Winterhoff, Boris

Abstract Purpose: Recent progress in understanding the molecular biology of epithelial ovarian cancer has not yet translated into individualized treatment for these women or improvements in their disease outcome. Gene expression has been utilized to identify distinct molecular subtypes, but there have been no reports investigating whether or not molecular subtyping is predictive of response to bevacizumab in ovarian cancer. Experimental Design: DASL gene expression arrays were performed on FFPE tissue from patients enrolled on the ICON7 trial. Patients were stratified into four TCGA molecular subtypes. Associations between molecular subtype and the efficacy of randomly assigned therapy with bevacizumab were assessed. Results: Molecular subtypes were assigned as follows: 122 immunoreactive (34%), 96 proliferative (27%), 73 differentiated (20%), and 68 mesenchymal (19%). In univariate analysis patients with tumors of proliferative subtype obtained the greatest benefit from bevacizumab with a median PFS improvement of 10.1 months [HR, 0.55 (95% CI, 0.34–0.90), P = 0.016]. For the mesenchymal subtype, bevacizumab conferred a nonsignificant improvement in PFS of 8.2 months [HR 0.78 (95% CI, 0.44–1.40), P = 0.41]. Bevacizumab conferred modest improvements in PFS for patients with immunoreactive subtype (3.8 months; P = 0.08) or differentiated subtype (3.7 months; P = 0.61). Multivariate analysis demonstrated significant PFS improvement in proliferative subtype patients only [HR, 0.45 (95% CI, 0.27–0.74), P = 0.0015]. Conclusions: Ovarian carcinoma molecular subtypes with the poorest survival (proliferative and mesenchymal) derive a comparably greater benefit from treatment that includes bevacizumab. Validation of our findings in an independent cohort could enable the use of bevacizumab for those patients most likely to benefit, thereby reducing side effects and healthcare cost. Clin Cancer Res; 23(14); 3794–801. ©2017 AACR.

Abstract Background: The heparan sulfate mimetic PG545 has been demonstrated to inhibit the function of vascular endothelial growth factor (VEGF) and heparanase (HPSE) in vitro whilst downregulating HPSE expression in tumor and metastatic tissue in vivo. Although there are reports of consistent drug-induced increases in plasma VEGF with VEGF inhibitors such as bevacizumab, similar data are lacking for HS mimetics. Given PG545's mechanism of action which involves inhibition of both angiogenesis and metastasis, the evaluation of plasma VEGF and HPSE may represent pharmacodynamic (PD) biomarkers for each of these key processes. Materials and Methods: The levels of plasma VEGF and HPSE were measured using ELISA in samples from preclinical models of ovarian cancer (syngeneic ID8 and xenogeneic A2780 models) and a small cohort of patients (dosed with PG545 by the subcutaneous route) with advanced solid tumors from a Phase I clinical study. In tumor models, PG545 was administered once weekly by intravenous or intraperitoneal routes and efficacy was determined by quantifying tumor burden using the following methods: measurement of palpable solid tumors, abdominal circumference, ascites volume and/or by tracking cells using bioluminescent reporter imaging (depending on the model). In the A2780 model and the clinical samples, the pharmacokinetics of PG545 was also evaluated using LC-MS/MS, which ensured multiple samples to facilitate the measurement of VEGF and HPSE in plasma over the duration of the dosing interval following treatment with PG545. Results: PG545 demonstrated potent antitumor and antimetastatic activity when administered once weekly via intravenous or intraperitoneal routes in preclinical studies. These studies also indicated that PG545 treatment led to increases in plasma VEGF and HPSE. Importantly, plasma samples from advanced cancer patients treated with PG545 also revealed increases in these PD markers with initial peaks often correlating with Cmax of PG545 but longer-term gradual increases also noted in some instances. We consider that these increases in plasma levels result from PG545 binding to, thereby inhibiting, VEGF and HPSE in the tumor microenvironment. Conclusion: Given the questionable utility of single time point measures of plasma PD markers (including VEGF), these preliminary data suggest that kinetic profiling can reveal the previously undetermined dynamics of circulating VEGF and HPSE following treatment with PG545. Thus, the measurement of VEGF and HPSE appears to represent potential markers of pharmacodynamic activity. A new Phase I trial with intravenously-administered PG545 has recently commenced and should further clarify the potential utility of these basic PD biomarker responses in advanced cancer patients. Citation Format: Keith Dredge, Edward Hammond, Boris Winterhoff, Shailendra Giri, Attila Teoman, Darryn Bampton, Michael Millward, Viji Shridhar. The utility of plasma vascular endothelial growth factor and heparanase as pharmacodynamic markers following treatment with PG545, a heparan sulfate mimetic: preliminary evidence from preclinical and clinical settings. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3695. doi:10.1158/1538-7445.AM2014-3695

