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.     

Neuron-specific enolase expression and neuroendocrine differentiation in carcinomas of the breast

The concentration of neuron-specific enolase (NSE, gamma-subunit of enolase) in tumor extracts of 25 nonargyrophilic and seven argyrophilic carcinomas of the female breast was measured by radioimmunoassay. The mean concentration of NSE in nonargyrophilic carcinomas was 89 ng/mg of protein, (range, 9 to 281 ng/mg), and the mean concentration of NSE in argyrophilic carcinomas was 153 ng/mg of protein (range, 4 to 420 ng/mg). All carcinomas, irrespective of the silver reaction, could be grouped in regular histopathologic terms. Neuron-specific enolase immunoreactivity in sections of the corresponding fixed tumors occurred in both silver-stained and unreactive tumors, although the reaction was more prominent in the argyrophilic tumors. The results indicate that NSE in considerable amounts may be present in both argyrophilic and nonargyrophilic carcinomas of the breast, although higher concentrations are found most often in the former tumor type.     

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.     

Primary neuroendocrine carcinomas of the thymus.

Primary neuroendocrine carcinomas of the thymus are rare and comprise a wide spectrum of lesions ranging from well-differentiated to poorly-differentiated neoplasms. The classification of such tumors in the thymus is still controversial. By convention, the better-differentiated examples have been traditionally designated as thymic carcinoids and thought to represent the mediastinal counterpart of carcinoid tumors in other foregut locations. However, recent studies have shown that such neoplasms, when arising in the thymus, exhibit a much more aggressive behavior than those originating at other locations. We therefore consider these lesions to represent fully malignant neoplasms that fall within the spectrum of neuroendocrine carcinomas. The designation of well-, moderately-, or poorly-differentiated thymic neuroendocrine carcinoma is therefore favored for these tumors in the present review. Because such tumors may often adopt unusual morphological appearances, it is important to distinguish them from other more common conditions presenting at this location that may exhibit similar histological features. The clinicopathologic, immunohistochemical, and differential diagnostic features of these tumors in the mediastinum are discussed.     

Immunohistochemical and ultrastructural analysis of bronchopulmonary neuroendocrine neoplasms. II. Well-differentiated neuroendocrine carcinomas

We have attempted to characterize a group of bronchopulmonary neoplasms that share certain structural features with true carcinoids but appear distinctly more pleomorphic and behave far more aggressively. In reviewing our files from 1973 to 1982, 11 such neoplasms were identified; the original diagnoses were "atypical bronchial carcinoid" (3 cases), "malignant carcinoid" (1 case), "bronchial carcinoid" (3 cases), "peripheral carcinoid" (2 cases), and "peripheral oat cell carcinoma" (2 cases). Of the 11 neoplasms, 5 were central and 6 were peripherally located. At presentation, 7 patients had lymph node metastases and 1 had a distant metastasis. No patient had a conventionally defined hormonal syndrome; however, 2 patients had a history of episodic flushing, one of which was associated with diarrhea. All cases were studied by light microscopy and light microscopic immunohistochemistry for NSE (neuron-specific enolase), serotonin, and broad-spectrum neuropeptides. Five cases were studied by electron microscopy. By light microscopy, the tumors were composed of solid clusters of polygonal to fusiform cells in an evident organoid arrangement. Foci of glandular and/or squamous differentiation were seen in 7 cases. Pleomorphism was moderate and mitoses were readily found. Focal necrosis was seen. By immunohistochemistry, 10 cases expressed NSE immunoreactivity. All cases demonstrated hormonal immunoreactivity; in 9 cases, immunoreactivity for more than one hormone was observed. The hormones most frequently expressed were serotonin, bombesin, gastrin, leu-enkephalin, and ACTH. By electron microscopy, all cases studied contained heterogeneous populations of neurosecretory granules; the latter, however, were not abundant and tended to aggregate either in the basal pole of the cells or, more frequently, interlacing "dendritelike" cytoplasmic processes. Aggregates of intermediate filaments were frequently seen. Basal lamina deposition was seen but gaps and larger areas of discontinuity were frequent. We believe that these neoplasms constitute a distinct pathologic entity for which the term "well-differentiated neuroendocrine carcinoma" has been proposed. Clinically, these tumors merit special attention since they are demonstrably more aggressive than true carcinoids but are distinctly less malignant than the intermediate or small cell variants of neuroendocrine carcinoma.     

