Immunohistochemical demonstration of pancreatic secretory trypsin inhibitor in normal and neoplastic colonic mucosa.

Specimens of normal and neoplastic colonic mucosa from 52 patients were analysed by immunohistochemistry using a monospecific polyclonal antiserum against human pancreatic secretory trypsin inhibitor (PSTI). In normal colonic mucosa PSTI was found in the goblet cells in the basal parts of the crypts. In adenomas of tubular, villous, and tubulo-villous types PSTI was also found in the upper parts of the polyps, usually occurring in the regeneration zone. There was a more intense staining reaction in polyps with increased atypia. Carcinomas of different types and of various grades of differentiation and of in situ type did not contain PSTI. These findings indicate that PSTI could be a marker for adenomatous rather than carcinomatous epithelium in the colon. Furthermore, the absence of the inhibitor in malignant cells might facilitate tissue invasion by malignant cells because of deficient protease inhibition.     

Immunohistochemical demonstration of lysozyme in normal, reactive and neoplastic cells of the mononuclear phagocyte system

Using the peroxidase antiperoxidase (PAP) method, lysozyme (LZM) was shown to exist in normal, reactive and neoplastic cells belonging to the mononuclear phagocyte system (MPS), but was not detected in histiocytosis X cells. Immunostaining for cytoplasmic LZM by the PAP method is useful for identification of mononuclear phagocytes and for diagnosis of the diseases in which these cells participate.     

Immunohistochemical distribution of S-100 protein and glial fibrillary acidic protein in normal and neoplastic salivary glands

Summary Immunohistochemical localization of S-100 protein, its and subunits, and glial fibrillary acidic protein (GFAP) in normal and neoplastic salivary glands was studied by the peroxidase-antiperoxidase method and immunoblot analysis. Positive immunostaining for S-100 protein was observed in pleomorphic adenoma, adenolymphoma, tubular adenoma, adenoid cystic carcinoma, acinic cell tumour, adenocarcinoma and carcinoma in pleomorphic adenoma. S-100 protein was localized in myoepithelial cells, epithelial cells of intercalated ducts and serous acinar cells of normal salivary gland. Both and subunits of S-100 protein showed almost identical distribution in normal and neoplastic salivary glands, but skeletal muscle cells were -positive/-negative whereas Schwann cells and fat cells were -negative/-positive in the stroma and neighbouring tissue. GFAP was only found in pleomorphic adenoma and its malignant counterpart. Immunoblot analysis showed that the GFAP-related antigen consisted of several polypeptide bands with a molecular weight ranging between 35,000 to 50,000 daltons.     

The synthesis of amylase in parotid glands of young and old rats

The age-related changes in the rate of synthesis of total and secretory proteins were examined in parotid glands of young (2 months) and old (24 months) rats. The differences in the rate of incorporation of radioactive leucine into acid-insoluble proteins of the gland indicate that the rate of protein synthesis declines with age in this gland. To determine whether the rate of synthesis of secretory proteins changes with age in this gland, the rates of incorporation of [3H]leucine into amylase, a major secretory protein of the gland, were compared by radioactivity determinations. For this comparison, amylase was precipitated with glycogen after incubating the gland slices in the presence of the labeled amino acid. The study shows that rate of synthesis of amylase declines significantly with age in this gland. The possible relationship between the decline in protein synthesis and the reduced level of secretory activity of the gland due to aging is discussed.     

