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.

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.

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.

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.

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.

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.

High-dose-rate intracavitary brachytherapy: results of analyses of late rectal complications.

PURPOSE: To examine the incidence of radiation-induced late rectal complications by analyzing the data of measured rectal doses in patients with cancer of the uterine cervix treated with high-dose-rate intracavitary brachytherapy. METHODS AND MATERIALS: We measured doses to the rectum in 105 patients with cancer of the cervix during high-dose-rate intracavitary brachytherapy with a semiconductor dosimeter that can measure five points in the rectum simultaneously. On the basis of these measurements, equivalent doses, to which the biologically equivalent doses were converted as if given as fractionated irradiation at 2 Gy/fraction, were calculated as components of the cumulative dose at five rectal points in intracavitary brachytherapy combined with the external whole pelvic dose. RESULTS: The calculated values of equivalent doses for late effects at the rectum ranged from 15 to 100 Gy (median 60 Gy for patients who did not develop complications and 76 Gy for patients who subsequently developed Grade II or III complications). When converted to a graph of absolute rectal complication probability, the data could be fitted to a sigmoid curve. The data showed a very definite dose-response relationship, with a threshold for complications at approximately 50 Gy and the curve starting to rise more steeply at approximately 60 Gy. The steepest part of the curve had a slope equivalent to approximately 4% incidence/1 Gy increase in equivalent doses. CONCLUSION: The radiation tolerance dose, 5% and 50% complication probability, was about 64 and 79 Gy, respectively. Our data almost agree with the prescribed dose for the rectum for the radiation tolerance doses on the basis of the recorded human and animal data. The probability of rectal complications increased drastically after the maximal rectal dose was >60 Gy.

A relationship between Matrix metalloproteinase-1 (MMP-1) promoter polymorphism and cervical cancer progression.

Single nucleotide polymorphism (SNP) of the promoter region of MMP-1 (at -1607 bp) creates Ets binding sites, and correlations between this SNP and cancer susceptibility have been reported for various cancers. In this study, we genotyped the SNP in 23 cervical intraepithelial neoplasias (CIN) and 86 cervical cancer specimens. We found a correlation between promoter polymorphism and MMP-1 expression, and that this SNP was correlated with the clinical stage of cervical cancer. These findings suggested that SNP of MMP-1 promoter might influence the ability in cervical cancer invasion via transcriptional activity of this gene.

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.

Reproductive function after treatment of malignant germ cell ovarian tumors.

The outcome and reproductive function were examined among patients with malignant ovarian germ cell tumors treated since 1980. Between 1980 and 2001, fertility-sparing surgery was performed in 26 women, 23 of whom received adjuvant chemotherapy. With a median follow-up of 66.6 months, all patients have been alive, with histological types of 6 immature teratomas, 8 dysgerminomas, 6 yolk sac tumors, and 6 mixed types. Clinical stages were involved of 17 early stage and 9 advanced stage patients. After treatment, 20 women out of 26 recovered menstruation within 6 months. During follow-up, two chemotherapy-untreated and one treated patients experienced 4 conceptions in total. A treated patient conceived but selected artificial termination by affection of chemotherapy. Conservative surgery with adjuvant chemotherapy is the standard approach to treat patients with malignant ovarian germ cell tumors. In these 20 years, we experienced no delivery, so that fertility seems to be seriously affected by treatments.

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.

Successful twin pregnancy in panhypopituitarism caused by suprasellar germinoma.

BACKGROUND: Pregnancy in a woman with hypopituitarism from a suprasellar germinoma is rare. CASE: A 27-year-old woman presented with panhypopituitarism from a suprasellar germinoma. She had diabetes insipidus, hypothyroidism, adrenal cortex dysfunction, and hypogonadotropic ovarian failure. When treated with thyroxin, cortisol, antidiuretic hormone, human menopausal gonadotropin, and human chorionic gonadotropin, she conceived and gave birth to healthy twins. CONCLUSION: Hormonal replacement therapy and ovulation induction resulted in a successful pregnancy in a woman with panhypopituitarism.

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.

Expression of matrix metalloproteinases in ovarian endometriomas: immunohistochemical study and enzyme immunoassay.

Like carcinoma, endometriosis has the unique characteristics, of invasion and metastasis, though pathologically, it is a benign tumor. However, the mechanism of destruction of the surrounding tissue in endometriosis is still unclear. In this study, the expression and localization of matrix metalloproteinases (MMP)-1, -2, -3, -7, -9 and tissue inhibitors of metalloproteinases-1 (TIMP-1) were evaluated by immunohistochemistry for 20 cases and the amounts of MMP-1, TIMP-1 and MMP-1/TIMP-1 complex in the fluid of endometrioma, were analyzed by ELISA and western blotting for 20 cases, which were analyzed by immunohistochemical study. MMP-1, -2 and -9 were detected strongly in both stromal and epithelial cells and MMP-7 in the epithelial cells in the menstrual period. MMP-3 was mainly expressed in macrophage containing hemosiderin but the change of expression was not clear. TIMP-1 was intensively detected in both stromal and epithelial cells in the menstrual period but the expression decreased in other stages of the menstrual cycle. ELISA for MMP-1 also showed results similar to immunohistochemistry, suggesting that it was released to the cyst in the menstrual period when it was released to the extracellular space from the cytoplasm. The expression of TIMP-1 was not clearly changed during the menstrual cycle. From these results, it was suggested that the destruction of the surrounding matrix by endometriosis might be caused by various MMPs, which are mainly produced in stromal cells.

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.

Usefulness of silver intensification of immunostaining for cytologic diagnosis of primary melanoma of the female genital organs.

OBJECTIVE: To improve the procedure for diagnosing vaginal melanoma with cytopathologic analysis of HMB-45. STUDY DESIGN: The study examined silver intensification of immunostaining of HMB-45 in nine cases of primary melanoma of the vagina and vulva using archival Papanicolaou-stained smears. RESULTS: All nine samples showed positive staining for HMB-45. Five cases showed intensive staining, two moderate and two weak. The positive staining was black in the cytoplasm of melanoma cells but was detected in neither the background nor normal squamous cells. Though destaining of Papanicolaou stain was not performed before immunostaining, the positivity of immunostaining was easily judged. CONCLUSION: After morphologic observation, immunocytochemical study of HMB-45 is possible even though time has passed since the cytologic specimen was obtained. When there is a suspicion of amelanotic melanoma or scantily pigmented melanoma of the vagina and vulva, cytogenesis with HMB-45 is helpful, especially because it involves little invasion.

[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.

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.