Metastatic properties of murine sarcomas and carcinomas I. Positive correlation with lung colonization and lack of correlation with s.c. tumor take

The spontaneous metastatic properties of six sarcomas and seven carcinomas syngeneic to C₃Hf/Kam mice were investigated and the correlation between spontaneous metastasis, the lung colony forming efficiency (LCFE) of i.v. injected tumor cells, and s.c. tumor take was determined. The incidence and number of spontaneous metastases in the lung were determined in mice that had primary tumors in the leg removed 17 to 120 days earlier, depending on tumor type. There was a significant positive correlation between spontaneous metastasis and LCFE when all 13 tumors were compared, but the significance was lost when carcinomas and sarcomas were considered separately. No significant correlation between spontaneous metastasis and the s.c. tumor take was observed. Also, no correlation was found between LCFE and the s.c. tumor take of carcinomas, but there was a strong inverse relationship between these two properties of sarcomas. The number of cells shed from primary tumors was estimated and found to be more extensive in tumors with higher metastatic properties. Thus, in general, highly metastatic tumors were characterized by a high LCFE and a significant cell shedding. Furthermore, LCFE was greatly increased by treatment of animals with cyclophosphamide and by admixing heavily irradiated tumor cells to viable cells, implying that local environmental factors are important in determining the establishment of tumor cell clonogens into metastasis.     

ABC-immunoperoxidase staining of cytologic preparations: improvement of specificity.

Immunoperoxidase (IP) methods perfected on formalin-fixed, paraffin-embedded tissues (FFPE) and then applied to aspirate smears may result in high background staining and a significant number of false-positive results. This is especially true if polyclonal primary antibodies are used or if aspirates and fluids contain a high interstitial or serum protein content. Because of a recurring problem with antiserum to alpha-fetoprotein (AFP), AFP was selected as the primary test antibody with which to evaluate our avidin-biotin complex (ABC)-IP method. The same method, with diaminobenzidine (DAB) or aminoethylcarbazole (AEC) chromogens, was performed on six types of cytologic preparations of a fresh liver specimen. The liver did not stain for AFP in FFPE and frozen tissue; therefore, it could be used to evaluate potential false-positive staining of direct touch imprints, washed aspirate smears, and cytospins that were both air-dried and alcohol-carbowax-fixed. Initial chromogen incubation times were standardized to give identical results on AFP-positive fixed hepatoma and fetal liver controls. Cytologic preparations immunostained with ABC-IP with AEC chromogen resulted in varying background and hepatocyte staining. In comparison, the ABC-IP method using DAB chromogen resulted in no false-positive results and a clean background. The ABC-IP method with AEC standardized for sensitivity on a fixed tissue control required a markedly shortened chromogen incubation time to preclude significant false-positive staining of cytology specimens. It appears that use of AEC chromogen for this antibody with incubation time standardized on a FFPE tissue control and then applied to cytologic preparations also amplifies nonspecific and background staining, contributing difficulty in assessing a true-positive result.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of chromosomal imbalances in neuroendocrine and non-small-cell lung carcinomas.

Lung carcinomas are represented by non-small-cell lung carcinomas (NSCLC) and neuroendocrine carcinomas (NE) which differ in their clinical presentation and prognosis. We used comparative genomic hybridization (CGH) to characterize and compare the chromosomal pattern of 11 NSCLC and 11 high-grade NE lung carcinomas. Overall, the total number of aberrations was higher in NSCLC than in high-grade NE lung tumors (p < 0.05) and gains predominated over losses in NSCLC (p < 0.0003). Gains common to both lung tumor phenotypes were detected in 1p, 1q, 3q, 5p, 6p, 8q, 12, 17q, 19p, 19q, 20p, 20q, and X, whereas common losses were found in 2q, 3p, 4p, 4q, 5q, 8p, 9p, 10p, 11p, 11q, 13q, and 17p. Major gains on 18q and losses on 2p and 16q were exclusively detected in high-grade NE lung tumors. On the other hand, major gains on 2p and 15q and losses on 21q were found only in NSCLC. Furthermore, gains within 22q11-q12 and 7p12-p15 were associated with NSCLC (p < 0.05). The differences in the pattern and distribution of genetic changes observed in NSCLC as opposed to high-grade NE lung carcinomas suggest the existence of distinct tumorigenic pathways between these two major classes of lung tumors.     

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.     

Efficacy and tolerability of pegylated IFN-alpha in patients with neuroendocrine gastroenteropancreatic carcinomas.

Interferon-alpha (IFN-alpha) is well established in the treatment of neuroendocrine carcinomas (NEC). Treatment is accompanied by fatigue and flu-like symptoms. In patients with chronic hepatitis C, pegylated IFN (PEGIFN) leads to improved antiviral efficacy and good tolerability. Our aim was to assess the efficacy and tolerability of PEG-IFN on the management of patients with well-differentiated NEC of the gastroenteropancreatic system. In 17 patients, the effect of PEG-IFN-alpha2b was studied. After first-line octreotide treatment, IFN-alpha was added at the time of tumor progression. Six patients were switched from conventional IFN-alpha, and 11 patients were IFN naive. Inhibition of tumor growth, including stabilization of disease, occurred in 13 of 17 patients, and biochemical and symptomatic responses were seen in 7 of 10 patients with functionally active tumors. Tolerability of PEG-IFN-alpha2b was much better than that of IFN-alpha. Fatigue occurred in 59% of all patients but was mild in severity. Eleven of thirteen patients who had a benefit remained on therapy for a median time of 20 months (range 6-30 months). PEG-IFN-alpha2b provides symptomatic and antiproliferative efficacy in patients with NEC. Better tolerability of PEG-IFN-alpha2b improved patients' compliance, justifying its use in patients who do not tolerate conventional IFN-alpha treatment.     

