Takashi Miyatake

Overexpression of LAMP3/TSC403/DC-LAMP Promotes Metastasis in Uterine Cervical Cancer

LAMP3 (DC-LAMP, TSC403, CD208) was originally isolated as a gene specifically expressed in lung tissues. LAMP3 is located on a chromosome 3q segment that is frequently amplified in some human cancers, including uterine cervical cancer. Because two other members of the LAMP family of lysosomal membrane glycoproteins, LAMP1 and LAMP2, were previously implicated in potentially modulating the interaction of vascular endothelial and cancer cells, we hypothesized that LAMP3 might also play an important part in metastasis. To clarify the metastatic potential of LAMP3 in cervical cancers, we transfected a LAMP3 expression vector into a human uterine cervical cancer cell line, TCS. In an in vitro invasion assay, the migration of LAMP3-overexpressing TCS cells was significantly higher than in control TCS cells. In an in vivo metastasis assay, distant metastasis was detected in 9 of 11 LAMP3-overexpressing TCS cell-injected mice and in only 1 of 11 control mice. Histologic study showed that LAMP3-overexpressing cells readily invaded into the lymph-vascular space. In clinical samples, quantitative real-time reverse transcription-PCR (RT-PCR) analyses showed that LAMP3 mRNA was significantly up-regulated in 47 of 47 (100%) cervical cancers and in 2 of 15 (13%) cervical intraepithelial neoplasias, compared with a low level of LAMP3 mRNA expressed in normal uterine cervixes. Interestingly, high LAMP3 expression was significantly correlated with the overall survival of patients with stage I/II cervical cancers. These findings indicate that LAMP3 overexpression is associated with an enhanced metastatic potential and may be a prognostic factor for cervical cancer.

Monoclonal Expansion with Integration of High-Risk Type Human Papillomaviruses Is an Initial Step for Cervical Carcinogenesis: Association of Clonal Status and Human Papillomavirus Infection with Clinical Outcome in Cervical Intraepithelial Neoplasia

To define the natural history of cervical intraepithelial neoplasia (CIN) as related to clonal status, we evaluated 20 cases of CIN1 and 18 cases of CIN2 that had been clinically followed for 7 to 48 months at Osaka University Hospital. These included 10 cases that progressed, 15 cases that persisted, and 13 cases that regressed. We analyzed the clonal status of each case by analysis of the pattern of X-chromosomal inactivation. Human papillomavirus (HPV) infection was detected by PCR-RFLP analysis. CINs that are monoclonal or infected by high-risk HPVs are more likely to progress or persist than cases that are polyclonal or negative for high-risk HPVs (p = 0.009 for monoclonal vs polyclonal, p = 0.024 for high-risk HPV positive vs negative p = 0.024). Eighteen (90%) of 20 monoclonal, high-risk HPV-associated CINs progressed or persisted, whereas 9 (60%) of 15 polyclonal or high-risk HPV-negative CINs regressed. Therefore, the combination of clonality status and high-risk type HPV infection was significantly correlated with clinical outcome (p = 0.003). The physical status of the HPV genome was evaluated in 17 cases of HPV-16 positive CINs by real-time PCR. Of those, the HPV viral genome was present in both episomal and integrated forms in 14 CINs (84%), and 12 of these cases (86%) were monoclonal in composition. By contrast, all three CINs in which the HPV genome was present in episomal form were polyclonal. In one CIN1 that was polyclonal, HPV-16 was originally present in episomal form but after 24 months, the patient developed a monoclonal CIN3 in which the HPV-16 genome was present in mixed form. These results may imply that HPV viral integration into the host genomic DNA is associated with progression from polyclonal to monoclonal status in CIN. These events may play a fundamental role in the progression from low-grade to higher grade dysplasia of the cervical mucosa.

Enhanced expression of Annexin A4 in clear cell carcinoma of the ovary and its association with chemoresistance to carboplatin

Clear cell carcinoma (CCC) of the ovary is known to be highly resistant to platinum‐based chemotherapy. The purpose of our study was to identify a candidate protein that is associated with chemoresistance of CCC and to investigate the specific mechanism of chemoresistance conferred by the identified protein. Enhanced expression of Annexin A4 (Anx A4) was identified in ovarian CCC cells using 2‐D differential gel electrophoresis (2D‐DIGE) and mass spectrometry. Anx A4 levels were elevated in CCC cells compared with non‐CCC cells as determined by real‐time RT‐PCR and Western blot analysis. Immunohistochemical analysis of Anx A4 was performed in 126 epithelial ovarian cancer tissue samples and demonstrated significantly elevated levels of Anx A4 protein levels in ovarian CCC tumors compared with ovarian serous and endometrioid tumors (p < 0.01). Anx A4‐transfected ovarian non‐CCC cells were more resistant to carboplatin (IC50 = 42 μM) compared with control cells (IC50 = 23 μM) as determined by modified MTT assay. Intracellular platinum levels were significantly lower in Anx A4‐transfected cells compared with control cells after carboplatin treatment (p = 0.0020) and after an additional 360 min of carboplatin‐free incubation (p = 0.0004), as measured by atomic absorption spectrophotometry. Expression of Anx A4 is elevated in ovarian CCC tumors and is associated with chemoresistance in cultured ovarian cancer cells. These results demonstrate that Anx A4 confers chemoresistance in ovarian cancer cells in part by enhancing drug efflux. Thus, Anx A4 may represent a novel therapeutic target of chemoresistance in patients with ovarian CCC.

