R. Yoshiyuki Osamura

In situ expression of corticotropin-releasing hormone (CRH) and proopiomelanocortin (POMC) genes in human skin

Systemic stresses induce corticotropin‐releasing hormone (CRH) expression in hypothalamus. CRH is released to the pituitary gland, where it stimulates proopiomelanocortin (POMC) production acting via the CRH receptor (CRH‐R). CRH and POMC peptides are also detected in sites outside of the central nervous system (CNS), such as the skin. However, it has not been elucidated whether these peptides detected in the skin are derived from CNS or are produced locally. Using immunohistochemical and in situ reverse‐transcription (RT)‐PCR techniques, we demonstrated coexpression of CRH and POMC mRNAs in the epidermis and pilosebaceous units of the human skin. This coexpression was confirmed by the combination of laser‐capture microdissection (LCM) with RT‐PCR, analyzing mRNA expressions in captured sebaceous cells. Immunoreactivities and expressions of CRH and POMC mRNAs were strong in inflammatory lesions, melanocytic nevus, seborrheic keratosis, and also in the periphery of the benign tumor. These findings suggest that CRH and POMC peptides are produced locally in the skin and are regulated by inflammatory cells as well as by autocrine mechanisms. The skin may have “a local stress response system,” whose activity is mediated by CRH and POMC peptides, in an equivalent to hypothalamus‐pituitary adrenal axis.

Pathology of pituitary tumors.

Pituitary adenomas are benign neoplasms originating in adenohypophysial cells. They represent the most common neoplasm of the sellar region, comprising approximately 15% of all primary intracranial tumors. Depending on the studies of unselected adult autopsy material, their frequency as an incidental finding varies between 5% and 20%. The first part of this article summarizes the immunohistochemistry of nontumorous human adenohypophysis. In the second part, the classification of pituitary tumors is discussed, followed by the immunohistochemical and electron microscopic findings of pituitary adenomas and pituitary carcinomas.

Quantitative immunohistochemical evaluation of HER2/neu expression with HercepTestTM in breast carcinoma by image analysis

HercepTestTM (DAKO A/S, Glostrup, Denmark) is an immunohistochemical assay that detects HER2/neu gene products, and evaluates the overexpression status of the HER2/neu protein in determining eligibility for the Trastuzumab (HerceptinR, Genentech, San Francisco, CA, USA) therapy. However, practically, interobserver variability of the HER2/neu interpretation of the immunostained results has caused marked disagreement with regard to the intensity of tumor staining. In this study, we quantitated HER2/neu expression by image analysis, and applied this analyzing system to help to minimize interobserver variability of the interpretation of the HercepTestTM. All the immunostained results were scored semiquantitatively on a range of 0 to 3+ in accordance with the criteria described as per the manufacturer’s instructions, and quantitatively evaluated using an image analyzing system with image processing software. Among the 92 cases, 15 were scored as 3+, six were 2+, and 32 were 1+ under intraobservers agreement. When the cases were quantitated, a high correlation was shown between the signal area extracted by image analysis and the corresponding score of staining intensity with the HercepTestTM. By converting the quantitatively extracted data into a scoring system based upon the criteria, the outcome demonstrated a strong concordance with the scoring data obtained from immunostaining. The results indicated that a quantitative scoring system performed by simple image analysis may provide to improve interobserver agreement of the interpretation of the HercepTest TM in clinical practice.

Ghrelin and growth hormone (GH) secretagogue receptor (GHSR) mRNA expression in human pituitary adenomas

OBJECTIVE The level of growth hormone (GH), growth hormone secretogogue (GHS) and GHS receptor (GHSR) messenger ribonucleic acid (mRNA) expression has been reported as being higher in GH‐producing pituitary adenomas than in other types of pituitary adenomas. Recently, ghrelin, an endogenous ligand specific for GHSR, was isolated. Therefore, we attempted to clarify whether ghrelin mRNA is expressed in various types of human pituitary adenoma by competitive reverse transcription‐polymerase chain reaction (RT‐PCR). We also examined the relationship between the levels of ghrelin or GHSR mRNA and hormonal and tumour characteristics in patients with pituitary adenomas.

