Francesca Bizzaro

Tumor Delivery of Chemotherapy Combined with Inhibitors of Angiogenesis and Vascular Targeting Agents

Numerous angiogenesis-vascular targeting agents have been admitted to the ranks of cancer therapeutics; most are used in polytherapy regimens. This review looks at recent progress and our own preclinical experience in combining angiogenesis inhibitors, mainly acting on VEGF/VEGFR pathways, and vascular targeting agents with conventional chemotherapy, discussing the factors that determine the outcome of these treatments. Molecular and morphological modifications of the tumor microenvironment associated with drug distribution and activity are reviewed. Modalities to improve drug delivery and strategies for optimizing combination therapy are examined.

Patient-derived ovarian tumor xenografts recapitulate human clinicopathology and genetic alterations

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. On the basis of its histopathology and molecular-genomic changes, ovarian cancer has been divided into subtypes, each with distinct biology and outcome. The aim of this study was to develop a panel of patient-derived EOC xenografts that recapitulate the molecular and biologic heterogeneity of human ovarian cancer. Thirty-four EOC xenografts were successfully established, either subcutaneously or intraperitoneally, in nude mice. The xenografts were histologically similar to the corresponding patient tumor and comprised all the major ovarian cancer subtypes. After orthotopic transplantation in the bursa of the mouse ovary, they disseminate into the organs of the peritoneal cavity and produce ascites, typical of ovarian cancer. Gene expression analysis and mutation status indicated a high degree of similarity with the original patient and discriminate different subsets of xenografts. They were very responsive, responsive, and resistant to cisplatin, resembling the clinical situation in ovarian cancer. This panel of patient-derived EOC xenografts that recapitulate the recently type I and type II classification serves to study the biology of ovarian cancer, identify tumor-specific molecular markers, and develop novel treatment modalities.

Combination therapy in cancer: effects of angiogenesis inhibitors on drug pharmacokinetics and pharmacodynamics

Validated preclinical studies have provided evidence that anti-vascular endothelial growth factor (VEGF) compounds enhance the activity of subsequent antitumor therapy, but the mechanism of this potentiation is far from clear. The most widespread explanation is enhanced delivery of therapeutics due to vascular remodeling, lower interstitial pressure, and increased blood flow. While the antiangiogenic effects on vascular morphology have been fairly consistent in both preclinical and clinical settings, the improvement of tumor vessel function is debated. This review focuses on the effect of anti-VEGF therapy on tumor microenvironment morphology and functions, and its therapeutic benefits when combined with other therapies. The uptake and spatial distribution of chemotherapeutic agents into the tumor after anti-VEGF are examined.

Expression of thrombospondin-1 by tumor cells in patient-derived ovarian carcinoma xenografts

Abstract Purpose: Thrombospondin-1 (TSP-1), a major regulator of cell interaction with the environment, is often deregulated in cancers, including ovarian carcinoma. Both the tumor and the host cells can release TSP-1 in the tumor microenvironment. The relative contribution of the two sources in determining TSP-1 levels in ovarian cancer remains to be elucidated. This study was designed to investigate the expression of tumor TSP-1 in a panel of 29 patient-derived ovarian adenocarcinoma xenografts (PDX), using analytical tools specific for human (tumor-derived) rather than murine (host-derived) TSP-1. Methodology: Human-specific microarray and ELISA were used to measure tumor TSP-1 expression and plasma levels. Results: Tumor-derived TSP-1 was heterogeneously expressed in PDX. Expression was higher in the corresponding original patients tumor, where stroma-derived TSP-1 is also analyzed, indicating that both the tumor and the host contribute to TSP-1 production. TSP-1 was differentially expressed according to tumor grade, but not affected by p53 expression or mutational status. Findings were confirmed in an external gene expression dataset (101 patients). In a functional enrichment analysis, TSP-1 correlated with genes related to angiogenesis, cell motility, communication and shape. Plasma TSP-1, detectable in 10/11 PDX, was not associated to its expression in the tumor. The possible association of plasma TSP-1 with p53 mutations and response to chemotherapy warrants further investigation. Conclusions: Ovarian carcinoma PDX are a useful tool to investigate the relative contribution of stroma and tumor cells in the production of tumor associated factors, in relation to the tumor behavior, molecular properties and response to therapy.

