The nature of breast dense core granules: chromogranin reactivity

Ultrastructural immunoreactivity for chromogranin with the LK2H10 antibody was examined in nine cases of breast carcinoma and one example of normal resting breast. These tissues were selected for study on the basis of argyrophilia or LK2H10 immunostaining by light microscopy. In two cases, positive reactivity with the chromogranin antibody was seen in breast neuroendocrine-like dense core granules. In a further six cases chromogranin reactivity was not seen in similar granules that showed the neuroendocrine properties of ultrastructural argyrophilia and uranaffinity. Inability to exhibit the full complement of neuroendocrine characteristics in breast carcinomas contrasts with the facility to demonstrate them in tissues of usual neuroendocrine differentiation. Furthermore, in two mucoid carcinomas and one example of normal resting breast ultrastructural cytoplasmic LK2H10 reactivity was evident, which was not localized to dense core granules, although these were present in two of the cases. Our findings demonstrate the heterogeneity of breast dense core granules and discourage acceptance of such characteristics as evidence of histogenesis of these carcinomas from a neuroendocrine cell type in breast tissue.     

Distribution of dense core granules in normal, benign and malignant breast tissue

In this study breast tissue from 114 patients has been examined ultrastructurally for dense core granules (DCG). The tissue included examples of normal 'resting', pregnant and lactating breast plus various benign and malignant lesions. DCG were observed in low numbers in the apical cytoplasm in a proportion of the examples of 'resting' and pregnant breast tissue but were absent in the lactating patients. The incidence appeared to relate to hormonal changes. They were present in 50 per cent of the benign lesions examined. DCG were also observed in a high proportion of the ductal, lobular and tubular carcinomas examined and were associated with luminal differentiation. In the mucoid carcinomas over half the tumours possessed some DCG with large numbers of DCG present within certain of the malignant cells in two cases. It is possible that the granules could be related to mucin secretion. Therefore, in normal, benign and malignant (with the exception of mucoid carcinoma) breast tissue the presence of DCG would appear to be related to hormonal changes and represent prelactational differentiation rather than providing evidence of neuroendocrine differentiation. We emphasize the need for a comprehensive knowledge of the normal morphological variations within a tissue before attempting to interpret its tumours.     

Neuroendocrine differentiation in male breast carcinomas.

The presence of neuroendocrine differentiation, as expressed by cellular chromogranin immunoreactivity, was investigated in paraffin-embedded tissue material from 51 consecutive cases of male breast carcinoma. From six of these cases electron microscopic studies were included. Chromogranin-immunoreactive cells were present in solid cords and delineated tubular structures. Ultrastructurally, dense core secretory granules could be detected. The expression of neuroendocrine differentiation was 45%, which is between two and eight times higher than reported for female breast carcinomas by other investigators. The present findings suggest that male breast carcinoma is an exclusive tumour disease showing both similarities and discrepancies when compared to its female counterpart.     

Ultrastructural Features of Neuroendocrine Differentiated Carcinomas of the Breast

The ultrastructural patterns of neuroendocrine (NE) differentiated breast carcinomas are analyzed and discussed. Reports in the literature describe wide variations in the size of observed dense-core membrane-bound granules and discrepancies in their interpretation. In the present study 24 cases of breast carcinoma with recognized morphologic, histochemical, and immunocytochemical features of NE tumors were investigated. Five different types of dense-core granules of neurosecretory (NS) type (confirmed by the ultrastructural localization of chromogranin A) and five different cell types were recognized. Some amphicrine cells were found to contain both mucin and NS granules. Another notable ultrastructural feature of breast NE carcinomas was the presence of clear vesicles of presynaptic type, which correlated with expression of synaptophysin.     

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.     

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.     

