Ioana Braicu

Prognostic impact of programmed cell death-1 (PD-1) and PD-ligand 1 (PD-L1) expression in cancer cells and tumor-infiltrating lymphocytes in ovarian high grade serous carcinoma

Aims Antibodies targeting the checkpoint molecules programmed cell death 1 (PD-1) and its ligand PD-L1 are emerging cancer therapeutics. We systematically investigated PD-1 and PD-L1 expression patterns in the poor-prognosis tumor entity high-grade serous ovarian carcinoma. Methods PD-1 and PD-L1 protein expression was determined by immunohistochemistry on tissue microarrays from 215 primary cancers both in cancer cells and in tumor-infiltrating lymphocytes (TILs). mRNA expression was measured by quantitative reverse transcription PCR. An in silico validation of mRNA data was performed in The Cancer Genome Atlas (TCGA) dataset. Results PD-1 and PD-L1 expression in cancer cells, CD3+, PD-1+, and PD-L1+ TILs densities as well as PD-1 and PD-L1 mRNA levels were positive prognostic factors for progression-free (PFS) and overall survival (OS), with all factors being significant for PFS (p < 0.035 each), and most being significant for OS. Most factors also had prognostic value that was independent from age, stage, and residual tumor. Moreover, high PD-1+ TILs as well as PD-L1+ TILs densities added prognostic value to CD3+TILs (PD-1+: p = 0.002,; PD-L1+: p = 0.002). The significant positive prognostic impact of PD-1 and PD-L1 mRNA expression could be reproduced in the TCGA gene expression datasets (p = 0.02 and p < 0.0001, respectively). Conclusions Despite their reported immune-modulatory function, high PD-1 and PD-L1 levels are indicators of a favorable prognosis in ovarian cancer. Our data indicate that PD-1 and PD-L1 molecules are biologically relevant regulators of the immune response in high-grade serous ovarian carcinoma, which is an argument for the evaluation of immune checkpoint inhibiting drugs in this tumor entity.

Molecular characterization of circulating tumor cells in patients with ovarian cancer improves their prognostic significance — A study of the OVCAD consortium

OBJECTIVE The study aims at identifying novel markers for circulating tumor cells (CTCs) in patients with epithelial ovarian cancer (EOC), and at evaluating their impact on outcome. METHODS Microarray analysis comparing matched EOC tissues and peripheral blood leucocytes (N=35) was performed to identify novel CTC markers. Gene expression of these novel markers and of EpCAM was analyzed using RT-qPCR in blood samples taken from healthy females (N=39) and from EOC patients (N=216) before primary treatment and six months after adjuvant chemotherapy. All samples were enriched by density gradient centrifugation. CTC positivity was defined by over-expression of at least one gene as compared to the healthy control group. RESULTS CTC were detected in 24.5% of the baseline and 20.4% of the follow-up samples, of which two thirds were identified by overexpression of the cyclophilin C gene (PPIC), and just a few by EpCAM overexpression. The presence of CTCs at baseline correlated with the presence of ascites, sub-optimal debulking, and elevated CA-125 and HE-4 levels, whereas CTC during follow-up occurred more often in older and platinum resistant patients. PPIC positive CTCs during follow-up were significantly more often detected in the platinum resistant than in the platinum sensitive patient group, and indicated poor outcome independent from classical prognostic parameters. CONCLUSIONS Molecular characterization of CTC is superior to a mere CTC enumeration or even be the rationale for CTC diagnostics at all. Ultimately CTC diagnostics may lead to more personalized treatment of EOC, especially in the recurrent situation.

Prognostic value of residual tumor size in patients with epithelial ovarian cancer FIGO stages IIA-IV: analysis of the OVCAD data.

Objective The objective of the study was to evaluate the prognostic impact of residual tumor size after cytoreductive surgery in patients with epithelial ovarian cancer. Methods In this prospective, multicenter study, 226 patients with epithelial ovarian cancer (International Federation of Gynecology and Obstetrics stages IIA–IV) were included. Patients were treated with cytoreductive surgery and adjuvant platinum-based chemotherapy. Univariate and multivariable survival analyses were performed to investigate the impact of residual tumor size on progression-free and overall survival. Results In 69.4% of patients, surgery resulted in complete tumor resection; minimal residual disease (≤1 cm) was achieved in 87.2% of patients. Advanced tumor stage was associated with a lower rate of complete tumor resection (P < 0.001). After cytoreductive surgery, 3-year overall survival rates were 72.4%, 65.8%, and 45.2% for patients without, with minimal, and with gross residual disease (>1 cm), respectively (P < 0.001). Multivariable survival analysis revealed residual tumor size (P = 0.04) and older patient age (P = 0.02) as independent prognosticators for impaired overall survival. Complete cytoreduction was predictive for a higher rate of treatment response (P = 0.001) and was associated with prolonged progression-free and overall survival (P < 0.001 and P = 0.001). Conclusions The size of residual disease after cytoreduction is one of the most crucial prognostic factors for patients with ovarian cancer. Patients after complete cytoreduction have a superior outcome compared with patients with residual disease. Leaving no residual tumor has to be the aim of primary surgery for ovarian cancer; therefore, patients should receive treatment at centers able to undertake complex cytoreductive procedures.

