Kousuke Yoshinaga

Vasohibin-1 in human breast carcinoma: a potential negative feedback regulator of angiogenesis.

Vasohibin‐1 is a recently identified negative feedback inhibitor or suppressor of angiogenesis induced by vascular endothelial growth factor (VEGF)‐A. The status of vasohibin‐1 in human breast carcinoma has not been examined. We examined 151 breast specimens including 98 cases of invasive ductal carcinoma (IDC), 12 of ductal carcinoma in situ (DCIS), 16 of fibroadenoma (FA), six of inflammatory lesion, nine of fibrocystic change and seven of non‐pathological breast tissue. We immunolocalized vasohibin‐1 and compared its immunoreactivity to that of VEGF‐A, basic fibroblastic growth factor (bFGF), VEGF receptor 2 (Flk‐1), CD31, CD34 and Ki‐67/MIB‐1. The correlation of vasohibin‐1 immunoreactivity with overall survival (OS), and disease‐free survival (DFS) of the patients with breast carcinoma was also evaluated. In addition, we evaluated Ki‐67 and CD31, and Ki‐67 and vasohibin‐1 double‐immunostaining for further characterization of neovascularization. Vasohibin‐1 was detected in endothelial cells of human breast and its immunodensity was significantly higher in IDC and inflammatory lesions than the other types (P < 0.001). In addition, a significant positive correlation was detected between vasohibin‐1 and VEGF‐A, bFGF or Flk‐1 (P < 0.001). There was also positive associations between vasohibin‐1 and OS (P = 0.004) and between vasohibin‐1 and DFS (P ≤ 0.001) in carcinoma cases. Results of double‐immunostaining demonstrated the ratio of Ki‐67‐positive cells among vasohibin‐1‐positive endothelial cells (46.5%) was significantly higher than those among CD31‐positive cells (23.5%). This is the first study demonstrating the status of vasohibin‐1 in human breast lesions, which indicates that vasohibin‐1 is associated with neovascularization and may especially play important roles in the regulation of intratumoral angiogenesis in human breast cancer. (Cancer Sci 2009; 100: 88–94)

Expression of vasohibin as a novel endothelium-derived angiogenesis inhibitor in endometrial cancer

We have previously reported on vasohibin as a novel endothelium‐derived vascular endothelial growth factor (VEGF)–inducible inhibitor of angiogenesis. The aim of our present study was to define the role of vasohibin in endometrioid endometrial adenocarcinoma. We collected 78 sections of endometrial carcinoma for assessment using immunohistochemistry. Twenty‐seven were well differentiated (G1), 25 were moderately differentiated (G2), and 26 were poorly differentiated endometrioid adenocarcinomas (G3). We also included 12 sections of normal cyclic endometria, six of which were in the proliferative phase and six were in the secretory phase. We investigated the expression of vasohibin, and compared it to VEGF receptor‐2 (VEGFR‐2: KDR/flk‐1), CD34, Ki‐67, VEGF‐A, and D2‐40 (as a lymphatic vessel marker). We assessed the ratio of vasohibin‐ and VEGFR‐2‐positive vessels in the stroma of endometrial carcinoma. Immunohistochemical assessment was classified as negative or positive based on staining intensity. Vasohibin was selectively expressed on vascular endothelial cells in both cyclic endometria and endometrial carcinomas. Vasohibin was highly expressed in the normal functional endmetrium of the secretory phase, especially in the spiral artery, and was highly expressed in all grades of endometrioid adenocarcinomas. The stromal endothelial cells in G3 expressed vasohibin and VEGFR‐2 more frequently than these in G1. In endometrioid adenocarcinomas, there was a significant correlation between the expression percentage of vasohibin and that of VEGFR‐2 (P < 0.0001, r2 = 0.591). This is the first study to elucidate the correlation between expression of vasohibin in the stromal endothelial cells and that of VEGFR‐2 in human carcinomas. (Cancer Sci 2008; 99: 914–919)

The PTEN, BAX, and IGFIIR Genes Are Mutated in Endometrial Atypical Hyperplasia

To pursue the pathogenesis of endometrial carcinogenesis, we investigated microsatellite instability, mutations in the PTEN, TGFβRII, IGFIIR, and BAX genes, and LOHs on 10q in 18 putative endometrial premalignant lesions (11 endometrial atypical hyperplasias (ATHs), 2 complex hyperplasias, and 5 simple hyperplasias) as well as 8 endometrial cancers (ECs). In the ATH cases, MSIs as well as LOHs at 10q were observed at frequencies similar to those in ECs. Mutations in PTEN, BAX, and IGFIIR were observed only in ATHs and ECs. These results suggest that (1) PTEN, BAX, and IGFIIR are already mutated in ATHs, and (2) ATH is one of the precursor lesions which could lead to EC.

