Sadaaki Hori

Immunohistochemical studies on oncogene products (c-erbB-2, EGFR, c-myc) and estrogen receptor in benign and malignant breast lesions. With special reference to their prognostic significance in carcinoma.

It is a matter of debate whether the amplification of c-erbB-2 oncogene or production of the oncoprotein in breast cancers correlate with the presence of lymph node metastasis and with a poor prognosis. This study was aimed at elucidating the immunohistochemical localization of oncogene products which are related to cell growth, c-erbB-2 product, epidermal growth factor receptor (EGFR), c-myc protein and estrogen receptor (ER), in benign and malignant lesions of the breast. Fresh frozen sections of 25 breast cancers and 11 fibroadenomas from Japanese women were studied by indirect immunoperoxidase method with proper fixation. C-erbB-2 product and EGFR were localized on the cell membrane whereas c-myc protein and ER were observed in the nuclei. Immunohistochemical expression of oncogene products and ER were not only observed in the mammary carcinomas but also in the fibroadenomas. However immunoreactivities of EGFR and ER were more frequently seen in the fibroadenomas (p<0.05). In breast cancers, the incidence of immunoreactivity for c-erbB-2 was higher in the cases with lymph node metastasis than cases without nodal metastasis (p < 0.05) and there was reciprocal correlation between the expressions of EGFR and ER (p<0.05). Regarding the size of the primary tumour, there was no statistically significant correlation with the expressions of c-erbB-2, EGFR, c-myc or ER. Histological grade correlated only with the expression of ER (p<0.05).

Adenovirus-mediated anti-K-ras ribozyme induces apoptosis and growth suppression of human pancreatic carcinoma.

Human pancreatic cancer is a lethal malignancy, and the lesions show a very high incidence of point mutations of the K-ras oncogene. These alterations can be used as potential targets for specific ribozyme (Rz)-mediated growth suppression of the cancer cells. We designed an anti-K-ras Rz against mutant K-ras gene transcripts (codon 12, GGT to GTT) and generated a recombinant adenovirus (rAd) to express the Rz (rAd/anti-K-ras Rz). More than 95% of Capan-1 human pancreatic cells were infected with rAd/anti-K-ras Rz when treated with the virus at 200 plaque-forming units/cell. The virus, rAd/anti-K-ras Rz, significantly suppressed mutant K-ras gene expression and inhibited the growth of Capan-1 cells. At 3 days postinfection, we observed maximum growth suppression of the cells, characteristic morphological changes of apoptosis such as nuclear condensation and oligonucleosomal DNA fragmentation, and suppression of bcl-2 oncoprotein. These changes were not found in control virus-infected cells. Our results indicated that the virus rAd/anti-K-ras Rz specifically down-regulated the K-ras/bcl-2 pathway and induced apoptotic changes in Capan-1 pancreatic carcinoma cells. High-efficiency adenovirus-mediated delivery of anti-K-ras Rz could become a significant gene therapy strategy against human pancreatic cancer.

Helicobacter pylori infection in gastric xanthomas: immunohistochemical analysis of 145 lesions.

A total of 145 paraffin‐embedded biopsy samples of gastric xanthoma were analyzed for the localization of Hellcobacter pylorl (HP) antigens. By the indirect immunoperoxidase method using a polyclonal antibody, HP lnfection was identffied on the surface of foveolar cells In 69 (48%) samples. In 38 (55%) of the 69 lesions, the HP antigens were demonstrated in the cytoplasm of xanthoma cells clustered in the actively inflamed lamina propria mucosae, Among the remalning 76 xanthoma lesions negative for HP lnfection on the epithellal surface, only eight (11%) showed the existence of HP antigens in the foamy histiacytes, and 39 (51 %) revealed mild inflammatory change. Monoclonal antibody study using 75 specimens also gave a comparable result. Pre‐embedding lmmunoelectron microscopy using paraffin sections revealed positively labeled rod‐shaped bacteria both on the epithelial surface and in the phagosome of the xanthoma cells. These findings strongly suggest that some of the xanthoma lesions are provoked by lamina proprial invasion of surface‐infected HP.

