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.     

Ectopic neural and neuroendocrine neoplasms

Although neural and neuroendocrine tissues are distributed virtually ubiquitously throughout the body, the occurrence of selected neoplasms related to those lineages is extremely uncommon in some topographic sites. This review considers the clinicopathologic characteristics of heterotopic pituitary adenomas; neuroendocrine carcinomas in non-organ-based locations; ectopic (extraneuraxial) meningiomas and gliomas; visceral neuroblastic neoplasms and primitive neuroectodermal tumors; and paragangliomas arising outside the sympathoadrenal neural network. Practical approaches to differential diagnosis are emphasized.     

Immunoreactivity of neurofilament proteins in neuroendocrine neoplasms.

We immunostained histologic sections of formalin-paraffin-processed specimens from 112 neuroendocrine tumors to evaluate the expression of intermediate filaments, especially neurofilament (NF) protein. The commercially available anti-human NF protein monoclonal antibody was raised against the Mr 200,000 and 70,000 components of neurofilaments. None of our tumors exhibited unequivocal immunoreactivity for Mr 200,000 or 70,000 neurofilament proteins. The failure of a tumor to express the Mr 200,000 or 70,000 component of NF protein does not question its neuroendocrine nature. However, the unequivocal positivity for Mr 200,000 or 70,000 components of NF in an undifferentiated tumor with a differential diagnosis between neuroendocrine small-cell carcinoma and a neurogenic tumor should support the latter consideration.     

Gastroenteropancreatic neuroendocrine neoplasms: Historical context and current issues

The digestive organs contain a large number of neuroendocrine cells as part of the diffuse neuroendocrine system. Neuroendocrine tumors can occur in every digestive organ. It has long been recognized that this is a diverse group of tumors with very different clinical outcomes; however, well-recognized prognostic parameters had been elusive until recently. Over the years, there have been several different classification schemes, each with different strengths and weaknesses. In an effort to standardize the classification and grading criteria for gastroenteropancreatic neuroendocrine tumors, the current World Health Organization classification includes a histologic grade based on proliferative rate (mitotic rate and Ki67 index) and a TNM stage that varies from organ to organ. The prognostic value of both the grade and stage has been validated in multiple studies. However, several issues remain, including the lack of standardized methods to assess proliferative rate, potential discrepancies between the mitotic count and the Ki67 index; intratumoral heterogeneity in proliferative rate; and the need for refinement in proliferative cut-points to define the grades. More studies are needed to further improve the classification of neuroendocrine tumors, thus guiding optimal treatment for these tumors.     

Combined polymerase chain reaction approach for clonality detection in lymphoid neoplasms.

The present study analyzes the efficiency of a combination of four immunoglobulin heavy chain (IgH) gene polymerase chain reaction (PCR) primer systems and a multiplex T-cell receptor gamma chain (TRG) gene PCR for detection of clonality in 409 samples (234 paraffin sections, 175 bone marrow aspirates) of different lymphomas. Using the four IgH PCR systems together, clonality was detected in all samples of B-cell chronic lymphocytic leukemias, hairy cell leukemias, common acute lymphoblastic leukemias, and Burkitt-like B-cell lymphomas. Clonality was detected in all bone marrow aspirates with lymphoplasmacytoid immunocytoma, mantle cell lymphoma, marginal zone B-cell lymphoma, and unclassifiable low-grade B-cell lymphomas. The combined IgH gene PCR approach allowed clonality detection in 78.2% of myelomas, 75% of Burkitt lymphomas, 74.4% of diffuse large B-cell lymphomas, 68.7% of follicular center lymphomas, 50% of posttransplant lymphomas, 28.6% of anaplastic large cell lymphomas, 29% of T-cell lymphomas, and 18.8% of Hodgkin diseases. The combination of the four IgH gene primer systems with the multiplex TRG gene PCR allowed detection of clonality in 84.2% of B-cell neoplasms, 92.1% of T-cell non-Hodgkin lymphomas, and 18.8% of Hodgkin diseases, which was much more efficient than single PCR protocols.     

Middle ear adenoma: tumour of mixed mucinous and neuroendocrine differentiation.

Two cases of progressive hearing loss due to middle ear tumours are described. The histological characteristics numbered intraluminal mucin production and neuroendocrine features, as shown by argyrophilia and ultrastructural demonstration of dense core granules. These tumours have been known by many different names, reflecting the controversies relating to their presumed histogenesis and differentiation. The currently preferred designation is middle ear adenoma, and these two cases provide further evidence for dual lines of differentiation.     

