Involvement of DPP9 in gene fusions in serous ovarian carcinoma.

BACKGROUND: A fusion gene is a hybrid gene consisting of parts from two previously independent genes. Chromosomal rearrangements leading to gene breakage are frequent in high-grade serous ovarian carcinomas and have been reported as a common mechanism for inactivating tumor suppressor genes. However, no fusion genes have been repeatedly reported to be recurrent driver events in ovarian carcinogenesis. We combined genomic and transcriptomic information to identify novel fusion gene candidates and aberrantly expressed genes in ovarian carcinomas. METHODS: Examined were 19 previously karyotyped ovarian carcinomas (18 of the serous histotype and one undifferentiated). First, karyotypic aberrations were compared to fusion gene candidates identified by RNA sequencing (RNA-seq). In addition, we used exon-level gene expression microarrays as a screening tool to identify aberrantly expressed genes possibly involved in gene fusion events, and compared the findings to the RNA-seq data. RESULTS: We found a DPP9-PPP6R3 fusion transcript in one tumor showing a matching genomic 11;19-translocation. Another tumor had a rearrangement of DPP9 with PLIN3. Both rearrangements were associated with diminished expression of the 3' end of DPP9 corresponding to the breakpoints identified by RNA-seq. For the exon-level expression analysis, candidate fusion partner genes were ranked according to deviating expression compared to the median of the sample set. The results were collated with data obtained from the RNA-seq analysis. Several fusion candidates were identified, among them TMEM123-MMP27, ZBTB46-WFDC13, and PLXNB1-PRKAR2A, all of which led to stronger expression of the 3' genes. In view of our previous findings of nonrandom rearrangements of chromosome 19 in this cancer type, particular emphasis was given to changes of this chromosome and a DDA1-FAM129C fusion event was identified. CONCLUSIONS: We have identified novel fusion gene candidates in high-grade serous ovarian carcinoma. DPP9 was involved in two different fusion transcripts that both resulted in deregulated expression of the 3' end of the transcript and thus possible loss of the active domains in the DPP9 protein. The identified rearrangements might play a role in tumorigenesis or tumor progression.

Vulvar carcinoma in Norway: A 50-year perspective on trends in incidence, treatment and survival.

OBJECTIVE: To explore trends in vulvar squamous cell carcinoma (SCC) incidence, age and stage at diagnosis, treatment and survival in Norway from 1961 to 2010. METHODS: From 1961 to 2010, 2233 cases of vulvar SCC were extracted from the Cancer Registry of Norway. Data on age at diagnosis, tumor morphology, stage of the disease and treatment were analyzed. Age-standardized incidence rates, adjusted to the Norwegian standard population, were computed. Relative survival was calculated as a ratio of the observed survival in the study population over the expected survival in the background population. Multivariate Cox model was fitted to estimate hazard ratios. RESULTS: The overall incidence of vulvar SCC increased >2.5 fold (from 1.70 to 4.66 per 100,000 women/year; P<0.01). Age-specific incidence rates increased among women aged ≤60years (by 150% in age group 0-39years, 175% in age group 40-49years and 68% in age group 50-59years). From 1971 to 2010, the percentage of patients receiving surgery as only treatment decreased from 81% to 61%, whereas the use of radiation and combination therapy (surgery and radiation) increased from 3% to 11% and 6% to 20%, respectively. 5-year relative survival increased significantly among women ≤80years (from 72% to 83% among women aged ≤60years and from 60% to 65% among women aged 61-80years). CONCLUSIONS: The incidence of vulvar SCC has increased since the sixties, particularly among women younger than 60years. Despite less aggressive surgical treatment, survival has improved.

Bilateral ovarian carcinomas differ in the expression of metastasis-related genes.

