Randomized phase III trial of pelvic radiotherapy versus cisplatin-based combined chemotherapy in patients with intermediate- and high-risk endometrial cancer: a Japanese Gynecologic Oncology Group study.

OBJECTIVE: To establish an optimal adjuvant therapy for intermediate- and high-risk endometrial cancer patients, we conducted a multi-center randomized phase III trial of adjuvant pelvic radiation therapy (PRT) versus cyclophosphamide-doxorubicin-cisplatin (CAP) chemotherapy in women with endometrioid adenocarcinoma with deeper than 50% myometrial invasion. METHODS: Among 385 evaluated patients, 193 patients received PRT and 192 received CAP. The PRT group received at least 40 Gy. The CAP group received cyclophosphamide (333 mg/m2), doxorubicin (40 mg/m2) and cisplatin (50 mg/m2) every 4 weeks for 3 or more courses. RESULTS: No statistically significant differences in progression-free survival (PFS) and overall survival (OS) were observed. The 5-year PFS rates in the PRT and CAP groups were 83.5% and 81.8% respectively, while the 5-year OS rates were 85.3% and 86.7% respectively. These rates were also not significantly different in a low- to intermediate-risk group defined as stage IC patients under 70 years old with G1/2 endometrioid adenocarcinoma. However, among 120 patients in a high- to intermediate-risk group defined as (1) stage IC in patients over 70 years old or with G3 endometrioid adenocarcinoma or (2) stage II or IIIA (positive cytology), the CAP group had a significantly higher PFS rate (83.8% vs. 66.2%, log-rank test P=0.024, hazard ratio 0.44) and higher OS rate (89.7% vs. 73.6%, log-rank test P=0.006, hazard ratio 0.24). Adverse effects were not significantly increased in the CAP group versus the PRT group. CONCLUSION: Adjuvant chemotherapy may be a useful alternative to radiotherapy for intermediate-risk endometrial cancer.

Identical changes in Bax expression, but not Fas ligand expression, occur in structural luteolysis in gonadotropin releasing hormone agonist- and prolactin-treated superovulated rats.

Structural luteolysis induced by gonadotropin releasing hormone agonist (GnRHa) or prolactin (PRL) is defined as histological involution of the corpus luteum. We reported that one of the mechanisms of structural luteolysis induced by PRL was tissue remodeling by matrix metalloproteinase (MMP) and also apoptosis in superovulated rats. We also reported that GnRHa induced structural luteolysis with elevation of MMP. In this study, we investigated whether GnRHa caused apoptosis in mature corpus luteum of superovulated rats and also examined the expression of apoptosis-related molecules (Fas, Fas ligand (FasL), Bcl-2, Bax). We gave 4-day GnRHa treatment 5 days after hCG injection to immature female rats treated with pregnant mare surum gonadotrophin (PMSG) and hCG to induce structural involution of mature corpus luteum. PMSG-hCG-treated rats without GnRHa treatment, rats treated with bromocryptine (Brom) to induce functional luteolysis and rats treated with Brom followed by PRL (Brom+PRL) to mimic the PRL surge to induce structural luteolysis as we previously reported were used for comparison. GnRHa treatment caused structural luteolysis characterized by structural involution, a decrease in the serum progestin level, and apoptotic bodies as well as structural luteolysis induced by Brom+PRL. FasL expression in corpora lutea was elevated after Brom treatment, but there was no elevation of FasL after GnRHa treatment started. FasL expression decreased and Bax expression increased in structural luteolysis induced by GnRHa as well as Brom+PRL treatment, although Fas and Bcl-2 expression did not change throughout the luteal phase. In summary, both GnRHa and Brom+PRL caused structural luteolysis, one of whose mechanisms was apoptosis with an increase in Bax expression, but not with an identical change in FasL expression. It is speculated that the significance in alteration of FasL may involve some mechanism other than apoptosis.

Hypermethylation in promoter region of retinoic acid receptor-beta gene and immunohistochemical findings on retinoic acid receptors in carcinogenesis of endometrium.

