Possible de novo clear cell carcinoma in the contralateral ovary 9 years after fertility-sparing surgery for Stage IA clear cell ovarian carcinoma.

A patient who underwent fertility-sparing surgery for Stage IA clear cell carcinoma may have developed de novo clear cell carcinoma in the contralateral ovary 9 years later. She underwent fertility-sparing surgery and postoperative adjuvant chemotherapy for right ovarian carcinoma at 33 years of age (when endometriosis was observed in the contralateral ovary). At the age of 41 years, a tumor was discovered in the left ovary. This was diagnosed pathologically as clear cell carcinoma with clear cell adenofibroma, which may have developed de novo. A consensus is currently taking shape that although fertility-sparing surgery is a therapeutic option for patients with Stage IA clear cell carcinoma, long-term outpatient monitoring is advised to watch for its recurrence or de novo development in the contralateral ovary.

Loss of PTEN expression is an independent predictor of favourable survival in endometrial carcinomas.

BACKGROUND: We and others previously reported the prognostic significance of PTEN mutational status on favourable survival in endometrial carcinomas. Here, we demonstrate that loss of PTEN expression in immunohistochemistry is an independent prognostic marker for favourable survival in endometrial carcinomas. METHODS: We conducted immunohistochemical analyses of PTEN, PIK3CA, phosphorylated Akt (p-Akt), and p27 in primary endometrial carcinomas from 221 patients. Mutation of PTEN was analysed further. RESULTS: Expression of PTEN was lost in 56 patients (25%), and PIK3CA was overexpressed in 159 patients (72%). Overexpression of PIK3CA was associated with p-Akt overexpression (P<0.001), which was in turn associated with loss of nuclear p27 expression (P=0.028). Loss of PTEN expression was found to be associated with endometrioid histology (P=0.03), and was inversely associated with the presence of lymphovascular space invasion (P=0.03). Univariate and multivariate survival analyses revealed that factors of PTEN loss, age <70, histological grade 1, early International Federation of Gynecology and Obstetrics (FIGO) stage, and absence of lymphovascular invasion were independent prognostic indicators for better overall survival (P=0.03, 0.04, 0.01, <0.001, and 0.03, respectively). The subset analysis showed a stronger tendency of PTEN loss towards favourable survival in advanced-stage (III and IV) disease than in early-stage (I and II) disease (P=0.05 vs 0.14). Moreover, our mutational analysis demonstrated that PTEN expression loss was associated with PTEN-truncating mutations (P=0.03). CONCLUSION: The current observations further support the prognostic significance of PTEN aberration on favourable outcome in endometrial carcinomas, providing useful implications for the individualised management of the disease.

Silent venous thromboembolism before treatment in endometrial cancer and the risk factors.

Venous thromboembolism (VTE) often occurs after surgery and can even occur before surgery in patients with gynaecological malignancies. We investigated the incidence of VTE before treatment of endometrial cancer and associated risk factors. Plasma D-dimer (DD) levels before initial treatment were examined in 171 consecutive patients with endometrial cancer. Venous ultrasound imaging (VUI) of the lower extremities was performed in patients with DD >or=1.5 microg ml(-1), as the negative predictive value of DD for VTE is extremely high. For patients with deep vein thrombosis (DVT), pulmonary scintigraphy was performed to ascertain the presence of pulmonary thromboembolism (PTE). Risk factors for VTE were analysed using univariate and multivariate analyses for 171 patients. Of these, 37 patients (21.6%) showed DD >or=1.5 microg ml(-1), 17 (9.9%) displayed DVT by VUI and 8 (4.7%) showed PTE on pulmonary scintigraphy. All patients with VTE were asymptomatic. Univariate analysis for various risk factors revealed older age, non-endometrioid histology and several variables of advanced disease as significantly associated with VTE before treatment. Obesity, smoking and diabetes mellitus were not risk factors. Multivariate analysis confirmed extrauterine spread and non-endometrioid histology as independently and significantly associated with risk of VTE. These data suggest that silent or subclinical VTE occurs before treatment in at least around 10% of patients with endometrial cancer. Risk factors for VTE before treatment might not be identical to those after starting treatment.

PTEN mutation located only outside exons 5, 6, and 7 is an independent predictor of favorable survival in endometrial carcinomas.