The time interval from surgery to start of chemotherapy significantly impacts prognosis in patients with advanced serous ovarian carcinoma — Analysis of patient data in the prospective OVCAD study

OBJECTIVE Cytoreductive surgery and platinum-based systemic therapy constitute the standard treatment of patients with advanced ovarian cancer. The aim of the present study was to evaluate whether the time interval from surgery to start of chemotherapy has an impact on clinical outcome. METHODS Data of 191 patients with advanced serous (FIGO III-IV) ovarian cancer from the prospective multicenter study OVCAD (OVarian CAncer Diagnosis) were analyzed. All patients underwent primary surgery followed by platinum-based chemotherapy. RESULTS The 25%, 50%, and 75% quartiles of intervals from surgery to start of chemotherapy were 22, 28, and 38 days, respectively (range, 4-158 days). Preoperative performance status (P<0.001), extent of surgery (P<0.001), and perioperative complications (P<0.001) correlated with intervals from surgery to initiation of chemotherapy. Timing of cytotoxic treatment [≤ 28 days versus >28 days; hazard ratio (HR) 1.73 (95% confidence interval 1.08-2.78), P=0.022], residual disease [HR 2.95 (95% confidence interval 1.87-4.67), P<0.001], and FIGO stage [HR 2.26 (95% confidence interval 1.41-3.64), P=0.001] were significant prognostic factors for overall survival in multivariate analysis. While the interval from surgery to start of chemotherapy did not possess prognostic significance in patients without postoperative residual disease (n=121), it significantly correlated with overall survival in patients with postoperative residual disease [n=70, HR 2.24 (95% confidence interval 1.08-4.66), P=0.031]. CONCLUSION Our findings suggest that delayed initiation of chemotherapy might compromise overall survival in patients with advanced serous ovarian cancer, especially when suboptimally debulked.

Outcome and clinical management of 275 patients with advanced ovarian cancer International Federation of Obstetrics and Gynecology II to IV inside the European Ovarian Cancer Translational Research Consortium-OVCAD.

Introduction The Sixth Framework Program European Union project OVCAD, “Ovarian Cancer—Diagnosis of a Silent Killer,” aimed to investigate new predictors for early detection of minimal residual disease in epithelial ovarian cancer (EOC). Here we present the main pathologic, surgical, and chemotherapy characteristics of the OVCAD patient cohort. Methods Between February 2005 and December 2008, 5 European gynecologic cancer centers (WP2 group) enrolled prospective 275 consecutive patients with EOC into this translational study. Inclusion criteria were as follows: advanced International Federation of Obstetrics and Gynecology II to IV stage, cytoreductive surgery, platinum-based chemotherapy, and collected tumor samples. WP2 coordinated the implementation, screening, and recruiting of the patients and tumor samples into a Web-based data bank according established standard operating procedures. Results Median age at the time of diagnosis was 58 years. Most patients presented advanced high-grade EOC: International Federation of Obstetrics and Gynecology III/IV (94.5%), grade 2/3 (96%), serous histology (86.2%), ascites (76%), peritoneal carcinomatosis (67.6%), and lymph node involvement (52%). The most common surgical procedures were omentectomy (92.4%), bilateral salpingo-oophorectomy (90.9%), hysterectomy (77.3%), pelvic (69.5%) and paraaortic (66.9%) lymphadenectomy, and large (37.7%) or small bowel resection (13.4%). Patients were treated commonly with platinum-based therapy (98.2%). The macroscopic cytoreduction rate was 68.4%. After a median follow-up of 37 months, 70 patients (25.5%) developed a platinum-resistant recurrence. Biological materials such as tumor and paraffin tissue, ascites, and blood samples were collected consecutively. Conclusions The implementation of the OVCAD cohort demonstrated the feasibility and advantages of an open, prospective, and multicenter recruitment inside a translational research study. Essential was the predefinition of all inclusion criteria, standard operating procedures, and Web-based software, which enabled the prospective patient recruitment and tissue sampling, minimizing institutional bias and variability in the quality of the biological samples. The translational concept of the OVCAD study does not conflict with the state-of-the-art surgical and chemotherapy management and guaranteed an improved outcome of patients with EOC.