Abstract 2994: Cediranib affects tumor progression and survival of mice bearing patient derived ovarian carcinoma xenografts (EOC-PDX)

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Vascular Endothelial Growth Factor (VEGFA/VEGFC) are over-expressed and secreted in large amount in epithelial ovarian cancer (EOC) (Maneneti et al, 2013; Decio et al, 2013). Cediranib, a potent inhibitor of VEGF receptor tyrosine kinases has recently shown to improve PFS and OS in women with recurrent platinum sensitive ovarian cancer (ICON6 trial). The aim of this study was to investigate the efficacy of cediranib (AZD2171; AstraZeneca, Alderley Park, Macclesfield, UK) on a platform of patient derived human ovarian carcinoma xenografts (EOC-PDX; n=12), which recapitulates the histopathological and fundamental genetic diversity of the patient tumors and the pharmacological heterogeneity of this type of cancer. The efficacy of cediranib alone and in combination with the-standard-of-care chemotherapy for ovarian cancer (platinum, DDP and paclitaxel, PTX) was investigated on EOC-PDX growing orthotopically and disseminating in the peritoneal cavity of nude mice. Tumor progression (tumor dissemination; soluble VEGFR) and overall survival were investigated. The response of EOC-PDX to cediranib was heterogeneous with some tumors being extremely sensitive, other marginally responsive to the treatment (ILS from 12% to 85%); the length of response depended on the duration of the treatment. Cediranib was significantly active also on advanced stage tumor. Cediranib added to DDP based therapy improved the response to chemotherapy treatment without increase of toxicity; the sensitivity of the EOC-PDX to DDP (platinum sensitive vs. platinum resistant) determined the final outcome. The histopathological analysis at the end of treatment (interim) shows that the combination affected the spread of solid tumor into the peritoneal cavity and ascites formation. The combination of cediranib with combined chemotherapy (DDP/PTX) was studied on one EOC-PDX platinum responsive. The addition of cediranib to DDP/PTX marginally increased the survival of the mice compared to chemotherapy; cediranib continued after chemotherapy (maintenance regimen for 60 days) significantly improved the survival of the mice, with 70% tumor free at the end of the treatment. These results show that the response to cediranib of EOC-PDX reflects the behavior in patients. The heterogeneous response of EOC-PDX to cediranib warrants further studies for better understanding the mechanism of response /resistance to this type of treatment. AD is a fellow of the Italian Foundation for Cancer Research (FIRC). Citation Format: Alessandra Decio, Marta Cesca, Francesca Bizzaro, Dorina Belotti, Raffaella Giavazzi. Cediranib affects tumor progression and survival of mice bearing patient derived ovarian carcinoma xenografts (EOC-PDX). [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 2994. doi:10.1158/1538-7445.AM2014-2994

Platinum sensitivity and DNA repair in a recently established panel of patient-derived ovarian carcinoma xenografts