Abstract Background: Mucinous ovarian carcinomas (MC) and mucinous borderline ovarian tumours (MBOT) are associated with ERBB2 amplification and KRAS activating mutations. These events occur in a near-mutually exclusive pattern, and KRAS mutations are more prevalent in MBOTs than MC's whereas ERBB2 amplification is more frequently seen in MCs. Frequencies of mutations in other oncogenes and tumour suppressor genes in MC are not well established though TP53, BRAF, NRAS, and CDKN2A mutations have all been reported. As these carcinomas are commonly chemo-resistant a definitive molecular categorization of this subtype is needed as treatment options are explored. Methods: We undertook review of pathology reports and after exclusion of potential gastric, appendiceal or pancreatic involvement over 100 mucinous ovarian tumours were identified. HPV testing was undertaken to exclude rare metastasis from endocervical primary site. All specimens were assayed for p53 expression via TMA and subject to sequencing of common cancer gene hotspots using Ion-Torrent or Illumina MiSeq. Previously derived data on ERBB2 status was combined in our analysis and, where available, samples showing heterogeneity of ERBB2 amplification and any other Ras-pathway activating mutation were microdissected with components evaluated independently for concurrent hotspot mutations. Results: All samples were consistent with mucinous tumours of ovarian origin and none were HPV positive. We detected mutations in KRAS, BRAF, CDKN2A, TP53, PTEN, PIK3CA as well as presumed somatic/detrimental, variants in APC that have not previously been reported in mucinous tumours. Despite some samples having enriched tumour content, lower than expected allelic frequencies of KRAS activating mutations suggested intra-tumoural heterogeneity. This was consistent with results observed previously with rare ERBB2 amplification coinciding KRAS mutations. Conclusions: The prevalence of Ras-pathway mutations, especially amongst borderline lesions, suggests these mutations are critical for tumour onset. However varying allelic frequencies of KRAS mutations suggest progression to carcinoma is strongly influenced by other molecular features. Citation Format: Michael S. Anglesio, Robertson Mackenzie, Stefan Kommoss, Boris J. Winterhoff, Benjamin Kipp, Jaoquin Garcia, Jesse S. Voss, Kevin Halling, Sarah Kerr, Janine Senz, Winnie Yang, Magnus von Knebel Doeberitz, Elena-Sophie Prigge, Miriam Reuschenbach, Anna V. Tinker, Blake Gilks, Jamie N. Bakkum-Gamez, David G. Huntsman, Jessica N. McAlpine. Defining ovarian mucinous tumors: Cancer genes and heterogeneity. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A18.

