Toshiharu Yasugi

Multicenter Phase II Study of Fertility-Sparing Treatment With Medroxyprogesterone Acetate for Endometrial Carcinoma and Atypical Hyperplasia in Young Women

PURPOSE To assess the efficacy of fertility-sparing treatment using medroxyprogesterone acetate (MPA) for endometrial carcinoma (EC) and atypical endometrial hyperplasia (AH) in young women. PATIENTS AND METHODS This multicenter prospective study was carried out at 16 institutions in Japan. Twenty-eight patients having EC at presumed stage IA and 17 patients with AH at younger than 40 years of age were enrolled. All patients were given a daily oral dose of 600 mg of MPA with low-dose aspirin. This treatment continued for 26 weeks, as long as the patients responded. Histologic change of endometrial tissue was assessed at 8 and 16 weeks of treatment. Either estrogen-progestin therapy or fertility treatment was provided for the responders after MPA therapy. The primary end point was a pathologic complete response (CR) rate. Toxicity, pregnancy rate, and progression-free interval were secondary end points. RESULTS CR was found in 55% of EC cases and 82% of AH cases. The overall CR rate was 67%. Neither therapeutic death nor irreversible toxicities were observed; however, two patients had grade 3 body weight gain, and one patient had grade 3 liver dysfunction. During the 3-year follow-up period, 12 pregnancies and seven normal deliveries were achieved after MPA therapy. Fourteen recurrences were found in 30 patients (47%) between 7 and 36 months. CONCLUSION The efficacy of fertility-sparing treatment with a high-dose of MPA for EC and AH was proven by this prospective trial. Even in responders, however, close follow-up is required because of the substantial rate of recurrence.

Expression profiling in ovarian clear cell carcinoma: Identification of hepatocyte nuclear factor-1β as a molecular marker and a possible molecular target for therapy of ovarian clear cell carcinoma

Of all of the epithelial ovarian cancers, clear cell carcinoma (CCC) of the ovary has the worst prognosis. We applied the oligonucleotide array technique to identify genes generally involved in CCC. Of the approximately 12,600 genes that were analyzed, 28 were expressed significantly differently between four CCC and seven non-CCC cell lines. Among 16 up-regulated genes in CCC, we further investigated a transcription factor, hepatocyte nuclear factor-1 beta (HNF-1 beta). We validated up-regulation of HNF-1 beta in CCC in terms of both mRNA and protein level using real-time quantitative reverse transcriptase-polymerase chain reaction and immunoblotting. Immunohistochemical analysis of 83 surgically resected ovarian cancers showed that almost all CCC specimens (21 of 22 cases) had nuclear staining for HNF-1 beta, whereas most non-CCC specimens (60 of 61 cases) showed no immunostaining or only focal and faint staining in the nucleus. Furthermore, we investigated the significance of HNF-1 beta expression in CCC using RNA interference. The reduction of HNF-1 beta expression by RNA interference induced apoptotic cell death in ovarian CCC cells, which was confirmed by terminal dUTP nick-end labeling and fluorescence-activated cell-sorting analyses. Our results suggest that HNF-1 beta is not only an excellent CCC-specific molecular marker but also a molecular target for therapy of ovarian CCC.

Assessment of metastases to aortic and pelvic lymph nodes in epithelial ovarian carcinoma. A proposal for essential sites for lymph node biopsy.

In staging epithelial ovarian carcinoma, it is necessary to assess the presence of lymph node metastases. However, the essential sites of selective lymph node biopsy have yet to be determined.

Analysis of the expression and localisation of a LAP protein, human scribble, in the normal and neoplastic epithelium of uterine cervix