Therapeutic strategy targeting the mTOR-HIF-1α-VEGF pathway in ovarian clear cell adenocarcinoma

Malignant tumors usually involve a relatively hypoxic state, which induces overexpression of hypoxia‐inducible factor‐1α (HIF‐1α) to satisfactorily enable the tumor to survive. Thus, inhibition of the mammalian target of rapamycin (mTOR) pathway including HIF‐1α is expected to play a major role in suppression of tumor cell growth, having recently drawn much attention as an anti‐cancer therapeutic strategy for various malignant tumors. In the present study, which compared clear cell adenocarcinoma (CLA) of the ovary with serous adenocarcinoma (SEA), the immunohistochemical expression of mTOR, phosphorylated‐mTOR (p‐mTOR), HIF‐1α, and vascular endothelial growth factor (VEGF) was examined in surgically resected specimens of 29 SEA and 47 CLA. There were no significant differences in expression of mTOR, HIF‐1α and VEGF between SEA and CLA, but it was noted that p‐mTOR expression was more prominent in CLA than SEA. Then, using the cell lines of CLA (RMG‐1 and W3uF), an experimental study was designed to clarify whether tumor suppression due to downregulation of mTOR activity could represent a promising therapeutic strategy for CLA. After treatment of an analogue of rapamycin (everolimus), expression of mTOR, p‐mTOR, HIF‐1α and VEGF was examined on western blot. As a result, although mTOR expression remained unchangeable, expression of p‐mTOR, HIF‐1α and VEGF was shown to be sharply depressed. The same expression alterations were demonstrated in the xenograft model treated with everolimus. In conclusion, mTOR‐targeted therapy through usage of drugs such as everolimus may be more effective for CLA of the ovary because of its significant expression of p‐mTOR.

DNA Mismatch Repair Protein (MSH6) Correlated With the Responses of Atypical Pituitary Adenomas and Pituitary Carcinomas to Temozolomide: The National Cooperative Study by the Japan Society for Hypothalamic and Pituitary Tumors

CONTEXT Temozolomide (TMZ) is an alkylating agent and was a first-line chemotherapeutic agent for malignant gliomas. Recently, TMZ has been documented to be effective against atypical pituitary adenomas (APAs) and pituitary carcinomas (PCs). OBJECTIVE The clinical and pathological characteristics of APAs and PCs treated with TMZ in Japan were surveyed and analyzed retrospectively. DESIGN Members of the Japan Society of Hypothalamic and Pituitary Tumors were surveyed regarding the clinical characteristics of APAs and PCs treated with TMZ. Stored tumor samples were gathered from the responders and were assessed by the immunohistochemistry of Ki-67, O(6)-methyl-guanine-DNA methyltransferase, p53, MSH6, and anterior pituitary hormones. Responses to TMZ treatment were defined as complete response (CR), partial response (PR), progressive disease (PD), and stable disease (SD) according to RECIST (Response Evaluation Criteria in Solid Tumors) version 2.0. SUBJECTS Three samples from 3 subjects with APA and 11 samples from 10 subjects with PC were available. RESULTS The 13 subjects had APAs and PCs consisting of 5 prolactin-producing tumors, 5 ACTH-producing tumors, and 3 null cell adenomas. The clinical response to TMZ treatment was as follows: 4 cases of CR and PR (31%), 2 cases of SD (15%), 6 cases of recurrence after CR and PR (46%), and 1 case of PD (8%). However, considerable subjects had recurrent disease after a response to TMZ. The immunohistochemical findings of Ki-67, O(6)-methyl-guanine-DNA methyltransferase, and p53 did not show any significant correlation with the efficacy of TMZ. However, the immunopositivity of MSH6 was positively correlated with TMZ response (P = .015, Fishers exact test). CONCLUSIONS This study showed that preserving MSH6 function was contributory to the effectiveness of TMZ in malignant pituitary neoplasms. It is necessary to survey more cases and evaluate multifactor analyses.