PIK3CA gene mutations and amplifications in uterine cancers, identified by methods that avoid confounding by PIK3CA pseudogene sequences

PIK3CA codes for a Class IA p110-alpha catalytic subunit of the PI3Ks (phosphatidylinositol 3-kinases) that regulate various signaling pathways important for neoplasia, including cell proliferation, motility, adhesion, and survival. Pro-oncogenic mutations in exons 9 and 20 of the PIK3CA gene have been frequently observed in numerous types of human malignancies. Amplification of the PIK3CA gene has been reported in uterine cervical cancers. In this study, we have done in depth analysis of uterine cervical and endometrial cancers for PIK3CA gene mutations and amplifications. In uterine cervical cancers, PIK3CA mutations were found in 3 of 22 cases (14%), all of them in exon 9. In endometrial cancers, a similar incidence of mutations was found, in 3 of 29 cases (10%), however they were all within exon 20. Amplification of the PIK3CA gene was also detected in 2 out of 22 (9%) cervical cancers and 3 out of 29 (10%) endometrial cancers. In this study, we were unable to find a clear association between PIK3CA mutations and gene amplifications, nor with tumor histological subtypes or staging. Mutations and amplifications of the PIK3CA gene are relatively infrequent in human cervical and endometrial cancers; however, PIK3CA gene alteration may still play a role in some subset of uterine cancers.

A retrospective analysis of ovarian endometriosis during pregnancy

OBJECTIVE To clarify the frequency of pregnancy complicated by ovarian endometriosis and to investigate the size change and outcome of ovarian endometriosis during pregnancy. DESIGN Retrospective study. SETTING Departments of obstetrics and gynecology of the Osaka University and Izumiotsu Municipal Hospitals of Osaka, Japan. PATIENT(S) Women who delivered between 1996 and 2007. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) The frequency of pregnancies complicated by ovarian endometriosis and the size change of the lesions during pregnancy. RESULT(S) The frequency of ovarian endometriosis-complicated pregnancy has almost quadrupled over the last 12 years, to become the most common adnexal mass detected during pregnancy; it was 0.14% (five cases among 3558 deliveries) during the 6-year period from 1996 to 2001, but has increased to 0.52% (19 cases among 3599 deliveries) during the 6-year period from 2002 to 2007, a statistically significant increase of 3.8-fold. Among 25 ovarian endometriotic lesions observed during pregnancy in 24 women (one case had two lesions), the size of the cyst decreased in 13 lesions (52%), went unchanged in 7 (28%), and increased in 5 (20%), that demonstrated decidualization, abscess and rupture. CONCLUSION(S) Ovarian endometriosis during pregnancy can be safely observed conservatively; however, further investigation is required to predict the occurrence of abscess formation or rupture of ovarian endometriosis, and to distinguish the enlargements due to malignant transformation from those related to decidualization.

Analysis of Clonality and HPV Infection in Benign, Hyperplastic, Premalignant, and Malignant Lesions of the Vulvar Mucosa

To elucidate the pathogenesis of vulvar carcinomas, we studied clonality and human papillomavirus (HPV) infection in vulvar epithelial diseases. Monoclonal composition was demonstrated in all 9 invasive tumors (squamous cell carcinoma [SCC], 6; basal cell carcinoma, 1; malignant melanoma, 2), 15 of 20 cases of vulvar intraepithelial neoplasia (VIN), 7 of 9 cases of Paget disease, 2 of 6 cases of lichen sclerosus (LS), and 2 of 3 cases of squamous cell hyperplasia (SCH); high-risk type HPV was revealed in 5 of 6 SCCs and 17 of 20 VINs. These observations might imply that a subset of cases of LS and SCH result from a neoplastic proliferation, similar to VINs but not related to infection with high-risk type HPV. In 1 case of SCC with concurrent VIN 3 in an adjacent lesion, both lesions showed the same pattern of X chromosome inactivation and the presence of HPV-16 in episomal and integrated forms, suggesting that monoclonal expansion triggered by high-risk type HPV integration is an early event for carcinogenesis of HPV-associated SCC.