Primary Large Cell Neuroendocrine Carcinoma of the Breast: Radiologic and Pathologic Findings

Some breast neoplasms are classified as primary neuroendocrine carcinomas because they are positive for neuroendocrine markers. Although neuroendocrine carcinomas can originate from various organs of the body, primary neuroendocrine carcinomas of the breast are extremely rare. The diagnosis of primary neuroendocrine carcinoma of the breast can only be made if nonmammary sites are confidently excluded or if an in situ component can be found. Here we report a primary large-cell neuroendocrine carcinoma (LCNL) involving the left breast. Breast ultrasonography revealed a lobulated, heterogeneous, low-echoic mass in the left breast, and the lesion ap-peared as a well-defined, highly-enhancing mass on a chest computed tomography scan. Ultrasound-guided core needle biopsy was performed on the mass, and primary LCNC was confirmed by histopathologic examination.     

Atypical endocrine granules in atypical endocrine tumor (AET) of the lung. An immunoelectron microscopic study

Highly dense granules are a hallmark for recognizing atypical endocrine tumor (AET) of the lung. We report a case of AET with many atypical neurosecretory-type granules: moderately dense granules (mean size 373.7 nm) and "target" granules with a central dense core (425.1 nm), both apparently larger than the highly dense granules (223.3 nm). Immunoelectron microscopical studies demonstrated that all three types of granule were positive for gastrin-releasing peptide (GRP), human chorionic gonadotropin alpha-subunit (hCG alpha), calcitonin or serotonin. Although the size profiles of positive granules were similar for calcitonin and hCG alpha, they were different from those of GRP or serotonin granules. The presence of atypical granules and the different size profiles of hormonal products in AET indicate that caution is required in ultrastructural evaluation of granules in lung carcinomas.     

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.     

Argyrophilic cells in carcinoma of the female breast

Summary Argyrophilic hormone storage granules were sought in the tissue specimens obtained from 52 benign breast lesions (13 normal breasts, 27 cases of fibrocystic disease, 9 fibroadenomas, 2 intraductal papillomas, and 3 cases of gynecomastia) and from 90 adenocarcinomas of the female breast. No argyrophilic cells were found in the normal breast tissue or in the benign lesions studied. In three of the carcinomas (3.3%) such granules were found in the tumor cells. Using electron microscopy, the argyrophilic granules were shown to be of moderate or high electron density with an average diameter of 165 to 170 nm. Ectopic hormone production was not observed clinically in any of these three patients. The absence of argyrophilic cells in normal and benign ductal and acinar epithelium, and their occasional presence in breast carcinomas favors the concept of the histogenesis of these cells through genomic derepression during the course of neoplastic transformation.This work was supported by the grant from E. Aaltonen Foundation     

Eosinophilic and granular cell tumors of the breast.

Eosinophilic and granular cell tumors of the breast are a heterogeneous group encompassing both epithelial and mesenchymal lesions. A granular appearance of the cytoplasm may be caused by the accumulation of secretory granules, mitochondria, or lysosomes. In the breast, mucoid carcinomas, carcinomas showing apocrine differentiation, and neuroendocrine carcinomas are well known entities, while tumors with oncocytic and acinic cell differentiation have been only recently recognized. An abundance of lysosomes is characteristic of Schwannian granular cell neoplasms, but smooth muscle cell tumors also may have this cytoplasmic feature. Awareness of all these possibilities when granular cells are found in breast lesions improves diagnostic accuracy and helps to avoid misdiagnosis of both benign lesions and malignant tumors.     

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.     

Ultrastructural characteristics and variations in human mucinous breast carcinomas

Nineteen human mucinous breast carcinomas have been studied by electron microscopy in order to investigate the variations in the fine structure of this type of tumor. All the investigated tumors are characterized by a well-developed, rough endoplasmic reticulum and prominent Golgi complexes. The majority of tumor cells contain secretory granules. Approximately 50% (9/19) of the tumors have cytoplasmic dense core granules that are morphological identical to the neurosecretory granules found in APUD-cell derived tumors. Six out of the nine tumors react positively in a Grimelius staining for light microscopic argyrophilic granules. Two of the investigated breast carcinomas contain tonofilaments that are normally regarded as characteristic of squamous epithelium. It is concluded that mucinous breast carcinomas--that to this time have been regarded as a morphological homogenous group of tumors--are ultrastructurally characterized by heterogeneity.     

