Abstract: 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.
Abstract: A 36-year-old nulliparous woman developed multiple extra-uterine fibroids in the pelvic cavity years after laparoscopic myomectomy. Molecular genetic analysis by methylation-specific polymerase chain reaction (MSPCR) of the human X-linked androgen receptor gene and loss of heterozygosity (LOH) analysis at 5 microsatellite loci was performed on the tumors. All tumors showed an identical non-random X-chromosome inactivation pattern by MSPCR and an identical pattern of LOH was found in all the tumors by LOH analysis. This demonstrated that 3 fibroids resected 2 years later and 14 fibroids resected 6 years later were all metastatic tumors originating from the uterine leiomyoma found during the initial surgery, suggesting that morcellation before removal of the leiomyoma nodule during laparoscopic myomectomy may have been associated with the pathogenesis of this case.
Abstract: 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 microM) compared with control cells (IC50 = 23 microM) 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.
Abstract: Cervical intraepithelial neoplasia (CIN) has its highest incidence during women's reproductive years. During 2 sequential 7-year periods, 1994 to 2000 and 2001 to 2007, 3695 and 3894 deliveries were performed, respectively, at Osaka University Hospital. CIN was detected in 21 cases (0.57%) during 1994-2000 and in 43 cases (1.1%) during 2001-2007. By comparison, cervical intraepithelial neoplasia-complicated pregnancies increased significantly in the latter period (P = .015 by Fisher exact test, Odds ratio = 1.95; 95%CI: 1.16-3.30). We observed CIN regression in 34 (76%) of 45 cases of vaginal delivery and in 6 (50%) of 12 cases of cesarean delivery, indicating that the outcome of an initially diagnosed CIN and the delivery routes appeared not to be significantly related. However, a different result was obtained when only those patients whose CIN lesions persisted until the delivery were analyzed. Among the 35 such cases in the vaginal delivery group, 24 cases (69%) regressed after the delivery; in 8 such cases from the cesarean delivery group, only 2 cases (25%) regressed afterward. Our study clearly shows that pregnancy complicated with CIN is increasing rapidly in Japan. We also find that there is a significantly more frequent postpartum regression of biopsy-proven CIN lesions following a vaginal delivery compared to cesarean section (P = .042 by Fisher exact test, Odds ratio = 6.55; 95% CI: 1.13-37.8).
Abstract: 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.
Abstract: We analyzed the clonality and human papillomavirus (HPV) infection status of concurrent glandular and squamous lesions and adenosquamous carcinomas of the uterine cervix to clarify their histogenesis. The glandular and squamous components were clonally different from each other in 7 informative concurrent lesions. HPV was episomal in 2 polyclonal glandular dysplasias (GDs). HPV was in a mixed integrated-episomal form in a monoclonal GD, an adenocarcinoma in situ, and an adenocarcinoma. Both tumor components were monoclonal in origin in 6 adenosquamous carcinomas, with identical patterns of X-chromosomal inactivation and types and physical status of HPV. These results imply that the concurrent glandular and squamous lesions are formed separately, whereas adenosquamous carcinoma is more likely to be a combination tumor of monoclonal origin, and that integration of HPV has an important role in the progression from polyclonal GD through monoclonal expansion to adenocarcinoma in situ and adenocarcinoma.
Abstract: 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 epithelia. 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.