Gastroenteropancreatic neuroendocrine neoplasms: selected pathology review and molecular updates

Gastroenteropancreatic (GEP) neuroendocrine neoplasms can be broadly separated into well‐ and poorly differentiated categories. Tumours within each category have similarities in morphology and immunophenotype, but vary in grade, behaviour, molecular signature and responses to therapy. The aetiology of these differences is multifactorial. Site of origin, mucosal milieu and hereditary influences are some of the currently known factors. Given these differences, staging and grading systems continue to evolve, and the most recent World Health Organization classification of pancreatic neuroendocrine neoplasms reflects this by introducing a grade 3 neuroendocrine tumour category for morphologically well‐differentiated tumours with an elevated Ki‐67 proliferation index and/or mitotic count. This review aims to highlight current classification guidelines with discussion of unique site‐specific features of selected GEP neuroendocrine neoplasms and an emphasis on practical issues related to daily reporting.     

Analysis of clonality of tumour infiltrating lymphocytes in breast cancer and uveal melanoma.

Fresh tumour infiltrating lymphocytes (TILs) from 6 uveal melanomas and 4 breast cancers were analysed by flow cytometry with a panel of 6 monoclonal antibodies to V beta regions of the T cell receptor (V betas 5a, 5b, 5c, 6, 8a and 12a). With a single exception where one TIL sample lacked V beta 12a, lymphocytes from both tumour and blood contained cells reactive with all 6 probes, and no probe was highly dominant or missing. The proportions of reactive cells differed between tumour and blood within each patient. The data indicate that while tumour infiltrating lymphocytes have a capacity to locate selectively within the tumour they nonetheless comprise a population expressing a diversity of TCR V beta genes.     

Myoclonus-dystonia: significance of large SGCE deletions

Myoclonus-dystonia (M-D) is an autosomal-dominant movement disorder caused by mutations in SGCE. We investigated the frequency and type of SGCE mutations with emphasis on gene dosage alterations and explored the associated phenotypes. We tested 35 M-D index patients by multiplex ligation-dependent probe amplification (MLPA) and genomic sequencing. Mutations were found in 26% (9/35) of the cases, all but three with definite M-D. Two heterozygous deletions of the entire SGCE gene and flanking DNA and a heterozygous deletion of exon 2 only were detected, accounting for 33% (3/9) of the mutations found. Both large deletions contained COL1A2 and were additionally associated with joint problems. Further, we discovered one novel small deletion (c.771_772delAT, p.C258X) and four recurrent point mutations (c.289C>T, p.R97X; c.304C>T, p.R102X; c.709C>T, p.R237X; c.1114C>T, p.R372X). A Medline search identified 22 articles on SGCE mutational screening. Sixty-four unrelated M-D patients were described with 41 different mutations. No genotype-phenotype association was found, except in patients with deletions encompassing additional genes. In conclusion, a rigorous clinical preselection of patients and careful accounting for non-motor signs should precede mutational tests. Gene dosage studies should be included in routine SGCE genetic testing.     

Coexpression of neuroendocrine markers and epithelial cytoskeletal proteins in bronchopulmonary neuroendocrine neoplasms

Neuroendocrine (NE) neoplasms of the human bronchopulmonary tract were examined by electron microscopy, immunocytochemistry, and gel electrophoresis of cytoskeletal proteins from microdissected tissue samples. All samples (carcinoids, well-differentiated NE carcinoma, NE carcinomas of intermediate type, NE carcinomas of the small cell type) contained significant numbers of cells that immunostained for one or more of the following neuroendocrine markers tested: bombesin, calcitonin, ACTH, leu-enkephalin, gastrin, serotonin, somatostatin, alpha-melanocyte-stimulating hormone, vasoactive intestinal peptide, glucagon, insulin, substance P, and neuron-specific enolase. Electron microscopy revealed typical NE cell features, including variable abundant and frequently heterogeneous neurosecretory granules. Tumor cells contained filaments specifically stained with different conventional and monoclonal antibodies to cytokeratins and displayed punctate plasma membrane staining with antibodies to desmoplakins, in agreement with the electron microscopic demonstration of tonofilament bundles and desmosomes. Immunocytochemistry for NE markers and cytoskeletal proteins on consecutive sections revealed both cytokeratins and neuroendocrine substances in single cells. Using gel electrophoresis of cytoskeletal proteins of tissue regions extracted with high salt buffer and detergent, we could detect, in the tumors tested, appreciable amounts of cytokeratin polypeptides 8, 18, and 19, i.e., major cytokeratins also found in certain other lung carcinomas such as adenocarcinomas. Tumor cells were not significantly stained with antibodies to other intermediate filament proteins such as vimentin, desmin, glial filament protein, and neurofilament protein. The results show that NE substances can be synthesized in cells containing a typical epithelial cytoskeleton, i.e., cytokeratin filaments and desmosomes. These findings support the notion of an epithelial character of these tumors and appear in contrast with recent reports that neurofilaments are the only type of intermediate filaments present in carcinoids and other pulmonary NE tumors. These observations may have important implications for the histogenesis of NE carcinomas and for diagnostic pathology.     