The mechanisms behind bilaterality of ovarian carcinomas are not fully understood, as the two tumors could possibly represent two primary tumors, a primary tumor and a metastasis, or two metastases. The gene expression profiles from bilateral high-grade serous carcinomas (HGSCs) and clear cell carcinomas (CCCs) of the ovary were compared to study the association between the tumors of the two sides. A separate analysis of genes from chromosome 19 was also performed, since this chromosome is frequently rearranged in ovarian carcinomas. Tumors from four patients were included (three pairs of HGSC and one pair of CCC). The gene expression was analyzed at the exon level, and bilateral tumors were compared to identify within-pair differences. Gene expression data were also compared with genomic information on the same tumors. Similarities in gene expression were observed between the tumors within each pair, as expected if the two tumors were clonally related. However, certain genes exhibited differences in expression between the two sides, indicating metastasis involvement. Among the most differently expressed genes, one gene was common to all four pairs: Immunoglobulin J. In all HGSC pairs, serpin peptidase inhibitor, clade B (ovalbumin), member 2, serpin family E member 1 and phospholipase A2, group IIA (platelets, synovial fluid) were also among the differentially expressed genes. The specific analysis of chromosome 19 highlighted expression differences in the zinc finger protein 36 gene. These results indicate that bilateral ovarian tumors represent different stages during progression of a single clonal process. Several of the genes observed to be differently expressed are known to be metastasis-related, and are likely to be also involved in spreading from one side to the other in the bilateral cancer cases examined.

A novel truncated form of HMGA2 in tumors of the ovaries.

Neoplasms of the ovary are the second most common tumor of the female reproductive system, and the most lethal of the gynecological malignancies. Ovarian tumors are divided into a copious number of different groups reflecting their different features. The present study analyzed 187 ovarian tumors (39 sex-cord stromal tumors, 22 borderline tumors and 126 carcinomas) for the expression of the high-mobility group AT-hook 2 (HMGA2) gene, for mutations in the isocitrate dehydrogenase (NADP(+)) 1, cytosolic (IDH1), isocitrate dehydrogenase (NADP(+)) 2, mitochondrial (IDH2) and telomerase reverse transcriptase (TERT) genes, and for methylation of the O6-methylguanine-DNA methyltransferase (MGMT) promoter. Reverse transcription-polymerase chain reaction analysis showed that HMGA2 was expressed in 74.5% of the samples (120/161). A truncated transcript of HMGA2 was identified in 11 cases. A novel truncated form of HMGA2 was found in 4 serous high-grade carcinomas. Only 4 tumors (4/185) showed the TERT C228T mutation. No IDH1 or IDH2 mutations were found. Methylation of the promoter of MGMT was found in 2 borderline tumors (2/185). HMGA2 was expressed, in its truncated and native form, in different ovarian tumors, even the less aggressive types, underscoring the general importance of this gene in ovarian tumorigenesis. Mutations involving TERT, as well as MGMT promoter methylation, are rare events in ovarian tumors.

HMGA2 expression pattern and TERT mutations in tumors of the vulva.

Malignant tumors of the vulva account for only 5% of cancers of the female genital tract in the USA. The most frequent cancers of the vulva are squamous cell carcinoma (SCC) and malignant melanoma (MM). Little is known about the genetic aberrations carried by these tumors. We report a detailed study of 25 vulva tumors [22 SCC, 2 MM, 1 atypical squamous cell hyperplasia (AH)] analyzed for expression of the high-mobility group AT-hook family member genes HMGA2 and HMGA1, for mutations in the IDH1, IDH2 and TERT genes, and for methylation of the MGMT promoter. The RT-PCR and immunohistochemistry analyses showed that HMGA2 was expressed in the great majority of analyzed samples (20 out of 24; SCC as well as MM) but not in the normal controls. HMGA1, on the other hand, was expressed in both tumors and normal tissues. Five of the 24 tumors (all SCC) showed the C228T mutation in the TERT promoter. Our results showed that HMGA2 and TERT may be of importance in the genesis and/or the progression of tumors of the vulva.

Low frequency of ESRRA-C11orf20 fusion gene in ovarian carcinomas.