Abstract: Atypical immature squamous metaplasia (ISM) of the uterine cervix often has histological features that overlap with the histological characteristics of high-grade cervical intraepithelial neoplasia. To identify the cellular basis and clinical significance of atypical immature metaplasia (AIM), 10 cases of AIM were analyzed for the clonal status, and the presence of human papillomavirus (HPV) infection. The physical status of HPV was also evaluated in HPV type 16 (HPV-16)-positive cases. Squamous metaplasias with no nuclear atypia (29 mature squamous metaplasias [SMs]) and a single case of ISM were analyzed as a control. Nine AIMs, 20 SMs, and a single case of ISM were informative for clonal analysis. Monoclonal composition of the lesion was demonstrated in 8 (89%) of 9 AIMs, but only in 2 (10%) of 21 cases of SM without atypia (AIM vs SM + ISM, 8/9 vs 2/21; P < 0.0001). High-risk HPV was detected in 6 (60%) of 10 AIMs, all were HPV-16, but only in 3 (13%) of 24 SMs with no atypia (2/23 SM and 1/1 ISM). The frequency of high-risk HPV infection was also significant between AIMs and SM with no atypia (6/10 vs 3/24; P < 0.001). Among the cases, which were informative for clonal analysis, all 5 AIMs positive for high-risk HPV were monoclonal in composition. Physical status of HPV was examined in HPV-16-positive cases. Human papillomavirus type 16 was present as a mixture of episomal form and integrated form in 4 of 6 AIMs. These observations imply that unlike SMs with no atypia, which arises as a result of reactive or inflammatory process, lesions with the histological characteristics of AIM may be indeed true precursors of cervical carcinoma.
Abstract: The prognostic significance of p53 mutation, microsattelite instability and DNA mismatch protein hMLH1 expression in suboptimally resected advanced ovarian carcinoma treated with the combination chemotherapy of paclitaxel and carboplatin was evaluated. The overall combination chemotherapy response rate and the complete remission rate were significantly higher among patients with mutant p53 tumors than those with wild-type p53 tumors (35/42 (83%) vs. 32/58 (55%); P=0.003 and 18/42 (43%) vs. 16/58 (28%); P=0.03, respectively). This tendency apparently existed in non-serous carcinoma, but not in serous carcinoma. Univariate analysis showed that the risk of death due to disease and risk of progression was significantly lower among patients with p53 mutation (P=0.0357 and 0.0281, respectively). However, the presence of microsattelite instability or loss of hMLH1 expression was not associated with either the clinical response or prognosis. Determining p53 mutational status can be useful in predicting therapeutic response to drugs in ovarian carcinoma, especially in non-serous tumors.
Abstract: We have discovered several protein biomarkers that are altered during carcinogenesis of the human uterine endometrium. Proteins prepared from 19 endometrial carcinomas (Group A), and 20 normal endometria obtained from benign gynecological conditions (Group B), were investigated by Surface Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS). Two proteins, EC1 and EC2, were consistently expressed differentially. EC1 had an increased level of expression in carcinomas (P<0.001), while EC2 was expressed at a lower level (P=0.004). The isoelectric points of EC1 and EC2 were approximately pH 5.0 and 7.0, respectively. These proteins are thus potential biomarkers for detection of endometrial carcinoma.
Abstract: 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.
Abstract: 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.
Abstract: The CDKN2A locus on human chromosome 9p21 encodes two tumor suppressors, p14(ARF) and p16(INK4A), which enhance the growth-suppressive functions of the retinoblastoma (Rb) and the p53 proteins, respectively. Conversely, the E6 and E7 oncoproteins of the high-risk human papillomaviruses (HPVs) causally associated with carcinogenesis of the uterine cervix contributes to tumor development by inactivating p53 and Rb. Nevertheless, a correlation between expression of p14(ARF)/p16(INK4A) and HPV infection in uterine cervix is less clear. To clarify this, we examined 25 cervical cancers and 11 normal uterine cervixes. HPV was detected in 21 of 25 cervical cancers (84%) and their subtype was determined by PCR-RFLP. Quantitative real-time RT-PCR assays showed overexpression of p14(ARF) mRNA in all 21 HPV-positive cases (100%). p16(INK4A) mRNA was overexpressed in 17 cases of the HPV-positive cases (81%). In four HPV-negative cancers, reduced expression of p14(ARF) mRNA was detected in two cases (50%) and reduced p16(INK4A) mRNA in three cases (75%). Our data indicate that the overexpression of p14(ARF) and p16(INK4A) strongly associates with HPV-positive cervical cancers and that reduced expression of p14(ARF) and p16(INK4A) correlates with HPV-negative cervical cancers. These findings may indicate that impaired p14(ARF) and p16(INK4A) mRNA expression contribute to tumor development in HPV-negative cervical cancers by failure to support p53 and Rb instead of their inactivation by HPV E6 and E7.