Intermediate filament expression in normal parotid glands and pleomorphic adenomas

Summary A comparative immunohistochemical study of intermediate filament expression in normal parotid glands and pleomorphic adenomas (PA) was performed using material fixed in a modified methacarn fixative. The normal myoepithelial cells of acini stained only with monoclonal antibodies 312C8-1 (cytokeratin (CK) 14) and 4.62 (CK 19) while myoepithelial/basal cells of ducts also reacted with antibodies 8.12 (CK 13, 16), 8.60 (CK 10, 11, +1), and PKK1 (CK 7, 8, 17, 18). Normal duct luminal cells showed a different CK profile, reacting consistently with ECK, a polyclonal antibody to epidermal prekeratin (CK 3,6), and monoclonal antibodies 4.62, PKK1 and 8.60. In PA, tumour cells at the periphery of ducts, in solid areas, and at the edge of myxoid regions all had CK profiles similar to normal myoepithelial/ basal cells except that antibody 4.62 was generally negative. Vimentin and glial fibrillary acidic protein (GFAP) were uniformly negative in normal parotids but showed variable (often strong) reactivity with some cells in chondroid, myxoid and solid areas of PA. A surprising feature of most PA was the variability of CK subtype expression not only from one case to another but also within morphologically similar areas of the same specimen. These results suggest that the morphology of PA is the result of diversity of tumour cell differentiation rather than the processes implicit in a reserve cell histogenetic model.     

Immunohistochemical demonstration of insulin-like growth factor I (IGF-1) in normal and pathological human pituitary glands.

The authors have studied the presence and distribution of Insulin-Like-Growth-Factor-1 (IGF-1) in 5 autopsied normal and 20 surgically removed human pituitary adenomas, employing a peroxidase-anti-peroxidase method. IGF-1 could be demonstrated in all cases, with variation of cells immunostaining from 60% in normal pituitary gland to 100% in corticotroph cell adenoma.     

Immunohistochemical analysis for desmin in normal and neoplastic ovarian stromal tissue

Ovarian stroma contains cells with the ultrastructural features of smooth-muscle cells. The purpose of this study was to analyze normal ovaries, ovarian stromal tumors (fibroma/thecomata and granulosa cell tumors), and ovarian leiomyomata for desmin reactivity. Groups of ovarian stromal cells that expressed desmin were noted in six of six normal ovaries. Desmin was also detected in two of six fibroma/thecomata and two of two ovarian leiomyomata. The number of tumor cells with detectable desmin was much greater in the leiomyomata. Desmin was not detected in any of six granulosa cell tumors. We conclude that stromal cells with an immunohistochemical feature of smooth-muscle cells are routinely found in normal ovaries. This study demonstrates the usefulness of immunohistochemistry in corroborating the diagnosis of ovarian leiomyomata, although desmin positivity per se is not diagnostic of ovarian leiomyomata, and also raises the possibility that some ovarian leiomyomata may be derived from stromal cells.     

Nucleolar organizer regions in normal,hyperplastic and neoplastic parathyroid glands

Summary Using a one-stage silver staining technique, nucleolar organizer regions (AgNORs) were studied in paraffin sections of parathyroid glands (and in two lymph node metastases) from patients operated upon because of hyperparathyroidism or thyroid disease. The parathyroids were microscopically differentiated into normal, hyperplastic, adenomatous and carcinomatous glands. AgNORs were observed as distinct black dots of varying size and somewhat varying configuration in the nuclei of all glands. The mean number of AgNORs in the hyperplastic and adenomatous glands was not significantly different from that in the normal glands, whereas the carcinomatous glands exhibited significantly increased mean AgNOR number. No evidence was obtained for a role of AgNOR counting in the differentiation between normal and hyperplastic or adenomatous parathyroids, but the results suggest a potential role of enumeration of AgNORs in the discrimination between benign and malignant parathyroid neoplasms.     

Immunohistochemical demonstration of nonspecific cross-reacting antigen in normal and neoplastic human tissues using a monoclonal antibody. Comparison with carcinoembryonic antigen localization