The identification of recurrent gene fusions in common epithelial cancers--for example, TMPRSS2/ERG in prostate cancer and EML4/ALK in nonsmall cell lung carcinomas--has raised the question of whether fusion genes are pathogenetically important also in ovarian carcinomas. The first recurrent fusion transcript in serous ovarian carcinomas was reported by Salzman et al. in 2011, who used deep paired-end sequencing to detect the fusion gene ESRRA-C11orf20 in 10 out of 67 (15%) serous ovarian carcinomas examined, a finding that holds great promise for our understanding of ovarian tumorigenesis as well as, potentially, for new treatment strategies. We wanted to test how frequent the ESRRA/C11orf20 fusion is in ovarian carcinomas of all subtypes, and therefore examined a series of 230 ovarian carcinomas of which 197 were of the serous subtype and 163 of the 197 were of stages III and IV--that is, the very same carcinoma subset where the fusion transcript had been found. We performed PCR and high-throughput sequencing analyses in search of the fusion transcript. We used the same primers described previously for the detection of the fusion and the same primer combination, but found no ESRRA/C11orf20 fusion in our series. A synthetic DNA plasmid containing the reported ESRRA/C11orf20 fusion was included as a positive control for our PCR experiments. Data from high-throughput sequencing of 23 ovarian carcinomas were screened in search of alternative partner(s) for the ESRRA and/or C11orf20 gene, but none was found. We conclude that the frequency of the ESRRA/C11orf20 gene fusion in serous ovarian carcinomas of stages III and IV must be considerable less than that reported previously (0/163 in our experience compared with 10/67 in the previous study). At the very least, it seems clear that the said fusion cannot be a common pathogenetic event in this tumor type.

DNA ploidy may be a prognostic marker in stage I and II serous adenocarcinoma of the endometrium.

In patients with serous adenocarcinoma (SAC) of the endometrium, we evaluated the prognostic importance of clinicopathological parameters, DNA ploidy, and immunoexpression of p53, estrogen receptor (ER), progesterone receptor (PR), and Ki-67. In a series of 73 stage I and II SAC, DNA ploidy analysis was performed on hysterectomy specimens using DNA image cytometry. Immunohistochemical analysis of p53, ER, PR, and Ki-67 expression was additionally performed. In the review of the histological slides by three gynecologic pathologists, the presence of a serous component was not agreed upon in 17 (23 %) cases. The remaining 56 cases, consisting of pure SAC or SAC mixed with endometrioid adenocarcinoma, were further analyzed. Tumor recurrence was observed in 14 patients, and 28 patients died during the follow-up period. Patients with diploid (n = 19), aneuploid (n = 29), and tetraploid (n = 8) tumor had 5-year recurrence rates of 10, 38, and 53 %, respectively (p = 0.09). A DNA ploidy parameter, 5c exceeding rate, was found to be a prognostic marker for recurrence (p = 0.03), progression-free survival (p < 0.01), and overall survival (p = 0.02). Immunoexpression of p53, ER, PR, and Ki-67 did not have prognostic value, and the same was true for FIGO stage, lymphovascular invasion, the extent of myometrial invasion, and lymphadenectomy. The histological diagnosis of SAC may be difficult in some cases. Established clinicopathological parameters do not seem to be strong prognosticators in stage I and II disease. A DNA ploidy parameter, 5c exceeding rate, may be a prognostic marker in this patient group and should be further validated in larger series.

The hypoxic microenvironment upgrades stem-like properties of ovarian cancer cells.

BACKGROUND: To study whether hypoxia influences the stem-like properties of ovarian cancer cells and their biological behavior under hypoxia. METHOD: Ovarian cancer cell lines ES-2 and OVCAR-3 were cultivated in different oxygen tensions for proliferation, cell cycling and invasion analyses. The clonogenic potential of cells was examined by colony formation and sphere formation assays. Stem cell surface markers, SP and CD44bright and CD44dim cells were analyzed by flow cytometry. Protein expression of HIF-1α, HIF-2α, Ot3/4 and Sox2 were investigated by Western blotting. RESULTS: Both cell lines cultivated at hypoxic condition grew relatively slowly with extended G0/G1 phase. However, if the cells were pre-treated under 1% O2 for 48 hrs before brought back to normoxia, the cells showed significantly higher proliferation rate with higher infiltration capability, and significant more colonies and spheres, in comparison to the cells always cultivated under normoxia. CD44bright cells expressed significantly higher levels of Oct3/4 and Sox2 than the CD44dim cells and formed significantly more clones and spheres examined in vitro. Hypoxic treatment of the cells resulted in stronger CD44 expression in both cell lines, and stronger CD133 expression in the OVCAR-3 cell line. In parallel with these findings, significantly increased number of side population (SP) cells and up-regulated expression of Oct3/4 and Sox2 in both ES-2 and OVCAR-3 cell lines were observed. CONCLUSION: We conclude that ovarian cancer cells survive hypoxia by upgrading their stem-like properties through up-regulation of stemness-related factors and behave more aggressively when brought back to higher oxygen environment.