Gastric small cell carcinoma with squamous and neuroendocrine differentiation.

A rare gastric carcinoma containing diverse components, that is, neuroendocrine (small cell carcinoma), squamous and gland-like elements in an 82 yr old woman is described. Radiologic examination revealed a large ulcerated tumor, and a Borrmann type II tumor, 6.5 x 5 cm, was found in the resected stomach. Histologically, the tumor was mainly composed of small cells with hyperchromatic nuclei and scant cytoplasm. Argyrophilic granules were seen in these cells. There were also scattered foci of large cells with features of squamous cells, and many intermediate cells with oncocytic cytoplasm. The small cancer cells were positive for chromogranin A and neuron specific enolase. Squamous cell nests were positive for high molecular cytokeratin (CK), and intermediate cells were positive for low molecular CK. Electron microscopic examination revealed secretory granules in the small cells and tonofilaments in the squamous cells. This tumor might have originated from the pluripotential stem cell in the gastric epithelium.     

Synaptophysin expression in neuroendocrine neoplasms as determined by immunocytochemistry.

Synaptophysin is an integral membrane glycoprotein originally isolated from presynaptic vesicles of bovine neurons. The authors have studied a wide spectrum of neuroendocrine (NE) neoplasms by immunofluorescence microscopy on cryostat sections of freshly frozen tissues using a monoclonal antibody to this protein (SY 38). Without exception, they found the identical--or a very similar--protein expressed in all neuroblastomas, ganglioneuroblastomas, ganglioneuromas, pheochromocytomas, and paragangliomas studied. In these "neural" type NE neoplasms, synaptophysin was coexpressed with neurofilament proteins. Synaptophysin was also demonstrated in NE neoplasms of "epithelial" type in which it was predominantly coexpressed with cytokeratins and desmoplakin. It was invariably found in all variants of islet cell neoplasms and in all medullary thyroid carcinomas. Synaptophysin was also demonstrated in several adenomas of the hypophysis and parathyroids, in the majority of carcinoids of the bronchopulmonary and gastrointestinal tracts, and in many, though not all, NE carcinomas of the same sites, and of the skin. Conversely, SY 38 did not immunostain any of a large number of benign and malignant non-NE epithelial neoplasms; nor was any immunostaining obtained in a group of mesenchymal tumors. It is remarkable that SY 38 did not immunostain a number of malignant melanomas, including several that were immunostained for neuron-specific enolase (NSE) and several neuropeptides. Parallel studies conducted on conventionally fixed, paraffin-embedded tissue sections immunostained by the use of the avidin-biotin complex technique yielded very similar results. The findings indicate that synaptophysin is expressed in the whole range of NE neoplasms without detectable relation to the expression of other NE markers such as NSE, serotonin, and neuropeptides. Nor could the expression of synaptophysin by these tumors be correlated with their epithelial and/or neural cytoskeletal characteristics, their clinical aggressiveness, or the presence or absence of endocrinologic abnormalities. While the consistent expression of synaptophysin by the "neural" type of NE neoplasms would seem predictable its presence in diverse benign and malignant NE tumors of "epithelial" type is remarkable. It is concluded that synaptophysin is a significant as well as novel NE marker, and the use of antibody SY 38 as a broad range marker for the study and diagnosis of NE neoplasms is proposed.     

Genetic alterations in combined neuroendocrine neoplasms of the lung.