Immunogold Detection of Chromogranin a in the Neuroendocrine Tumor

Immunogold ultrastructural localization of chromogranin A in secretory granules within tumor cells provides convincing evidence of endocrine or neuroendocrine differentiation. A modified immunogold method (designed for use on osmicated tissue) produced positive labeling of small granules not only in well-differentiated tumors but also in poorly differentiated small cell tumors primary in lung, cervix, and skin; only a proportion of granules in some of the tumor cells were positively labeled. Many non-small cell lung tumors often stain focally positive for chromogranin A at the light microscopy level, and such tumors may also contain sparse, small, dense granules. Because positive labeling could not be demonstrated over small granules in non-small cell lung tumors, the theory that such tumors are neuroendocrine in type may be erroneous.     

Usefulness of Electron Microscopy in the Diagnosis of “Small” Round Cell Tumors of the Sinonasal Region

The sinonasal region is known to harbor several types of tumors that belong to the general category of “small” round cell tumors and offer considerable diagnostic challenges. This study evaluated 33 cases of such tumors by electron microscopy to characterize their ultrastructural features in conjunction with immunohistochemistry, in an attempt to define diagnostic criteria of various types. Electron microscopy was useful in the proper classification of tumors in 27 cases: esthesioneuroblastoma (EN), 12; undifferentiated carcinoma, 6; melanoma, 3; lymphoma, 3; melanotic neuroectodermal tumor, 1; rhabdomyosarcoma, 1; and pituitary adenoma, 1. In the remaining six cases, the ultrastructural features were those of poorly differentiated carcinomas. They usually exhibited some epithelial characteristics as well as neuroendocrine features by immunohistochemistry and electron microscopy. These tumors could be best described as poorly differentiated neuroendocrine carcinomas (malignant neuroepitheliomas). The most controversial diagnostic problems existed between the tumors categorized as esthesioneuroblastomas and neuroendocrine (NE) carcinomas. Esthesioneuroblastomas were characterized by uniform round nucleated cells with variable amounts of dendritic processes containing numerous dense core granules ranging from 150 to 350 nm in the perikarya and dendritic processes. Dendritic processes contained longitudinally arranged neural tubules and revealed an occasional synaptic junction. In three of the 12 cases of EN, cells with the appearance of sustentacular cells were recognized by electron microscopy. The NE carcinomas usually consisted of closely packed round cells with scanty cytoplasm that lacked any feature of neuroblastic cells. The tumor cells in this category often were epithelioid in appearance and exhibited a varying degree of cytokeratin positivity. Neuron-specific enolase was also positive in all cases, further suggesting their neuroepithelial nature. The greatest difference between EN and NE carcinomas was the absence of sustentacular cells in NE carcinomas. Immunohistochemical and electron microscopic studies are essential in the differential diagnosis of EN and NE carcinomas, because their microscopic appearance is very similar. The study indicates that EM is useful in the diagnostic categorization of sinonasal tumors of uncertain nature, particularly when it is used in conjunction with immunohistochemistry.     

Ultrastructural and electron immunohistochemical features of medullary thyroid carcinoma

Summary An ultrastructural study, both morphological and immunohistochemical, has been carried out on eight thyroglobulin-positive and nine thyroglobulin-negative medullary carcinomas of the thyroid. The morphometric analysis of granule size showed that all tumours contained cells with small granules and cells with medium size granules, whereas eight tumours had additional cells with large granules. The small granules had an electron dense core, while the medium and large sized granules were both pale-cored and dense-cored. The cells with small, medium or large secretory granules were all immunoreactive for calcitonin and CGRP. No ultrastructural differences were observed between thyroglobulin-positive and thyroglobulin-negative cases of medullary carcinoma of the thyroid.     

