrointestinal autonomic nerve (GAN) tumours. In all of them the diagnosis was based on the presence of dendritic processes with dense neuroendocrine granules. Immunohistochemically, the seven tumours were negative for smooth-muscle markers. All stained positively for vimentin. NSE, chromogranin, and synaptophysin were positive in most of them, while S-100 protein was positive only in two cases. We present the ultrastructural and immunohistochemical features of seven GANT against the background of the GISTs of our series. We conclude that GAN tumours cannot be diagnosed by light microscopy alone but this tumour group displays characteristic electron microscopic and immunohistochemical features and appears to represent a distinct type of GIST.     

肺癌组织神经内分泌分化的免疫组化研究

目的 了解非小细胞肺癌（ＮＳＣＬＣ）的神经内分泌（ＮＥ）分化特征。方法 采用ＳＰ超敏免疫组化法对１２０例ＮＳＣＬＣ和２４例ＳＣＬＣ进行了神经烯醇化酶（ＮＳＥ）、嗜铬粒素Ａ（ＣｇＡ）和突触素（ＳＹＮ）的检测。并与各型肺癌的分型及分化程度地行了相关分析。结果 三种神经内分泌标记物均为胞浆当色。ＮＳＥ和ＳＹＮ的阳性染色部分为弥漫性,部分为灶性分布。而ＣｇＡ均为小灶性分布。在ＮＳＣＬＣ中ＣｇＡ和ＳＹＮ阳性的病例ＮＳＥ均阳性,除２例外ＣｇＡ阳性的ＳＹＮ均阳性。ＮＳＥ的阳性染色除分布癌细胞外,部分间料可见非特异染色。在１２０例ＮＳＣＬＣ中,５２．５％表达ＮＳＥ,９．２％表达ＣｇＡ,２８．３％表达ＳＹＮ。在ＳＣＬＣ中１００％表达ＮＳＥ,３３．３％表达ＣｇＡ,５４．２％表达ＳＹＮ。三种标记物的阳性率与ＮＳＣＬＣ的分型及分化程度     

Expression of lectin binding in cutaneous papillomas of animals

A group of spontaneously occurring animal papillomas which were negative or positive for papillomavirus group-specific antigen were examined with a battery of biotinylated lectins including Con A, WGA, succinylated-WGA, PNA and UEA-I. Canine papillomas, equine papillomas, white-tailed deer fibromas, mule deer fibromas, and bovine fibropapillomas were examined. Each lectin had a specific staining pattern. No obvious differences in staining patterns between normal skin, viral antigen-positive and -negative neoplasms were identified. This may be due to the well-differentiated and organized nature of these tumours.     

A comparative study of immunohistochemical staining for neuron-specific enolase, protein gene product 9.5 and S-100 protein in neuroblastoma, Ewing's sarcoma and other round cell tumours in children

A comparative study of immunohistochemical staining for neuron-specific enolase (NSE), protein-gene product 9.5 (PGP 9.5) and S-100 was made in 71 undifferentated round cell tumours from 65 children using formalin-fixed tissues and a standard alkaline phosphatase-anti-alkaline phosphatase method. All of 29 neuroblastomas marked for NSE and 27 for PGP 9.5; staining was diffuse and usually strong in all tumour elements, irrespective of the degree of differentiation. Patterns of staining remained consistent in primary, recurrent and metastatic tumours and were not modified by previous chemotherapy. S-100 staining was weak and confined to cell processes and schwannian elements in less than half of the tumours studied. Two primitive neuroectodermal tumours both stained strongly for NSE and PGP 9.5. Staining for NSE was observed in single maturing cells in 3/12 rhabdomyosarcomas and in tubular elements in 2/4 Wilms' tumours; primitive rhabdomyoblasts and undifferentiated renal blastema were negative; seven lymphomas were negative. Six of 17 skeletal Ewing's sarcomas showed light to moderate cytoplasmic staining for NSE and PGP 9.5. The site, histology and clinical course of these marker-positive Ewing's sarcomas showed no distinctive features. Staining for PGP 9.5 is a useful additional marker for neural differentiation in round cell tumours.     

Neurone specific enolase immunostaining in the diagnosis of breast carcinomas with neuroendocrine differentiation. Its usefulness and limitations

Thirty-eight infiltrating ductal carcinomas, nine infiltrating lobular carcinomas, two tubular carcinomas and one papillary carcinoma were studied by light microscopy, immunocytochemistry and electron microscopy. Seventeen cases showed immunoreactivity for NSE. Immunostaining for different peptide-hormones was observed in 12 of these 17 cases and in none of the 10 NSE-negative cases used for controls. Scattered cells were positive for gastrin in five cases, pancreatic polypeptide in five, leu-enkephalin in three, sub-P in two, ACTH in one, bombesin in one and beta-endorphin in one case. Four cases revealed immunoreactivity for more than one peptide-hormone. Typical neuroendocrine granules were seen in five cases (all positively stained for NSE). Small, electron dense granules of possible neuroendocrine nature were not found in any of the 33 NSE-negative tumours. Our results confirm that immunoreactivity for NSE is present in a high proportion of breast carcinomas, but that neuroendocrine differentiation cannot be proved to be present in all these cases.