Kenji Kashiwabara

Expression Status of p16 Protein Is Associated with Human Papillomavirus Oncogenic Potential in Cervical and Genital Lesions

The p16 protein (p16) is a cyclin-dependent kinase (CDK) inhibitor that decelerates the cell cycle by inactivating the CDKs that phosphorylate retinoblastoma (Rb) protein. Recent biological studies have revealed that p16 expression is markedly influenced by the status of Rb expression, and p16 overexpression has been demonstrated in cervical cancers because of functional inactivation of Rb by human papillomavirus (HPV) E7 protein. To clarify the relationship between p16 overexpression and HPV infection in cervical carcinogenesis, immunohistochemical analysis of p16 and detection of HPV by in situ hybridization and polymerase chain reaction were performed on 139 formalin-fixed and paraffin-embedded samples of cervical and genital condylomatous and neoplastic lesions. Marked overexpression of p16 protein, ie, diffuse and strong immunostaining, was observed in all cervical cancers and preneoplastic lesions with infection by high- and intermediate-risk HPVs, ie, subtypes 16, 18, 31, 33, 52, and 58. Condylomata acuminata and low-grade squamous intraepithelial lesions with infection by low-risk HPV such as HPV-6/11 showed focal and weak immunohistochemical staining for p16. Our results clearly showed that the mode of p16 expression in lesions with high- and intermediate-risk HPVs differed from its expression in lesions with low-risk HPVs and thus might be attributable to differences in functional inactivation of Rb protein by different HPVs.

Immunohistochemical overexpression of p16 protein associated with intact retinoblastoma protein expression in cervical cancer and cervical intraepithelial neoplasia.

Both p16 and retinoblastoma (Rb) proteins are important tumor supprsssors that regulate the cell cycle. The status of both proteins In lnvasive cervical cancer and cervical intraepithelias neopiasis (CIN) has not yet been examined. The aim of this study was to investigate the expression of p16 and Rb proteins by immunohlstochemistry using 98 formalln‐fixed and paraffin‐embedded samples of various cervical neoplastic lesions. Strong immunoreactiyity for the p16 protein was observed in both the nuclei and cytoplasm of all CIN and lnvasive cancer cases except several low‐grade CIN lesions. Expression of Rb protein was also demonatrated In the scattered nuclel of neoplastic and normal cells in all cases Investigated. The results suggest that the deletion or mutational inactivity of both p16 and Rb proteins may be a rare event In cervical carcinogenesls. Moreover, overexpression of the p16 protein may be a useful dlagnostic marker for cervical neoplastic lesions on routine laboratory screening.

Correlation between methylation status of the p16/CDKN2 gene and the expression of p16 and Rb proteins in primary non-small cell lung cancers.

In order to clarify the frequency of p16 gene inactivation and its relationship with Rb expression, immunohistochemical analysis of p16 and Rb proteins was carried out on 82 paraffin‐embedded sections of primary non‐small cell lung cancers (NSCLCs). From immunohistochemical results, abnormal p16 expression was observed in 66% of NSCLCs, 80% in squamous cell carcinomas and 46% in adenocarcinomas. An inverse correlation between p16 and Rb expressions was noted. Moreover, the methylation status of the p16 gene was investigated by the methylation‐specific polymerase chain reaction (MS‐PCR) using 29 frozen samples of NSCLCs. MS‐PCR revealed the methylation of the p16 gene in 10 (34%) of 29 NSCLCs. All NSCLCs exhibiting methylation exhibited abnormal p16 expression and were positive for Rb. In NSCLCs, no difference in methylation status was observed with respect to clinico‐pathological characteristics including histological subtype and tumor stage. Our results demonstrate that abnormality of p16 expression is frequent in primary NSCLCs and methylation of the promoter of the p16 gene occurs in 34% of primary NSCLCs, which might play a significant role in the inactivation of the p16<0R> gene. Int. J. Cancer (Pred. Oncol.) 79:215–220, 1998.© 1998 Wiley‐Liss, Inc.

