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 rnetastases 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 immu-noreactivity. All cases demonstrated hormonal immu-noreactivity; 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, in 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 carcinomay” 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.     

Reliability of Criteria for Ultrastructural Identification of Neuroendocrine Granules

For full diagnostic use to be made of the neurosecretory granule, the range of sizes, forms, and staining qualities for this cytoplasmic organelle, along with the extent of its expression in various neoplasms, must be established. Neurosecretory type granules occasionally occur in nonneuroendo-crine tumors. A series of carcinoids of the lung provides a model for assessing the morphologic types of cytoplasmic granules identified by antibodies to chromogranin A and immunogold labeling. The results show that granule structure in tumors is pleomorphic. Despite having sizes within the expected range, many labeled and, indeed, unlabeled secretory granules are atypical, particularly in structural form. Cell-to-cell variation in the proportion of even typical neurosecreotry granules labeling for chromogranin A is the rule. Studies correlating biochemical, immunohistochemical, electron microscopic, and perhaps in situ hybridization characteristics are required to define better the criteria for unequivocal identification of neurosecretory granules in tumors.     

Breast Carcinomas with Neuroendocrine Differentiation

Twenty-two breast carcinomas with membrane bound granules by electron microscopy were tested for the presence of neuron specific enolase (NSE), neuropeptides and serotonin by immunohistochemistry. By light microscopy the cases studied included infiltrating ductal carcinomas, intraductal carcinomas, apocrine carcinomas, infiltrating lobular carcinomas of both classical and alveolar types, mixed lobular/colloid carcinomas, carcinoid growth pattern and one unclassified carcinoma. Ten cases showed immunoreactivity for 1 or 2 neuropeptides in scattered cells whereas all cases were positively and rather diffusely stained with anti-NSE. Immunohistochemical staining at the ultrastructural level was carried out; the presence of neuropeptides could not be confirmed. Scattered granules were marked with gold particles when antiserum against casein was used.

We conclude that neither argyrophilia, nor NSE immunoreactivity nor membrane bound granules seen by electron microscopy constitute at present sufficient evidence to designate a breast carcinoma as neuroendocrine. However, our study indicates that certain breast carcinomas of several types do include cells with neuroendocrine features demonstrable convincingly by light microscopic immunohistochemistry. We have no evidence that these breast carcinomas with neuroendocrine features behave differently from their counterparts lacking such features. The intriguing speculation is that neuropeptides produced by certain breast carcinomas may act as local modulators of tumor growth and differentiation.     

Ultrastructural and Immunocytochemical Features of a Case of Neuroendocrine Carcinoma Developing in a Prior Ileostomy Site

A patient who developed a mixed neuroendocrine carcinoma and adenocarcinoma at the site of a previous long-standing ileostomy is reported. The neuroendocrine features are documented by both ultrastructural and immunocytochemical findings. Carcinoma arising in an ileostomy site is rare but has been recorded in patients with long-standing ileostomies after colectomy for chronic inflammatory bowel disease, as in this patient. Neuroendocrine carcinoma developing in this setting apparently has not been described before, however.     

Intriguing Case: Argyrophilic Neuroendocrine Carcinoma of the Male Breast

A case of breast carcinoma with neuroendocrine features occurring in an elderly male patient is reported. Histologically, the tumor was characterized by solid growth with nests and ribbons of small to medium-size uniform neoplastic cells. Argyrophilia, expression of chromogranins at both protein and gene level, and the presence of dense granules of the neurosecretory type by electron microscopy were demonstrated.     

Small Cell Neuroendocrine Carcinoma of the Urinary Bladder: Report of Three Cases with Ultrastructural Analysis

Three cases of small cell neuroendocrine carcinoma of the urinary bladder were studied by light and electron microscopic techniques. Dense-core, membrane-bound granules were identified in the cytoplasm of all 3 cases, substantiating the light microscopic impression of a morphologic similarity to other neuroendocrine carcinomas, such as small cell (oat cell) carcinoma of the lung. Two of the three cases showed clinical evidence of distant metastases, suggesting an aggressive biologic potential of this tumor similar to oat cell carcinoma of the lung. A partial remission was induced in these 2 cases using chemotherapy protocols similar to the drug regimens for small cell (oat cell) carcinomas of the lung. Recognition of this distinct entity has important clinical implications regarding therapeutic approach.     

Small Cell Neuroendocrine Carcinoma of the Urinary Bladder: Report of Three Cases with Ultrastructural Analysis

Three cases of small cell neuroendocrine carcinoma of the urinary bladder were studied by light and electron microscopic techniques. Dense-core, membrane-bound granules were identified in the cytoplasm of all 3 cases, substantiating the light microscopic impression of a morphologic similarity to other neuroendocrine carcinomas, such as small cell (oat cell) carcinoma of the lung. Two of the three cases showed clinical evidence of distant metastases, suggesting an aggressive biologic potential of this tumor similar to oat cell carcinoma of the lung. A partial remission was induced in these 2 cases using chemotherapy protocols similar to the drug regimens for small cell (oat cell) carcinomas of the lung. Recognition of this distinct entity has important clinical implications regarding therapeutic approach.     

