Thomas Bauknecht

Molecular and prognostic distinction between serous ovarian carcinomas of varying grade and malignant potential

Profiles of gene transcription have begun to delineate the molecular basis of ovarian cancer, including distinctions between carcinomas of differing histology, tumor progression and patient outcome. However, the similarities and differences among the most commonly diagnosed noninvasive borderline (low malignant potential, LMP) lesions and invasive serous carcinomas of varying grade (G1, G2 and G3) have not yet been explored. Here, we used oligonucleotide arrays to profile the expression of 12 500 genes in a series of 57 predominantly stage III serous ovarian adenocarcinomas from 52 patients, eight with borderline tumors and 44 with adenocarcinomas of varying grade. Unsupervised and supervised analyses showed that LMP lesions were distinct from high-grade serous adenocarcinomas, as might be expected; however, well-differentiated (G1) invasive adenocarcinomas showed a strikingly similar profile to LMP tumors as compared to cancers with moderate (G2) or poor (G3) cellular differentiation, which were also highly similar. Comparative genomic hybridization of an independent cohort of five LMP and 63 invasive carcinomas of varying grade demonstrated LMP and G1 were again similar, exhibiting significantly less chromosomal aberration than G2/G3 carcinomas. A majority of LMP and G1 tumors were characterized by high levels of p21/WAF1, with concomitant expression of cell growth suppressors, gadd34 and BTG-2. In contrast, G2/G3 cancers were characterized by the expression of genes associated with the cell cycle and by STAT-1-, STAT-3/JAK-1/2-induced gene expression. The distinction between the LMP-G1 and G2–G3 groups of tumors was highly correlated to patient outcome (χ2 for equivalence of death rates=7.681189; P=0.0056, log-rank test). Our results are consistent with the recent demonstration of a poor differentiation molecular ‘meta-signature’ in human cancer, and underscore a number of cell-cycle- and STAT-associated targets that may prove useful as points of therapeutic intervention for those patients with aggressive disease.

Expression and potential role of Fas-associated phosphatase-1 in ovarian cancer.

Fas-associated phosphatase-1 (FAP-1) is a protein-tyrosine phosphatase that binds the cytosolic tail of Fas (Apo1, CD95), presumably regulating Fas-induced apoptosis. Elevations of FAP-1 protein levels in some tumor cell lines have been correlated with resistance to Fas-induced apoptosis. To explore the expression of FAP-1 in ovarian cancer cell lines and archival tumor specimens, mouse monoclonal and rabbit polyclonal antibodies were generated against a FAP-1 peptide and recombinant FAP-1 protein. These antibodies were used for immunoblotting, immunohistochemistry, and flow-cytometry analysis of FAP-1 expression in the Fas-sensitive ovarian cancer lines HEY and BG-1, and in the Fas-resistant lines OVCAR-3 FR and SK-OV-3. All methods demonstrated high levels of FAP-1 in the resistant lines OVCAR-3 FR and SK-OV-3, but not in the Fas-sensitive lines HEY and BG-1. Furthermore, levels of FAP-1 protein also correlated with the amounts of FAP-1 mRNA, as determined by reverse transcriptase-polymerase chain reaction analysis. FAP-1 protein levels were investigated by immunoblotting in the National Cancer Institutes panel of 60 human tumor cell lines. Although FAP-1 failed to correlate with Fas-resistance across the entire tumor panel, Fas-resistance correlated significantly with FAP-1 expression (P: < or = 0.05) and a low Fas/FAP-1 ratio (P: < or = 0.028) in ovarian cancer cell lines. FAP-1 expression was also evaluated in 95 archival ovarian cancer specimens using tissue-microarray technology. FAP-1 was expressed in nearly all tumors, regardless of histological type or grade, stage, patient age, response to chemotherapy, or patient survival. We conclude that FAP-1 correlates significantly with Fas resistance in ovarian cancer cell lines and is commonly expressed in ovarian cancers.

An Integrated Clinical-Genomics Approach Identifies a Candidate Multi-Analyte Blood Test for Serous Ovarian Carcinoma

