Takaaki Sano

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.

Overexpression of p16 and p14ARF is associated with human papillomavirus infection in cervical squamous cell carcinoma and dysplasia.

The CDKN2 gene encodes two structurally different proteins: a cyclin‐dependent kinase inhibitor, p16, which regulates retinoblastoma protein (pRb)‐dependent G1 arrest, and a cell cycle inhibitor, p14ARF, which blocks MDM2‐induced p53 degradation resulting in an increase in p53 levels that leads to cell cycle arrest. Recent studies have revealed that expression of p16 and p14ARF is influenced markedly by the status of pRb and p53, and p16 overexpression has been demonstrated in cervical neoplasia because of functional inactivation of pRb by the human papillomavirus (HPV) E7 protein. To clarify the p14ARF status and the relationship between p16/p14ARF and other cell cycle molecules in cervical carcinogenesis, immunohistochemical analysis of p16, p14ARF, p53 and MDM2 was performed on 65 samples of cervical and genital condylomatous and neoplastic lesions, including nine HPV‐negative tumors. In most cervical cancers and preneoplastic lesions with HPV infection of high and intermediate risk, a marked overexpression of p14ARF as well as the p16 protein (i.e. dotted nuclear immunostaining) was observed. All condyloma acuminata except one and low‐grade dysplasia with HPV infection of low risk, such as HPV 6, immunohistochemically showed completely negative staining for p14ARF, also seen in non‐neoplastic and mesenchymal cells. Our results clearly show that the mode of p14ARF overexpression in cervical neoplastic cells with HPV association differs from that in cancers of other organs without HPV association, and the p14ARF overexpression may be attributable to a negative feedback result in the functional inactivation of the pRb and p53 proteins by HPV oncoproteins.

Gastrointestinal stromal tumors and KIT-positive mesenchymal cells in the omentum

Gastrointestinal stromal tumor (GIST) is currently considered to be derived from the interstitial cells of Cajal (ICC). To test the hypothesis that omental mesenchymal tumor is also a type of GIST, we evaluated the expression of specific molecules in GIST, and c‐kit gene mutation in omental mesenchymal tumors, and we identified a possible counterpart of ICC in the omentum. Immunohistochemically, all of the omental mesenchymal tumors (n = 5) were positive for both KIT and CD34, and three of the five tumors were also positive for an embryonic form of smooth‐muscle myosin heavy chain (SMemb). Polymerase chain reaction–single‐strand conformational polymorphism analysis (PCR–SSCP) and direct sequencing revealed mutations in c‐kit gene exon 11 in all five tumors. As for the ICC counterparts in the omentum, there were some KIT‐positive mesenchymal cells resembling ICC at the surface of the omentum. Double fluorescence immunostaining, using anti‐KIT polyclonal antibodies and monoclonal antibodies against other molecules, demonstrated that KIT‐, CD34‐ and SMemb‐positive cells were present just beneath the mesothelial cells of the omentum. These results show that omental mesenchymal tumor corresponds to GIST of the omentum, and that KIT‐positive bipolar mesenchymal cells may be a counterpart of ICC in the gastrointestinal tract. Identification of a new type of KIT‐positive mesenchymal cell in the omentum may lead to the discovery of a new physiological role for this organ.

PKC theta, a novel immunohistochemical marker for gastrointestinal stromal tumors (GIST), especially useful for identifying KIT‐negative tumors

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor in the digestive tract and the majority of GIST has characteristic gain‐of‐function mutations of the c‐kit gene, which encodes the KIT receptor for stem cell factor. The present study aimed to establish the usefulness of protein kinase C theta (PKC θ) as an immunohistochemical marker for GIST in comparison with KIT immunohistochemistry. PKC θ immunohistochemistry was carried out not only on 48 cases of GIST and another 40 cases of gastrointestinal mesenchymal tumors, but also on 24 cases of various tumors known to be immunohistochemically positive for KIT. Immunohistochemically, 41 out of 48 cases (85%) of GIST were positive for PKC θ, and its expression was confirmed by Western blot analysis using six cases of surgically resected GIST. In the present study there were six GIST immunohistochemically negative for KIT, which histologically revealed a myxoid epithelioid appearance characteristic to that of GIST with platelet‐derived growth factor receptor alpha mutation. All six GIST were immunohistochemically positive for PKC θ. No PKC θ immunoreactivity was observed in other gastrointestinal mesenchymal tumors and various KIT‐positive tumors except for three cases (14%) of gastrointestinal schwannomas. The present study revealed that PKC θ is an immunohistochemically novel and useful marker for GIST, especially for GIST negative for KIT.

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.