In the present study, we analyzed the immunohistochemical findings for the RA receptor (RAR), retinoic X receptor (RXR) and hypermethylation of promoter-region CpG island methylation of RAR-beta2. Immunohistochemistry indicated that though RXR-alpha and -gamma were present in endometrial hyperplasia and cancer, other retinoid receptors were only weakly detected. The hypermethylation of RAR-beta2 was found in 75.0% of endometrial hyperplasia samples and 92.2% of carcinomas. No normal endometria had methylation. This evidence may point to one of the reasons why endometrial hyperplasia acquires high proliferative capacity without differentiation, and the hypermethylation of RAR-beta2 may occur in the early stage of endometrial carcinogenesis.

Cytology of vaginal and uterine sarcomas.

OBJECTIVE: To retrospectively review, based on cytologic and histopathologic findings, the diagnoses of 13 patients with uterine sarcoma and 1 with vaginal sarcoma. STUDY DESIGN: There were 8 cases of uterine carcinosarcoma (CS), 2 of leiomyosarcoma, 2 of endometrial stromal sarcoma (ESS), 1 of endocervical stromal sarcoma (ECSS) and 1 of malignant fibrous histiocytoma (MFH) of the vagina. The presence of sarcomatous components was retrospectively investigated by microscopic observation of preoperative specimens from the endocervical canal and endometrial cells. Characteristic features of sarcomatous cells were then investigated by cytodiagnostic micrometry of malignant cells. RESULTS: Of the 14 patients, 1 with low grade ESS and 1 with homologous CS were diagnosed as negative for sarcomatous components. One case of high grade ESS had been overlooked, as were 4 cases of CS. Thus, 7 cases (50%) were diagnosed as positive for sarcomatous cells by preoperative cytologic observation. Based on these findings, 12 of the 14 cases (85.7%) were positive for sarcomatous elements on retrospective reexamination of the specimens. CONCLUSION: Careful attention should be paid to small sarcomatous cells since cases of ESS or ECSS with such cells show morphologic characteristics similar to those of stromatous cells. Furthermore, careful microscopic observation of an entire specimen is required to avoid misdiagnosis as carcinoma since it is easy to overlook sarcomatous elements in smears with carcinosarcoma if there are only a few sarcomatous cells.

Preoperative diagnosis and treatment results in 106 patients with uterine sarcoma in Hokkaido, Japan.

OBJECTIVE: The aim of this study was to evaluate the clinicopathological features of uterine sarcoma in Hokkaido, Japan, between 1990 and 1999, and to identify prognostic factors of patients with such malignancies in this area and period. METHODS: One hundred and six patients with histologically proven uterine sarcoma were evaluated retrospectively. RESULTS: 93.5% of the patients with carcinosarcoma (CS) were diagnosed as having malignant disease preoperatively, while 65% of those with leiomyosarcoma (LMS) and 75% of those with endometrial stromal sarcoma (ESS) were preoperatively diagnosed as benign leiomyoma. When patients had no residual disease postoperatively, 5-year survival rates in patients with CS and LMS were 78.8 and 73.0%, respectively. ESS cases had a better prognosis (94.7% for stage I cases). In patients with early-stage sarcoma, pelvic lymphadenectomy and adjuvant chemotherapy, with or without cis-diamminedichloroplatinum, failed to show a survival benefit in both CS and LMS cases. Distant metastasis, myometrial invasion, and no residual disease at surgery were significantly associated with risk of death or recurrence in CS and LMS cases. CONCLUSION: Accurate preoperative diagnosis of uterine sarcoma was difficult, and no residual disease at surgery was the most important prognostic factor in patients with this disease. Postoperative adjuvant therapy had little effect on survival, especially in early-stage disease.

E1AF/PEA3 reduces the invasiveness of SiHa cervical cancer cells by activating serine proteinase inhibitor squamous cell carcinoma antigen.

E1AF/PEA3, a member of the Ets family of transcription factors, is associated with the malignant characteristics of cancer cells. The initial aim of our study was to test whether the invasiveness of SiHa cervical cancer cells could be diminished by transfection with antisense E1AF. Using an in vitro invasion assay in which cells penetrate a layer of Matrigel, we found that this was not the case; indeed, the invasiveness of the transfectants was enhanced. To better understand the mechanism of this enhancement, we used the cDNA microarray technique to search for genes whose expression was altered in the antisense E1AF-transfected SiHa cells. Among several genes affected, we found that expression of squamous cell carcinoma antigen (SCCA), a member of the ovalbumin serine proteinase inhibitor family, was significantly reduced. Forced expression of E1AF enabled activation of SCCA expression, and Luciferase reporter assays revealed that E1AF activates the SCCA promoter. Introduction of antisense SCCA into SiHa cells inhibited production of SCCA protein and markedly increased the invasiveness of the cells. Taken together, these results suggest that E1AF suppresses the invasiveness of SiHa cervical cancer cells through transcriptional activation of the SCCA serine proteinase inhibitor gene.