Reticular crypt epithelium and intra‐epithelial lymphoid cells in the hyperplastic human palatine tonsil: An immunohistochemical analysis

Extensive immunohistochemical analyses of the hyperplastic human palatine tonsil disclosed variegated B cell phenotypes on the lymphoid cells among the crypt epithelium. The reticular epithelial network was evident by cytokeratin immunostaining. The reticular epithelium near the crypt Iumen was positive for Iysozyme. Secretory component was negative, while HLA‐DR was frequently expressed. Intramucosal small Iymphocytes, densely distributed in the Iuminal side, consisted mainly of B cells expressing CD19, CD20, CD21, CD22, CD45R, CD74, DBB42, HLA‐DR, HLA‐DQ, bcl‐2 protein and surface lgM. Some B cells revealed mantle zone phenotypes (surface IgD+, CD5+, CD24+, DBA44+, CD10‐‐, DNA7‐‐). Cells of germinocyte phenotype (CD10+, DNA7+) were sparsely seen. A good number of intramucosal lymphoid cells were further labeled for CD11b, a phenotype of so‐called B‐1 cells. Plasma cells were clustered within the basal half. IgG was their major immunoglobulin class, followed by IgA, IgM and lgD classes. A smaller number of T cells (CD2+, CD3+, CD5+, CD45RO+, TCR αβ+) were identified among the epithelium. CD4+ cells predominated over CD8+ cells. TCR γΔ+ cells were rare. Macrophages (CD68+), dendritic histio‐cytes (S‐100 protein+, CD1+), and natural killer cells (CD16+ or CD57+) were also dispersed. Another unique feature of this lymphoepithelial complex was the existence of HLA‐DR intramucosal microvasculature, where lymphocyte recirculation was suggested. Proliferating cell nuclear antigen was detected commonly in the epithelial cells but rarely in the lymphoid cells. Possible lymphoepithelial interactions and morphologic similarities to the thymic medulla are discussed.

Histological differentiation between chlamydial and bacterial epididymitis: Nondestructive and proliferative versus destructive and abscess forming—Immunohistochemical and clinicopathological findings

Chlamydia trachomatis (CT) and Escherichia coli (EC) antigens were sought in routinely prepared paraffin-embedded sections from 31 cases of acute and chronic epididymitis by indirect immunoperoxidase staining. Chlamydia trachomatis antigens were detected in epithelial cells as cytoplasmic inclusions in samples from six patients (mean age, 43 years) with severe epididymitis, characterized by minimally destructive, periductal, and intraepithelial inflammation with active epithelial proliferation. Squamous metaplasia and formation of lymphoepithelial complexes occasionally were noted. Escherichia coli antigens, common to other pyogenic bacteria, were observed in the cytoplasm of foamy histiocytes in samples from seven patients (mean age, 60 years), characterized by highly destructive epididymitis forming large abscesses and xanthogranulomas. Specimens from 18 patients were negative for either antigen. Pre-embedding immunoelectron microscopy on paraffin-embedded sections demonstrated positive reactions on the cell wall of the chlamydial bodies and rod-shaped bacteria. Escherichia coli-positive cases were accompanied by scrotal pain, pyuria, positive bacterial culture, leukocytosis, accelerated erythrocyte sedimentation rate, and a positive C-reactive protein test. Chlamydia trachomatis-positive cases were clinically indolent and manifested by an epididymal tumor. Chlamydial epididymitis can be distinguished from bacterial epididymitis not only clinically and immunohistochemically but also histologically.

Perivascular epithelioid cell tumor (PEComa) of the pancreas: Immunoelectron microscopy and review of the literature

A perivascular epithelioid tumor (PEComa) is a rare tumor probably arising from the perivascular epithelioid cells. Only three cases of pancreatic PEComa have been reported in the English‐language literature. The present report describes an extremely rare case of pancreatic PEComa. A 47‐year‐old Japanese woman complained of lower abdominal pain and a well‐demarcated solid tumor was found in the pancreatic head. There was no history of tuberous sclerosis complexes. Pylorus‐preserving pancreaticoduodenectomy was thus performed. There was a well‐demarcated, solid tumor measuring 17 mm in the pancreatic head. The tumor was composed of a diffuse proliferation of epithelioid tumor cells with many blood vessels but no adipose tissue. The tumor cells expressed HMB45 and α‐smooth muscle actin. Ultrastructurally, the tumor cells possessed many membrane‐bound granules that were positive for HMB45 on immunoelectron microscopy. The results of immunoelectron microscopy show that some PEComas possess not only typical melanosomes or premelanosomes but also aberrant melanosomes.

