To investigate the aetiological role of human papillomavirus (HPV) in breast cancer, we examined the presence, genotype, viral load, and physical status of HPV in 124 Japanese female patients with breast carcinoma. Human papillomavirus presence was examined by PCR using SPF10 primers, and primer sets targeting the E6 region of HPV-16, -18, and -33. The INNO-LiPA HPV genotyping kit was used to determine genotype. Human papillomavirus DNA was detected in 26 (21%) breast carcinomas. The most frequently detected HPV genotype was HPV-16 (92%), followed by HPV-6 (46%), HPV-18 (12%), and HPV-33 (4%). In 11 normal epithelium specimens adjacent to 11 HPV-16-positive carcinomas, 7 were HPV-16-positive. However, none of the normal breast tissue specimens adjacent to HPV-negative breast carcinomas were HPV-positive. The real-time PCR analysis suggested the presence of integrated form of viral DNA in all HPV-16-positive samples, and estimated viral load was low with a geometric mean of 5.4 copies per 10 4 cells. In conclusion, although HPV DNA was detected in 26 (21%) breast carcinomas and, in all HPV-16-positive cases, the HPV genome was considered integrated into the host genome, their low viral loads suggest it is unlikely that integrated HPV is aetiologically involved in the development of Japanese breast carcinomas that we examined.

When the human prostate cancer cell line, LNCaP 104-S, the growth of which is stimulated by physiological levels of androgen, is cultured in androgen-depleted medium for >100 and androgen-independent LNCaP 104-R2t sublines were isolated as described (6). The characteristics of these cells in vitro were confirmed before injection into nude mice. Briefly, proliferation of LNCaP 104-S cells increased 10-to 13-fold in medium containing charcoal-treated fetal bovine serum (FBS) and 0.1 nM synthetic androgen R1881, compared with cells cultured in medium containing charcoal-treated FBS without added androgen. LNCaP 104-R2 cells grew in medium supplemented with charcoal-treated FBS without additional androgen. Their proliferation was not stimulated but was repressed by 0.1 nM R1881. LNCaP 104-S cells were maintained in DMEM (GIBCO) supplemented with 1 nM S5t-dihydrotestosterone and 10% FBS (Summit Biotechnology, Ft. Collins, CO), and LNCaP 104-R2 cells were maintained in DMEM supplemented with 10% FBS treated with charcoal to remove steroid (6). PC-3 and MCF-7 cell lines were obtained from the American Type Culture Collection (Rockville, MD) and were maintained in DMEM supplemented with 10% FBS.Animals. BALB/c athymic (nude) male (LNCaP, PC-3 cell lines) and female (MCF-7 cell line) mice (Taconic, Germantown, NY), 5 to 7 weeks old, were used. Mice were housed in a pathogen-free environment, four to five mice per cage. Cages (filter top), bedding, and water were autoclaved before use. Feed was irradiated Pico Lab Mouse Chow 20 5058 (Purina).Abbreviations: AR, androgen receptor; FBS, fetal bovine serum; PSA, prostate-specific antigen; TP, testosterone propionate.