Changed expression of heat shock proteins in various pathological findings in placentas with intrauterine fetal growth restriction.

Heat shock proteins (HSPs) are activated in the cells of most organisms in response to sublethal heat shock and other stressors. It has been reported that HSP27, HSP60, HSP70, and HSP90 are expressed in normal human placenta, and it was thought that these HSPs play a role in the demonstration of cell viability and function. In this study, we performed an immunohistochemical (IHC) study of these HSPs for 27 placentas that had complicated intrauterine fetal growth restriction (IUGR) and compared the IHC findings with the pathological findings. To quantify HSP27, HSP60, HSP70, and HSP90, immunoreacted cells in the chorionic villi, syncytiotrophoblasts (ST), and cytotrophoblasts (CT) were counted. In thrombus, excessive syncytial knots, and avascular villi, the expression of HSPs was higher in the pathological sections compared to control in both ST and CT. In contrast, all HSPs decreased in both ST and CT around the infarction region. The data suggested that chorionic villi cells locally responded to some stresses, e.g., hypoxia and increase or decrease in the expression of HSPs. Although the villous cells around the infarction histologically appear viable, they may have received lethal damage, and as a result the expression of HSPs was decreased. These results are expected to improve our understanding of the pathological findings of IUGR in placentas, including the quality, damage, and function of the chorionic villi.

Identification of SCN3B as a novel p53-inducible proapoptotic gene.

Tumor suppressor p53 is a transcription factor that induces growth arrest and/or apoptosis in response to cellular stress. To identify novel p53-inducible genes, we compared the expression of genes in normal mouse embryo fibroblasts (MEFs) to p53-null cells by cDNA representational difference analysis. We report here that expression of endogenous sodium channel subunit beta 3 (SCN3B) is upregulated in mouse embryonic fibroblasts by DNA damage in a p53-dependent manner. In addition, we found that SCN3B levels are upregulated in human cancer cell lines by DNA damaging agents, as well as by overexpression of p53, but not significantly by p63 or p73. Furthermore, we identified two putative p53-binding sites upstream of the first exon (RE1) and in the third intron (RE2). The p53 protein can directly interact with the putative p53-binding sites in vivo, as assessed by chromatin immunoprecipitation. A reporter gene assay revealed that these two p53-binding sites are functional response elements. The SCN3B protein appears to be localized to the endoplasmic reticulum (ER). Introduction of the SCN3B gene into T98G and Saos2 cells potently suppressed colony formation. Furthermore, we found that adenovirus-mediated transfer of SCN3B induced apoptosis when combined with anticancer agents. The results presented here suggest that SCN3B mediates a p53-dependent apoptotic pathway and may be a candidate for gene therapy combined with anticancer drugs.

Analysis of the prevalence of bacterial vaginosis and Chlamydia trachomatis infection in 6083 pregnant women at a hospital in Otaru, Japan.

AIM: Our aims were to evaluate the prevalence of bacterial vaginosis (BV), Chlamydia trachomatis (C. trachomatis) infection and Mobiluncus spp. infection among pregnant women in Otaru, Hokkaido, Japan according to the month and year of the first prenatal visit, and to evaluate their risk factors. METHODS: Six thousand and eighty-three pregnant women who were seen consecutively at our hospital between 1993 and 2000, were enrolled in the study. Vaginal and endocervical swabs were subjected to Gram stain and detection of C. trachomatis. Univariate and multivariate methods were used to investigate the association between each infection and potential risk factors including age, gravidity, parity, history of dilatation and curettage (D & C), and history of natural abortion. RESULTS: The annual rate of BV increased from 13.6% in 1993 to 21.4% in 2000. The annual rate of C. trachomatis infection was relatively constant. The prevalence of bacterial vaginosis, C. trachomatis infection and Mobiluncus spp. infection over the 8-year period was 18.2%, 4.2%, and 4.1%, respectively. The prevalence of the three infections was significantly higher among teenagers and among women with a history of D & C. The prevalence of C. trachomatis and Mobiluncus spp. infections was significantly higher among women with no history of delivery. BV was not associated with parity on multivariate analysis. The monthly prevalence of BV was significantly higher in May than in December, and the monthly prevalence of C. trachomatis infection was high in August. CONCLUSION: The differences in the annual and monthly infection patterns between BV and C. trachomatis infection suggest that the etiologies of the two infections differ.

