Model Of High-grade Serous Carcinoma Research Articles

Targeted Microchip Capillary Electrophoresis-Orbitrap Mass Spectrometry Metabolomics to Monitor Ovarian Cancer Progression.

The lack of effective screening strategies for high-grade serous carcinoma (HGSC), a subtype of ovarian cancer (OC) responsible for 70–80% of OC related deaths, emphasizes the need for new diagnostic markers and a better understanding of disease pathogenesis. Capillary electrophoresis (CE) coupled with high-resolution mass spectrometry (HRMS) offers high selectivity and sensitivity for ionic compounds, thereby enhancing biomarker discovery. Recent advances in CE-MS include small, chip-based CE systems coupled with nanoelectrospray ionization (nanoESI) to provide rapid, high-resolution analysis of biological specimens. Here, we describe the development of a targeted microchip (µ) CE-HRMS method, with an acquisition time of only 3 min and sample injection volume of 4nL, to analyze 40 target metabolites in serum samples from a triple-mutant (TKO) mouse model of HGSC. Extracted ion electropherograms showed sharp, baseline resolved peak shapes, even for structural isomers such as leucine and isoleucine. All calibration curves of the analytes maintained good linearity with an average R2 of 0.994, while detection limits were in the nM range. Thirty metabolites were detected in mouse serum with recoveries ranging from 78 to 120%, indicating minimal ionization suppression and good accuracy. We applied the µCE-HRMS method to biweekly-collected serum samples from TKO and TKO control mice. A time-resolved analysis revealed characteristic temporal trends for amino acids, nucleosides, and amino acid derivatives. These metabolic alterations are indicative of altered nucleotide biosynthesis and amino acid metabolism in HGSC development and progression. A comparison of the µCE-HRMS dataset with non-targeted ultra-high performance liquid chromatography (UHPLC)–MS results showed identical temporal trends for the five metabolites detected with both platforms, indicating the µCE-HRMS method performed satisfactorily in terms of capturing metabolic reprogramming due to HGSC progression while reducing the total data collection time three-fold.

Aging accelerates while multiparity delays tumorigenesis in mouse models of high-grade serous carcinoma

ObjectivesThe “incessant ovulation” hypothesis links increased risk for tubo-ovarian high-grade serous carcinoma (HGSC) due to more ovulations and reduced risk conferred by pre-menopausal exposures like oral contraceptive use, multiparity, and breastfeeding. However, most women diagnosed with HGSC are postmenopausal, implying age is a major risk factor for HGSC. Our mouse model for HGSC, based on tamoxifen (TAM)-induced somatic inactivation of the Brca1, Trp53, Rb1, and Nf1 (BPRN) tumor suppressor genes in oviductal epithelium, recapitulates key genetic, histopathologic, and biological features of human HGSCs. We aimed to credential the model for future efforts to define biological and risk modification factors in HGSC pathogenesis. MethodsBPRN mice were treated with TAM to induce tumors at defined ages and parity status. ResultsBPRN mice aged 9-months prior to tumor induction had markedly shorter survival than 6–8 week old mice induced to form tumors (median 46.5 weeks versus 61.5 weeks, log-rank test P = 0.0006). No significant differences in cancer phenotypes were observed between multiparous versus nulliparous BPRN mice. However, using a modified tumor model with one wild-type Nf1 allele (BPRNfl/+), nulliparous mice had more advanced tumors than multiparous mice (Mantel-Haenszel Chi-square test of association, P = 0.01). ConclusionsOur findings show aging is associated with significantly shortened survival post tumor induction in the BRPN model and multiparity delays development and/or progression of HGSC in certain genetic contexts. The findings support relevance of our mouse model to gain mechanistic insights into how known factors exert their protective effects and to test novel approaches for HGSC prevention.

Altering the Microbiome Inhibits Tumorigenesis in a Mouse Model of Oviductal High-Grade Serous Carcinoma.