Validating the impact of a molecular subtype in ovarian cancer on outcomes: A study of the OVCAD Consortium

Most patients with epithelial ovarian cancer (EOC) are diagnosed at advanced stage and have a poor prognosis. However, a small proportion of these patients will survive, whereas others will die very quickly. Clinicopathological factors do not allow precise identification of these subgroups. Thus, we have validated a molecular subclassification as new prognostic factor in EOC. One hundred and ninety‐four patients with Stage II–IV EOC were characterized by whole‐genome expression profiling of tumor tissues and were classified using a published 112 gene set, derived from an International Federation of Gynecology and Obstetrics (FIGO) stage‐directed supervised classification approach. The 194 tumor samples were classified into two subclasses comprising 95 (Subclass 1) and 99 (Subclass 2) tumors. All nine FIGO II tumors were grouped in Subclass 1 (P = 0.001). Subclass 2 (54% of advanced‐stage tumors) was significantly correlated with peritoneal carcinomatosis and non‐optimal debulking. Patients with Subclass 2 tumors had a worse overall survival for both serous and non‐serous histological subtypes, as revealed by univariate analysis (hazard ratios [HR] of 3.17 and 17.11, respectively; P ≤ 0.001) and in models corrected for relevant clinicopathologic parameters (HR 2.87 and 12.42, respectively; P ≤ 0.023). Significance analysis of microarrays revealed 2082 genes that were differentially expressed in advanced‐grade serous tumors of both subclasses and the focal adhesion pathway as the most deregulated pathway. In the present validation study, we have shown that, in advanced‐stage serous ovarian cancer, two approximately equally large molecular subtypes exist, independent of classical clinocopathological parameters and presenting with highly different whole‐genome expression profiles and a markedly different overall survival. Similar results were obtained in a small cohort of patients with non‐serous tumors. (Cancer Sci 2012; 103: 1334–1341)

Fibroblast growth factor receptor 4 gene (FGFR4) 388Arg allele predicts prolonged survival and platinum sensitivity in advanced ovarian cancer

FGFR4 has been shown to play an important role in the etiology and progression of solid tumors. A single nucleotide polymorphism (SNP) within the FGFR4 gene has previously been linked to prognosis and response to chemotherapy in breast cancer and other malignancies. This study evaluates the relevance of this SNP in advanced ovarian cancer. FGFR4‐genotype was analyzed in 236 patients recruited as part of the OVCAD project. Genotyping was performed on germ‐line DNA using a TaqMan based genotyping assay. Results were correlated with clinicopathological variables and survival. The FGFR4 388Arg genotype was significantly associated with prolonged progression‐free and overall survival (univariate: HR 0.68, p = 0.017; HR 0.49, p = 0.005; multivariate: HR 0.69, p = 0.025; HR 0.49, p = 0.006) though the positive prognostic value was restricted to patients without postoperative residual tumor. Indeed, there was a significant interaction between FGFR4 genotype and residual tumor for overall survival. Furthermore, the FGFR4 388Arg genotype significantly correlated with platinum sensitivity in the same subgroup (multivariate OR 3.81 p = 0.004). FGFR4 Arg388Gly genotype is an independent and strong context specific prognostic factor in patients with advanced ovarian cancer and could be used to predict platinum‐sensitivity.

Treatment reality in elderly patients with advanced ovarian cancer: a prospective analysis of the OVCAD consortium

BackgroundApproximately one third of women diagnosed with ovarian cancer is 70 years or older. Information on the treatment reality of these elderly patients is limited.Methods275 patients with primary epithelial ovarian cancer FIGO stage II-IV undergoing cytoreductive surgery and platinum-based chemotherapy were prospectively included in this European multicenter study. Patients <70 and ≥70 years were compared regarding clinicopathological variables and prognosis.ResultsMedian age was 58 years (18–85); 47 patients (17.1%) were 70 years or older. The postoperative 60-day-mortality rate was 2.1% for elderly and 0.4% for younger patients (p < 0.001). Elderly patients were less likely to receive optimal therapy (no residual disease after surgery and platinum combination chemotherapy) compared to patients <70 years (40.4% vs. 70.1%, p < 0.001) and their outcome was less favorable regarding median PFS (12 vs. 20 months, p = 0.022) and OS (30 vs. 64 months, p < 0.001). However, in multivariate analysis age itself was not a prognostic factor for PFS while the ECOG performance status had prognostic significance in elderly patients.ConclusionsElderly patients with ovarian cancer are often treated less radically. Their outcome is impaired despite no consistent prognostic effect of age itself. Biological age and functional status should be considered before individualized treatment plans are defined.