Overexpressed progesterone receptor form B inhibit invasive activity suppressing matrix metalloproteinases in endometrial carcinoma cells.

In this study, we focused on the influence of progesterone and its receptor in invasion and MMPs on endometrial carcinoma cells. The growth of Ishikawa cells, to which an progesterone receptor form B (PR-B) expressing vector was transfected, was inhibited by progesterone as was the inhibition of the expression of cyclin D1. By invasion assay, in conditions with progesterone, the invasiveness of Ishikawa cells was inhibited as well as the expression of (metalloproteinase) MMP-1, -2, -7 and -9 and Ets-1 decreased. These results suggest that activation of PR-B by progesterone results in tumor suppression by inhibiting cell growth and invasiveness via suppression of the expression of MMPs.

Ultrastructural study of benign, low-malignant potential (LMP), and malignant ovarian tumors.

Ultrastructural characteristics of benign, low-malignant potential (LMP), and malignant ovarian tumors were investigated, considering the aspects of histologic subtypes and histologic grading. In addition, the histogenesis of ovarian cancer was histologically investigated in an attempt to elucidate whether malignant tumor was generated from benign or LMP tumor, or whether it was generated de novo from normal tissues. Although all the benign, LMP, and malignant tumors appeared to be derived from Mullerian duct in serous tumors, the origin of endometrioid or mucinous tumor could not be ultrastructurally clarified. However, there was ultrastructural similarity between benign and malignant tumors among serous, endometrioid, and mucinous tumors, and it was suggested that benign adenoma may be the developmental origin of malignant tumors regardless of the histologic subtype. In addition, the investigation of endometrioid tumors revealed that the differences of histologic grading in malignant tumors reflected the ultrastructural differences, and that G1 tumor had an ultrastructure that was more similar to that of benign and LMP tumors than to that of G2 tumor.

[A pilot study of combined chemotherapy with paclitaxel, doxorubicin and cisplatin for endometrial cancer].

A pilot trial of combined chemotherapy with paclitaxel, doxorubicin and cisplatin was conducted in patients with advanced endometrial cancer. Between June 2000 and March 2002 8 patients were treated with combined chemotherapy, consisting of paclitaxel, 135 mg/m2; doxorubicin, 30 mg/m2; and cisplatin, 50 mg/m2 (TAP therapy). Patients received 3 to 5 courses of TAP therapy every 4 weeks. The major adverse effect was myelosuppression. All patients had grade 3 or 4 neutropenia, but did not have any severe infection with uncontrollable fever. Only 1 patient discontinued additional therapy due to grade 3 thrombocytopenia after 3 cycles. Grade 2 neurotoxicity occurred in 5 patients, but grade 3 was not observed. Among 5 patients with measurable tumors, 4 achieved partial response and 1 had no change of tumor size, indicating a response rate of 80.0%. We found that TAP therapy was feasible with G-CSF support and shows potential for high efficacy in advanced endometrial cancer.

Gene expression profiling in two morphologically different uterine cervical carcinoma cell lines derived from a single donor using a human cancer cDNA array.