Studies have shown bacteria influence the initiation and progression of cancers arising in sites that harbor rich microbial communities, such as the colon. Little is known about the potential for the microbiome to influence tumorigenesis at sites considered sterile, including the upper female genital tract. The recent identification of distinct bacterial signatures associated with ovarian carcinomas suggests microbiota in the gut, vagina, or elsewhere might contribute to ovarian cancer pathogenesis. Here, we tested whether altering the microbiome affects tumorigenesis in a mouse model of high-grade serous carcinoma (HGSC) based on conditional oviduct-specific inactivation of the Brca1, Trp53, Rb1, and Nf1 tumor suppressor genes. Cohorts of control (n = 20) and antibiotic-treated (n = 23) mice were treated with tamoxifen to induce tumor formation and then monitored for 12 months. The antibiotic cocktail was administered for the first 5 months of the monitoring period in the treatment group. Antibiotic-treated mice had significantly fewer and less advanced tumors than control mice at study endpoint. Antibiotics induced changes in the composition of the intestinal and vaginal microbiota, which were durable in the fecal samples. Clustering analysis showed particular groups of microbiota are associated with the development of HGSC in this model. These findings demonstrate the microbiome influences HGSC pathogenesis in an in vivo model that closely recapitulates the human disease. Because the microbiome can modulate efficacy of cancer chemo- and immunotherapy, our genetically engineered mouse model system may prove useful for testing whether altering the microbiota can improve the heretofore poor response of HGSC to immunotherapies. SIGNIFICANCE: This study provides strong in vivo evidence for a role of the microbiome in ovarian cancer pathogenesis.

Abstract B11: Collective extrusion initiates dissemination in organotypic model of high-grade serous carcinoma

Abstract Development of serous tubal intraepithelial carcinoma (STIC) from transformed fallopian tube secretory epithelial cells (FTSEC) is considered to be an initiating event for a significant proportion of high-grade serous carcinomas (HGSCs). Some regions of STIC lesions can form multicellular loosely cohesive outgrowths that apically extend out of the tubal epithelium. These outgrowths can detach and disseminate within the peritoneal cavity, causing organ obstruction. Immunohistochemical analysis of STICs suggests the possibility that molecular pathways associated with proliferation, DNA repair, cytoskeletal dynamics, and extracellular matrix adhesion contribute to outgrowth formation. Due to limited availability of transformed FTSEC in vitro culture models that faithfully capture outgrowth dynamics, the role of these pathways in STIC/HGSC outgrowth formation remains unknown. Here we present the development of an organotypic model of outgrowth formation. Long-term live-cell imaging of transformed human, and mouse FTSEC and STIC-like multicellular organotypic assemblies, have shown that outgrowths are initiated by collective apical extrusion of less-cohesive, loosely attached cell populations that can proliferate outside of the organotypic assembly. mRNA and immunofluorescence analysis of loosely attached cell populations revealed: (i) activation of Rho pathway, (ii) increased expression of several alpha integrin subunits that form heterodimers with beta 1 integrin, and (iii) nonpolarized apical and junctional localization of activated integrin beta 1. Pharmacologic or genetic inhibition of Rho-associated kinase (ROCK) blocked outgrowth formation without affecting cell proliferation. Conversely, activation of Rho significantly accelerated outgrowths, indicating that Rho-ROCK is a critical regulator of early steps of HGSC dissemination. Quantitative image-based evaluation of ROCK target phosphorylated myosin light chain expression in patient-derived xenograft cells revealed the presence of cell populations with high ROCK activation. Blocking integrin beta 1 activity resulted in the disintegration of already formed outgrowths. Our data indicate that cell populations with high Rho/ROCK activity remodel integrin beta 1-dependent adhesion to initiate apical extrusion and peritoneal dissemination. Citation Format: Sarah Alshehri, Sadaf Farsinejad, Douglas Kung, Estefan Santi, Yali Zhai, Thomas Cattabiani, Hongjun Wang, Kathleen Cho, Marcin Iwanicki. Collective extrusion initiates dissemination in organotypic model of high-grade serous carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B11.

Abstract B65: Defining the tumor immune landscape in a mouse model of high-grade serous carcinoma