Cyclin E1 (CCNE1) as independent positive prognostic factor in advanced stage serous ovarian cancer patients - A study of the OVCAD consortium

Cyclin E, coded by the genes CCNE1 and CCNE2, is the main regulator for transition from G1 to S phase determining cell division. CCNE1 and CCNE2 are known oncogenes in many cancer entities. Especially CCNE1 has frequently been associated with gene amplifications in various malignancies, emphasising its role as a putative oncogene. We determined gene expression and copy number of CCNE1 and CCNE2 by quantitative polymerase chain reaction (PCR) from 172 International Federation of Obstetrics and Gynecology (FIGO) II/III/IV stage serous epithelial ovarian cancer (EOC) tissues and analysed its impact on outcome. Furthermore, whole transcriptome gene expression changes correlating with CCNE1 expression were determined by microarray technology, interpreted by Signalling Pathway Impact Analysis (SPIA), Tool for Inferring Network of Genes (TINGe), and illustrated by hive plots. Protein-protein interaction (PPI) networks were also used for the interpretation. Interestingly, and contradictory to most reports and intuitive expectations, high CCNE1 expression correlated with better overall survival (p=0.005) if corrected for usual clinicopathologic parameters and a molecular subclassification. Using different grading systems or only high graded tumours had no impact on this correlation. Copy number of CCNE1 was increased in 25% of cases which correlated highly significantly with expression but showed no impact on outcome. CCNE2 had no impact on outcomes at all. Whole genome transcriptome analysis revealed 1872 differentially expressed genes correlated to CCNE1 expression, which were significantly enriched with genes from five pathways (e.g. cell cycle and viral carcinogenesis pathway were up-regulated and the Fanconi anaemia pathway was down-regulated). High CCNE1 gene expression is a significant and independent predictor for prolonged overall survival in FIGO III/IV EOC patients. This upside down impact of CCNE1 on survival probably reflects the special characteristic of EOC with tumour dissemination in the near anaerobic peritoneal cavity as the predominant cause of death, compared to other cancer entities where distant metastasis are predominantly lethal.

Quaternary cytoreductive surgery in ovarian cancer: does surgical effort still matter?

Background:To evaluate surgical outcome and survival benefit after quaternary cytoreduction (QC) in epithelial ovarian cancer (EOC) relapse.Methods:We systematically evaluated all consecutive patients undergoing QC in our institution over a 12-year period (October 2000–January 2012). All relevant surgical and clinical outcome parameters were systematically assessed.Results:Forty-nine EOC patients (median age: 57; range: 28–76) underwent QC; in a median of 16 months (range:2–142) after previous chemotherapy. The majority of the patients had an initial FIGO stage III (67.3%), peritoneal carcinomatosis (77.6%) and no ascites (67.3%). At QC, patients presented following tumour pattern: lower abdomen 85.7%; middle abdomen 79.6% and upper abdomen 42.9%. Median duration of surgery was 292 min (range: a total macroscopic tumour clearance could be achieved. Rates of major operative morbidity and 30-day mortality were 28.6% and 2%, respectively.Mean follow-up from QC was 18.41 months (95% confidence interval (CI):12.64–24.18) and mean overall survival (OS) 23.05 months (95% CI: 15.5–30.6). Mean OS for patients without vs any tumour residuals was 43 months (95% CI: 26.4–59.5) vs 13.4 months (95% CI: 7.42–19.4); P=0.001. Mean OS for patients who received postoperative chemotherapy (n=18; 36.7%) vs those who did not was 40.5 months (95% CI: 27.4–53.6) vs 12.03 months (95% CI: 5.9–18.18); P<0.001.Multivariate analysis indentified multifocal tumour dissemination to be of predictive significance for incomplete tumour resection, higher operative morbidity and lower survival, while systemic chemotherapy subsequent to QC had a protective significant impact on OS. No prognostic impact had ascites, platinum resistance, high grading and advanced age.Conclusion:Even in this highly advanced setting of the third EOC relapse, maximal therapeutic effort combining optimal surgery and chemotherapy appear to significantly prolong survival in a selected patients ‘group’.