A xenobank of patient-derived (PDX) ovarian tumor samples has been established consisting of tumors with different sensitivity to cisplatin (DDP), from very responsive to resistant. As the DNA repair pathway is an important driver in tumor response to DDP, we analyzed the mRNA expression of 20 genes involved in the nucleotide excision repair, fanconi anemia, homologous recombination, base excision repair, mismatch repair and translesion repair pathways and the methylation patterns of some of these genes. We also investigated the correlation with the response to platinum-based therapy. The mRNA levels of the selected genes were evaluated by Real Time-PCR (RT-PCR) with ad hoc validated primers and gene promoter methylation by pyrosequencing. All the DNA repair genes were variably expressed in all 42 PDX samples analyzed, with no particular histotype-specific pattern of expression. In high-grade serous/endometrioid PDXs, the CDK12 mRNA expression levels positively correlated with the expression of TP53BP1, PALB2, XPF and POLB. High-grade serous/endometrioid PDXs with TP53 mutations had significantly higher levels of POLQ, FANCD2, RAD51 and POLB than high-grade TP53 wild type PDXs. The mRNA levels of CDK12, PALB2 and XPF inversely associated with the in vivo DDP antitumor activity; higher CDK12 mRNA levels were associated with a higher recurrence rate in ovarian patients with low residual tumor. These data support the important role of CDK12 in the response to a platinum based therapy in ovarian patients.

Tumor progression and metastatic dissemination in ovarian cancer after dose-dense or conventional paclitaxel and cisplatin plus bevacizumab

The efficacy of therapeutic regimens incorporating weekly or every‐3‐weeks paclitaxel (PTX) for ovarian cancer is debated. We investigated the addition of bevacizumab in regimens of chemotherapy with different PTX doses and schedules in preclinical models. Treatments were cisplatin (DDP) with weekly PTX (conventional), or dose‐dense‐equi (every other day to the conventional cumulative dose), or dose‐dense‐high (total dose 1.5 times higher), with or without bevacizumab. Treatment efficacy was evaluated analyzing tumor growth in different time‐windows in two patient‐derived ovarian cancer xenografts with different sensitivity to cisplatin. Tumor progression, metastasis and survival were studied in ovarian cancer models growing orthotopically and disseminating in the mouse peritoneal cavity. Short‐term effects on cell cycle, tumor cell proliferation/apoptosis and vasculature were evaluated by flow cytometry and immunohistochemistry. PTX dose‐dense (with/without DDP) was superior to the conventional scheme in a dose‐dependent manner; the high efficacy was confirmed by the lower ratio of tumor to normal cells. All schemes benefited from bevacizumab, which reduced tumor vessels. However, DDP/PTX dose‐dense‐high (only chemotherapy) was at least as active as DDP/PTX conventional plus bevacizumab. DDP/PTX dose‐dense‐high plus bevacizumab was the most effective in delaying tumor progression, though it did not prolong mouse survival and the continuous treatment with bevacizumab was associated with a malignant disease. These findings indicate that the effect of bevacizumab in combination with chemotherapy may depend on the schedule‐dose of the treatment and help to explain the unclear benefits after bevacizumab.

Abstract 2816: Patient derived ovarian cancer xenograft (OC-PDX) to study the response of the PARP inhibitor olaparib