Abstract: Minimal deviation adenocarcinoma (MDA) is a well-differentiated variant of mucinous adenocarcinoma of the uterine cervix and is found relatively infrequently in the general population. However, MDA is strongly associated with Peutz-Jeghers syndrome (PJS), a rare hereditary autosomal disorder characterized by benign hamartomatous polyposis in the gastrointestinal tract and mucocutaneous pigmentation. A serine threonine kinase gene, STK11, has been identified as the tumor suppressor gene responsible for the PJS. In this study we investigated the possible direct role of STK11 in the development of MDA of the uterine cervix. Eleven rare cases of mucinous MDA, not known to be associated with PJS, were screened for the presence of mutations in the STK11 gene by single-strand conformation polymorphism analysis of PCR-amplified DNA fragments. Subsequently our findings were confirmed with cloning and sequencing. As a control, 24 cases of endocervical adenocarcinomas of other histologic subtypes, with no family history of PJS (19 mucinous adenocarcinomas, 4 endometrioid adenocarcinomas, and 1 clear cell adenocarcinoma), 15 cases of squamous cell carcinomas of the uterine cervix, 5 cases of endocervical glands with pyloric gland metaplasia, and 2 deeply situated nabothian cysts were investigated. Somatic mutations of the STK11 gene were confirmed in 6 (55%) of the 11 mucinous MDAs and 1 (5%) of the 19 mucinous adenocarcinomas, but not in the 5 nonmucinous adenocarcinomas, the 15 squamous cell carcinomas, nor the 5 endocervical glands with gastric metaplasia. MDAs with the STK11 mutation had a significantly poorer prognosis than MDAs without the STK11 mutation (p = 0.039). A germline mutation of STK11 was detected in one PJS patient with mucinous adenocarcinoma of the uterine cervix. These results suggest that mutations in the STK11 gene may play an important role in the etiology of MDA of the uterine cervix and may distinguish this rare tumor from other common types of adenocarcinoma of the uterine cervix.
Abstract: 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.
Abstract: The patterns of X chromosome inactivation and mutations of PTEN and K-ras were evaluated in cases of endometrial hyperplasia to determine the presence of potentially premalignant neoplastic versus polyclonal benign cell populations. Endometrial glandular epithelial cells were collected by laser capture microdissection, and genomic DNAs were extracted. Following treatment with the methylation sensitive restriction endonuclease Hha I, polymerase chain reaction amplification was performed targeting a highly polymorphic short tandem repeat of the human androgen receptor gene (HUMARA). PTEN and K-ras gene mutations were evaluated by analysis of single-strand conformation polymorphism. Two pathologists performed histologic diagnosis of the lesions independently. Monoclonal composition was demonstrated in 13 of 15 (87%) endometrial hyperplasias with atypia and 17 of 31 (55%) complex hyperplasias without atypia. Cytological atypia is significantly associated with the clonal status of the endometrial hyperplasia (13/15 vs 17/31, P = 0.049). In contrast, all 14 normal endometrial tissue samples were polyclonal. PTEN gene mutations were detected in 4 of 13 (30%) monoclonal endometrial hyperplasias with atypia and 2 of 17 (12%) monoclonal endometrial hyperplasias without atypia but were not detected in polyclonal endometrial hyperplasias, with or without atypia. K-ras gene mutations were present in 3 of 13 (23%) monoclonal endometrial hyperplasias with atypia but not in 2 cases of polyclonal endometrial hyperplasia with atypia or in 26 cases of endometrial hyperplasia without atypia. K-ras mutation is thus significantly more frequently found in endometrial hyperplasias with atypia than those without atypia (3/15 vs 0/31, P = 0.030). This study indicates that most cases of endometrial hyperplasia with atypia and a high proportion of cases of endometrial hyperplasias without atypia originate from a single progenitor cell, possibly as a result of genetic alterations, rather than as a result of benign reactive processes.