Abstract Clinical efficacy of immune checkpoint therapy in high-grade serous carcinoma (HGSC) has been disappointing to date and highlights the need to better understand the relationship between HGSC and its associated immune microenvironment. Tractable model systems for interrogating the tumor microenvironment in HGSC could be used to address this unmet need. We have generated genetically engineered mouse models (GEMMs) of HGSC utilizing oviduct-restricted tamoxifen-regulated Cre recombinase to drive conditional inactivation of key tumor suppressor genes (Brca1, Trp53, Rb1, Nf1) observed to be recurrently altered in human HGSC samples. These autochthonous tumors arise from the murine fallopian tube (oviduct) in the context of an intact immune system. We employed RNA sequencing (RNA-seq) of bulk tumors from 19 mouse HGSCs to compare their gene expression profiles to those of normal oviduct and ovary tissues (4 each), and 6 mouse endometrioid-like oviductal carcinomas based on conditional inactivation of Apc and Pten. We employed immunogenomic analysis of the RNA-seq data to characterize the intratumoral infiltrate of 28 immune cell types in these samples in silico. The analysis revealed a prevalence of gene expression signatures associated with immunosuppressive cell types, including myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) in the mouse HGSCs, not observed in the other sample groups. Human HGSCs with available RNA-seq data (from TCGA samples) showed similar enrichment of MDSC and Treg gene signatures in the subset of human HGSCs classified as mesenchymal or immunoreactive expression subtypes. To further validate the findings in the mouse HGSCs, we used immunohistochemistry to detect subsets of tumor-infiltrating lymphocytes (TILs) in the tumor tissues. Specifically, CD8+ TIL infiltrates recapitulated epithelial (E-TIL), stromal (S-TIL) and combined epithelial/stromal (ES-TIL) patterns described in human HGSCs. Moreover, FoxP3+ Tregs were frequently observed to be co-infiltrated with CD8+ TILs as expected in an immunosuppressive tumor microenvironment. Gene set enrichment analysis showed that NF-kappa B signaling is among the top five pathways upregulated in HGSCs versus normal oviducts, suggesting a plausible mechanism by which tumor cells can alter and respond to the microenvironment during tumor development. These studies provide an experimental paradigm for improving our understanding of the role of the tumor microenvironment in HGSC pathogenesis and may aid in efforts to enhance the efficacy of immunotherapies in this lethal disease. Citation Format: Kevin W. McCool, Rork Kuick, Yali Zhai, Zach Freeman, Rong Wu, Eric Fearon, Kathleen R. Cho. Defining the tumor immune landscape in a mouse model of high-grade serous carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B65.

Abstract IA14: Integrated molecular characterization of the genome, transcriptome, and immune microenvironment in oviductal high-grade serous carcinomas arising in genetically engineered mice

Abstract Robust preclinical models of ovarian high-grade serous carcinoma (HGSC) are needed to advance our understanding of HGSC pathogenesis and to test novel strategies aimed at improving clinical outcomes for women with the disease. Genetically engineered mouse models of HGSC have been developed that recapitulate the likely cell of origin (fallopian tube epithelium), underlying genetic defects, histology, and biologic behavior of human HGSCs, but the degree to which the mouse tumors acquire the somatic genomic changes, gene expression profiles, and immune microenvironment that characterize human HGSCs remains unclear. We used integrative molecular characterization of oviductal HGSCs arising in the context of Brca1, Trp53, Rb1, and Nf1 (BPRN) inactivation to determine whether the mouse tumors recapitulate human HGSCs across multiple domains of molecular features. Targeted DNA sequencing showed that the mouse BPRN tumors, but not endometrioid carcinoma-like tumors based on different genetic defects (e.g., Apc, Pten, Arid1a), acquire somatic mutations and widespread copy number alterations similar to those observed in human HGSCs. RNA sequencing showed that the mouse HGSCs most closely model the immunoreactive and mesenchymal subsets of human HGSCs. A combined immunogenomic analysis showed that the immune microenvironment of BPRN tumors models key aspects of tumor-immune dynamics in human HGSC, with enrichment of immunosuppressive cell subsets (myeloid-derived suppressor cells and regulatory T cells) as observed in the immunoreactive and mesenchymal subsets of human tumors. These findings, as well as early proof-of-principle studies, support the BPRN model as a robust preclinical experimental platform to help overcome barriers to clinical success in preventing, diagnosing, and treating this lethal cancer. Citation Format: Kathleen R. Cho. Integrated molecular characterization of the genome, transcriptome, and immune microenvironment in oviductal high-grade serous carcinomas arising in genetically engineered mice [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr IA14.

Murine Oviductal High-Grade Serous Carcinomas Mirror the Genomic Alterations, Gene Expression Profiles, and Immune Microenvironment of Their Human Counterparts.

