Targeted DNA sequencing of high-grade serous ovarian carcinoma reveals association of TP53 mutations with platinum resistance when combined with gene expression.

High-grade serous ovarian carcinoma (HGSC) is the most common subtype of ovarian cancer and is among the most fatal gynecological malignancies worldwide, due to late diagnosis at advanced stages and frequent therapy resistance. In 47 HGSC patients, we assessed somatic and germline genetic variability of a custom panel of 144 known or suspected HGSC-related genes by high-coverage targeted DNA sequencing to identify the genetic determinants associated with resistance to platinum-based therapy. In the germline, the most mutated genes were DNAH14 (17%), RAD51B (17%), CFTR (13%), BRCA1 (11%), and RAD51 (11%). Somatically, the most mutated gene was TP53 (98%), followed by CSMD1/2/3 (19/19/36%), and CFTR (23%). Results were compared with those from whole exome sequencing of a similar set of 35 HGSC patients. Somatic variants in TP53 were also validated using GENIE data of 1287 HGSC samples. Our approach showed increased prevalence of high impact somatic and germline mutations, especially those affecting splice sites of TP53, compared to validation datasets. Furthermore, nonsense TP53 somatic mutations were negatively associated with patient survival. Elevated TP53 transcript levels were associated with platinum resistance and presence of TP53 missense mutations, while decreased TP53 levels were found in tumors carrying mutations with predicted high impact, which was confirmed in The Cancer Genome Atlas data (n = 260). Targeted DNA sequencing of TP53 combined with transcript quantification may contribute to the concept of precision oncology of HGSC. Future studies should explore targeting the p53 pathway based on specific mutation types and co-analyze the expression and mutational profiles of other key cancer genes.

Identification of a four-gene methylation biomarker panel in high-grade serous ovarian carcinoma.

Background The lack of effective biomarkers for the screening and early detection of ovarian cancer (OC) is one of the most pressing problems in oncogynecology. Because epigenetic alterations occur early in the cancer development, they provide great potential to serve as such biomarkers. In our study, we investigated a potential of a four-gene methylation panel (including CDH13, HNF1B, PCDH17 and GATA4 genes) for the early detection of high-grade serous ovarian carcinoma (HGSOC). Methods For methylation detection we used methylation sensitive high-resolution melting analysis and real-time methylation specific analysis. We also investigated the relation between gene hypermethylation and gene relative expression using the 2-ΔΔCt method. Results The sensitivity of the examined panel reached 88.5%. We were able to detect methylation in 85.7% (12/14) of early stage tumors and in 89.4% (42/47) of late stage tumors. The total efficiency of the panel was 94.4% and negative predictive value reached 90.0%. The specificity and positive predictive value achieved 100% rates. Our results showed lower gene expression in the tumor samples in comparison to control samples. The more pronounced downregulation was measured in the group of samples with detected methylation. Conclusions In our study we designed the four-gene panel for HGSOC detection in ovarian tissue with 100% specificity and sensitivity of 88.5%. The next challenge is translation of the findings to the less invasive source for biomarker examination, such as plasma. Our results indicate that combination of examined genes deserve consideration for further testing in clinical molecular diagnosis of HGSOC.

Aberrant methylation of PCDH17 gene in high-grade serous ovarian carcinoma.

BACKGROUND: Aberrant DNA methylation of protocadherins (PCDHs) has been associated with development and progression of various types of cancer. It could represent possible direction in the search for critically needed tumor biomarkers for ovarian cancer. OBJECTIVE: To investigate methylation of δ2 group of non-clustered PCDHs in high-grade serous ovarian carcinoma (HGSOC) tissue in comparison with control tissue. METHODS: We used next-generation sequencing for detecting regions with the most altered methylation. For further confirmation of discovered alterations we used methylation-sensitive high-resolution melting analysis. RESULTS: PCDH17 methylation was detected in almost 70% of HGSOC patients without any methylation in the group of control samples and was found both in the late stage tumors as well as in the early stage ones. Other selected PCDHs did not show any relevant changes in methylation. Subsequent gene expression analysis of PCDH17 revealed decreased expression in all of the tumor samples in comparison to the control ones. Statistically significant negative correlation was found between methylation and levels of expression suggesting potentially methylation-based silencing. CONCLUSIONS: Methylation of PCDH17 could play an important role in development and progression of HGSOC and has potential to become a target in the search for new clinical biomarkers.

Comparative study of various subpopulations of cytotoxic cells in blood and ascites from patients with ovarian carcinoma.

AIM OF THE STUDY: A number of observations have indicated that the immune system plays a significant role in patients with epithelial ovarian cancer (EOC). In cases of EOC, the prognostic significance of tumour infiltrating lymphocytes has not been clearly explained yet. The aim is to determine the phenotype and activation molecules of cytotoxic T cell and NK cell subpopulations and to compare their representation in malignant ascites and peripheral blood in patients with ovarian cancer. MATERIAL AND METHODS: Cytotoxic cells taken from blood samples of the cubital vein and malignant ascites were obtained from 53 patients with EOC. Their surface and activation characteristics were determined by means of a flow cytometer. Immunophenotype multiparametric analysis of peripheral blood lymphocytes (PBLs) and tumour infiltrating lymphocytes (TILs) was carried out. RESULTS: CD3(+) T lymphocytes were the main population of TILs (75.9%) and PBLs (70.9%). The number of activating T cells was significantly higher in TILs: CD3(+)/69(+) 6.7% vs. 0.8% (p < 0.001). The representation of (CD3(-)/16(+)56(+)) NK cells in TILs was significantly higher: 11.0% vs. 5.6% (p = 0.041); likewise CD56(bright) and CD-56(bright) from CD56(+) cells were higher in TILs (both p < 0.001). The activation receptor NKG2D was present in 45.1% of TILs vs. 32.3% of PBLs (p = 0.034), but we did not find a significant difference in the numbers of CD56(+)/NKG2D(+) in TILs and PBLs. CONCLUSIONS: These results prove that the characteristics and intensity of anti-tumour responses are different in compared compartments (ascites/PBLs). The knowledge of phenotype and functions of effector cells is the basic precondition for understanding the anti-tumour immune response.