Impact of histological subtype on survival of patients with surgically-treated stage IA2–IIB cervical cancer: Adenocarcinoma versus squamous cell carcinoma

OBJECTIVES To evaluate the significance of adenocarcinoma (AC) compared with squamous cell carcinoma (SCC) with regard to the survival of surgically-treated early stage cervical cancer patients. METHODS We retrospectively reviewed the medical records of 520 patients with FIGO stage IA2-IIB cervical cancer who were treated with radical hysterectomy with or without adjuvant radiotherapy between January 1998 and December 2008. The patients were classified according to (i) pathological risk factors (low-, intermediate-, or high-risk group) and (ii) adjuvant radiotherapy (concurrent chemoradiotherapy [CCRT group] or radiotherapy alone [RT group]). Survival outcomes were examined by Kaplan-Meier method and compared with Log-rank test. Multivariate analysis for disease-specific survival (DSS) was performed using Cox proportional hazards regression model to investigate the prognostic significance of histological subtype. RESULTS AC histology was associated with significantly decreased DSS compared with SCC histology in the intermediate- and high-risk groups (hazard ratio: 3.06 and 2.88, respectively, both P<0.05) while there was no survival difference in the low-risk group (P=0.1). Among patients who received any types of adjuvant radiotherapy, DSS of AC histology patients were significantly poorer than SCC histology. Multivariate analysis demonstrated AC histology to be an independent predictor of decreased DSS in both CCRT and RT groups. Moreover, pelvic nodal metastasis significantly predicted the poor survival of patients with AC histology who received CCRT in multivariate analysis CONCLUSIONS Adenocarcinoma is an independent prognostic indicator of poor survival in early stage cervical cancer patients with intermediate- and high-risk factors, regardless of the type of adjuvant radiotherapy after radical hysterectomy.

Serum Biomarkers for Early Detection of Gynecologic Cancers

Ovarian, endometrial, and cervical cancers are three of the most common malignancies of the female reproductive organs. CA 125, historically the most reliable serum marker for ovarian cancer, is elevated in 50% of early-stage ovarian tumors. For endometrial cancers, there are no established serum markers. SCC, which is the best studied serum marker for squamous cell carcinomas, has been unreliable; SCC is elevated in cervical squamous cell carcinomas ranging from 28–85% of the time. Recent proteomics-based analyses show great promise for the discovery of new and more useful biomarkers. In this review, we will discuss the currently utilized serum tumor markers for gynecologic cancers and the novel biomarkers that are now under investigation.

Frequent inactivation of RUNX3 in endometrial carcinoma

OBJECTIVE Our objective was to determine whether RUNX3 tumor suppressor is inactivated in endometrial carcinoma. METHODS We have investigated 24 endometrial carcinomas, 3 endometrial carcinoma cell lines, and 9 normal endometria for genetic and epigenetic alterations of RUNX3. Reverse-transcription PCR (RT-PCR), methylation-specific PCR (MS-PCR) analysis, and loss of heterozygosity (LOH) analysis were performed. We also tested RUNX3 protein expression by immunohistochemistry. RESULTS Using RT-PCR technique, we observed a significant loss of RUNX3 mRNA expression in nine of 24 endometrial carcinomas (38%) and in all 3-cell lines (100%). In contrast, all nine of the normal endometria showed an abundant expression of RUNX3 mRNA. Methylation-specific PCR (MS-PCR) analysis of the CpG islands of RUNX3 showed the promoter region to be hypermethylated in 18 of 21 analyzed carcinomas (86%), whereas only two of nine normal endometria (22%) were methylated (p<0.01). By using two polymorphic microsatellite markers, D1S199 and D1S1676, we detected 1p36 LOH in 7 of 21 carcinomas (33%). We observed a significant relationship between the loss of RUNX3 mRNA expression and this regional LOH (p<0.01). Immunohistochemical staining showed that RUNX3 protein expression was lost in 12 of 21 endometrial carcinomas (57%). We observed a significantly more frequent loss of RUNX3 protein expression in the histologically higher-grade tumors (Grade 3) than in Grade 1 or 2 tumors (p<0.01). CONCLUSION These findings indicate that RUNX3 inactivation may play an important role in carcinogenesis of the endometrium, especially in high-grade endometrial carcinoma.

Down‐regulation of insulin‐like growth factor binding protein‐5 (IGFBP‐5): Novel marker for cervical carcinogenesis

To better understand the underlying pathways of cervical carcinogenesis, cDNA microarray analysis was performed on 2 sets of squamous cell carcinomas (SCCs) and their adjacent normal squamous epitheliums. Consistently altered expression was detected for 32 genes. Real‐time RT‐PCR analysis was conducted on a selected subset of these genes (S100A2, GPC4, p72, IGFBP‐5, TRIM2 and NAB2) for 14 additional SCCs and 10 normal epithelia. This found that, of the 6 candidate genes, only the insulin‐like growth factor binding protein‐5 (IGFBP‐5) mRNA was generally and significantly under‐expressed in SCCs (p < 0.001). All normal cervical epithelia (30 of 30) stained positively for IGFBP‐5 protein, with 70% showing strong staining, whereas 65% (17/26) of SCC had complete loss of IGFBP‐5, and only 8% (2/26) SCC retained strong expression (p < 0.001). Immunohistochemistry of premalignant cervical intraepithelial neoplasia (CIN) lesions shows a significantly weaker or negative staining in advanced CIN3 lesions compared with normal squamous epithelia (p = 0.001). This is the first study to show that down‐regulation of IGFBP‐5 protein correlates with cervical carcinogenesis and does so at a preneoplastic stage.