Coexpression of HGF and c-Met/HGF receptor in human bone and soft tissue tumors

To understand the interaction between hepatocyte growth factor (HGF) and its receptor c‐Met on various bone and soft tissue tumors, their expressions were investigated by western blot analysts, immunohistochemistry and enzyme immunoassay. Western blot analysis revealed that c‐Met protein was expressed in 21 (38.8%) of 54 tumors, which detailed to seven (25.9%) of 27 bone tumors and 14 (51.8%) of 27 soft tissue tumors. Most malignant fibrous histiocy‐tomas (MFH) and all neurofibromas expressed c‐Met protein. The highest expression of c‐Met protein was seen in a case of biphasic synovial sarcoma, where its immunoreac‐tivity was localized only on the epithelial component and not on the sarcomatous component. By enzyme immunoassay for HGF, all but one MFH showed HGF production and the mean level of HGF was the highest among the tumors investigated. Neurofibmmas and osteosarcomas had the next highest mean levels of HGF production, respectively. Coexpression of HGF and c‐Met was obsewed in 19 (35.2%) of 54 tumors and was frequently observed in neurofibroma, followed by MFH and synovial sarcoma. Although the mode of interaction between HGF and c‐Met varies among the various bone and soft tissue tumors including MFH, their signaling system may play an Important role in the development and progression of bone and soft tissue tumors.

Gastric adenoma-carcinoma sequence with special reference to p53 and Ki-ras gene alterations

With the aim of detecting the timing of p53 and Ki-ras gene alterations in the gastric adenoma-carcinoma sequence, 19 early gastric adenocarcinomas arising from adenomas were studied. Immunohistochemically, 5 adenocarcinomas were positive for p53; 3 focally and 2 diffusely. The p53 point mutations were detected in a focal area with p53 immunoreactivity in 2 of the 5 p53-positive adenocarcinomas. This indicated that p53 point mutations may play a less crucial part in malignant conversion of adenoma to adenocarcinoma in the stomach than in the colon. No Ki-ras gene mutations at codons 12 and 13 were detected in any lesion. These results suggest that the adenoma-carcinoma sequence in the stomach has a different mechanism from that in the colon.

A Case of Esophageal Sarcomatoid Carcinoma with Molecular Evidence of a Monoclonal Origin

A case of polypoid tumor of the esophagus consisting of a sarcomatous tumor partly covered with superficial squamous cell carcinoma is described. The sarcomatous component consisted of anaplastic spindle and pleomorphic tumor cells that mimicked malignant fibrous histiocytoma (MFH). Both the sarcomatous and carcinomatous components were positive for p53 immunohistochemically. Further molecular analysis revealed that the two components had the same somatic mutation in the p53 gene. These results suggest a monoclonal origin of this biphasic tumor.

Significance of contracted cholecystitis lesions as high risk for gallbladder carcinogenesis.

A precancerous change has been identified incidentally in resected specimens from patients who have undergone cholecystectomy. We focused on chronic cholecystitis, showing a thick and sclerotic wall caused by recurrent inflammation, e.g. contracted cholecystitis, and examined the malignant potential of these lesions. We studied 88 patients who had undergone cholecystectomy. Contracted cholecystitis was diagnosed, using our criteria, in 28 of these cases. Ordinary chronic cholecystitis was diagnosed in 50 cases and gallbladder carcinoma in ten cases. We examined the expression of p53, Ki-67, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) immunohistochemically. Severe dysplasia or carcinoma in situ in a very small portion of the specimen was identified with hematoxylin-eosin staining in four cases (14.3%) of contracted cholecystitis. These specimens revealed a positive expression of not only p53, but also Ki-67, iNOS, and COX-2. Statistical significance was shown among the three disease groups in terms of the incidence of p53 overexpression, respectively (P<0.05). The results of this study suggest that contracted cholecystitis could be an early change leading to carcinogenesis.