Robust preclinical models of ovarian high-grade serous carcinoma (HGSC) are needed to advance our understanding of HGSC pathogenesis and to test novel strategies aimed at improving clinical outcomes for women with the disease. Genetically engineered mouse models of HGSC recapitulating the likely cell of origin (fallopian tube), underlying genetic defects, histology, and biologic behavior of human HGSCs have been developed. However, the degree to which the mouse tumors acquire the somatic genomic changes, gene expression profiles, and immune microenvironment that characterize human HGSCs remains unclear. We used integrated molecular characterization of oviductal HGSCs arising in the context of Brca1, Trp53, Rb1, and Nf1 (BPRN) inactivation to determine whether the mouse tumors recapitulate human HGSCs across multiple domains of molecular features. Targeted DNA sequencing showed the mouse BPRN tumors, but not endometrioid carcinoma-like tumors based on different genetic defects (e.g., Apc and Pten), acquire somatic mutations and widespread copy number alterations similar to those observed in human HGSCs. RNA sequencing showed the mouse HGSCs most closely resemble the so-called immunoreactive and mesenchymal subsets of human HGSCs. A combined immuno-genomic analysis demonstrated the immune microenvironment of BPRN tumors models key aspects of tumor-immune dynamics in the immunoreactive and mesenchymal subtypes of human HGSC, with enrichment of immunosuppressive cell subsets such as myeloid-derived suppressor cells and regulatory T cells. The findings further validate the BPRN model as a robust preclinical experimental platform to address current barriers to improved prevention, diagnosis, and treatment of this often lethal cancer. SIGNIFICANCE: The acquired gene mutations, broad genomic alterations, and gene expression and immune cell-tumor axis changes in a mouse model of oviductal serous carcinoma closely mirror those of human tubo-ovarian high-grade serous carcinoma.

Abstract GMM-061: MOLECULAR CHARACTERIZATION OF MOUSE MODELS OF HIGH-GRADE SEROUS CARCINOMA ARISING IN THE OVIDUCT

Abstract We recently developed genetically engineered mouse models (GEMMs) of oviductal high-grade serous carcinoma (HGSC) based on conditional inactivation of several different combinations of tumor suppressor genes (Brca1, Trp53, Rb1, and Nf1) that are recurrently mutated in human HGSCs. The histopathological features of the mouse tumors closely mimic their human tumor counterparts. In order to characterize how well the mouse tumors recapitulate the molecular characteristics of human HGSCs, targeted exome sequencing was used to analyze the 32 most commonly mutated genes in human HGSC, in 60 mouse tumors arising in the context of Brca1, Trp53, Rb1, and/or Nf1 inactivation. We employed the sequence data to assess DNA copy number alterations (CNAs) and single nucleotide variants (SNVs) in the mouse tumors. Compared to 14 normal tissues and 8 oviductal tumors arising in the context of Apc, Pten, ± Arid1a inactivation, the mouse HGSCs showed a high level of genomic instability, with many widely distributed CNAs – very similar to the widespread CNAs observed in human HGSCs. Targeted exome sequencing also showed that a subset of the mouse tumors acquired alterations observed in human HGSCs, including amplification of cMyc, and deletion of Pten. Variant analysis identified nonsynonymous SNVs in Csmd3, Crebbp, Pten, Mettl17, and Zymynd8 and a frameshift deletion of Pten. Sanger sequencing confirmed the presence of these somatic mutations in the mouse tumors and their absence in matched normal tissues. Loss of PTEN expression was observed in those tumors that acquired somatic Pten alterations. These data show that HGSCs arising in our GEMMs have very similar molecular characteristics to their human tumor counterparts. The somatic alterations are likely acquired during the relatively lengthy period (several months) between tumor initiation and progression to overt malignancy. These features render the models particularly well suited for studying the early phases of HGSC development and for translational applications aimed at identifying effective strategies for HGSC prevention and early detection. Citation Format: Yali Zhai, Yisheng Wang, Stephanie M. Schulman, Kevin Hu, Albert Liu, Scott A. Tomlins, Eric R. Fearon, and Kathleen R. Cho. MOLECULAR CHARACTERIZATION OF MOUSE MODELS OF HIGH-GRADE SEROUS CARCINOMA ARISING IN THE OVIDUCT [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-061.