Immunochemical analysis of HPV L1 capsid protein and p16 protein in liquid-based cytology samples from uterine cervical lesions.

HPV L1 capsid protein is expressed together with the production of infectious viral particles, but its expression and relation to p16 expression, which has been a surrogate marker for human papilloma virus (HPV) infection in cervix, are little studied in cytology samples. The authors aimed to elucidate the relation between L1 capsid protein and p16 protein expressions in liquid‐based samples from uterine cervical lesions.

Expression of an activated mammalian target of rapamycin in adenocarcinoma of the cervix: A potential biomarker and molecular target therapy

Alterations of the Akt/mTOR pathway have been observed in numerous types of cancer, thus this pathway represents an exciting new target for molecular therapeutics. We investigated the expression of activated Akt (p‐Akt) and mTOR (p‐mTOR) in patients with adenocarcinoma of the cervix and the involvement of the p‐Akt/p‐mTOR pathway in response to combination of inhibitor agents, rapamycin and LY294002, with conventional therapy, cisplatin, in vitro. Immunohistochemistry analysis of p‐Akt and p‐mTOR was conducted in 26 patients with adenocarcinoma of the cervix. Western blot analysis was performed to determine the protein expression involved in response to chemotherapy in cervical cancer cell lines. The results showed that p‐Akt and p‐mTOR were identified in 50% and 53.8% of adenocarcinoma of the cervix. The expression of p‐mTOR was a significant independent marker for prognosis. A significant correlation between p‐Akt and p‐mTOR was observed. There was no correlation between their expressions with any of clinicopathological factors. In the in vitro study, cisplatin at CPI50 targets both the apoptosis and survival pathway by activating the caspase‐cascade; inhibiting Akt, mTOR, p70S6K, and 4EBP1. Combination of rapamycin with cisplatin induced synergistic interaction. On the other hand, combination with LY294002 resulted in either synergistic or antagonistic effect depending on the doses given. Rapamycin pretreatment potentiated cisplatin‐induced apoptosis cell death and enhanced blocking of the survival pathway. Overall, the expression of p‐mTOR is a significant prognostic marker of adenocarcinoma of the cervix and a potential molecular target for the treatment of cervical cancer. Inhibition of the mTOR pathway contributes to cisplatin‐induced apoptosis in cervical cancer cell lines.

A Comparison of Interobserver Reproducibility of Gleason Grading of Prostatic Carcinoma in Japan and the United States

CONTEXT Gleason grading is now the sole prostatic carcinoma grading system recommended by the World Health Organization. It is imperative that there be good interobserver reproducibility within this system worldwide. To our knowledge, there are no studies, using the same specimens, that compare the interobserver reproducibility of Gleason grading in Japan and the United States. OBJECTIVE To compare the interobserver reproducibility of Gleason grading of prostatic carcinoma in Japan and the United States using, in Japan, images from the identical biopsy glass slides that were originally graded in the United States. DESIGN Microsopic images from 37 needle biopsies of prostatic carcinoma were placed on CD-ROM and distributed to 14 Japanese pathologists for grading. These 14 physicians included 8 general pathologists and 6 pathologists with a special interest in urologic pathology. The needle biopsies had been previously reviewed so that a consensus diagnosis could be formed by a panel of urologic pathologists in the United States and Canada. Interobserver agreement with the consensus diagnoses was calculated by determining the overall kappa coefficient for the Japanese pathologists and then compared to the interobserver agreement among American general pathologists who had previously graded identical needle biopsies from which the CD-ROM images had been taken. RESULTS The interobserver agreement with the consensus diagnoses for the 4 Gleason grading groups (Gleason grades 2-4, 5-6, 7, and 8-10) among the Japanese urologic pathologists in this series of cases was substantial (overall kappa = 0.68), and for the Japanese general pathologists, it was moderate (overall kappa = 0.49), similar to that reported in the earlier study of American general pathologists (overall kappa = 0.44). The major interobserver reproducibility problem for both Japanese and American general pathologists is undergrading. The major areas of undergrading are the underdiagnosis of Gleason scores 5-6 as Gleason scores 2-4, and the underdiagnosis of cribriform sheets and fragments of cribriform Gleason pattern 4 carcinoma as Gleason pattern 3. CONCLUSIONS The interobserver reproducibility of the Gleason grading for this collection of specimens was similar among Japanese and American general pathologists. The overall kappa values for these generalists of 0.44 and 0.49 are only in the moderate (0.41-0.60) range of interobserver agreement when compared to 0.68, substantial (0.61-0.80) agreement, for Japanese urologic pathologists. Educational efforts to improve Gleason grading have been shown to be effective and are clearly warranted.