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The tumor suppressor gene maspin has been reported to inhibit the invasiveness and motility of breast cancer cells. It has been reported that maspin is expressed in normal human mammary epithelial cells but is downregulated during cancer progression, and that p53 could induce maspin expression by transcriptional activation. However, to date, the clinical significance of maspin expression and its correlation with p53 protein expression in human breast cancer patients have not been elucidated. One hundred and sixty-eight female patients diagnosed with invasive ductal carcinoma, who had undergone a mastectomy (154 patients) or breast-conserving surgery (14 patients), were followed up for 15-119 months (median: 87 months) postoperatively. Immunoreactivity for maspin and p53 antibodies with paraffin-embedded carcinoma tissue was investigated using labeled streptavidin-biotin methods. Tumors with more than 20% of positive cells were considered positive for the expression of maspin. The expression of maspin in carcinoma cells was found in 27.4% (46 of 168) and significantly correlated with larger tumor size (p = 0.008), higher histologic grade (p = 0.0001) and positive p53 status (p = 0.003). A significant inverse relationship was observed between the expression of maspin and estrogen receptor (p = 0.0004) or progesterone receptor status (p = 0.02). Univariate analysis by log-rank test revealed a significant association between the expression of maspin and shorter relapse-free survival (p < 0.0001) and overall survival (p < 0.0001). According to Cox's multivariate analysis, the expression of maspin had the most significant effect in relapse-free survival (p < 0.0001) and overall survival (p < 0.0001) followed by lymph node status. In turn, the expression of maspin in 58 cases of ductal carcinoma in situ were also investigated to explore whether the downregulation of maspin through cancer progression are true or not. However, there were no positive cases in our series. These results seem to be contrary to previous reports defining maspin as a tumor suppressor gene. Although more precise characterization of the maspin expression, especially gene analysis is essential, the present investigation suggests that the expression of maspin is not downregulated through malignant progression and that the immunohistochemic detection of maspin in carcinoma cells may be helpful for selecting the group of breast cancer patients with an aggressive phenotype.

Severe left ventricular dysfunction with associated conduction disturbances and ventricular arrhythmias occur in patients with terminal stage cardiac Fabry disease. Furthermore, LVH is present and associated with thinning of the base of the left ventricular posterior wall. In contrast to typical Fabry disease, accumulation of glycosphingolipids was observed in myocardial cells but not in other organs.

These results and our previous investigation in which 27.4% of IDC were positive for maspin suggest that the expression of maspin in epithelial cells could be up-regulated during the progression of ductal carcinoma, and that it could be correlated with the acquisition of an aggressive phenotype.

CyclinD1 plays a critical role in regulating cell cycle progression. CyclinD1 mRNA and protein are overexpressed in approximately 50% of primary breast cancer cases. However, its clinical significance as a predictive factor remains unclear. One hundred and seventy-three female patients diagnosed with invasive ductal carcinoma who had undergone a mastectomy (161 patients) or breast-conserving surgery (12 patients) were followed up for 6 -119 months (median 86 months) postoperatively. Immunoreactivity for monoclonal anti-cyclinD1 antibody (clone DCS-6) with paraffin-embedded carcinoma tissues was investigated using a labeled streptavidin-biotin method. Overexpression of cyclinD1 was found in 42% (73 of 173), and strongly correlated with estrogen receptor (ER) expression (p < 0.000001). Univariate analysis revealed no association between overexpression of cyclinD1 and overall survival or relapse-free survival in all patient groups. However, in the ER-negative subgroup (n ‫؍‬ 75), overexpression of cyclinD1 was significantly correlated with shorter overall survival (p ‫؍‬ 0.018) and relapse-free survival (p ‫؍‬ 0.014) as well as the lymph node status and tumor size. In contrast, there were no significant associations between overexpression of cyclinD1 and clinical outcome in the ER-positive subgroup. According to Cox's multivariate analysis in the ER-negative subgroup, overexpression of cyclinD1 had the most significant effect on overall survival (p ‫؍‬ 0.02) and relapse-free survival (p ‫؍‬ 0.0058), followed by nodal status and histologic grade. These findings suggest that overexpression of cyclinD1 is an independent prognostic indicator in ER-negative breast cancer patients.

This article reviews the current status of hormone receptor evaluation in and out of Japan, and introduces the proposed working protocol of the task force for Adequate immunohistochemical evaluation in routine practice for breast cancer by the Japanese Breast Cancer Society. Understanding the principles and the developmental process of immunohistochemistry helps us to utilize a scoring system adequately. Methodologies of hormone receptor examination and immunohistochemical procedures are briefly introduced. Each scoring system is based on different principles. The proposed working protocol takes into account the reproducibility of results among observers, institutions and staining procedures, which is justified based on the current situation in Japan. Future directions for the standardization of immunohistochemical hormone receptor evaluation are also described.