Large-cell neuroendocrine and small-cell lung carcinomas are highly aggressive neuroendocrine tumors that can be associated in a variant of 'small-cell lung carcinoma combined with large-cell neuroendocrine carcinoma'. Little is known about this rare tumor type with biphenotypic neuroendocrine differentiation. The aim of the present study was to genetically characterize each component of a series of combined small-cell/large-cell neuroendocrine carcinomas, to gain information on their histogenesis and to compare the alterations observed with those found in their respective pure forms. To this end, 22 formalin-fixed, paraffin-embedded lung neuroendocrine tumors obtained from surgical resections were investigated: six combined small-cell/large-cell carcinomas, eight pure large-cell carcinomas and eight pure small-cell carcinomas. For the combined neuroendocrine neoplasms, DNA was extracted separately from each of the two cytologically different populations. Allelic imbalance was investigated by PCR amplification of 30 highly polymorphic microsatellite markers located at 11 different chromosomal regions. A common background of genetic alterations, similar in both components of the combined neoplasms, was demonstrated at 17p13.1, 3p14.2-3p21.2, 4q12-4q24, 5q21 and 9p21. In fact, the two components appeared to be more similar to each other than to their respective pure forms. In addition, allelic imbalances preferentially involving one of the two components were found. These alterations often appeared to be specific for this histological variant, as compared with those observed in pure forms or in the literature. In conclusion, this is the first report in which a molecular characterization of the variant of small-cell lung carcinoma combined with large-cell neuroendocrine carcinoma was performed. The finding of common alterations in the two phenotypically different neuroendocrine cell components suggests a close genetic relationship and supports the hypothesis of a monoclonal origin from a common ancestor. The genetic differences observed provide the basis for the divergent differentiation and parallel the morphological differences in the two components of these combined neuroendocrine neoplasms.     

Pulmonary neuroendocrine neoplasms: A review of clinicopathologic and cytologic features

Neuroendocrine tumors form a distinct group of lung neoplasms sharing characteristic cytohistologic, immunohistochemical, ultrastructural, and molecular features. The objective of this review article is to discuss the diagnostic classifications and the morphologic cytologic–histologic features for the different categories of neuroendocrine tumors of the lung. An accurate characterization of the neuroendocrine tumors of the lung requires knowledge of specific criteria separating the major categories, which is highly essential for determining prognosis and treatment options for these patients. Diagn. Cytopathol. 2010;38:607–617. 2009 Wiley‐Liss, Inc.     

Neuroectodermal neoplasms of the head and neck with emphasis on neuroendocrine carcinomas.

Tumors exhibiting neuroectodermal differentiation occur throughout the body, and the diverse tissues of the head and neck give rise to a wide assortment of these neoplasms. Neuroectodermal neoplasms may be divided into lesions showing primarily epithelial differentiation (Group I, neuroendocrine carcinomas) and a more diverse group (Group II) of nonepithelial neoplasms. This article reviews these neuroectodermal tumors of the head and neck with emphasis on the neuroendocrine carcinomas and their nomenclature. The author believes that with regard to Group I tumors, the older terminology of carcinoid, atypical carcinoid, and small cell carcinoma should be replaced by subclassifications of well-differentiated, moderately differentiated, and poorly differentiated neuroendocrine carcinoma. The latter category should be further subdivided into small cell and large cell variants. Neuroendocrine carcinomas, particularly the moderately differentiated subtype, are often underdiagnosed in the head and neck region. In the larynx, these tumors are the most common form of nonsquamous carcinoma. Poorly differentiated neuroendocrine carcinoma of small cell type is most common in the salivary glands but can occur elsewhere in the region. The large cell subtype of poorly differentiated neuroendocrine carcinoma has not been well documented in this region. However, the most likely candidate for this tumor category is the so-called sinonasal undifferentiated carcinoma. Group II tumors discussed include olfactory neuroblastoma, malignant melanoma, and Ewing's sarcoma. In addition, differential diagnostic problems related to Group I and II tumors are reviewed in detail. This article reviews and updates our understanding of neuroectodermal neoplasms arising in the head and neck. The focus is on tumors that exclusively involve this region or show a strong predilection to occur here.     

Cytopathology-histopathology of the mediastinum II. Mesenchymal, neural, and neuroendocrine neoplasms

An appraisal of the cytopathology and corresponding histopathology of epithelial, germ cell, and lymphoid lesions of the mediastinum was previously reported in the Journal. This report aims to complete that topic with a discussion focusing on the correlative cyto-histopathology of the major mesenchymal, neural, and neuroendocrine neoplasms from this anatomic site. As previously stated, the mediastinum remains an uncommon site for fine needle aspiration (FNA) biopsy when compared with other anatomic sites. Yet, the recent use of endoscopic ultrasound-guided FNA for sampling mediastinal masses may increase this use. The purpose of this review is to focus on the shared and dissimilar morphologic features of this subset of neoplasms to improve diagnostic correlation between the two.