Purpose: Cancer of the ovary confers the worst prognosis among women with gynecologic malignancies, underscoring the need to develop new biomarkers for detection of early disease, particularly those that can be readily monitored in the blood. Experimental Design: We developed an algorithm to identify secreted proteins encoded among ∼22,500 genes on commercial oligonucleotide arrays and applied it to gene expression profiles of 67 stage I to IV serous papillary carcinomas and 9 crudely enriched normal ovarian tissues, to identify putative diagnostic markers. ELISAs were used to validate increased levels of secreted proteins in patient sera encoded by genes with differentially high expression. Results: We identified 275 genes predicted to encode secreted proteins with increased/decreased expression in ovarian cancers (<0.5- or >2-fold, P < 0.001). The serum levels of four of these proteins (matrix metalloproteinase-7, osteopontin, secretory leukoprotease inhibitor, and kallikrein 10) were significantly elevated in a series of 67 independent patients with serous ovarian carcinomas compared with 67 healthy controls (P < 0.001, Wilcoxon rank sum test). Optimized support vector machine classifiers with as few as two of these markers (osteopontin or kallikrein 10/matrix metalloproteinase-7) in combination with CA-125 yielded sensitivity and specificity values ranging from 96% to 98.7% and 99.7% to 100%, respectively, with the ability to discern early-stage disease from normal, healthy controls. Conclusions: Our data suggest that this assay combination warrants further investigation as a multi-analyte diagnostic test for serous ovarian adenocarcinoma.

Evaluation of methods to detect p53 mutations in ovarian cancer

Objective: The p53 status is increasingly regarded as a marker predictive of response to particular cancer therapies, but for this approach it is self-evident that the p53 status must be determined correctly. Methods: We have tested ovarian cancers with single-strand conformation polymorphism analysis (SSCP), immunohistochemical staining with DO-1 anti-p53 antibody (IHC), and yeast p53 functional assay (FASAY). Results: These techniques commonly used to detect p53 mutations showed important differences in their sensitivity. Of 53 tumors tested with three indirect techniques, 27 (50%), 33 (62%) and 41 (77%) were positive by SSCP, IHC, and FASAY, respectively. In a subset of 32 tumors strongly suspected of containing mutations, 25 (78%), 26 (81%), 29 (91%) and 30 (94%) were positive by SSCP, immunostaining, DNA sequencing and yeast assay, respectively. Conclusions: Under comparable routine conditions, the FASAY reached the highest sensitivity. Since no single technique detected all mutations, we recommend the use of at least two different techniques in situations where the p53 status will affect patient management.

A phase II trial of pemetrexed in combination with carboplatin in patients with recurrent ovarian or primary peritoneal cancer

OBJECTIVE Carboplatin-based combinations are commonly used in platinum-sensitive ovarian cancer (PSOC). Pemetrexed in combination with carboplatin has been shown to be feasible in a phase I study in PSOC. The primary objective of this subsequent phase II study was to determine the overall response rate (ORR; defined by Response Evaluation Criteria in Solid Tumors) of this combination in patients with recurrent PSOC. Secondary objectives included progression-free survival (PFS), overall survival (OS), and toxicity. METHODS Patients with PSOC (defined by recurrence ≥ 6 months after completion of up to two lines of prior platinum-based therapy), measurable disease, Eastern Cooperative Oncology Group performance status of 0 to 2, and adequate organ function were eligible. Pemetrexed 500 mg/m(2) was administered as a 10-minute infusion followed by carboplatin AUC 6 as a 30-minute infusion on day 1 of a 21-day cycle. RESULTS Sixty-six patients were treated. Of the 61 patients evaluable for response, there were 20 responders (one complete response and 19 partial responses), for an ORR of 32.8% (95% CI: 21.3%, 46.0%). For the intent-to-treat population (all 66 patients), the median PFS was 9.4 months (95% CI: 8.3, 11.1), with 22.7% censoring. Median OS was not reached due to the high censoring rate. There was one drug-related death (multi-organ failure). The most common drug-related grade 3/4 toxicities were neutropenia (39.4%), thrombocytopenia (24.2%), carboplatin hypersensitivity (9.1%), nausea (6.1%), and vomiting (6.1%). CONCLUSIONS Carboplatin AUC 6 and pemetrexed 500 mg/m(2) has a low incidence of serious toxicities. Defining the platinum-based combination with the best therapeutic index would require a prospective phase III study.

Randomized, Phase II, Placebo-Controlled, Double-Blind Study With and Without Enzastaurin in Combination With Paclitaxel and Carboplatin As First-Line Treatment Followed by Maintenance Treatment in Advanced Ovarian Cancer