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)

Simultaneous immunohistochemical localization of gastrin releasing peptide (GRP) and calcitonin (CT) in human bronchial endocrine-type cells

Formalin-fixed paraffin-embedded sections of fetal and adult human lungs were examined for the localization of gastrin releasing peptide (GRP) and calcitonin (CT) in bronchial endocrine-type cells with the indirect immunoperoxidase method. In fetal lungs, the appearance of CT was much later than that of GRP, and CT-containing cells were less frequent than GRP-containing cells which, in later fetal life, formed “neuroepithelial bodies (NEB)”. NEB revealed little CT immunoreactivity. The serial section technique demonstrated that all CT immunoreactants in fetal and neonatal lungs were present within GRP-containing cells. An increase of CT immunoreactivity in GRP-containing cells was observed in perinatal lungs. The lung of a neonate who died of hyaline membrane disease contained the most abundant CT immunoreactants. In adult lungs, CT immunoreactivity was identified in some GRP-containing cells but cells containing only GRP or CT were also present. Cells containing both hormones occasionally formed hyperplastic foci in the bronchi of fibrotic lungs. Most cells of pulmonary tumorlets consistently showed GRP immunoreactivity, but the number of CT immunoreactive cells in them varied greatly.

Immunohistochemical studies on oncogene products (c-erbB-2, EGFR, c-myc) and estrogen receptor in benign and malignant breast lesions. With special reference to their prognostic significance in carcinoma.

It is a matter of debate whether the amplification of c-erbB-2 oncogene or production of the oncoprotein in breast cancers correlate with the presence of lymph node metastasis and with a poor prognosis. This study was aimed at elucidating the immunohistochemical localization of oncogene products which are related to cell growth, c-erbB-2 product, epidermal growth factor receptor (EGFR), c-myc protein and estrogen receptor (ER), in benign and malignant lesions of the breast. Fresh frozen sections of 25 breast cancers and 11 fibroadenomas from Japanese women were studied by indirect immunoperoxidase method with proper fixation. C-erbB-2 product and EGFR were localized on the cell membrane whereas c-myc protein and ER were observed in the nuclei. Immunohistochemical expression of oncogene products and ER were not only observed in the mammary carcinomas but also in the fibroadenomas. However immunoreactivities of EGFR and ER were more frequently seen in the fibroadenomas (p<0.05). In breast cancers, the incidence of immunoreactivity for c-erbB-2 was higher in the cases with lymph node metastasis than cases without nodal metastasis (p < 0.05) and there was reciprocal correlation between the expressions of EGFR and ER (p<0.05). Regarding the size of the primary tumour, there was no statistically significant correlation with the expressions of c-erbB-2, EGFR, c-myc or ER. Histological grade correlated only with the expression of ER (p<0.05).

Thyrotropin-secreting pituitary adenomas. Clinical and biological heterogeneity and current treatment

Thyrotropin (TSH)-secreting pituitary adenomas represent about 1–2% of all pituitary adenomas and cause secondary or central hyperthyroidism. TSH-secreting adenomas are part of the syndrome of ‘inappropriate secretion of TSH’ (SITSH). The hormonal profile is characterized by nonsuppressed TSH in the presence of high levels of free thyroid hormones (FT3 and FT4). Previous reports have described the surgical cure of TSH adenoma to be more difficult than other functional adenomas because of large and invasive features. However, with the current introduction of ultrasensitive immunometric assays, TSH-secreting adenomas are more often recognized. Early diagnosis of TSH-secreting adenomas leads to a high rate of remission of hyperthyroidism after surgery. However, some of those type of adenomas have clinical heterogeneity, and subsequently cannot be cured by surgery alone. We present our experiences and review reported cases of TSH-secreting adenomas to direct current nobreak management.