Abstract To reveal biologic mechanisms underlying clinical outcome of high-grade serous (HGS) epithelial ovarian carcinomas (EOC), we evaluated the association between tumor epigenetic changes and time to recurrence (TTR). We assessed methylation at approximately 450,000 genome-wide CpGs in tumors of 337 Mayo Clinic (Rochester, MN) patients. Semi-supervised clustering of discovery (n = 168) and validation (n = 169) sets was used to determine clinically relevant methylation classes. Clustering identified two methylation classes based on 60 informative CpGs, which differed in TTR in the validation set [R vs. L class, P = 2.9 × 10−3, HR = 0.52; 95% confidence interval (CI), 0.34–0.80]. Follow-up analyses considered genome-wide tumor mRNA expression (n = 104) and CD8 T-cell infiltration (n = 89) in patient subsets. Hypomethylation of CpGs located in 6p21.3 in the R class associated with cis upregulation of genes enriched in immune response processes (TAP1, PSMB8, PSMB9, HLA-DQB1, HLA-DQB2, HLA-DMA, and HLA-DOA), increased CD8 T-cell tumor infiltration (P = 7.6 × 10−5), and trans-regulation of genes in immune-related pathways (P = 1.6 × 10−32). This is the most comprehensive assessment of clinical outcomes with regard to epithelial ovarian carcinoma tumor methylation to date. Collectively, these results suggest that an epigenetically mediated immune response is a predictor of recurrence and, possibly, treatment response for HGS EOC. Cancer Res; 74(11); 3084–91. ©2014 AACR.

Abstract Here, we investigate the potential role of the PARP inhibitor rucaparib (CO-338, formerly known as AG014699 and PF-01367338) for the treatment of sporadic ovarian cancer. We studied the growth inhibitory effects of rucaparib in a panel of 39 ovarian cancer cell lines that were each characterized for mutation and methylation status of BRCA1/2, baseline gene expression signatures, copy number variations of selected genes, PTEN status, and sensitivity to platinum-based chemotherapy. To study interactions with chemotherapy, we used multiple drug effect analyses and assessed apoptosis, DNA fragmentation, and γH2AX formation. Concentration-dependent antiproliferative effects of rucaparib were seen in 26 of 39 (67%) cell lines and were not restricted to cell lines with BRCA1/2 mutations. Low expression of other genes involved in homologous repair (e.g., BCCIP, BRCC3, ATM, RAD51L1), amplification of AURKA or EMSY, and response to platinum-based chemotherapy was associated with sensitivity to rucaparib. Drug interactions with rucaparib were synergistic for topotecan, synergistic, or additive for carboplatin, doxorubicin or paclitaxel, and additive for gemcitabine. Synergy was most pronounced when rucaparib was combined with topotecan, which resulted in enhanced apoptosis, DNA fragmentation, and γH2AX formation. Importantly, rucaparib potentiated chemotherapy independent of its activity as a single agent. PARP inhibition may be a useful therapeutic strategy for a wider range of ovarian cancers bearing deficiencies in the homologous recombination pathway other than just BRCA1/2 mutations. These results support further clinical evaluation of rucaparib either as a single agent or as an adjunct to chemotherapy for the treatment of sporadic ovarian cancer. Mol Cancer Ther; 12(6); 1002–15. ©2013 AACR.

Abstract The recent identification of activating fibroblast growth factor receptor 2 (FGFR2) mutations in endometrial cancer has generated an opportunity for a novel target-based therapy. Here, we explore the therapeutic potential of 2 FGFR inhibitors, the multikinase inhibitor dovitinib (TKI258) and the more selective FGFR inhibitor NVP-BGJ398 for the treatment of endometrial cancer. We examined the effects of both inhibitors on tumor cell growth, FGFR2 signaling, cell cycle, and apoptosis using a panel of 20 molecularly characterized human endometrial cancer cell lines. Anchorage-independent growth was studied using soft agar assays. In vivo studies were conducted using endometrial cancer xenograft models. Cell lines with activating FGFR2 mutations (S252W, N550K) were more sensitive to dovitinib or NVP-BGJ398 when compared with their FGFR2 wild-type counterparts (P = 0.073 and P = 0.021, respectively). Both agents inhibited FGFR2 signaling, induced cell-cycle arrest, and significantly increased apoptosis in FGFR2-mutant lines. In vitro, dovitinib and NVP-BGJ398 were both potent at inhibiting cell growth of FGFR2-mutant endometrial cancer cells, but the activity of dovitinib was less restricted to FGFR2-mutant lines when compared with NVP-BGJ398. In vivo, dovitinib and NVP-BGJ398 significantly inhibited the growth of FGFR2-mutated endometrial cancer xenograft models. In addition, dovitinib showed significant antitumor activity in FGFR2 wild-type endometrial cancer xenograft models including complete tumor regressions in a long-term in vivo study. Dovitinib and NVP-BGJ398 warrant further clinical evaluation in patients with FGFR2-mutated endometrial cancer. Dovitinib may have antitumor activity in endometrial cancer beyond FGFR2-mutated cases and may permit greater flexibility in patient selection. Mol Cancer Ther; 12(5); 632–42. ©2013 AACR.