Nonspecific cross-reacting antigen (NCA) immunoreactivity was localized in normal and neoplastic human tissues using a monoclonal antibody to 55, 90 and 95 kDa molecules of NCA. This was compared to the localization of immunoreactive carcinoembryonic antigen (CEA) as demonstrated by polyclonal and monoclonal antibodies. In frozen sections, CEA was localized in normal surface epithelium of the stomach and colon where NCA was only weakly detected. Type 1 and type 2-like pneumocytes were positive for NCA, while CEA was localized only in type 2-like pneumocytes. CEA and NCA were both demonstrated in ductal cells of frozen pancreatobiliary and mammary tissues. The antigenicity of CEA and NCA in normal tissues was significantly lost after paraffin embedding as compared to frozen sections. NCA was consistently demonstrated in eccrine sweat glands embedded in paraffin. In various tumor tissues, CEA and NCA were colocalized and expression increased sufficiently to be detected in paraffin sections. Adenocarcinomas of the stomach and colon and cystadenocarcinoma of the pancreas, as well as neuroendocrine carcinomas of the lung and thyroid, showed a CEA predominance over NCA. In ductal adenocarcinomas of the pancreas and breast and in cholangiocarcinoma, NCA reactivity was greater than CEA. Keratinizing foci of most squamous cell carcinomas of mucosal origin and some adenocarcinomas equally expressed both. Hepatocellular carcinoma, lobular mammary carcinoma and papillary thyroid carcinoma were positive only with unabsorbed polyclonal antibody which widely recognizes CEA-related substances. Renal cell carcinoma, prostatic adenocarcinoma, transitional cell carcinoma, anaplastic carcinomas, choriocarcinoma and basal cell carcinomas showed little or no immunoreactivity. Hence the relative ratio of CEA/NCA expression in tumors was dependent on the tissue of origin and histologic type. The cytoplasmic granular staining of NCA in cancer cells was a noteworthy difference from the plasma membrane-associated localization of CEA.     

Immunohistochemical studies of beta-hexosaminidase in neoplastic and normal tissues with monoclonal antibodies.

A total of 87 human specimens with 10 histological types of primary neoplasm were studied immunohistochemically with monoclonal antibodies specific for beta-hexosaminidase (Hex). High levels of Hex were found in malignant neoplasms of the skin, cervix, colorectum and in benign as well as neoplastic plasma cells, while no activity was detected in normal epidermis, normal colorectal epithelium or benign naevi. The strongest immunohistochemical reaction was revealed in tumor cells of malignant melanoma. Adenomas and adenocarcinomas of the colorectum showed high levels of Hex with a basal pattern of immunoreactivity more frequent in the tumor cells of adenocarcinomas than adenomas. Fibroblasts and macrophages in the tumors often disclosed immunoreactivity. In most of the sections (including those from plasma cell neoplasms), 7E4 antibody showed low immunoreactivity compared to 2E3, except for non-neoplastic plasma cells, which were as a rule positive with 7E4 and largely negative with 2E3 antibody. This result probably indicated different isoenzymes in benign and neoplastic plasma cells.     

Immunohistochemical demonstration of keratins in the cysts of thyroid glands,parathyroid glands,and C-cell complexes of the dog

Cyst structures were often detected in and around thyroid glands of the dog. The present study revealed the frequency of occurrence, the light microscopic features, and the immunoperoxidase reactions to anti-keratin and anti-ldS-thyroglobulin antisera of each cyst located in parathyroid III, parathyroid IV, thymus IV, C-cell complexes, and thyroid parenchyma from 112 dogs. In each location, cysts showed characteristic features. In parathyroid III, the cysts were covered with single or pseudostratified epithelium composed of ciliated cells; whereas in parathyroid IV they were covered with keratinizing stratified squamous epithelium. In C-cell complexes, small cysts lined with small packed cells were predominant, and large cysts lined with single cuboidal cells or stratified squamous cells were also present. In thymus IV located in the close vicinity of parathyroid IV, cyst epithelium consisted of several types of cells showing variable futures. In thyroid parenchyma, there were several types of cysts: some were covered with ciliated columnar cells, and others were covered with two or multilayers of small packed cells or cuboidal cells. In spite of these differences in appearance of the cysts located in different tissues, all their epithelia were immunoreactive to the keratin antisera, except for small cysts in C-cell complexes, which were regarded as immature structures. Thus, the presence of keratin filaments in epithelial cells seems to be a characteristic feature of all cysts. The lumens of each cyst contained variable amounts of amorphous materials, which showed colloid-like, flocculent, foamy, and granular features and were periodic acid-Schiff-positive in variable degrees, from weak to intense. Although the lumenal contents of the cysts in parathyroid III revealed no immunoreactivity for 19S-thyrogIobulin, those in thyroid parenchyma, C-cell complexes, parathyroid IV, and thymus IV reacted strongly with the 19S-thyroglobulin antiserum.     