Ultrastructural morphometry distinguishes Burkitt's-like lymphomas from neuroendocrine neoplasms: useful criteria applied to the evaluation of a poorly differentiated neuroendocrine neoplasm of the nasal cavity masquerading as Burkitt's-like lymphoma

The present study describes the potential usefulness of ultrastructural morphometry in diagnosis. Ultrastructural morphometric criteria were applied to the evaluation of a poorly differentiated neuroendocrine neoplasm of the nasal cavity that was initially thought to be a Burkitt's-like lymphoma (BLL). Although the nasal lesion in question failed to stain with over 50 cell lineage-relevant antibodies, it did stain for vimentin (an intermediate filament protein) and Ki-67 (a nuclear antigen associated with cell proliferation). Routine electron microscopy revealed a primitive neoplasm with abundant cytoplasmic lipid droplets and sparse dense granules with no intercellular junctions. Treatment options, which included extensive facial surgery, prompted more study. Tissue processed for the uranaffin reaction revealed sparse uranaffin-positive granules indicating the presence of true neurosecretory granules. An ultrastructural morphometric analysis of the neoplastic nuclei of this patient placed the tumor outside the morphometric domains for BLLs (18 cases) and neuroblastomas (11 cases) and within the morphometric domain of neuroendocrine carcinomas (9 cases). A greater mean standard deviation (P less than 0.05) and mean coefficient of variation (P less than 0.02) of nuclear perimeter in the neuroendocrine (NE) group related to the BLL group indicated greater nuclear pleomorphism within the NE group as illustrated in bivariate graphic displays. The possible origin of the neoplasm within the nasal mucosa is discussed.     

Chromogranin-reactive endocrine cells in argyrophilic carcinomas ("carcinoids") and normal tissue of the breast

Breast carcinomas, either positive or negative with the Grimelius' silver procedure, benign fibroadenomas, duct papillomas, and areas of histologically normal breast tissue were tested immunocytochemically with the mouse monoclonal antibody LK2H10 directed against human chromogranin. This is regarded as a general stain for polypeptide-hormone-producing cells and tumors. In 3 of the 9 cases of argyrophilic carcinoma, but in none of 12 ductal infiltrating carcinomas, chromogranin-positive cells were found: the number of reactive cells was very low in 1 case, while in the other 2 carcinomas about 50% of the argyrophilic cells appeared stained. In areas of histologically normal breast tissue, rare argyrophilic chromogranin-positive cells were detected. This study is the first reported evidence concerning the presence of endocrinelike cells probably belonging to the diffuse neuroendocrine system in the normal mammary parenchyma. Our data are consistent with the endocrine nature of at least some of the breast argyrophilic carcinomas.     

Gastric neuroendocrine neoplasms: tumour clonality and malignancy-associated large X-chromosomal deletions.

Type I gastric carcinoid tumours associated with corporal (body of stomach) atrophic gastritis (CAG) are benign tumours developing as the final step of a hyperplastic precursor sequence. The neoplastic nature of these tumours has been assumed but never proved. Type III gastric carcinoid tumours and neuroendocrine carcinomas are malignant neoplasms without known precursor lesions. To assess the neoplastic nature of type I carcinoids, the clonal status of 35 tumours from 23 female patients was investigated using the human androgen receptor (HUMARA) gene test, which is based on the pattern of X-chromosome inactivation. For comparison, the same test was also performed on four type III carcinoids and two neuroendocrine carcinomas. DNA extracted from paraffin sections was digested with Hha I restriction enzyme and then amplified by polymerase chain reaction (PCR) using established HUMARA primers. The PCR products were analysed in an automated DNA sequencer. In a complementary analysis of the same tumours, loss of heterozygosity (LOH) on the X chromosome was studied using three polymorphic markers (DXS989, DXS1003, DXS1192) in a PCR-microsatellite-based technique. After exclusion of non-informative cases, 14 of 16 type I carcinoids were found to be monoclonal on the basis of the pattern of X-chromosome inactivation. Monoclonality was also documented in one of three type III carcinoids and in the single neuroendocrine carcinoma, on the basis of LOH at the HUMARA locus, which per se can be regarded as evidence for clonality. Extensive LOH of the X chromosome involving at least two markers, was found in all metastasizing tumours (two type III carcinoids and two neuroendocrine carcinomas), but in none of the 27 benign carcinoids of types I and III. These results indicate that most type I carcinoids are true monoclonal neoplasms and that malignant evolution in gastric neuroendocrine tumours is associated with extensive allelic deletion of one X chromosome.