Recently, a LAP protein, scribble, was identified in Drosophila epithelia as a basolateral protein that controls the apical-basolateral polarity. Loss of scribble causes disorganisation and overgrowth of the epithelia. Scribble has a human homologue, human scribble (hScrib), which is a substrate of ubiquitin-mediated degradation by human papillomavirus E6 and the E6AP ubiquitin-protein ligase. In the present study, we revealed that hScrib localised to the basolateral regions of the epithelial cell line MDCK and human uterine cervical epithelial tissues by immunofluorescence. Human scribble colocalised rather with the adherens junction protein E-cadherin, but not with the tight junction protein ZO-1. Histochemical analysis showed a dramatic decrease in the expression of hScrib with the progression of disease from normal uterine cervical tissues to invasive cervical cancers through the precursor lesions. In contrast, the expression of hScrib was retained in the throughout epithelial layer of the HPV-negative cervical high-grade squamous intraepithelial lesions (H-SIL). Although quantitative RT–PCR revealed no significant downregulation of hScrib mRNA expression in the H-SIL, it revealed a clear downregulation in the invasive cancers. These results suggest the possibility that degradation by HPV E6 is one of the causal roles for the progressive decrease of hScrib expression during the disease progression from low-grade squamous intraepithelial lesions to H-SIL, and a cooperative role of downregulation of hScrib mRNA expression and ubiquitin-mediated degradation of hScrib by E6 and E6AP led to the complete decrease of hScrib expression during the process of carcinogenesis from H-SIL to invasive cancer. These data underscore the importance of hScrib in the construction of tissue architecture and prevention of cancer development.

Secondary cytoreductive surgery for recurrent epithelial ovarian carcinoma: proposal for patients selection

The value of secondary cytoreductive surgery (SCS) for recurrent ovarian cancer is still controversial. The aim of this study was to clarify candidates for SCS. Between January 1987 and September 2000, we performed SCS in 44 patients with recurrent ovarian cancer, according to our selection criteria, disease-free interval (DFI) >6 months, performance status <3, no apparent multiple diseases, age <75years and no progressive disease during preoperative chemotherapy, if undertaken. The variables were investigated by univariate and multivariate analyses. Of 44 patients, 26 (59.1%) achieved complete removal of all visible tumours at SCS. Secondary cytoreductive surgery outcome, complete or incomplete resection, was significantly related to overall survival (P=0.0019). As for variables determined before SCS, DFI >12 months, no liver metastasis, solitary tumour and tumour size <6 cm were independently associated with favourable overall survival after recurrence in the multivariate analysis. Patients with three or all four variables (n=31) had significantly better survival compared with the other patients (n=13) (47 vs 20 months in median survival, P<0.0001). In these patients, fairly good median survival (40 months) was obtained even in patients with incomplete resection. Secondary cytoreductive surgery had a large impact on survival of patients with recurrent ovarian cancer when they had three or all of the above-mentioned four factors at recurrence. These patients should be considered as ideal candidates for SCS.

Prevalence of ovarian endometriosis in epithelial ovarian cancer

Objective: The objective of this study was to examine the occurrence of ovarian endometriosis in epithelial ovarian cancer in Japan. Method: The presence of ovarian endometriosis was determined by reviewing the sections of resected specimens in 172 epithelial ovarian cancers. Results: The incidence of ovarian endometriosis in ovarian cancer (14.5%) was higher than that in Western countries. The rank order of incidence of endometriosis in each histologic type was clear cell (40.6%)>endometrioid (23.1%)>serous (8.7%)>mucinous (2.9%). The incidence in serous type was higher when compared with that reported in Western countries. The higher incidence of endometriosis in Japan can be explained by a greater proportion of clear cell type, comprising 18.6% of all the cases and a higher incidence of endometriosis in the serous type. Conclusion: The association of ovarian endometriosis with epithelial ovarian cancer was more frequently found in Japan.

Prevalence of Endometriosis in Ovarian Cancer

Endometriosis may be the precursor of clear cell or endometrioid ovarian cancer. In this review, we focus on the prevalence of endometriosis in ovarian cancer and related clinical and epidemiological issues. According to 15 published reports, the rank order of the prevalence of endometriosis in each histologic type was clear cell (39.2%) > endometrioid (21.2%) > serous (3.3%) > mucinous type (3.0%). The high prevalence of endometriosis in clear cell and endometrioid types is a consistent finding in Japan and western countries. However, the incidence of the clear cell type is much higher (15–20% vs. 7–8%), and that of the endometrioid type is lower (7–16% vs. 18–26%), in Japan compared with western countries. This review is also concerned with the relationship between the presence of ovarian endometriosis and clinical features such as age, parity, menopausal status, clinical stage, and survival in ovarian cancer patients.