The expression and localization of amylase in normal and malignant glands of the endometrium and endocervix

Amylase activity was studied in 70 specimens of normal endometrium, 21 normal endocervices, 19 endometrial carcinomas, and 20 endocervical adenocarcinomas. Amylase was observed in the secretory (8.7 per cent) but not in the proliferative phase of the menstrual cycle. It is possible that the presence of amylase activity may serve a functional role in the degradation of glycogen to glucose in the secretory endometrium. The great majority (90.5 per cent) of uterine cervices showed strong and extensive staining of the endocervical glands for amylase. No glycogen was demonstrated and the role of amylase in endocervical glands remains obscure. Amylase was observed in one (5.3 per cent) out of 19 cases of endometrial carcinoma, and the presence of this enzyme may be considered a eutopic rather than an ectopic expression. Amylase was not detected in any of the endocervical adenocarcinomas examined. This study has shown a complete loss of amylase activity in malignant transformation of endocervical glands and this could be attributable to the immature nature of de-differentiated neoplastic cells.     

Homology between the female paraurethral (Skene's) glands and the prostate. Immunohistochemical demonstration

Homology between female paraurethral glands and the prostate has often been suggested. A means was developed that would lend histochemical support to this hypothesis. Female urethra from autopsy and surgical material was serially sectioned and studied in 19 patients ranging in age from newborn to 86 years. Paraurethral glands were identified in 18 of these. The tissue was stained with antibodies to prostate-specific antigen (PSA) and prostate-specific acid phosphatase (PSAcPh) using the peroxidase-antiperoxidase method. Of those cases in which paraurethral glands were seen, 83% were positive for PSA and 67% for PSAcPh. Intensity of staining was semi-quantitatively evaluated. In addition, intraluminal secretions and urethral columnar epithelium showed positive enzyme and antigen staining. There was no discernible variation of glandular development or pattern of staining with patient age. This study demonstrates the homologous nature of the female paraurethral glands and the prostate and supports speculations about functional similarity.     

Immunohistochemical analysis of Mcl-1 and Bcl-2 proteins in normal and neoplastic lymph nodes.

The Bcl-2 protein blocks programmed cell death and becomes overproduced in many follicular non-Hodgkin's lymphomas as the result of t(14; 18) translocations involving the Bcl-2 gene. Mcl-1 is a recently discovered gene whose encoded protein has significant homology with Bcl-2 but whose function remains unknown. In this study, we compared the in vivo patterns of Bcl-2 and Mcl-1 protein production in normal and neoplastic lymph node biopsies by immunohistochemical means using specific polyclonal antisera. Intracellular Mcl-1 immunoreactivity was located primarily in the cytosol in a punctate pattern and was also seen in association with the nuclear envelope in many cases, similar to the results obtained for Bcl-2, which resides in the outer mitochondrial membrane, nuclear envelope, and endoplasmic reticulum. In 4 of 4 reactive tonsils and 28 of 28 nodes with reactive follicular hyperplasia, reciprocal patterns of Bcl-2 and Mcl-1 protein expression were observed. Bcl-2 immunostaining was highest in mantle zone lymphocytes and absent from most germinal center cells, whereas Mcl-1 immunoreactivity was highest in germinal center lymphocytes and absent from mantle zone lymphocytes. Mcl-1 was also expressed in some interfollicular lymphocytes, particularly those that had the appearance of activated lymphocytes. Similar to the patterns of Bcl-2 and mcl-1 expression seen in reactive nodes, Mcl-1 protein was largely absent from the malignant cells in 2 of 2 mantle cell lymphomas, whereas strong Bcl-2 immunostaining was found in these cells. In contrast to normal nodes, however, the neoplastic follicles of t(14;18) containing follicular non-Hodgkin's lymphomas immunostained positively for both Bcl-2 and Mcl-1 in 24 of 27 cases. Intense immunostaining for Mcl-1 was also observed in Reed-Sternberg cells in 2 of 2 cases of Hodgkin's disease but Bcl-2 immunoreactivity was present at much lower levels. These findings demonstrate that the levels of Mcl-1 and Bcl-2 proteins are differentially regulated in normal and neoplastic cells in lymph nodes and thus suggest different roles for these proteins in the control of cell life and death in these tissues.     