Borderline ovarian tumours.

Borderline ovarian tumours account for 10-20% of all epithelial ovarian cancer. Historically, standard primary surgery has included borderline ovarian tumours, omentectomy, peritoneal washing and multiple biopsies. As one-third of borderline ovarian tumours are diagnosed in women under the age of 40 years, fertility-sparing treatment has been more frequently used in the past 10 years. Fertility drugs are well tolerated in women with infertility after fertility-sparing surgery. Careful selection of candidates is necessary. Laparoscopic techniques can be used, but should be reserved for oncologic surgeons. This conservative treatment increases the rate of recurrence, albeit with no effect on survival. The pregnancy rate is nearly 50%, and most are achieved spontaneously. These women should be closely followed up. The question is whether this is acceptable from a gynaecologic oncologic point of view. For this reason, we will discuss recently published studies and gynaecologic oncologic concerns about the mode of fertility-sparing surgery and its consequences.

DNA ploidy heterogeneity in endometrial carcinoma: comparison between curettage and hysterectomy specimens.

DNA ploidy has been reported to be a prognostic marker for patients with endometrial carcinoma. In this study, DNA ploidy and histologic heterogeneity were evaluated by comparing curettage and hysterectomy specimens in 99 consecutive patients diagnosed with endometrial carcinoma. High-resolution DNA ploidy image analysis and review of histologic specimens were performed. The histologic subtypes were identical in 77 (78%) and differed in 22 (22%) cases. The DNA ploidy results were concordant in the curettage and hysterectomy specimens in 72 (72.7%) and discordant in 27 (27.3%) cases. Histologic heterogeneity was significantly associated with DNA ploidy heterogeneity (P=0.03). On the basis of histologic heterogeneity, DNA ploidy-discordant cases were divided into 2 groups. One group (16.2% of cases) consisted of specimens with similar histology in curettage and hysterectomy, all belonging to the endometrioid subtype. This group showed DNA ploidy discordance because of a DNA diploid peak in 1 specimen and an aneuploid peak (DI=1.05-1.2) in the other. The other group (11.1% of cases) consisted of cases with different histologic subtype or grade and showed a more pronounced DNA ploidy difference (diploid vs. aneuploid with DI>1.2). Our results suggest that the DNA ploidy results of the hysterectomy and curettage specimens are not identical. The difference observed, which we believe reflects the intratumoral heterogeneity, should be taken into account when applying DNA ploidy to endometrial carcinoma specimens.

Gross genomic alterations differ between serous borderline tumors and serous adenocarcinomas--an image cytometric DNA ploidy analysis of 307 cases with histogenetic implications.

Our objective was to study the gross genomic alterations in serous borderline tumors and serous adenocarcinomas of the ovary. A retrospective analysis of 245 serous borderline tumors and 62 serous adenocarcinomas from 249 patients was performed using high-resolution image cytometric DNA ploidy analysis. DNA ploidy status, S-phase fraction, and DNA index were evaluated. The majority of serous borderline tumors were diploid (225/245 cases, 92%). The remaining 8% showed an aneuploid peak predominantly with DNA index of less than 1.4. Grades 2 and 3 serous adenocarcinomas were more often (80%) nondiploid, mostly with DNA index exceeding 1.4. Grade 1 serous adenocarcinomas were an intermediate group, more similar to serous borderline tumors. The S-phase fraction increased from serous borderline tumors (mean = 0.6%) through grade 1 serous adenocarcinomas (mean = 2.8%), being highest in grades 2 and 3 adenocarcinomas (mean = 6.8%). Our findings support the hypothesis that serous borderline tumors and grades 2 and 3 serous adenocarcinomas are genomically different lesions, with grade 1 serous adenocarcinomas being an intermediate group more close to borderline tumors.