Shinobu Umemura

Lactogenesis: The Transition from Pregnancy to Lactation

The most important factors in initiation of the cascade of changes in the mammary epithelium that constitute lactogenesis stage II seem to be a prepared mammary epithelium, progesterone withdrawal, maintained plasma prolactin (in most species), and removal of milk from the breast within an undefined interval after birth. Although the molecular mechanisms by which prolactin regulates milk protein synthesis are the subject of intense and productive studies, the specific mechanisms by which progesterone and milk removal interact with the mammary epithelial cell at parturition have not been studied, perhaps because no in vitro model system exists that mimics lactogenesis stage II, or because of the complexity of the changes that must be coordinated during this process, or because of a lack of general understanding of the complex progression of changes in the function of the breast as it goes from the quiescent state of pregnancy to the active secretory state of lactation. With new technologies designed to investigate the biology of complex systems arising from the growing knowledge of the genome of human and animal species and the growing availability of animal and tissue culture models for these processes, physicians can expect a rapid increase in the molecular understanding of lactogenesis in the near future. These fundamental studies must be coupled with good prospective clinical studies if physicians are to obtain a useful, comprehensive understanding of lactogenesis in women.

Increased phosphorylation of Akt in triple-negative breast cancers

Cells from breast cancers lacking hormone receptors (estrogen receptor [ER], progesterone receptor [PgR]) and human epidermal growth factor receptor (HER) 2 strongly express the cell proliferation marker Ki‐67. However, the mechanisms of and stimulus signals involved in cell proliferation of this type of breast cancer are not well understood. The aim of the present study was to examine the characteristics of signal transduction in triple‐negative (ER‐, PgR‐, and HER2‐negative) breast cancers. For 44 tumor samples, western blotting analysis was conducted to examine the phosphorylation of HER2, external signal‐regulated kinase (ERK)1 and ‐2 and Akt, and the immunohistochemical phenotypes of the samples with respect to ER and HER2 were also assessed. Phosphorylation of HER2 was detected in 4 of 15 immunohistochemically HER2‐positive tumor samples (26.7%). ERK1/2 was more highly phosphorylated in triple‐negative breast cancers. Phosphorylation of Akt kinase was significantly higher in triple‐negative breast cancers. Triple‐negative breast cancers are characterized by increased phosphorylation of Akt kinase. In the present study, we found for the first time that there is a population with a significantly activated Akt pathway in this type of breast cancer. (Cancer Sci 2007; 98: 1889–1892)

Ten cases of sebaceous carcinoma arising in nevus sebaceus.

Although nevus sebaceus is known to develop various types of secondary neoplasms, it rarely causes carcinoma and only 14 cases of secondary sebaceous carcinoma have been reported. In this study, 10 cases of sebaceous carcinoma arising in nevus sebaceus were collected. The clinicopathological features and results of immunohistochemical examinations with adipophilin, perilipin and p53 were summarized. Sebaceous carcinoma arising in nevus sebaceous predominantly occurred on the scalp (8/10) of elderly women (mean age, 67.7 years). No case was associated with Muir–Torre syndrome. We found several pathological features of sebaceous carcinoma; that is, made up mainly of germinative cells, moderate nuclear atypia without pleomorphism and many mitoses (4–28/10 high‐power field). Adipophilin and perilipin antibodies highlighted lipid drops in the cytoplasm of the malignant cells in all cases. Overexpression of p53 was seen in all cases. In two cases there were coexisting benign‐looking sebaceous lesions at the periphery of the main cancer nodule, and in these lesions p53 showed low positivity compared with the clearly malignant area. There was co‐occurrence of another neoplasm in three cases with trichoblastoma, sebaceoma and syringocystadenoma papilliferum, respectively. All cases were treated by excision of the malignant lesion, with or without inclusion of the nevus sebaceus. In a follow‐up period of 1–7 years, there was no case of recurrence, lymph node metastases or distant metastases. With these specific pathological and immunohistochemical findings using adipophilin, perilipin and p53, we have to consider the possibility that there is a tendency to underdiagnose secondary sebaceous carcinomas in nevus sebaceus. These clinicopathological features of sebaceous carcinomas developing in the nevus sebaceus seem to indicate different biological entities from de novo sebaceous carcinoma.

Immunohistochemical evaluation for hormone receptors in Breast Cancer: A practically useful evaluation system and handling protocol

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 immunohistochemisty 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.