AbstractMucinous ovarian carcinomas (MCs) typically do not respond to current conventional therapy. We have previously demonstrated amplification of HER2 in 6 of 33 (18.2%) mucinous ovarian carcinomas (MCs) and presented anecdotal evidence of response with HER2‐targeted treatment in a small series of women with recurrent HER2‐amplified (HER2+) MC. Here, we explore HER2 amplification and KRAS mutation status in an independent cohort of 189 MCs and 199 mucinous borderline ovarian tumours (MBOTs) and their association to clinicopathological features. HER2 status was assessed by immunohistochemistry (IHC), FISH, and CISH, and interpreted per ASCO/CAP guidelines, with intratumoural heterogeneity assessment on full sections, where available. KRAS mutation testing was performed with Sanger sequencing. Stage and grade were associated with recurrence on both univariate and multivariate analysis (p < 0.001). Assessment of HER2 status revealed overexpression/amplification of HER2 in 29/154 (18.8%) MCs and 11/176 (6.2%) MBOTs. There was excellent agreement between IHC, FISH, and CISH assessment of HER2 status (perfect concordance of HER2 0 or 1+ IHC with non‐amplified status, and 3+ IHC with amplified status). KRAS mutations were seen in 31/71 (43.6%) MCs and 26/33 (78.8%) MBOTs, and were near mutually exclusive of HER2 amplification. In the 189 MC cases, a total of 54 recurrences and 59 deaths (53 of progressive disease) were observed. Within MCs, either HER2 amplification/overexpression or KRAS mutation was associated with decreased likelihood of disease recurrence (p = 0.019) or death (p = 0.0041) when compared to cases with neither feature. Intratumoural heterogeneity was noted in 26% of HER2‐overexpressing cases. These data support the stratification of MCs for the testing of new treatments, with HER2‐targeted therapy as a viable option for HER2+ advanced or recurrent disease. Further research is required to delineate the molecular and clinical features of the ∼34% of MC cases with neither HER2 amplification nor KRAS mutations.