Solid Papillary Carcinoma of Breast: An Ultrastructural Study

Solid papillary carcinoma of the breast is a subset of papillary carcinoma, which occurs in older women and usually has a favorable prognosis. It is primarily intraductal but also is often associated with invasive carcinoma, especially mucinous carcinoma. Intracellular and extracellular mucin is also found in the in situ stage, in most tumors. In addition to forming solid papillary masses, the cells palisade around vessels in pseudo-rosettes and show minimal nuclear atypia. Some cells show neuroendocrine differentiation, based on argyrophilia with Grimelius staining. Four examples of this neoplasm were studied electron microscopically. Myoepithelial cells were not found. Neoplastic cells had an ultrastructure that was generally similar to that of other types of mammary carcinoma. There were extracellular microlumens, but intracellular lumens and pseudolumens were few or absent. Secretory activity varied among cells, and those cells appearing active had a variety of granule types, including typical flocculent and “bull's-eye” mucinous granules, small dense-core granules, and large serous-like granules. Some of the dense-core granules were interpreted as neuroendocrine in nature, based on their abluminal or juxtavascular location, whereas others that were apical and subluminal were probably mucinous in type. The large serous-appearing granules were subluminal in some cells and diffuse in others and may also have represented a variant of mucinous granules. The results support earlier opinions that accurate interpretation of specific granular function at the electron microscopic level depends on cytochemical studies using uranaffin as a marker of neuroendocrine activity. Although mucinous granules are identified by their lack of staining with uranaffin, the nature of the serous-appearing granules would still not be answered by this method; that is, a negative reaction would not define whether the granules are truly serous, or simply another form of mucin. Regardless of limitations of this type, correlation and extrapolation of histo-chemical (Grimelius and Alcian blue) and immunohistochemi-cal (chromogranin and synaptophysin) results with subcellular structure are still very useful in establishing cell type.     

Neuroendocrine differentiation in basal cell carcinoma

Basal cell carcinoma (BCC) is the prototypical basaloid tumor of the skin. It may show various patterns simulating other cutaneous tumors due to its pleomorphism. It may have an unusal pattern of differentiation such as squamous, sebaceous, apocrine, eccrine, pilar, and endocrine differentiation. In order to establish the relative frequency of neuroendocrine differentiation in BCC, we performed a retrospective study of 33 consecutive BCCs using conventional immunohistochemistry with two neuroendocrine antibodies: Chromogranine A and synaptophysine. The age of the patients ranged from 17–83 years with mean of 65 years. The male to female ratio was 16:17. In immunohistochimestry, Chromogranine A was seen in 72.2% (24/33) while Synaptophysine was positive in 9.09% (3/33). Their expression was cytoplasmic and membranous and was seen in the periphery of these tumors in the overlying cells. Positive staining of chromogranine A was high (75–100% of tumors cells) in 9%, intermediate (25–75% of tumors cells) in 33% of cases and relatively low (<25%) in 30.3% of cases.     

Glycogen-rich Clear Cell Carcinoma of the Urethra: An Ultrastructural Study

A 49-year-old black woman developed a urethral glycogen-rich clear cell carcinoma. She was treated with anterior pelvic exenteration. The resected lymph nodes, vagina, uterine cervix, endometrium, ovaries, and urinary bladder were free of neoplasm. Histologically the neoplasm consisted of clear cells growing in sheets and occasional papillary structures. In some areas, hobnail cells were present. Ultrastructurally, the cells had apical caps, short microvilli, and complex cell bases, and contained abundant glycogen. These features were identified in one, but not the other of two previously reported cases. Because glycogen-rich clear cell carcinomas of the lower urinary tract do not resemble ultrastructurally mesonephric remnants or carcinomas known to arise from them, these glycogen-rich clear cell carcinomas should not be called “me-sonephromas” as has been the practice.     

Neuroendocrine Carcinomas of the Skin: Light Microscopic,Ultrastructural, and Immunohistochemical Analysis

Three primary skin carcinomas were analyzed by light microscopy, immunohistochemistry, and electron microscopy. In all cases, local recurrences, regional lymph node metastases, distant metastases, or all three developed. One patient had elevated serum calcitonin levels that did not decrease after thyroidectomy but did return to normal after removal of the skin tumor recurrences, its metastases, or both.

The tumor cells were arranged in solid clusters; a trabecular arrangement was occasionally seen. In 2 cases the cells were of intermediate size and showed vesicular central nuclei and pale, moderately abundant cytoplasm. In the remaining case the cells were distinctly smaller and either round or fusiform. Mitoses were more abundant in the latter case than in the former two.