Metastin Inhibits Migration and Invasion of Renal Cell Carcinoma with Overexpression of Metastin Receptor

BACKGROUND Metastin, the final peptide of the KiSS-1 gene, has been proposed to suppress cell motility. OBJECTIVE This study investigated whether renal cell carcinoma (RCC) tissue expresses metastin or its receptor, and clarified whether metastin can suppress migration and/or invasion and/or proliferation of RCC cells in vitro. DESIGN, SETTING, AND PARTICIPANTS Twenty-five RCC samples were submitted. Fresh RCC tissues were prepared for real-time RT-PCR, and formalin-fixed and paraffin-embedded tissues blocks were examined by immunohistochemistry. RCC cell lines Caki-1 and ACHN were supplied for cell migration, invasion, and proliferation assays. MEASUREMENTS Real-time RT-PCR was performed by using Taq Man gene expression system. ENVISION system was used in immunohistochemistry. Wound-healing assay and matrigel assays were used to identify migration and invasion abilities of RCC cell lines. Cell Counting Kit-8 was applied to measure the cell proliferation. Cell morphology was examined under a META system. Statistical analysis was performed with SPSS15.0J. RESULTS AND LIMITATIONS In twenty-five RCC samples, the mRNA level of metastin receptor was identified to be significantly higher than non-neoplastic renal cortex by real-time RT-PCR (p=0.011). Immunohistochemical study also detected metastin receptor protein in all RCC tumors. In vitro, this study showed that metastin inhibited migration and invasion of Caki-1 and ACHN cells. In contrast, it had no effects on cell proliferation. Metastin (10 micromol/l) induced excessive formation of focal adhesions and stress fibers in Caki-1 and ACHN cells; this phenomenon was inhibited by pretreating pharmacological Rho-kinase inhibitor (Y-27632) to those cells. CONCLUSION This is the first report regarding overexpression of the metastin receptor hOT7T175 in human RCC. We demonstrate that metastin can inhibit migration and invasion of the RCC cell line, which is regulated by a Rho-kinase inhibitor. Metastin and its receptor are therefore probable targets for suppressing RCC.

Immunohistochemical analysis of GCDFP-15 and GCDFP-24 in mammary and non-mammary tissue

BackgroundGross cystic disease fluid protein (GCDFP)-15, a major constituent protein in breast cysts, is known to be a marker of breast cancer, while the diagnostic value of GCDFP-24, a protein with a molecular weight of 24 000 daltons, has not been determined. The aim of this study was to elucidate the usefulness of GCDFP-24 for the differential diagnosis of breast cancer in combination with GCDFP-15 and to characterize the histologic features of GCDFP-24-positive breast cancer.MethodsA total of 326 samples including non-neoplastic tissue and benign and malignant tumors from several anatomic sites were examined using commercially available monoclonal antibodies against GCDFP-15 and GCDFP-24.ResultsIn non-mammary tissue, GCDFP-15 was detected in skin, salivary gland, bronchial gland, prostate and seminal vesicle, and GCDFP-24 was detected in apocrine glands and peripheral nerve. Thirty-seven (44.6%) and 22 (26.5%) samples of 83 breast cancers were positive for GCDFP-15 and-24, respectively. Combined assays of GCDFP-15 and -24 raised the positive rate to 50.6%. The markers were not detected in tumors originating from gastrointestinal tract, bronchopulmonary structures or the genitourinary system. Breast cancers positive for both GCDFP-15 and GCDFP-24 were of lower histologic grade according to Bloom & Richardson’s scoring system (p<0.05).ConclusionImmunohistochemical analysis of GCDFP-24 in combination with GCDFP-15 expression was useful for definitive diagnosis of breast cancers, and the expression of these markers correlated with low grade breast cancer.

Comparison of Evaluations for Hormone Receptors in Breast Carcinoma Using Two Manual and Three Automated Immunohistochemical Assays

The aims of this study were to compare the quality of immunohistochemical assays of estrogen receptor (ER) and progesterone receptor (PR) and to compare the intermethod variability of assays from different manufacturers. immunohistochemical staining was entrusted to the following laboratories in Japan: Kyowa Medex, dealing with the products of BioGenex (Mishima, Shizuoka), DAKO Japan (Kyoto) and Ventana Japan (Yokohama). All slides were semiquantitatively evaluated according to the Allred score. Intermethod variability showed fair to moderate multirater kappa values for ER and PR (for total score, ER, kappa = 0.34; PR, kappa = 0.45). Another scoring system was also applied in which, irrespective of the intensity of nuclear staining, the proportion of cells stained in each specimen was recorded as 0; less than 1%; 1% or more and less than 10%; or 10% or more. Intermethod variability showed substantial multirater kappa values for ER and PR (according to percentage of positive cells, ER, kappa = 0.67; PR, kappa = 0.72). Concerning intermethod consistency, the scoring system based on the percentage of positive cells was advantageous over other scoring systems.