Abstract PG545 is a synthetic heparan sulfate (HS) mimetic purported to inhibit angiogenesis via the blockade of VEGF and FGF-2 signaling, and block the enzymatic activity of heparanase - a molecule closely associated with metastatic dissemination. The attenuation of VEGF and FGF-2 signaling is supported by recent clinical data from the first four advanced cancer patients with multiple tumor types treated with PG545 which exhibited increases in plasma VEGF and FGF-2 levels, often in close association with the drug treatments/exposure profile during the first treatment cycle over 4 weeks. Unfortunately, due to local injection site reactions associated with subcutaneous (SC) administration, the trial was recently terminated. However, to support a re-entry to the clinic using the intravenous route, we tested PG545 in a preclinical model of ovarian cancer.The rationale to investigate PG545 in ovarian cancer is derived from recently generated data which found PG545 (5 µM) almost completely inhibited migration of SKOV3 ovarian carcinoma cells stimulated by each of the following HS binding growth factors: VEGF, HGF, FGF-2, SDF-1 and HB-EGF. Because HB-EGF is thought to play a major role in the spread of this cancer, PG545 was also tested against HB-EGF dependant SKOV3 cell invasion and was found to be a potent inhibitor of this process. The effect of PG545 on HB-EGF signaling pathways in these experiments were investigated and the results indicated that PG545 reduces activation of the HB-EGF receptor (EGFR) and, consequently, both AKTand MAPK signaling are also reduced. Given the strong mechanism of action data for ovarian carcinoma, PG545 was next tested in the A2780 ovarian xenograft model. The signficant antitumor activity using PG545 at 7.5 mg/kg twice weekly intravenous (IV) and 15 mg/kg once weekly IV was similar to a 20 mg/kg once weekly SC dose. Pharmacokinetic analysis confirmed that drug exposure (AUC) over the course of a week was consistent between groups. PG545 significantly reduced tumor burden in a cisplatin resistant ovarian tumor model (C200) when given intraperitoneally at the dose of 20mg/kg, once a week until the end of the study. Most importantly, PG545 completely reduced tumor mass and reduced ascites accumulation in a syngeneic immunocompetent mouse model of ovarian cancer (ID8). Given the known effects of PG545 on heparanase and in metastatic models, these emerging data on growth factors in the clinic and HB-EGF in preclinical models provide a strong rationale to re-enter the clinic using the IV route and to focus on ovarian cancer as the clinical indication. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-309. doi:1538-7445.AM2012-LB-309

Abstract Background: Endometrial carcinoma is the most common gynecological malignancy in the western world. Activating mutations of the fibroblast growth factor receptor 2 (FGFR2) have been described in 12-16% of endometrial cancers. Moreover, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) and their receptors are involved in the neovascularization, invasiveness, and metastatic potential of endometrial cancer. Therefore, suppression of FGFR/VEGFR/PDGFR signaling by TKI258 may represent a novel approach for treatment of endometrial cancer. Experimental Design: The aims of this study were 1.) to assess the effect of TKI258 on tumor cell growth in two-dimensional (2D) and three-dimensional (3D) culture assays using a panel of 20 human endometrial cancer cell lines, 2.) to identify candidate molecular markers predicting sensitivity using baseline gene expression profiling and mutational analyses, and 3.) to determine the in vivo antineoplastic activity of TKI258 in endometrial cancer xenograft models. In addition, we screened 200 fresh frozen endometrial cancer specimens to comprehensively assess the distribution pattern of PI3K, PTEN, and FGFR mutations in endometrial cancer. Results: Concentration-dependent anti-proliferative effects of TKI285 using 2D assays were seen in all endometrial cancer cell lines tested, but varied significantly between individual cell lines (IC50 range: 0.42µM – 3.06 µM). The three most sensitive endometrial cancer cell lines demonstrated activating FGFR2 mutations (MFE296: N549K, IC50 0.42µM; AN3CA: N549K and K310R, IC50 0.50µM; MFE280: S252W, IC50 0.66µM). Assessment of TKI258 responses in 3D assays demonstrated similar results to those observed in 2D culture assays in that the 3 cell lines harboring FGFR2 mutations were the most sensitive to TKI258 achieving 100% growth inhibition at a concentration of 1µM. AN3CA and MFE296 endometrial cancer cells formed xenografts in nude mice and antitumor activity was studied in both models. Inhibition of p-FGFR, p-PDGFR, p-VEGFR-2, pAKT and p-ERK1/2 were observed. Comprehensive data on the pattern of PI3K, PTEN and FGFR mutations in endometrial cancer will be provided. Conclusion: TKI258 demonstrates significant antitumor activity in endometrial cancer cells with FGFR2 mutations. This study provides a strong rationale for the clinical evaluation of TKI258 in patients with endometrial cancer, specifically in those harboring FGFR2 mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3589. doi:10.1158/1538-7445.AM2011-3589