Tetsunari Oyama

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.

Intraoperative identification of sentinel lymph nodes by near-infrared fluorescence imaging in patients with breast cancer

We present a novel method for sentinel lymph node (SLN) identification by fluorescence imaging that provides a high detection rate and a low false-negativity rate. Twenty-five breast cancer patients with tumors less than 3 cm in diameter were enrolled. A combination of indocyanine green and indigo carmine was injected subdermally in the areola. Subcutaneous lymphatic channels draining from the areola to the axilla were immediately showed by fluorescence imaging. After incising the axillary skin near the point of disappearance of the fluorescence, the SLN was dissected under fluorescence guidance. In all patients, the lymphatic channels and SLN were successfully visualized. The mean number of fluorescent SLN and blue-dyed SLN were 5.5 and 2.3. Eight patients were found to have lymph node metastases pathologically. All of them were recognized by fluorescence imaging. This method is feasible and safe for intraoperative detection of SLN allowing real-time observation without any need for training.

Overexpression of p53 as a possible prognostic factor in human thyroid carcinoma.

A total of 110 cases of thyroid carcinomas were examined immunohistochemically to evaluate the overexpression of mutant forms of p53 protein in the light of their relationship with their histological subtypes. A polyclonal antibody, CM-1, was applied to the routine formalin-fixed, paraffin-embedded tissues for this survey. Overall, immunohistochemically detected p53 expression confined to the nucleus was identified in 22.7% of the thyroid carcinomas. A significant difference in the positivity of p53 among histological subtypes was noted; the positivity was 11.1% of the cases in well-differentiated papillary carcinoma, 14.3% in well-differentiated follicular carcinoma, 40.9% in poorly differentiated carcinoma, and 63.6% in undifferentiated carcinoma. No immunohistochemical positivity was found in adjacent non-neoplastic tissues or in benign lesions, including follicular adenoma, adenomatous goiter, and chronic thyroiditis. These results suggest that overexpression of p53 is not a responsible factor for the oncogenesis itself, but rather that it plays a crucial role in aggressive subtypes of thyroid carcinomas. Additionally, the distinct entity of poorly differentiated carcinoma, previously categorized in the well-differentiated carcinoma under the name of papillary or follicular carcinoma, was statistically confirmed.

Stepwise participation of p53 gene mutation during dedifferentiation of human thyroid carcinomas.

The spectrum of p53 gene mutations was investigated in thyroid carcinomas with respect to histopathological classification. In all histological subtypes of thyroid carcinoma that had previously revealed positivity in immunohistochemical staining for p53 protein, single-stranded conformation polymorphism analysis and direct sequencing were performed to detect point mutations between exons 5 and 8. In well differentiated papillary and follicular carcinomas, in which we had already known that 11.1 and 14.3% of the cases, respectively, revealed p53 overexpression as determined by immunohistochemistry, genetic aberrations were undetectable. In poorly differentiated carcinoma, in which 40.9% had revealed overexpression, two of six cases revealed point mutations at codon 244 in exon 7 and at codon 278 in exon 8. In undifferentiated carcinoma, in which 63.6% had revealed overexpression, four of six cases examined showed point mutations at codon 157 in exon 5, at codon 248 in exon 7, and at codon 273 and a two-base insertion between codons 266 and 267 in exon 8. These results strongly suggest the crucial role of p53 gene aberration and protein overexpression in a biologically aggressive subtype, possibly as a stepwise participation in the process of tumor dedifferentiation in human thyroid carcinomas.

NOTCH3 Signaling Pathway Plays Crucial Roles in the Proliferation of ErbB2-Negative Human Breast Cancer Cells