C-erbB-2 protein and neuroendocrine expression in intraductal carcinomas of the breast.

A total of 52 intraductal carcinomas were classified according to nuclear size and histologic subtype and immunostained for neuron-specific enolase (NSE) and c-erbB-2 oncoprotein. Eleven out of 13 cases of the large cell comedo variant of intraductal carcinoma exhibited strong c-erbB-2 protein immunoreactivity, while only one was NSE positive. Nineteen out of 23 intraductal carcinomas of small cell type exhibited NSE immunoreactivity. None of these was c-erbB-2 protein positive. Some 72% of NSE positive cases were also immunoreactive for other neuroendocrine screening markers and/or hormones. We conclude that there is an inverse relationship between NSE immunoreactivity and c-erbB-2 protein expression in intraductal carcinomas.     

Comprehensive genetic features of gastric mixed adenoneuroendocrine carcinomas and pure neuroendocrine carcinomas

We aimed to determine the pathogenesis of gastric mixed adenoneuroendocrine carcinoma (MANEC) and pure neuroendocrine carcinoma (NEC), which is largely unknown. Targeted DNA sequencing was performed on 34 tumor samples from 21 patients – 13 adenocarcinoma (ADC)/NEC components from MANECs and eight pure NECs – and 21 matched non‐neoplastic gastric tissues. Mutational profiles of MANECs/NECs were compared with those of other tumors using public databases. The majority (64.1%; 59/92) of mutations in MANEC were shared by both ADC and NEC components. TP53 was the most commonly mutated gene in MANEC (69.2%, 9/13) and pure NEC (87.5%, 8/9). All TP53 mutations in MANEC were pathogenic mutations and were shared by both ADC and NEC components. A subset of TP53^WT MANECs had a microsatellite‐unstable phenotype or amplifications in various oncogenes including ERBB2 and NMYC, and the only TP53^WT pure NEC harbored MYC amplification. Compared to NEC in other organs, NECs arising from the stomach had unique features including less frequent RB1 mutations. Differentially altered genes of MANEC ADC components were significantly associated with receptor tyrosine kinase signaling pathways, while differentially altered genes of MANEC NEC components were significantly associated with the NOTCH signaling pathway. Our data provide evidence suggesting a possible clonal origin of ADC and NEC components of MANEC, and we found that gastric MANECs and pure NECs are distinct entities with unique mutational profiles and underlying protein networks. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

CD117 immunoreactivity in high-grade neuroendocrine tumors of the lung: a comparative study of 39 large-cell neuroendocrine carcinomas and 27 surgically resected small-cell carcinomas

Little is known about CD117 prevalence and clinicopathological implications in pulmonary large-cell neuroendocrine carcinoma. We studied CD117 immunoreactivity in surgical specimens from 39 large-cell neuroendocrine carcinomas of stages I–III and 27 limited-disease small-cell carcinomas, 56 typical and atypical carcinoids of the lung, and 10 neuroendocrine tumorlets, including the membrane and cytoplasmic immunostaining patterns. Membrane CD117 immunoreactivity in 5% or more tumor cells was documented in 30 (77%) large-cell neuroendocrine carcinomas and 18 (67%) small-cell carcinomas and 4 (7%) carcinoids, whereas cytoplasmic labeling was seen in 17 (44%) large-cell neuroendocrine carcinomas, 19 (70%) small-cell carcinomas, and 3 (5%) carcinoids. None of the neuroendocrine cells of the normal bronchial epithelium and of 10 tumorlets showed any CD117 immunoreactivity. Cytoplasmic immunostaining was more prevalent in small-cell carcinomas, whereas membrane labeling did not differ between the two types of high-grade carcinomas. Downregulation of CD117 by neoadjuvant chemotherapy was seen in large-cell neuroendocrine carcinomas but not small-cell carcinomas. Multiple linear regression analysis demonstrated a marginal association between cytoplasmic CD117 immunoreactivity and regional lymph node metastasis in small-cell carcinomas but not large-cell neuroendocrine carcinomas. There was no association between CD117 immunoreactivity and survival in either small-cell carcinoma or large-cell neuroendocrine carcinoma patients.