Immunohistochemical localization of chromogranins A and B and secretogranin II in normal, hyperplastic and neoplastic prostate

Routinely processed normal, hyperplastic and neoplastic prostatic tissue was immunohistochemically investigated with antibodies against chromogranin A and B and secretogranin II. In normal and hyperplastic prostates all three peptides were immunolocalized in scattered neuroendocrine cells situated within the glandular epithelium. In 17 prostatic carcinomas with pronounced neuroendocrine differentiation and in a case of prostatic carcinoid, chromogranin B was the major component whereas chromogranin A and secretogranin II were virtually absent in poorly differentiated (grade III) tumours. Neuroendocrine differentiation in prostatic cancer is most likely to be associated with a poor clinical outcome; thus, chromogranin B appears to be a useful marker in the histopathological diagnosis of these neoplasms.     

Prostate secretory granules in normal and neoplastic prostate glands: a diagnostic aid to needle biopsy

The recent increased efficacy of diagnosing prostate carcinoma from needle biopsy can be attributed to the accelerated biopsy rate as a result of cancer screening, the greater number of core samples per set, and the increased ability to identify malignancy in progressively smaller gland foci. This improvement in histological judgement has been facilitated by more sophisticated histological criteria, which in turn depend largely on an increasing knowledge of normal histological features and their abnormal counterparts. The recent discovery of the prostate secretory granule (PSG) as part of the normal secretory mechanism has prompted our study of the PSG as a possible additional criterion for distinction between benign and malignant cells in biopsy samples. The proper delineation of PSG required glutaraldehyde-based fixation, but this change in fixation showed additional diagnostic advantages. We quantitated PSG depletion in 150 sequential core biopsy samples, evaluating benign epithelium, dysplasia (PIN), Gleason grade 3, and grade 4 carcinoma separately. Overall, 80% of carcinomas and 63% of high-grade dysplasias were markedly depleted of PSG such that no granules were seen at low-power magnification with routine haematoxylin and eosin stains. This contrast between benign and malignant epithelium was especially prominent in small carcinoma foci greatly assisting in cancer recognition. Comparison between all groups showed an advantage of glutaraldehyde-based tissue fixation over formalin fixation for prostate needle biopsy specimens, providing clear resolution of cytological detail a well as an additional histologic criterion for cancer diagnosis. HUM PATHOL 31:1515-1519.     

Immunoelectron microscopy of amylase in the human parotid gland

Summary Specimens of the human parotid gland were studied by immuno-electron microscopy for the presence of amylase. Both the protein A-gold technique and the biotin-avidin-gold technique were used on the same specimens. Different fixations were tried. Amylase was detected in the zymogen granules in high amounts. This enzyme could even be seen in glutaraldehyde fixed and routinely embedded material. The subcellular localization of this enzyme opens a new field of functional morphological studies and studies in special tumours including acinic cell carcinomas.Supported by the Deutsche Forschungsgemeinschaft and by the Hamburger Stiftung zur Förderung der KrebsbekämpfungDedicated to Prof Dr. med. Dres. h.c. Wilhelm Doerr, Heidelberg, in honour of his 70th birthday