PURPOSE: To examine the differentially expressed cancer-related genes in two morphologically different uterine cervical carcinoma cell lines derived from the same patient by an Affymetrix Human Cancer G110 Array carrying 1700 cancer-associated genes. In addition, to investigate specific gene expression depending on histological type, we examined expression of the selected genes in a panel of established cervical carcinoma cell lines derived from cervical adenocarcinoma and squamous cell carcinoma (SCC). EXPERIMENTAL DESIGN: Two distinct human uterine cervical carcinoma cell lines SKG-IIIa and SKG-IIIb derived from a single donor were screened using a cDNA microarray. The array results were additionally validated using semiquantitative RT-PCR. Expressions of the 10 selected genes were analyzed in the nine established cervical carcinoma cell lines using RT-PCR. RESULTS: The cDNA microarray analysis showed that 16 genes in SKG-IIIa were upregulated more than 10-fold compared to SKG-IIIb, and seven genes in SKG-IIIb were upregulated. Semiquantitative RT-PCR analysis of a subset of these differentially expressed genes gave results consistent with microarray findings. Among the 10 selected genes, insulin-like growth factor-binding protein-3, inhibitor of apoptosis protein 1, and cadherin-13 were more frequently expressed in SCC cell lines. 1-8D gene of interferon-inducible genes, Sno oncogenes, and transforming growth factor-beta II receptor were expressed in both SCC and adenocarcinoma cell lines. CONCLUSIONS: Our experimental data demonstrated that multiple genes are differentially expressed in uterine cervical carcinoma cell lines. It is suggested that these genes are involved with the differences in morphological characteristics and carcinogenesis of cervical carcinoma.

Adjuvant oral 5-fluorouracil for cervical cancer: Japanese Gynecologic Oncology Group report.

Japanese women with low-stage cervical cancer receiving radical hysterectomy and radiotherapy have a good 5-year survival rate. However, women with risk factors such as nodal metastasis may benefit from adjuvant chemotherapy, which was studied in women having surgery alone or surgery plus radiotherapy. Patients having surgery alone (S) (n=623) or surgery and radiotherapy (SR) (n=919) were randomly assigned to receive or not receive oral 5-fluorouracil (5-FU) for 1 year. The effect of various factors on survival was studied by multivariate analysis. Patients who received S obtained no benefit from 5-FU, whereas 5-FU-treated SR patients had significantly better 5-year survival than those not receiving chemotherapy (P=0.043). The SR patients without nodal metastases had a better survival rate if they received 5-FU (P<0.001), whereas those with nodal metastases did not. Oral 5-FU after radical hysterectomy with radiotherapy appears useful for patients with low-stage cervical cancer who have some risk factors but not for those with pelvic lymph node metastases.

Mechanisms of paclitaxel-induced apoptosis in an ovarian cancer cell line and its paclitaxel-resistant clone.

BACKGROUND: To understand the complicated network of paclitaxel (PTX)-induced apoptosis pathways and to elucidate mechanisms of drug resistance in ovarian cancer, we looked at PTX-induced apoptosis by using cDNA microarray. We also quantitated the changes in apoptosis-related proteins in the process of apoptosis. METHODS: An ovarian cancer cell line KF, and its PTX-resistant clone KFTX, were treated with PTX or carboplatin (CBDCA). After exposure to PTX or CBDCA, the induction of apoptosis was examined by internucleosomal DNA fragmentation. Changes in mRNA expression after 12 h of exposure to PTX were studied using cDNA microarray and RT-PCR. Changes in P53 and Bcl-2 levels were also measured over 24 h by ELISA. RESULTS: With increased doses of PTX or CBDCA, an increase in apoptosis was noted in both cell lines. cDNA microarray revealed that PTX treatment upregulated expression of caspase 1, 2, 3, 4, 6, 9, 10, their activator apaf-1, and stress reaction-related genes, gadd34, gadd153 in KF, although most of them were unchanged or downregulated in KFTX. bag-1 and hsc70 were markedly upregulated in KFTX. p53 and bcl-2 were not upregulated in either cell line. Results from protein studies also supported the cDNA microarray data. CONCLUSIONS: p53-independent mitochondrial pathways and stress-reaction-induced pathways play critical roles in PTX-induced apoptosis in ovarian cancer cells. Suppression of those pathways and upregulation of bag-1 and hsp-70 played an important role in acquiring resistance to PTX.

Epigenetic inactivation of TMS1/ASC in ovarian cancer.