Lymphoplasmacytic Infiltrate of Regional Lymph Nodes in Küttner's Tumor (Chronic Sclerosing Sialadenitis): A Report of 3 Cases

Regional lymph nodes of Küttners tumor from 3 patients showed reactive follicular hyperplasia and prominent interfollicular plasmacytosis. The patients were 71-, 57-, and 73-year-old Japanese men. The polytypic nature of plasma cells was demonstrated by immunohistochemistry. There were numerous IgG-positive plasma cells with scattered IgA-positive or IgM-positive plasma cells. IgG4-positive cells comprised 25% to 40% of IgG-positive plasma cells. Prominent polyclonal hyperimmunoglobulinemia was demonstrated on laboratory test in 2 cases examined. An elevated serum IgG4 level (16%) was also demonstrated in 1 patient. The present 3 cases indicated that regional lymph node of Küttners tumor may show reactive follicular hyperplasia and prominent interfollicular plasmacytosis and should be differentiated from various benign and malignant lymphoproliferative disorders including systemic rheumatic disease, plasma cell type of Castleman disease, and lymph node involvement of marginal B-cell lymphoma of the mucosa-associated lymphoid tissue type showing prominent plasma cell differentiation.

Synergistic decline in expressions of p73 and p21 with invasion in esophageal cancers

The significance of the p73 gene, a homologue of the p53 gene, in esophageal cancers is not fully understood. In order to clarify the role of p73 expression in esophageal cancers, p73 expression was immunohistochemically investigated in 106 surgically resected esophageal cancers and the results were compared with various clinicopathological factors. In normal esophageal epithelium, the expression of p73 was observed only in the nuclei of basal cells. In esophageal cancers, p73 immunoreactivity was observed in all intraepithelial lesions except one cancer, and was reduced with cancer invasion, to 78% and 64% at superficial invasion and deep invasion sites, respectively. However, p73 expression was not correlated with any other clinicopathological factor. The expressions of p53 and p21 were also investigated in esophageal cancer. To evaluate the status of the p53 gene mutation immunohistochemically, two monoclonal antibodies (DO7 and PAb240) were used. There seemed to be an inverse correlation between p73 expression and p53 mutation. Moreover, the expression of p21 was highly correlated with p73 expression irrespective of the p53 mutation status. In human esophageal cancers, p73 expression decreased with increasing degree of tumor invasion, and its decreased expression in local advanced tumor caused down‐regulation of p21 expression, which might reflect tumor progression.

c‐Met expression of thyroid tissue with special reference to papillary carcinoma

It has become clear that papillary carclnomas of the thyroid often express the receptor for c‐Met/hepatocyte growth factor (HGF) receptor, but little Is known about the role of the HGF and c‐Met system In the pathogenesis of thyroid carcinoma. In this study, the expression of c‐MeUHGF receptor was evaluated in thyroid tlssue by western blot and immunohistochemistry, and compared with the concentration of HGF. Clinicopathologlcal characteristics were also compared. Fmeen of 20 papillary Carcinomas (75%) showed eMet bands of 145 kDa. No or only a low frequency of c‐Met expression was detected in healthy thyroid tissue (0/5), thyroiditis or Basedows disease (0/2), adenomatous goiters (0/8), follicular adenomas (119, 11%) and undifferentiated carcinomas (0/2). These results were confirmed by immunohistochemistry, but a relatively higher frequency of c‐Met expression was detected in adenomatous goiters (25%), follicular adenoma (44%) ancl papillary Carcinoma (100%) using formalin‐fixed and paraffin‐embedded materials. A strong immunoreaction for c‐Met was observed In the tumor cytoplasm of paplllary carcinomas among the fibrous tissues situated at the perlphery of the tumor. The densito‐metrically measured expression of c‐Met had no relation to tumor stage in papillary carcinoma, but did correlate to the concentration of HGF in papillary carcinomas. In conclusion, in thyroid lesions, c‐Met was highly expressed specifically in the cytoplasm of papillary carcinomas. c‐Met expression was not related to the aggressiveness of the tumor but was related to the concentration of HGF, which was probably derived from the stroma. Also, the c‐Met system might play a role in the pathogenesis of papillary carcinoma of the thyroid.