Clinical significance of the resistance proteins LRP, Pgp, MRP1, MRP3, and MRP5 in epithelial ovarian cancer.

OBJECTIVE: This study aimed to evaluate the correlation between the expressions of lung resistance protein (LRP), P-glycoprotein (Pgp), multidrug resistance-associated protein (MRP)-1, MRP3, and MRP5 and histopathological parameters and clinical outcome, and to determine the predictive and prognostic value of these transport proteins in patients with ovarian cancer. METHODS: Tumor samples from 111 chemonaive patients with epithelial ovarian cancer who underwent primary surgery from 2006 to 2010 were immunohistochemically stained for LRP, Pgp, MRP1, MRP3, and MRP5 expressions. RESULTS: MRP1 expression was greater among patients with late disease than among patients with early stage ovarian cancer [International Federation of Gynecology and Obstetrics (FIGO) I + II, 71.6% (confidence interval, 60-100); FIGO III + IV, 83.6% (confidence interval, 100-100); P = 0.03]. The histological subtype correlated with the expressions of LRP, Pgp, MRP1, and MRP3. Relapse of disease during the next 24 months occurred more often among patients with higher Pgp and MRP1 than among patients with lower Pgp and MRP1 expressions. FIGO stage, histological type, debulking efficiency, strong Pgp expression, and strong MRP1 expression correlated significantly with shorter progression-free survival (log-rank test, P = 0.001, P = 0.004, P = 0.001, P = 0.051, and P = 0.046, respectively). FIGO stage, histological type, debulking efficiency, and strong MRP1 expression correlated with poor patient survival (log-rank test, P = 0.001, P = 0.042, P = 0.005, and P = 0.018, respectively). CONCLUSIONS: Pgp and MRP1 expressions were clinically significant in patients with ovarian cancer. Pgp and MRP1 may be reliable, independent predictive and prognostic factors regarding the clinical outcome of ovarian cancer. MRP3 is less important as a predictive and prognostic factor than MRP1 expression. MRP5 and LRP expressions were not applicable prognostic parameters regarding ovarian cancer.

Lysophosphatidic acid (LPA)­a perspective marker in ovarian cancer.

To compare plasma lysophosphatidic acid (LPA) levels in ovarian cancer patients in women with benign ovarian tumors and in women with no ovarian pathology. We correlated clinico-pathological parameters with plasma LPA levels. Capillary electrophoresis with indirect ultraviolet detection was used to analyze the plasma LPA levels of 159 patients (81 patients with ovarian cancer, 27 women without ovarian or uterine pathologies, and 51 patients with benign ovarian tumors) during a 5-year period. Patients with ovarian cancer had a significantly higher plasma LPA level (n=81; median (med), 11.53 µmol/l; range, 1.78-43.21 µmol/l) compared with controls with no ovarian pathology (n=27; med, 1.86 µmol/l; range, 0.94-9.73 µmol/l), and patients with benign ovarian tumor (n=51; med, 6.17 µmol/l; range, 1.12-25.23 µmol/l; P<0.001). We found that plasma LPA levels were associated with the International Federation of Gynecology and Obstetrics stage. The histological subtype and grade of ovarian cancer did not influence the plasma LPA levels in this study. The plasma LPA level can be a useful marker for ovarian cancer, particularly in the early stages of the disease.

Prognostic significance of CD3+ tumor-infiltrating lymphocytes in ovarian carcinoma.

OBJECTIVE: Epithelial ovarian cancer (EOC) is the most common cause of death among gynecological malignancies in Europe and North America. Although the presence of tumor-infiltrating mononuclear cells has been documented in EOC, the association of tumor-infiltrating mononuclear cells with clinical outcome remains controversial. The aim of the present study was to investigate the prognostic significance of CD3+ tumor-infiltrating T lymphocytes (TIL) on overall survival of EOC patients. METHODS: We evaluated retrospectively by immunohistochemistry the distribution of CD3+ TIL in tumor specimens of 116 EOC patients. The expression of estrogen and progesterone receptors, Ki-67, DNA topoisomerase IIalpha, p21, p53, HER-2/neu, bax and bcl-2 was also evaluated by immunohistochemistry. The prognostic significance of CD3+ TIL and other immunohistochemical and clinical parameters was evaluated with log-rank test. Multivariate analysis was performed using the Cox regression. RESULTS: CD3+ TIL were observed in all tumor samples, both in cancer stroma and within cancer epithelium (intraepithelial TIL). The median counts of stromal TIL and intraepithelial TIL were 338 lymphocytes/mm2 (range 81-2094 lymphocytes/mm2) and 125 lymphocytes/mm2 (range 7-481 lymphocytes/mm2), respectively. In univariate analysis, age, stage, grade, presence of residual tumor, expression of progesterone receptors, Ki-67, DNA topoisomerase IIalpha and intraepithelial CD3+ TIL count were significant predictors of overall survival. On multivariate analysis, only the presence of residual tumor, stage, expression of progesterone receptors and intraepithelial CD3+ TIL count were found to be significant independent predictors of overall survival. CONCLUSION: Present data indicate that the intraepithelial CD3+ TIL count is a significant prognostic factor in EOC.