PURPOSE: The purpose of this work was to explore the role of epigenetic inactivation of apoptotic pathways in ovarian cancer by examining the DNA methylation and expression status of four proapoptotic genes in primary ovarian cancers and cancer cell lines and to correlate those findings with the clinicopathological features of ovarian cancer patients. EXPERIMENTAL DESIGN: Genomic DNA was isolated from 15 ovarian cancer cell lines, 80 primary ovarian cancer specimens, and 4 normal ovary specimens using phenol-chloroform extraction. The methylation status of the DNA was evaluated using combined bisulfite restriction analysis, gene expression was evaluated using reverse transcription-PCR, and histone acetylation was evaluated using chromatin immunoprecipitation. RESULTS: Of the four proapoptotic genes studied, expression of TMS1/ASC was absent in six ovarian cancer cell lines. Dense methylation of the 5' region of TMS1/ASC was detected in cells not expressing TMS1/ASC. Treating methylated cells with 5-aza-deoxycytidine restored gene expression, confirming the role of methylation in silencing the gene. Chromatin immunoprecipitation revealed histone to be deacetylated in cells not expressing TMS1/ASC, indicating that histone deacetylation is also involved in silencing TMS1/ASC. Aberrant methylation of TMS1/ASC was detected in 15 of 80 ovarian cancer tissues (19%) but in none of the normal ovary specimens. Aberrant methylation of TMS1/ASC was observed significantly more often in clear cell-type ovarian cancers than in other tumor types (P < 0.0001). CONCLUSIONS: Methylation-mediated silencing of TMS1/ASC confers a survival advantage to tumor cells by enabling them to escape apoptosis. The role for aberrant methylation in human ovarian tumorigenesis may be particularly important for ovarian cancers with the clear cell phenotype.

Gonadotropin-releasing hormone agonist administration reduced vascular endothelial growth factor (VEGF), VEGF receptors, and vascular permeability of the ovaries of hyperstimulated rats.

OBJECTIVE: Using hyperstimulated rats, to elucidate the mechanisms of gonadotropin-releasing hormone agonist (GnRH-a) treatment to prevent early ovarian hyperstimulation syndrome (OHSS). DESIGN: Descriptive study of hyperstimulated rats as an early OHSS model with Western blot analysis, Northern blot hybridization, and vascular permeability assay. SETTING: Experimental laboratory research. ANIMAL(S): Sprague-Dawley female rats were used for collecting ovarian samples. INTERVENTION(S): Hyperstimulated rats received consecutive GnRH-a treatment from the start of pregnant mare serum gonadotropin (PMSG) treatment through 2 days after hCG administration. MAIN OUTCOME MEASURE(S): Expressions of vascular endothelial growth factor (VEGF), VEGF receptor-1 (VEGFR-1: Flt-1), VEGF receptor-2 (VEGFR-2: KDR/Flk-1), and vascular permeability by Evans blue leakage. RESULT(S): GnRH-a treatment significantly reduced expressions of VEGF, VEGFR-1, and VEGFR-2 both in mRNA and protein levels in the ovaries of hyperstimulated rats. GnRH-a treatment also reduced vascular permeability in the ovaries of hyperstimulated rats. CONCLUSION(S): It is speculated that GnRH-a treatment may prevent early OHSS by reducing vascular permeability through the decrease in VEGF and its receptors.

Overexpression of estrogen receptor-alpha gene suppresses gap junctional intercellular communication in endometrial carcinoma cells.

Stimulation of the endometrium by estrogens without the differentiating effect of progestins is the primary etiological factor associated with the development of endometrial hyperplasia and adenocarcinoma. However, the correlation between sex steroids and gap junctional intercellular communication (GJIC), which is considered to play an important role in the control of cell growth and differentiation, is not well known in endometrial carcinoma. In this study, we focused on the influence of estrogen and its receptor in connexin (Cx) expression and GJIC in endometrial carcinoma cells, established stable clone IK-ER1 overexpressing ER-alpha to transfect the expression vector and analysed them in various hormonal conditions. The growth of IK-ER1 was accelerated by 17beta-estradiol and the acceleration of the 5-bromo-25-deoxyuridine labeling index was observed. GJIC was assayed by scoring the number of dye-coupled cells after microinjection of single cells with Lucifer-Yellow, and subcellular localization of Cx26 and Cx32 was analysed by immunocytochemistry. In the presence of estradiol, dye-coupled cells of IK-ER1 were significantly reduced compared to those without estradiol and the reduction was completely inhibited by adding ICI182.780, a pure antiestrogen substrate. Cxs were detected as only small spots by immunocytochemistry, and Western blotting showed that the expression was decreased. These results suggest that activation of ER-alpha by estrogen results in tumor progression by stimulating cell growth and suppressing GJIC via suppression of the expression of Cxs in endometrial carcinogenesis.