Abstract TMEM-018: IN VITRO MODELS OF HGSC BASED ON MURINE OVIDUCTAL EPITHELIAL ORGANOIDS

Abstract Mouse models of high-grade serous carcinoma (HGSC) arising from oviductal epithelium provide excellent in vivo systems with which to define the cellular and molecular events associated with HGSC development and progression. However, tumors in mice typically develop over months rather than weeks, and tumor progression in vivo cannot be easily monitored. Murine organoid models that closely mimic the biology of fallopian tube epithelium would facilitate research exploring HGSC pathogenesis. We have developed Ovgp1-iCreERT2 mice in which the Ovgp1 promoter controls expression of tamoxifen (TAM)-inducible Cre recombinase in the oviductal epithelium – the murine equivalent of human fallopian tube epithelium. We have established long-term, 3-dimensional oviductal epithelial organoid cultures from Ovgp1-iCreERT2;R26LSL-EYFP double transgenic mice, and show that the organoids contain both ciliated and secretory cells, and express oviductal epithelial markers including cytokeratin 8, ER, PAX8 and OVGP1. Organoids treated in vitro with 4-hydroxy-tamoxifen (4-OH-TAM) activate the EYFP reporter. Oviductal epithelial organoids established from Ovgp1-iCreERT2;Brca1del/fl; Trp53mut/fl;Rb1fl/fl (BPR) mice and treated in vitro with 4-OH-TAM show selection for the recombined tumor suppressor gene alleles by passage 6, accompanied by morphological and behavioral changes associated with neoplastic transformation. Stable organoid cultures can also be established from the oviductal epithelium of BPR mice treated in vivo with Tamoxifen (TAM). We show that long-lived 3-dimensional organoids can be generated from normal oviductal tissue; can undergo Cre-mediated recombination in vitro to inactivate tumor suppressor genes relevant to human HGSC pathogenesis; and can acquire properties associated with neoplastic transformation in a relatively short period of time (weeks). As we have already generated Ovgp1-iCreERT2 mice with various combinations of constitutional and Cre-inducible mutant tumor suppressor gene (Trp53, Rb1, Brca1, Pten, Nf1, Apc) and oncogene (PIK3CA, KRAS) alleles, the roles of specific genetic alterations in HGSC pathogenesis can be explored in a relatively rapid and controlled experimental system. Citation Format: Yali Zhai, Eric R. Fearon, and Kathleen R. Cho. IN VITRO MODELS OF HGSC BASED ON MURINE OVIDUCTAL EPITHELIAL ORGANOIDS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-018.

Abstract AP24: CRISPR/CAS9–MEDIATED TRP53, BRCA1, BRCA2 AND PTEN KNOCKOUT TO GENERATE IMPROVED MURINE MODELS OF OVARIAN HIGH GRADE SEROUS CARCINOMA

Abstract BACKGROUND: Ovarian high grade serous carcinoma (HGSC) is the commonest subtype of human ovarian cancer, and prognosis remains poor. Transplantable murine models of HGSC that recreate key mutations seen in the human disease are greatly needed. These models would assist investigation of the relationships between tumor genotype, chemotherapy response and immune microenvironment. ID8 is the most widely-used mouse model of ovarian cancer, but it has not been characterized in light of current understanding of HGSC biology. METHODS: We performed whole exome sequencing of ID8, covering 194,000 exons at a mean depth of 400x with 90% exons sequenced >50x. Using CRISPR/Cas9 gene editing, we have generated novel ID8 derivatives with single (Trp53-/-) and double (Trp53-/-;Brca2-/-, Trp53-/-;Brca1-/- and Trp53-/-;Pten-/-) deletions. We have characterized intra-peritoneal growth of these novel mutants, and investigated platinum and PARP inhibitor sensitivity and immune cell infiltration into the tumor microenvironment. RESULTS: Whole exome sequencing showed no functional mutations in genes characteristic of HGSC (Trp53, Brca1, Brca2, Nf1, Rb1). In addition, mutations typical of clear cell (Arid1A, Pik3ca), low grade serous (Braf), endometrioid (Ctnnb1) and mucinous (Kras) carcinomas were notably absent. ID8 also demonstrated intact p53 function and homologous recombination in functional assays. We show that loss of p53 alone significantly increases tumor growth rate within the peritoneal cavity of C57Bl/6 mice, and has marked effects upon immune microenvironment. In particular, loss of p53 increases CCL2 expression and induces immunosuppressive myeloid cell infiltration into tumor and ascites. Trp53-/-;Brca2-/- and Trp53-/-;Brca1-/- cells show significantly increased sensitivity to the PARP inhibitor rucaparib in vitro compared to parental and Trp53-/- cells. In vivo, Trp53- /-;Brca2-/- tumors are more sensitive to platinum chemotherapy than Trp53-/-. They also exhibit slower intraperitoneal growth, with appearance of intra-tumoral CD3+ cell-rich tertiary lymphoid structures, a phenotype observed in human tumors with high mutational burden. Mice bearing Trp53-/-;Pten-/- tumors have significantly reduced survival compared to Trp53-/-. Full in vivo assessment of the Trp53-/-;Brca1-/- and new tripleTrp53-/-;Brca2-/- ;Pten-/- lines is on-going. CONCLUSIONS: These models represent a new and simple tool to investigate the biology of HGSC. All cells will be made available to other researchers upon request. Citation Format: Josephine Walton, Julianna Blagih, Malcolm Farquharson, Darren Ennis, Elaine Leung, Suzanne Dowson, Laura A. Tookman, Clare Orange, Dimitris Athineos, Susan Mason, David Stevenson, Karen Blyth, Douglas Strathdee, Frances R. Balkwill, Karen Vousden, Michelle Lockley, Iain A. McNeish. CRISPR/CAS9–MEDIATED TRP53, BRCA1, BRCA2 AND PTEN KNOCKOUT TO GENERATE IMPROVED MURINE MODELS OF OVARIAN HIGH GRADE SEROUS CARCINOMA [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP24.