Localized amyloidosis of the seminal vesicle : Identification of lactoferrin immunoreactivity in the amyloid material

Three specimens of localized amyloidosis of the seminal vesicle surgically removed for prostatic cancer were immuno‐histochemically analyzed to clarify the nature of the permanganate‐sensitive congophilic subeplthelial deposition. A variety of known amyloidogenic substances and secretory products in the seminal fluid were screened using the indirect immunoperoxldase method. In addition to reactivities with antibodies to amyloid P component and human seminal plasma, the amyloid material was immunoreactive for lactoferrin using a rabbit antiserum and two of three mouse monoclonal antibodies. All the antibodies labeled some of the normal seminal vesicle epithelial cells for this iron‐binding, bacteriostatJc glycoprotein. In the prostate without accompanying amyloid deposition, a considerable proportion of the glandular epithelium and secretory material were positive for lactoferrin. Pre‐embedding immunoelectron microscopy showed lactoferrin immunoreactivity on the amyloid fibrils. Focal staining of the amyloid for gross cystic disease fluid protein‐15 was also observed in two lesions. These findings strongly suggest that lactoferrin is the major constituent in localized senile amyloidosis of the seminal vesicle.

Immunohistochemical Study of Transforming Growth Factor β, Fibronectin, and Fibronectin Receptor in lnvasive Mammary Carcinomas

Thirty‐one cases of mammary carcinoma were examined immunohistochemically for the expression of transforming growth factor (TGF) β, fibronectin (FN) and fibronectin receptor (FNR) in order to clarify the reason for the reported relationship between TGF β expression and a high incidence of lymph node metastasis. It was revealed that TGF β expression is closely related to the expression of FN, an intercellular matrix protein, and its cellular receptor FNR, one of the integrins. The interaction between FN and FNR in a tumor is considered to form the basis of the invasive nature of carcinoma cells. Thus, it is suggested that TGF β expression in carcinoma cells induces the interaction between FN and FNR, which may lead to carcinomatous invasion resulting in lymph nodal metastasis. Aeta Pathol Jpn 42: 645–650, 1992.

Secretory pathways and processing of human proopiomelanocortin (POMC) using transformed mouse cultured fibroblasts (L cells) and AtT20 cells by human POMC gene--preembedding immunoelectron microscopic studies.

In order to elucidate the relationship between secretory pathway and processing for precursor molecule of peptide hormones, we performed immunoelectron microscopic studies to localize POMC-derived peptides in mouse cultured L cells (fibroblasts without secretory granules) and in mouse AtT20 cells (ACTH secreting pituitary tumor cells with secretory granules) which had been transformed with human POMC gene. From the electron microscopic localization patterns, L42 cells were considered to serve as a model of constitutive pathway without processing of POMC, and A53 cells were considered to serve as a model of transgranular (regulated) pathway with processing of POMC. Immunoblotting supported these interpretations.

Immunohistochemical localization of catecholamine-synthesizing enzymes in human pheochromocytomas

A total of 17 (10 adrenal and 7 extraadrenal) pheochromocytomas were examined immunohistochemically for the localization of catecholamine-synthesizing enzymes-tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT)-as well as the marker peptides for pheochrornocytomas (i.e., met-enkephalin-arggly-leu [MEAGL]). Normal adult, fetal, and newborn human adrenal medullas were also examined. Six adrenal pheochromocytomas showed immunohistochernically positive staining for PNMT; 5 of these cases demonstrated elevated serum adrenalin levels, This indicated morphofunctional correlation. PNMT-positive cells were mostly positive for TH but did not show co-localization of MEAGL except in 1 case, Absence of co-localization was considered to reflect the physiological condition of the specimen, based on the similar staining in the normal adult adrenal medulla. Primary culture and immunoelectron microscopy suggested the processing and synthesis of adrenalin and MEAGL in the secretory granules.Seven extraadrenal pheochromocytornas were negative for PNMT and suggested the lack of adrenalin synthesis.