PCNA and Ki-67 as Prognostic Markers in Human Parathyroid Carcinomas

Background: It is widely accepted that histological diagnosis of parathyroid tumors is established with great difficulty. Carcinomas cannot be reliably separated from adenomas by histology alone. In this study, immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and Ki-67 was determined in 10 cases of parathyroid carcinomas, labeling indices (LIs) were calculated, and the results were correlated with the clinical outcomes.Methods: Ten cases of formalin-fixed, paraffin-embedded tissue with surgically resected parathyroid carcinoma were used. Immunohistochemical staining for PCNA and Ki-67 was performed and the LIs were calculated. We also examined whether LI could become a useful marker for parathyroid carcinomas.Results: Although nine patients with minimally invasive growth without recurrence of the tumor showed a low LI for both markers, one patient with a widely invasive neoplasm, and who died, had a high LI.Conclusions: These results suggested that the LI of PCNA and Ki-67, in addition to the histological appearance, may be markers of the biological behavior of parathyroid carcinomas. However, this study was on a small scale, so it may be valuable to repeat these studies in a larger group of patients with better defined histological criteria.

Overexpression of E2F-5 correlates with a pathological basal phenotype and a worse clinical outcome

The purpose of the present study is to identify genes that contribute to cell proliferation or differentiation of breast cancers independent of signalling through the oestrogen receptor (ER) or human epidermal growth factor receptor 2 (HER2). An oligonucleotide microarray assayed 40 tumour samples from ER(+)/HER2(−), ER(+)/HER2(+), ER(−)/HER2(+), and ER(−)/HER2(−) breast cancer tissues. Quantitative reverse transcriptase PCR detected overexpression of a cell cycle-related transcription factor, E2F-5, in ER-negative breast cancers, and fluorescence in situ hybridisation detected gene amplification of E2F-5 in 5 out of 57 (8.8%) breast cancer samples. No point mutations were found in the DNA-binding or DNA-dimerisation domain of E2F-5. Immunohistochemically, E2F-5-positive cancers correlated with a higher Ki-67 labelling index (59.5%, P=0.001) and higher histological grades (P=0.049). E2F-5-positive cancers were found more frequently in ER(−)/progesterone receptor (PgR)(−)/HER2(−) cancer samples (51.9%, P=0.0049) and in breast cancer samples exhibiting a basal phenotype (56.0%, P=0.0012). Disease-free survival in node-negative patients with E2F-5-positive cancers was shorter than for patients with E2F-5-negative cancers. In conclusion, we identify, for the first time, a population of breast cancer cells that overexpress the cell cycle-related transcription factor, E2F-5. This E2F-5-positive breast cancer subtype was associated with an ER(−)/PgR(−)/HER2(−) status, a basal phenotype, and a worse clinical outcome.

What Causes Discrepancies in HER2 Testing for Breast Cancer? A Japanese Ring Study in Conjunction With the Global Standard

We assessed interinstitutional and interobserver consistency of human epidermal growth factor receptor type-2 (HER2) testing using immunohistochemical analysis and fluorescence in situ hybridization (FISH) in a set of 20 breast cancer samples among 10 institutions in Japan and a Herceptin adjuvant study participating laboratory in Germany and identified factors that may lead to discordant results.We found a good agreement in immunohistochemical HER2 scoring between the coordinating institution and 10 participating laboratories (kappa = 0.718) and excellent agreement for FISH (kappa = 0.900). The results of a comparison between 10 Japanese laboratories and the German laboratory was good for immunohistochemical studies (kappa = 0.713) and excellent for FISH (kappa = 0.887). FISH retesting of equivocal samples (2+ immunohistochemically) improved agreement. Discrepancies between results were attributed to the evaluation process in 33.0% of the samples, staining procedures in 25.0%, and a combination of the two in 41.7%. Evaluation of samples according to the American Society of Clinical Oncology/College of American Pathologists guideline increased the number of 2+ immunohistochemical scores. By performing FISH retesting for these samples, consistency among multiple institutions could be archived. The quality of the staining procedures performed and the consistency of evaluations require regular assessment.