PURPOSE Enzastaurin is an oral serine/threonine kinase inhibitor antitumor agent. Our phase II trial tested the efficacy and safety of enzastaurin added to a standard carboplatin/paclitaxel chemotherapy regimen in patients with newly diagnosed advanced ovarian cancer. PATIENTS AND METHODS This was a randomized, placebo-controlled study in patients with International Federation of Gynecology and Obstetrics stage IIB to IV ovarian, fallopian tube, or peritoneal epithelial carcinoma. Patients were randomly assigned to six cycles of chemotherapy (paclitaxel/carboplatin ± enzastaurin [PCE/PC]) followed by maintenance therapy (enzastaurin/placebo). Primary end point was progression-free survival (PFS). Secondary measures included response rate, safety assessment, and translational research. RESULTS A total of 142 patients were randomly assigned to PCE (n = 69) or PC (n = 73). Patients in the PCE group had a 3.7-month longer median PFS compared with patients in the PC group; this was not statistically significant (hazard ratio [HR], 0.80; 95% CI, 0.50 to 1.29; P = .37). Safety profiles of the treatment arms were comparable. Frequency of discontinuation because of adverse events was similar (PCE, 11.9%; PC, 9.7%). Multivariate analyses confirmed the importance of optimal debulking with regard to PFS (debulking optimal v suboptimal: HR, 0.51; 95% CI, 0.30 to 0.85; P = .009). HR for covariate stage (stage IIB to IIIB v IIIC to IV) was not statistically significant (0.75; 95% CI, 0.38 to 1.47; P = .40). Translational research of immunohistochemistry protein assays did not identify any markers significantly associated with treatment difference regarding PFS. CONCLUSION The PCE combination increased PFS, but it was not significantly superior to PC in this phase II study.

Carboplatin and paclitaxel in combination with oral enzastaurin in advanced ovarian or primary peritoneal cancer: results from a safety lead-in study.

Introduction: This safety lead-in study examined the pharmacokinetic and adverse event profile of combining enzastaurin with paclitaxel plus carboplatin as first-line therapy for the treatment of advanced-stage ovarian cancer and primary peritoneal carcinoma. The specific objectives of this study were to assess safety and tolerability after 2 cycles of treatment, to determine if enzastaurin alters paclitaxel and carboplatin pharmacokinetics, and to determine if enzastaurin pharmacokinetics is affected by paclitaxel and carboplatin. Methods: After debulking surgery, patients with previously untreated epithelial ovarian or primary peritoneal carcinoma received sequential paclitaxel (175 mg/m2) and carboplatin (area under the curve, 5 mg × min/mL) on day 1 every 3 weeks for 6 cycles. Patients ingested an oral loading dose of 1125 mg enzastaurin on day 4 of cycle 1, followed by oral 500-mg enzastaurin daily until the end of therapy. Adverse events were graded according to the Common Terminology Criteria for Adverse Events v3.0. Results: There were 5 serious adverse events in 4 of 11 patients: soft tissue injury, wound infection, intestinal fistula, clostridial infection, and anemia. Coadministration with enzastaurin did not significantly alter paclitaxel and carboplatin pharmacokinetics (area under the curve ratio of treatment comparison ≈ 1.05 and 1.06, respectively). Enzastaurin exposures were unchanged (C av,ss ratio of treatment comparison ≈ 0.95 for average steady-state total analyte concentrations of enzastaurin and its metabolite). Conclusions: Adding enzastaurin to paclitaxel plus carboplatin chemotherapy is feasible for advanced ovarian cancer after radical cytoreduction. Enzastaurin did not alter paclitaxel or carboplatin pharmacokinetics, and enzastaurin exposures were not significantly changed by carboplatin and paclitaxel.

Genetik und Prävention des Ovarialkarzinoms

ZusammenfassungDas Ovarialkarzinom zählt zu den Genitalkarzinomen mit der ungünstigsten Prognose. Keimbahnmutationen der Gene BRCA1 und BRCA2 liegen ursächlich der erblichen Variante des Ovarialkarzinoms zu Grunde. Seine multifaktorielle Genese bedingt unterschiedliche phänotypische Ausprägungen und ist damit nicht nur auf das Ovarialkarzinom beschränkt. Die klinische Bedeutung dieser Syndrome wächst mit der Möglichkeit der prädiktiven Diagnostik der prädisponierenden Gene. Mutationsträgerinnen besitzen ein bis zu 60%iges Risiko für das Auftreten einer Ovarialneoplasie.Die klinische Umsetzung der Identifikation von Hochrisikopatientinnen erfordert die optimale Nutzung der primären Prävention und eine intensivierte Krebsfrüherkennung im Rahmen des von der Deutschen Krebshilfe geförderten Projekts “Familärer Brust- und Eierstockkrebs”.AbstractOvarian cancer has the poorest prognosis of all gynaecological neoplasia. Germline mutations of the genes BRCA1 and BRCA2 are considered to be of substantial relevance in hereditary ovarian cancer. The multifactorial aetiology of this phenomenon accounts for the various phenotypes which are not only relevant in ovarian cancer. The clinical relevance of these syndromes increases with the option of predictive genetic testing for the predisposing genes. The lifetime-risk for ovarian cancer in women with mutations of the BRCA1 and/or BRCA2 genes is increased by upto 60%.The clinical implications of identifying patients at high risk necessitates the optimal use of primary or secondary prevention as proposed by the federally funded “Project for Familiar Breast and Ovarian Cancer”.