Up to 50% of High Grade Serous (HGS) ovarian cancer patients exhibit homologous recombination deficiency (HRD) through various mechanisms including germline and somatic mutations in BRCA-1 or 2. Olaparib, a poly(ADP-ribose)polymerase (PARP) inhibitor, was recently approved for the treatment of patients with germline BRCA-mutated-advanced ovarian cancer. We have analyzed our panel of patient derived ovarian cancer xenografts (OC-PDX) 1 to investigate the association of the somatic BRCA1/2 mutational status with the response to olaparib. Whole Exome Sequencing (Illumina HiSeq4000) was performed on a panel (n=26) of OC-PDX to identify mutations in BRCA1/2. Next Generation Sequencing (NGS) results were confirmed by Sanger sequencing on tumor DNA and RNA. Thirteen OC-PDX were selected and treated with 100mg/kg olaparib for 4 weeks or as a maintenance regimen until progression. Cisplatin was used as reference drug. Efficacy was evaluated as the best T/C% (best growth inhibition) for subcutaneous (s.c) tumors. To mimick patient disease, OC-PDX were also established as intra-peritoneal models (i.p.) and best ILS% (best increment of lifespan) calculated. Response to olaparib varied among the models with distinct responsive and non-responsive groups associated with BRCA mutational status. OC-PDX with a homozygous frameshift mutation in BRCA1/2 (n=5), loss of BRCA1 (n=1) or no expression of BRCA1/2 (n=1) showed a sustained response to olaparib after 4 week treatment (T/C values from 2% to 40%; ILS of one representative OC-PDX = 74%), with complete responses following a longer term maintenance treatment. One BRCA1 mutated OC-PDX model was resistant, progressing rapidly under treatment. In this orthotopic model ILS% and tumor dissemination score of treated mice were similar to vehicle. Models responsive to olaparib were in general also sensitive to cisplatin. BRCA1/2 wild type OC-PDX models (n=4 s.c and n=1 i.p) did not respond to olaparib, even following a longer term treatment (T/C from 41% to 86%; ILS = 5%). NGS revealed a heterozygous mutation in BRCA2 in one OC-PDX. Sanger sequencing of the RNA confirmed the presence of both the wild type and the mutated BRCA2 transcripts. This OC- PDX was poorly responsive to olaparib (T/C = 57%). Our data showed that in general tumors with a homozygous mutation, loss or no expression of BRCA1/2 responded to olaparib; the drug was not active on those with mutation in heterozygosis (BRCA2 +/- ) or wild type. These findings indicate that tumor somatic mutations play a role in the response to olaparib. Models of OC-PDX moderately responsive to olaparib offer the opportunity to assess the potential of combination treatments. 1 Ricci, F. et al. Patient-derived ovarian tumor xenografts recapitulate human clinicopathology and genetic alterations. Cancer Res 74, 6980–90 (2014) Citation Format: Francesca Bizzaro, Alessia C. Marchetti, Alessandra Decio, Francesca Ricci, Mark J. O9Connor, Molly A. Taylor, Zhongwu Lai, Simon T. Barry, Maria R. Bani, Raffaella Giavazzi. Patient derived ovarian cancer xenograft (OC-PDX) to study the response of the PARP inhibitor olaparib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2816.

Abstract A40: Profile of DNA repair status in a recently established panel of patient-derived ovarian carcinoma xenografts

Background and Purpose: A xenobank of patient derived (PDX) ovarian tumor samples has recently been established and shown to recapitulate the biological, histological, molecular and pharmacological features of human epithelial ovarian cancer (EOC). This EOC-xenobank consists of tumors with different sensitivity to cisplatin (DDP) from very responsive, responsive to resistant tumors. As DNA repair pathway represents an important driver in tumor response to DDP, we analyzed the mRNA expression of 20 genes involved in Nucleotide Excision Repair (NER), in Fanconi Anemia (FA), in Homologous Recombination (HR), in Base Excision Repair (BER), in Mismatch Repair (MMR) and Translesion Repair (TLR) pathways and the methylation pattern of some of these genes. The correlation with the response to a platinum-based therapy was also investigated. Methods: The genes selected have a key role in the BER (OGG1 and PARP1); in the NER pathway (ERCC1, XPA, XPF, XPD and XPG); the FA pathway (BRCA1, FANCA, FANCC, FANCD2 and FANCF); in TLR (PolEta), in MMR (MLH1), in MMEJ (PolQ) and in the transcription of some genes (CDK12). The mRNA levels were evaluated by RT-PCR with ad hoc validated primers. The promoter methylation of BRCA1, XPA, ERCC1 and MLH1 was studied by pyrosequencing, while for XPG and FANCF no predesigned assays were available and we evaluated their methylation status following reported assays. Results: We demonstrated: i) all the DNA genes considered were variably expressed in all the 44 PDX analyzed, with no specific istotype-specific cluster of expression; ii) in high grade seros/endometriod PDXs, the CDK12 mRNA expression levels positively correlated with the expression of TP53BP1, PALB2, XPF and PolB; iii) high grade serous/endometriod PDXs with TP53 mutation had statistically significant higher levels of PolQ, FANC-D2, RAD51 and Pol beta than high grade TP53 wild type PDXs; iv) BRCA1 was found to be hypermethylated in 48% of the xenografts. However, no correlation between mRNA levels and methylation status of the considered CpG islands was found; v) only the mRNA levels of CDK12, PALB2 and XPF inversely correlated with the in vivo DDP antitumor activity. Conclusions: These data suggest the mRNA expression levels of targeted DNA repair pathway-related genes as PALB2, XPF and CDK12 predict the response to a platinum based therapy in ovarian cancer, even if they need to be prospectively validated in cohort of ovarian cancer patients. Citation Format: Federica Guffanti, Maddalena Fratelli, Monica Ganzinelli, Francesca Ricci, Maria Rosa Cappelletti, Damiele Generali, Francesca Bizzaro, Raffaella Giavazzi, Giovanna Damia. Profile of DNA repair status in a recently established panel of patient-derived ovarian carcinoma xenografts [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A40.