Although the prognostic impact of PTEN mutation in endometrial carcinoma is beginning to be analyzed, the prognostic significance of mutated PTEN exons has not ever been described. Sixty-seven endometrial carcinomas were analyzed for PTEN mutations using single-strand conformation polymorphism analysis and DNA sequencing. First, survival rates were compared according to PTEN status and mutated PTEN exons. Subsequently, univariate and multivariate analyses of various favorable prognostic factors for survival were conducted. The associations between PTEN mutation and clinicopathological features were also statistically evaluated. PTEN mutations were detected in 37 of 67 (55%) specimens. Among 47 mutations, frameshifts (57%) and mutations in exon 8 (38%) were most frequent. In univariate analysis, a factor of PTEN mutation only outside exons 5-7 was associated with significantly better survival (P = 0.02), although mutation in any exon of PTEN was not (P = 0.33). Subsequent multivariate analysis revealed that factors of mutation only outside exons 5-7 of PTEN, stage I/II, and G1 were significant and independent prognostic indicators for favorable survival (P = 0.004, 0.004, and 0.0006, respectively). In the subset of advanced-stage disease, mutation only outside exons 5-7 was associated with a trend toward better survival (P = 0.13). No significant correlation was observed between PTEN mutation and estrogen-related clinicopathological features. In conclusion, we find that PTEN mutation located only outside exons 5-7 is a significant and independent positive prognostic indicator for survival. The current observation has prognostic and therapeutic implications for the management of patients with endometrial carcinoma.

Growth and gene expression profile analyses of endometrial cancer cells expressing exogenous PTEN.

The PTEN tumor suppressor gene encodes a multifunctional phosphatase that plays an important role in inhibiting the phosphatidylinositol-3-kinase pathway and downstream functions that include activation of Akt/protein kinase B, cell survival, and cell proliferation. Enforced expression of PTEN in various cancer cell lines decreases cell proliferation through arrest of the cell cycle, accompanied in some cases by induction of apoptosis. We used cDNA microarrays containing 4009 cDNAs to examine changes in gene-expression profiles when exogenous PTEN was induced in PTEN-defective cells. The microarrays and subsequent semi-quantitative reverse transcription-PCR analysis revealed transcriptional stimulation of 99 genes and repression of 72 genes. Some of the differentially expressed genes already had been implicated in cell proliferation, differentiation, apoptosis, or cell cycle control, e.g., overexpression of PTEN-induced transactivation of cyclin-dependent inhibitor 1B (p27Kip1) and 2B (p15INK4B), members of the TNF receptor family, tumor necrosis factor-associated genes, and members of the Notch-signaling and Mad families. To our knowledge this is the first report of transactivation of those genes by PTEN. The genes differentially expressed in our experiments also included many whose correlation with cancer development had not been recognized before. Our data should contribute to a greater understanding of the broad spectrum of ways in which PTEN affects intracellular signaling pathways. Analysis of expression profiles with microarrays appears to be a powerful approach for identifying anticancer genes and/or disease-specific targets for cancer therapy.

A single nucleotide polymorphism in the matrix metalloproteinase-1 promoter in endometrial carcinomas.

Recent studies demonstrated that a single guanine insertion polymorphism in a matrix metalloprotease-1 promoter created an Ets binding site and affected the elevation of the transcriptional level of matrix metalloproteinase-1 (MMP-1). Furthermore, in tumor cell lines derived from melanoma and breast cancer, the incidence of the 2G / 2G genotype was significantly higher than that in the normal population. To evaluate the contribution of this polymorphism in endometrial carcinomas, we genotyped 100 endometrial carcinomas and then analyzed immunoexpression of MMP-1 in these carcinomas. We found that endometrial carcinoma patients showed a significantly higher rate of 1G / 2G or 2G / 2G genotype than control individuals, and that tumors containing the 2G allele(a) expressed MMP-1 protein more frequently than those with 1G / 1G genotype. Therefore, the single nucleotide polymorphism at the MMP-1 promoter affected the expression level of the MMP-1 protein, which may result in the association with more aggressive character in endometrial carcinoma. Our result suggests that the presence of 2G polymorphism at the MMP-1 promoter may be one of the risk factors for the development and / or progression of endometrial carcinoma.

Correlation between expression of the matrix metalloproteinase-1 gene in ovarian cancers and an insertion/deletion polymorphism in its promoter region.

Matrix metalloproteinases (MMPs), a family of closely related enzymes that degrade the extracellular matrix, are likely to be involved in invasion and metastasis of tumor cells. A guanine (G) insertion/deletion polymorphism within the promoter region of MMP-1 influences the transcription of this gene; i.e., the 2G (insertion-type) promoter possesses greater transcriptional activity than the 1G (deletion-type) promoter. To investigate whether this feature contributes to cancer development and/or progression, we genotyped 163 ovarian cancer patients for the polymorphism and then analyzed levels of expression of the MMP-1 gene in their tumors. The proportion of patients who were either heterozygotes or homozygotes for the 2G allele was significantly higher than that observed among 150 individuals without cancer (P = 0.028). Moreover, the levels of MMP-1 expression in cancer tissues among the patients carrying 2G alleles were elevated significantly in comparison with 1G homozygotes (P = 0.0038). By stimulating degradation of extracellular matrix, an excess of MMP-1 production may enhance development and/or rapid progression of ovarian cancers.

Complete DNA sequence and characterization of a 330-kb VNTR-rich region on chromosome 6q27 that is commonly deleted in ovarian cancer.