Roles of intrinsic angiogenesis inhibitor, vasohibin, in cervical carcinomas.

The aim of the present study is to clarify the critical roles of vasohibin in cervical carcinomas. We investigated the expression ratios of vasohibin and vascular endothelial growth factor (VEGF) receptor‐2 on endothelium and microvessel density, lymphatic vessel density (LVD) by immunohistochemistry. Sixty‐one squamous cell carcinoma (SCC), 18 mucinous adenocarcinoma (Adenocarcinoma), 38 carcinoma in situ (CIS), and 35 normal cervical epithelium were collected. We investigated the expression of vasohibin and compared it with the expression of VEGF receptor‐2 (VEGFR‐2, KDR/flk‐1), and CD34 in the stromal endothelium. Expression of VEGF was counted using the histological score (H score). D2‐40 was used as a marker for lymphatic endothelial cells to investigate LVD. The microvessel density of the normal cervical epithelium was significantly lower than that of CIS, SCC, and Adenocarcinoma (P < 0.05). The expression ratio of vasohibin in the normal cervical epithelium was significantly lower than that of SCC and Adenocarcinoma (P < 0.05). The expression ratio of VEGFR‐2 of the normal cervical epithelium was significantly lower than that of SCC and Adenocarcinoma (P < 0.05). The LVD of the normal cervical epithelium was significantly lower than that of CIS, SCC, and Adenocarcinoma (P < 0.05). For normal cervical epithelium, CIS, and SCC, there was a moderate correlation between the expression percentage of vasohibin and the expression percentage of VEGFR‐2 (P < 0.05, r2 = 0.3018). This is the first study to elucidate the correlation between the expression of vasohibin in the stromal endothelial cells and the expression of VEGFR‐2 in human cervical carcinomas. (Cancer Sci 2011; 102: 446–451)

Management of ovarian hyperstimulation due to follicle-stimulating hormone-secreting gonadotroph adenoma

Terguride therapy was administered and a brain MRI was planned due to mild hyperprolactinaemia and galactorrhoea. The MRI revealed a pituitary tumour. Visual field examination, which was performed after the MRI, revealed bitemporal hemianopsia. The levels of other pituitary hormones were within the normal ranges and were as follows: growth hormone (GH), 1.68 ng/mL; thyrotropinstimulating hormone (TSH), 1.05 AIU/mL; and adenocorticotropic hormone (ACTH), 36.7 pg/mL. Terguride therapy was cancelled in a week, and transsphenoidal removal of the pituitary adenoma was performed as the initial therapy. Her ovarian cysts were not treated because she did not have severe abdominal pain and there was a low possibility of malignancy. Immunohistochemical staining of the pituitary tumour for pituitary hormones revealed that the tumour cells expressed LH, FSH and PRL but not GH, TSH or ACTH. The patient was diagnosed with gonadotroph adenoma. Her post-operative course was uneventful. She had withdrawal bleeding for several days after the operation. The bilateral huge ovarian cysts dramatically decreased in size and her hormone levels of FSH, PRL, oestradiol and LH improved within one month after the operation. Other pituitary hormone levels were not influenced by the transsphenoidal surgery. Her menstrual cycle resumed, and her visual field deficits improved gradually. At 26 years of age, five years after excision of the pituitary tumour, she had no recurrent signs of gonadotroph adenoma. She conceived spontaneously during the following year. She delivered a female baby weighing 2488 g by spontaneous vaginal delivery at 37 weeks of gestation.