Abstract 508: DNA repair status in a patient derived ovarian cancer xenobank

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy with a 5-year relative survival rate of 45%. The high mortality rate is in part due to the development of platinum chemoresistance occurring in more than 70% of patients after the first-line therapy. DNA repair capacity has been reported to be a key determinant for the cellular response to platinum agents. Since half of the high grade serous EOCs lacks Homologous Recombination repair, we aimed to profile the DNA repair status in a panel of well characterized 42 ovarian patient derived xenografts (PDXs) recently established in our laboratory and to correlate it with the in vivo response to a platinum based therapy. We evaluated by real time PCR (ABI-7900, Applied Biosystems) the mRNA levels of genes with a key role in Base Excision Repair (OGG1, POLB and PARP1), Homologous Recombination (BRCA1, PALB2, TP53BP1 and RAD51), Nucleotide Excision Repair (ERCC1, XPA, XPF, XPD and XPG), Fanconi Anemia pathway (FANCA, FANCC, FANCD2 and FANCF), Translesion Repair (POLEta), Mismatch Repair (MLH1), Microhomology End Joining (POLQ), Non Homologous End Joining (XRCC4, XRCC5, XRCC6 and XRCC7) and CDK12, a kinase regulating the transcription of some DNA repair genes. The methylation status of BRCA1, ERCC1, MLH1, XPA, XPG and FANCF was investigated by standard techniques. Our results show that the DNA repair genes considered were variably expressed in all the 42 PDXs analyzed, with no specific histotype-specific cluster of expression. The expression of PALB2, FANCC, FANCD2, OGG1, POLQ and RAD51 was found to correlate with the expression of at least six other genes. In high grade serous/endometrioid PDXs, the CDK12 mRNA expression levels positively correlated with the expression of TP53BP1, PALB2, XPF and POLB. BRCA1 was found to be hypermethylated in 51% of the xenografts. TP53 mutated PDXs showed statistically significant higher levels of POLQ, FANCD2, RAD51, and POLB genes. The expression of CDK12 [p=0.017], PALB2 [p=0.019] and XPF [p= 0.016] was negatively associated with the in vivo response to DDP, with resistant PDXs showing higher mRNA levels than responsive ones. We looked for association with overall survival in the TCGA data set and we found that high levels of CDK12 were associated with a worse overall survival in patients with a residual tumor after surgery minus than 2cm. These data suggest that some DNA repair genes can have a role in EOC patients’ response to DDP therapy. Particularly, CDK12 was significantly able to predict worse survival in patients undergoing optimal debulking surgery. Our xenobank will be a valid instrument to set up functional DNA repair assays, as suggested by preliminary data on primary cultures. Citation Format: Federica Guffanti, Maddalena Fratelli, Monica Ganzinelli, Francesca Ricci, Roberta Affatato, Maria Rosa Cappelletti, Daniele Generali, Francesca Bizzaro, Massimo Broggini, Raffaella Giavazzi, Giovanna Damia. DNA repair status in a patient derived ovarian cancer xenobank [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 508. doi:10.1158/1538-7445.AM2017-508