Methylation-associated silencing of the Wnt antagonist SFRP1 gene in human ovarian cancers

The SFRP1 gene on chromosome 8p11.2 encodes a Wnt signaling antagonist, and was recently demonstrated to be a new tumor suppressor that is inactivated by promoter methylation in human colon cancers. Here, we analyzed promoter methylation of the SFRP1 gene in human ovarian cancers, in which loss of heterozygosity in 8p is frequently observed and involvement of the Wnt signaling pathway has been suggested. Methylation‐specific PCR (MSP) analysis showed that four of 13 ovarian cancer cell lines and two of 17 primary ovarian cancers had methylated SFRP1, while an immortalized ovarian epithelial cell line, HOSE, and seven ovarian endometrial cyst samples did not. In the four ovarian cancer cell lines with the methylation, SFRP1 was not expressed at all as determined by quantitative RT‐PCR analysis. A cell line with SFRP1 methylation, MCAS, was treated with a demethylating agent, 5‐aza‐2′‐deoxycytidine, and demethylation of the promoter and re‐expression of SFRP1 were observed. These results show that SFRP1 is inactivated by promoter methylation in human ovarian cancers, as well as colon cancers.

Safety and immunogenicity of a peptide containing the cross-neutralization epitope of HPV16 L2 administered nasally in healthy volunteers

Amino acid (aa) 108-120 of L2 protein of human papillomavirus (HPV) type 16 contains a cross-neutralization epitope against genital HPV. We designed a placebo-controlled trial in healthy adults to evaluate the safety and immunogenicity of a synthetic peptide consisting of the aa 108-120 of HPV16 L2 (L2-108/120) region. A total of 13 volunteers were given nasal inoculations with 0.1 (n=5) or 0.5mg (n=5) doses of the peptides or placebo (n=3) without adjuvant at weeks 0, 4, and 12. Sera were collected before inoculation and at 6, 16 and 36 weeks. The inoculation caused no serious local and systemic complications. The inoculation generated anti-L2 antibodies binding to both HPV16 and 52 L1/L2-capsids in four of the five recipients in the 0.5mg group. Sera of the four recipients showed neutralizing activities against HPV16 and 52. Serological responses to the peptides were not found in the 0.1mg group and the placebo group recipients. This study suggests the L2-108/120 peptide is tolerable in humans and has the potential as a broad-spectrum prophylactic vaccine against genital HPV.

Ubiquitous presence of E6 and E7 transcripts in human papillomavirus—Positive cervical carcinomas regardless of its type

The presence of human papillomavirus (HPV) DNA in almost all of the cervical carcinomas is one of the most compelling evidence for the viral carcinogenesis. HPVs are thought to induce cervical carcinoma most likely through the expression of E6 and E7 genes presumably by inactivating the tumor suppressor proteins, p53 and pRb, respectively. Thus far, the presence of HPV E6 and E7 transcripts have been identified only in cervical carcinoma‐derived cell lines harboring type 16 or 18, and in a limited number of cervical neoplasia specimens positive for type 16, 18, 33 or 51. To see whether the expression of E6 and E7 genes is an essential finding in HPV‐positive cervical carcinoma and cervical intraepithelial neoplasia (CIN), we constructed a reverse transcription‐polymerase chain reaction (RT‐PCR) assay using a pair of consensus primers in the E6 and E7 regions. Using the assay, E6 transcripts (full‐length E6/E7 transcripts) and E7 transcripts (spliced E6/E7 transcripts, E6* mRNA) were identified in 97% (30/31) and 100% (all 31) of cervical carcinomas and in 100% (all 23) and 74% (17/23) of CINs, respectively. This assay also revealed unknown splice donor and acceptor sites of E6* mRNA of less frequent HPV types 31, 35, 52, 56, 58 and 59 based on sequence analyses of the PCR products. Thus, the present study demonstrates that E6 and E7 transcripts of HPV exist in virtually all HPV‐positive cervical neoplasia specimens except for the absence of E7 transcripts in some of CINs. J. Med. Virol. 62:251–256, 2000.