By immunohistochemistry, calcitonin- and somatostatin-containing cells were demonstrated in all cases and ACTH in one. By electron microscopy, the cases consisting of intermediate-size cells displayed moderately abundant neurosecretory-type granules irregularly dispersed throughout the cytoplasm. The case consisting of smaller cells displayed fewer and smaller granules that tended to concentrate in slender cytoplasmic processes.

We conclude that these tumors constitute parts of the broadening spectrum of neuroendocrine skin carcinomas that may derive from Merkel cells.     

Glycogen-rich Clear Cell Carcinoma of the Urethra: An Ultrastructural Study

A 49-year-old black woman developed a urethral glycogen-rich clear cell carcinoma. She was treated with anterior pelvic exenteration. The resected lymph nodes, vagina, uterine cervix, endometrium, ovaries, and urinary bladder were free of neoplasm. Histologically the neoplasm consisted of clear cells growing in sheets and occasional papillary structures. In some areas, hobnail cells were present. Ultrastructurally, the cells had apical caps, short microvilli, and complex cell bases, and contained abundant glycogen. These features were identified in one, but not the other of two previously reported cases. Because glycogen-rich clear cell carcinomas of the lower urinary tract do not resemble ultrastructurally mesonephric remnants or carcinomas known to arise from them, these glycogen-rich clear cell carcinomas should not be called “me-sonephromas” as has been the practice.     

Neuroendocrine Carcinoma of the Skin (Merkel Cell Carcinoma): Ultrastructural and Immunohistochemical Demonstration of Neurofilaments

In this study we characterized a skin tumor that grew in the temporal region of a 69-year-old woman. On the basis of tumor morphology, a metastasis from a small cell carcinoma of the lung was initially suggested, but X-ray and bronchoscopic studies were negative. The tumor recurred twice within a year, yet no tumors were found elsewhere in the body. Ultrastructurally, cytoplasmic organelles compatible with neuroendocrine storage granules and perinuclear aggregates of intermediate-sized (8-10 nm) filaments were found in many tumor cells. Indirect immunofluorescence microscopy revealed neurofilament-type intermediate filaments in the tumor cells but no keratin-or vimentin-type filaments. Our results further demonstrate neural properties of this tumor type, which is generally considered to have its origin from Merkel cells, the cutaneous neuroendocrine cells.     

Neuroendocrine Carcinoma of the Skin (Merkel Cell Carcinoma): Ultrastructural and Immunohistochemical Demonstration of Neurofilaments

In this study we characterized a skin tumor that grew in the temporal region of a 69-year-old woman. On the basis of tumor morphology, a metastasis from a small cell carcinoma of the lung was initially suggested, but X-ray and bronchoscopic studies were negative. The tumor recurred twice within a year, yet no tumors were found elsewhere in the body. Ultrastructurally, cytoplasmic organelles compatible with neuroendocrine storage granules and perinuclear aggregates of intermediate-sized (8–10 nm) filaments were found in many tumor cells. Indirect immunofluorescence microscopy revealed neurofilament-type intermediate filaments in the tumor cells but no keratin-or vimentin-type filaments. Our results further demonstrate neural properties of this tumor type, which is generally considered to have its origin from Merkel cells, the cutaneous neuroendocrine cells.     

Medullary Carcinoma of the Breast: An Ultrastructural Morphometric Study of Nine Cases

Ultrastructural and morphometric features of 10 medullary carcinomas of the breast (MC) were investigated. Cases with a long follow-up were selected by applying stringent histologic criteria. All tumors had a homogeneous appearance by light microscopy. Under transmission electron microscopy, they showed occasional intracellular lumen formation or keratinization. In one tumor squamous differentiation was prominent and diffuse. Tumors with lymph node metastases possessed over 40% more desmosomes than nonmetastatic tumors. The number of cells with three or more nucleoli per nuclear section was significantly higher in metastatic than in nonmetastatic tumors (p = .02). Classic cases of MC of the breast display a relatively uniform appearance. However, subtle differences can be identified between metastatic and nonmetastatic tumors by ultrastructural morphometry. Although these differences are not associated with changes in the outcome of patients in this study, they seem to bear some relationship to the peculiar behavior of MC.     

Medullary Carcinoma of the Breast: An Ultrastructural Morphometric Study of Nine Cases

Ultrastructural and morphometric features of 10 medullary carcinomas of the breast (MC) were investigated. Cases with a long follow-up were selected by applying stringent histologic criteria. All tumors had a homogeneous appearance by light microscopy. Under transmission electron microscopy, they showed occasional intracellular lumen formation or keratinization. In one tumor squamous differentiation was prominent and diffuse. Tumors with lymph node metastases possessed over 40% more desmosomes than nonmetastatic tumors. The number of cells with three or more nucleoli per nuclear section was significantly higher in metastatic than in nonmetastatic tumors (p = .02). Classic cases of MC of the breast display a relatively uniform appearance. However, subtle differences can be identified between metastatic and nonmetastatic tumors by ultrastructural morphometry. Although these differences are not associated with changes in the outcome of patients in this study, they seem to bear some relationship to the peculiar behavior of MC.     