We report the complete genomic DNA sequence and the characterization of a 330-kb region on chromosome 6q27 that is often deleted in ovarian cancers. Using computer programs to predict exonic sequences, we isolated four novel genes, HGC6.1-4, as well as the known AF-6 gene. None of the deduced products of the novel genes exhibited significant homology to previously known proteins. We also identified ten microsatellites and 12 different VNTR sequences within the target region. HGC6.3 contained a VNTR within a coding exon, each repeat consisting of 42 nucleotides; the predicted 14-amino-acid consensus unit is MTPTVFSSQHTAGG. At least nine different sizes of this VNTR locus were detected among 20 unrelated DNA samples from caucasians. The polymorphic markers and the transcript map documented here may contribute to identification of novel genes or allelic aberrations associated with the development of ovarian cancers.

Growth suppression of human ovarian cancer cells by adenovirus-mediated transfer of the PTEN gene.

A tumor suppressor gene on chromosome 10q23, PTEN, encodes a phosphatidylinositol phosphatase that antagonizes activation of the phosphatidylinositol 3'-kinase-mediated pathway involved in cell growth. A gene encoding the catalytic subunit of phosphatidylinositol 3'-kinase (PIK3CA) is frequently activated in ovarian cancers; therefore, overexpression of the PTEN product through gene transfer might be an effective strategy for treating ovarian cancers. To test the potential for this type of gene therapy, we constructed a recombinant adenovirus encoding wild-type PTEN and examined its effects on nine cell lines derived from human ovarian carcinomas. Transduction of the PTEN gene significantly inhibited growth of six of these cell lines compared with infection with virus alone, and the degree of inhibition correlated with the efficiency of gene transfer as determined by beta-galactosidase assay. Results of flow cytometry suggested that the observed effects were mediated by two mechanisms, apoptosis and/or arrest in the G1 phase of the cell cycle, and that high adenoviral transduction efficiency of cells was associated with induction of apoptosis. We also found that the level of transcription of Integrin alpha(v) in ovarian cancer cells correlated with the efficiency of transduction (P = 0.014) and with the degree of growth inhibition after PTEN gene transfer (P = 0.009). These findings carry significant implications for adenovirus vector-based PTEN gene therapies for ovarian cancers.

No evidence of correlation between polymorphism at codon 72 of p53 and risk of cervical cancer in Japanese patients with human papillomavirus 16/18 infection.

Human papillomavirus (HPV)-16 and -18 encode E6 oncoprotein, which binds to and induces degradation of the tumor suppressor protein p53. A common polymorphism of p53, encoding either proline or arginine at position 72, affects the susceptibility of p53 to E6-mediated degradation in vivo; Caucasian women homozygous for arginine 72 reportedly are about seven times more susceptible to HPV-associated carcinoma of the cervix than heterozygotes. To examine whether arginine 72 could be a risk factor for HPV-associated cervical carcinomas in the Japanese population, we used the same PCR-based assay to analyze p53 genotypes of HPV-positive invasive cervical carcinomas from 103 Japanese women versus 110 control samples. Inasmuch as we detected no significant difference in the frequencies of proline or arginine alleles between the two groups, p53 polymorphism at residue 72 does not seem to be involved in the development of HPV-associated cervical carcinomas in women of Japanese ethnicity.

Complete genomic structure DNA polymorphisms, and alternative splicing of the human AF-6 gene.

In our previous work, detailed deletion mapping of ovarian cancers indicated that a 300-kb region of chromosome 6q27 was likely to contain one or more putative tumor suppressor genes associated with development of this type of cancer. DNA sequencing in the region disclosed the presence of AF-6, a gene that had been identified as the ALL-1 fusion partner involved in acute myeloid leukemias with t(6;11)(q27;q23) translocations. In the work reported here, we determined the complete genomic sequence of the AF-6 gene, including exon-intron boundaries, and found six DNA polymorphisms. One of them, an insertion/deletion polymorphism, determined the presence or absence of seven amino acids in the AF-6 product. We also identified two alternatively spliced forms of the gene; the two novel transcripts would encode additional C-terminal peptides in comparison to the reported protein. Sequencing of seven cosmid clones that covered the entire gene revealed 32 exons (not including one exon involved in the insertion/deletion polymorphism), spanning approximately 140 kb of genomic DNA. These results may contribute to an understanding of the mechanism causing chromosomal translocations in leukemic cells.

Isolation, mapping, and functional analysis of a novel human cDNA (BNIP3L) encoding a protein homologous to human NIP3.

We have isolated a novel cDNA that encodes a product showing significant sequence homology (56% identity) to human NIP3, a protein thought to interact with adenovirus E1B19kD and human BCL2 proteins. This cDNA contains an open reading frame of 657 nucleotides encoding a 219 amino acid polypeptide. The gene, designated BNIP3L, was expressed in all 16 normal human tissues examined; we mapped it to chromosome band 8p21 by fluorescence in situ hybridization. Introduction of the BNIP3L gene into six different cancer-cell lines caused significant growth suppression in each of them, while no such effect occurred when the antisense cDNA or the vector DNA was transfected, indicating that BNIP3L may function as a tumor suppressor.