CASE REPORT: Management of ovarian hyperstimulation due to follicle‐stimulating hormone‐secreting gonadotroph adenoma

Terguride therapy was administered and a brain MRI was planned due to mild hyperprolactinaemia and galactorrhoea. The MRI revealed a pituitary tumour. Visual field examination, which was performed after the MRI, revealed bitemporal hemianopsia. The levels of other pituitary hormones were within the normal ranges and were as follows: growth hormone (GH), 1.68 ng/mL; thyrotropinstimulating hormone (TSH), 1.05 AIU/mL; and adenocorticotropic hormone (ACTH), 36.7 pg/mL. Terguride therapy was cancelled in a week, and transsphenoidal removal of the pituitary adenoma was performed as the initial therapy. Her ovarian cysts were not treated because she did not have severe abdominal pain and there was a low possibility of malignancy. Immunohistochemical staining of the pituitary tumour for pituitary hormones revealed that the tumour cells expressed LH, FSH and PRL but not GH, TSH or ACTH. The patient was diagnosed with gonadotroph adenoma. Her post-operative course was uneventful. She had withdrawal bleeding for several days after the operation. The bilateral huge ovarian cysts dramatically decreased in size and her hormone levels of FSH, PRL, oestradiol and LH improved within one month after the operation. Other pituitary hormone levels were not influenced by the transsphenoidal surgery. Her menstrual cycle resumed, and her visual field deficits improved gradually. At 26 years of age, five years after excision of the pituitary tumour, she had no recurrent signs of gonadotroph adenoma. She conceived spontaneously during the following year. She delivered a female baby weighing 2488 g by spontaneous vaginal delivery at 37 weeks of gestation.

Identification of a 100‐kb region of common allelic loss on chromosome bands 10q25–q26 in human endometrial cancer

In human endometrial cancer, we have previously identified a 790‐kb region of common allelic loss in chromosome bands 10q25–q26, flanked by D10S587 and D10S1723. We constructed a contig covering the entire deleted region using YACs, PACs, and BACs. Five overlapping cosmid clones derived from YAC clones completely covered the entire deleted region: its size was estimated to be no larger than 200 kb. We further performed two‐color fluorescence in situ hybridization (FISH) analysis to confirm the deletion and narrowed down the deleted region to 100 kb or less; it was covered by three overlapping cosmid clones that were included in one BAC clone. Restriction endonuclease mapping identified a region in which NotI, SalI, SmaI, and XhoI were clustered, suggesting the possible existence of a CpG island. Genes Chromosomes Cancer 23:74–77, 1998.

Abstract 316: Keap1 and Nrf2 genetic mutations and polymorphisms: Associations with clinical outcome in endometrial cancer

Background: Constitutive activation and increased expression of Nrf2 as a result of oncogenic mutations or other endogenous factors that cause derepression of Nrf2 by its cytosolic inhibitor, Keap1, contribute to tumor resistance to adjuvant treatment. As endometrial cancer generally has a lower response rate to adjuvant treatment, Keap1 and Nrf2 genes were sequenced to investigate their roles in endometrial cancer. Materials and Methods: Tumor specimens from 105 individuals with endometrioid endometrial adenocarcinoma (Type I endometrial cancer) were sequenced. The specimens comprised 44 well-differentiated (FIGO grade 1), 38 moderately differentiated (FIGO grade 2) and 23 poorly differentiated (FIGO grade 3) tumors. Statistical analyses using the Fisher9s exact test, Pearson9s Chi-square test and Cox proportional hazards model were used to investigate the association of genetic mutations and haplotypes with patients’ annotated clinicopathologic characteristics. Results: A total of ten novel Keap1 mutations were found in nine individuals, and two novel Nrf2 mutations were found in three individuals. One patient had two simultaneous Keap1 mutations, whereas two patients had the same type of Nrf2 genetic mutation. Two single nucleotide polymorphisms, rs1048290 and rs11545829 were found in high frequency in the present cohort. The non-synonymous genetic mutations and rs11545829 were not associated with patients’ clinicopathologic characteristics. In the adjuvant treatment subgroup, the rs1048290 c.1413C/G genotype was significantly associated with a better progression-free survival (hazard ratio, 0.25; 95% confidence interval [CI], 0.068 to 0.94; P=0.041). On multivariate analysis, rs1048290 was an independent prognostic factor for progression-free survival (hazard ratio, 0.16; 95% CI, 0.036 to 0.69; P=0.014). Conclusion: Keap1 single nucleotide polymorphism rs1048290 may be a potential biomarker that enables individualized therapy using cytotoxic adjuvant treatment in endometrial cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 316. doi:10.1158/1538-7445.AM2011-316