Intraluminal Calcium Hydroxyapatite Crystals in Breast Carcinoma: An Ultrastructural Study

Sixty-three breast carcinomas were examined by electron microscopy to determine the frequency of calcium hydroxyapatite (apatite) within tumor lumina. Twenty-one adenocarcinomas contained apatite in intracytoplasmic and/or intercellular lumina. Well-differentiated tumors exhibited a higher incidence of apatite (44%), while only 20% of the poorly differentiated tumors contained apatite (7 = +.22). There was no apparent correlation between the presence of apatite and a positive estrogen receptor assay. Ninety-eight adenocarcinomas of other than breast origin (previously processed for electron microscopy) were examined, revealing 2 cases containing apatite in the appropriate locations. The tissue of origin in one case was determined to be ovarian, while the origin of the second case remains undetermined. The ultrastructural finding of apatite in lumina of adenocarcinoma appears to be unusual in that it has only been observed in breast carcinomas and certain ovarian tumors. The presence of apatite within the lumen in addition to other characteristics of an adenocarcinoma may suggest the breast as the primary site.     

Gastric Carcinomas With Unusual Cytoplasmic Ultrastructural Features

Alterations in tannic acid (TA) binding capacity of cell surface carbohydrates in normal, premalignant, and malignant squamous epithelium of the human uterine cervix have been studied using electron microscopic visualization in combination with microdensitometric evaluation.

While in normal epithelium there is distinct binding in four to five cell layers of the deep intermediate zone, cells of carcinoma in situ and invasive cancer lesions lack TA binding. In moderate dysplasia an intermediate reacting pattern is found.

Deep intermediate cells in areas bordering the carcinoma in situ lesions do not show any binding, although their ultrastructure cannot be distinguished from similar cells in normal tissue.

The TA deposition within the deep intermediate zone is probably related to the presence here of glycoprotein-containing membrane-coating granules.

The finding that TA binding discriminates between cells in normal squamous epithelium and morphologically normal cells in juxtaposition with lesional areas in premalignant and malignant epithelium opens the possibility for a more reliable cytologic diagnosis of cervical epithelial neoplasia.     

Primary Small Cell Neuroendocrine Carcinoma of the Kidney

The histological, immunohistochemical, and ultrastructural features of a primary small cell neuroendocrine carcinoma of the renal parenchyma are described. Tumor cells were positive for cytokeratin, neuron-specific enolase, and Leu 7, but Grimelius- and chromogranin-negative. They also exhibited positivity with anti-MIC2 antibody. By electron microscopy, tonofibrils, primitive desmosomes, and dense-core granules with a neuroendocrine appearance were present. This is only the 7th recorded example of such a tumor at this site, which showed an aggressive course characterized by widespread bony metastases.     

Surface Topography and Ultrastructural Changes of Mucinous Carcinoma Breast Cells

Mucinous carcinoma of the breast (MCB) is histologically classified into 2 groups: (1) pure MCB and (2) mixed MCB. Pure MCB carries a better diagnosis than mixed MCB. This research relates to the cell surface topography and ultrastructure of the cells in the above cases and aims to find the differences between them, by means of two methods: scanning electron microscopy (SEM) and transmission electron microscopy (TEM). For the SEM examination, it was necessary to initially culture the MCB tissues and then proceed with the usual SEM method. In contrast, for the TEM technique, MCB tissues were initially fixed followed by the classic TEM method. The authors found the topography of pure MCB cases to be without nodes. The cell membrane was smooth, with numerous pores and small ruffles that covered the entire cell. The ultrastructural appearance of the same cases was with a normal cell membrane containing abundant collagen fibers. They also had many small vesicles containing mucin as well as secretory droplets. In contrast the mixed MCB had a number of lymph nodes and their cell surface topography showed stronger changes such as microvilli, numerous blebs, ruffles and many long projections. Their ultrastructure showed very long microvilli with large cytoplasmic inclusions and extracellular mucin collections, electron-dense material vacuoles, and many important cytoplasmic organelles. An important fact is that mixed MCB also contains areas of infiltrating ductal carcinoma. These cells of the cytoplasmic organelles are clearly responsible for the synthesis, storage, and secretion of the characteristic mucin of this tumor type. Evidently, this abnormal mucin production and the abundance of secretory granules along with the long projections observed in the topographical structure might be responsible for transferring tumor cells to neighboring organs, thus being responsible for metastatic disease.