ErbB2-negative breast tumors represent a significant therapeutic hurdle because of a lack of effective molecular targets. Although NOTCH proteins are known to be involved in mammary tumorigenesis, the functional significance of these proteins in ErbB2-negative breast tumors is not clear. In the present study, we examined the expression of activated NOTCH receptors in human breast cancer cell lines, including ErbB2-negative and ErbB2-positive cell lines. Activated NOTCH1 and NOTCH3 proteins generated by gamma-secretase were detected in most of the cell lines tested, and both proteins activated CSL-mediated transcription. Down-regulation of NOTCH1 by RNA interference had little or no suppressive effect on the proliferation of either ErbB2-positive or ErbB2-negative cell lines. In contrast, down-regulation of NOTCH3 significantly suppressed proliferation and promoted apoptosis of the ErbB2-negative tumor cell lines. Down-regulation of NOTCH3 did not have a significant effect on the ErbB2-positive tumor cell lines. Down-regulation of CSL also suppressed the proliferation of ErbB2-negative breast tumor cell lines, indicating that the NOTCH-CSL signaling axis is involved in cell proliferation. Finally, NOTCH3 gene amplification was detected in a breast tumor cell line and one breast cancer tissue specimen even though the frequency of NOTCH3 gene amplification was low (<1%). Taken together, these findings indicate that NOTCH3-mediated signaling rather than NOTCH1-mediated signaling plays an important role in the proliferation of ErbB2-negative breast tumor cells and that targeted suppression of this signaling pathway may be a promising strategy for the treatment of ErbB2-negative breast cancers.

Expression and Mutations of KCNJ5 mRNA in Japanese Patients with Aldosterone-Producing Adenomas

CONTEXT Mutations of the KCNJ5 gene have recently been identified in patients with aldosterone-producing adenomas (APA). OBJECTIVE Our objective was to investigate the expression and mutations of the KCNJ5 gene in Japanese patients with APA. DESIGN AND PATIENTS We sequenced KCNJ5 cDNA and measured KCNJ5 mRNA levels in 23 patients with APA operated on at Gunma University Hospital. MAIN OUTCOME MEASURES Mutations and mRNA levels of the KCNJ5 gene were examined and compared to those in cortisol-producing adenomas (Cushings syndrome) and pheochromocytomas. RESULTS Of the 23 patients with APA, 15 (65.2%) had two somatic mutations of the KCNJ5 gene: 12 cases of p.G151R (eight with c.451G>A, and four with c.451G>C) and three cases of p.L168R (c.503T>G). Levels of KCNJ5 mRNA were significantly higher in the APA with mutations than those without. Immunohistochemistry also showed a stronger staining of KCNJ5 on the cell membrane in the tumor with a mutation. Furthermore, a PCR-restriction fragment length polymorphism assay with c.503T>G revealed the mutant mRNA to be expressed at a similar level to the wild type. The level of KCNJ5 mRNA in cortisol-producing adenomas was approximately 30% of that in APA, and almost no expression was observed in pheochromocytomas. CONCLUSION We found that: 1) a significant number of patients with APA had somatic mutations of the KCNJ5 gene; 2) KCNJ5 mRNA levels were higher in the APA with KCNJ5 mutations; and 3) the expression of KCNJ5 mRNA was significantly higher in APA than cortisol-producing adenomas and pheochromocytomas.

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.

Atypical cystic lobules: an early stage in the formation of low-grade ductal carcinoma in situ

Abstract Evidence from many studies has established the neoplastic potential of ductal carcinoma in situ, but the origin and the morphological characteristics of the early stages of this proliferation remain unidentified. Workers writing in the early twentieth century observed a cystic transformation of lobules and proposed that it represented one such early stage, and contemporary European and Japanese pathologists have reached the same conclusion. We describe the characteristics of this cystic transformation, which we call us “atypical cystic lobules,” and present evidence to support the proposal that the alteration is a step in the formation of low-grade ductal carcinoma in situ. Atypical cystic lobules are a proliferation of luminal cells showing low-grade cytological atypia without architectural atypia. The study group comprised 21 cases of atypical cystic lobules from specimens also showing conventional low-grade ductal carcinoma in situ or lobular neoplasia. Immunohistochemical staining for hormone receptors, keratin 19, and cyclin D1 revealed that atypical cystic lobules demonstrated a consistent immunophenotype, which differs from the pattern shown by normal lobules and benign lesions and matches that of low-grade ductal carcinoma in situ. In about 40% of the cases, atypical cystic lobules merged with fully established micropapillary/cribriform ductal carcinoma in situ. The similarities in the cytological and immunohistochemical features and the proximity of the two types of proliferation suggest that atypical cystic lobules represent an early stage in the formation of certain types of low-grade ductal carcinoma in situ.

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.