CRISPR/Cas9-Mediated Trp53 and Brca2 Knockout to Generate Improved Murine Models of Ovarian High-Grade Serous Carcinoma.

There is a need for transplantable murine models of ovarian high-grade serous carcinoma (HGSC) with regard to mutations in the human disease to assist investigations of the relationships between tumor genotype, chemotherapy response, and immune microenvironment. In addressing this need, we performed whole-exome sequencing of ID8, the most widely used transplantable model of ovarian cancer, covering 194,000 exomes at a mean depth of 400× with 90% exons sequenced >50×. We found no functional mutations in genes characteristic of HGSC (Trp53, Brca1, Brca2, Nf1, and Rb1), and p53 remained transcriptionally active. Homologous recombination in ID8 remained intact in functional assays. Further, we found no mutations typical of clear cell carcinoma (Arid1a, Pik3ca), low-grade serous carcinoma (Braf), endometrioid (Ctnnb1), or mucinous (Kras) carcinomas. Using CRISPR/Cas9 gene editing, we modeled HGSC by generating novel ID8 derivatives that harbored single (Trp53-/-) or double (Trp53-/-;Brca2-/-) suppressor gene deletions. In these mutants, loss of p53 alone was sufficient to increase the growth rate of orthotopic tumors with significant effects observed on the immune microenvironment. Specifically, p53 loss increased expression of the myeloid attractant CCL2 and promoted the infiltration of immunosuppressive myeloid cell populations into primary tumors and their ascites. In Trp53-/-;Brca2-/- mutant cells, we documented a relative increase in sensitivity to the PARP inhibitor rucaparib and slower orthotopic tumor growth compared with Trp53-/- cells, with an appearance of intratumoral tertiary lymphoid structures rich in CD3+ T cells. This work validates new CRISPR-generated models of HGSC to investigate its biology and promote mechanism-based therapeutics discovery. Cancer Res; 76(20); 6118-29. ©2016 AACR.

Abstract B22: Establishment of Patient-Derived Xenograft (PDX) Models of Ovarian Cancer

Abstract Ovarian cancer is the most lethal gynecological malignancy. The majority of ovarian cancer patients are diagnosed at an advanced stage of disease, and the lack of good early screening and diagnostic tests is one of the reasons for the low overall survival rate. Moreover, there is no good marker to predict patients' response to chemotherapy. Patient-derived xenografts (PDXs) have become the most promising preclinical model for gynecologic malignances. We have successfully established 22 PDX models derived from ovarian cancer patients' surgical specimens implanted into severe combined immunodeficient (SCID) mice, non-obese diabetic (NOD)-SCID or Nude mice. The majority of the PDX models were generated from patients' samples diagnosed with high-grade serous carcinoma including 3 with BRCA1/2 mutations, as well as less common types of ovarian cancers such as low-grade serous, endometrioid, mucinous carcinomas, and carcinosarcoma. PDXs have been established from solid masses as well as from ascites. We have optimized protocol for establishing ovarian PDXs including implantation and collection methods, tumor type (solid versus ascites), and type of mouse strains used. Using SCID mice, our success rate exceeded 80% and recent data indicated that the success rate in NOD/SCID mice was higher. The link between the pathology and PDX core, the “triage protocol” and ongoing PARP inhibitor trials will provide an important array of PDXs including pre- and post-treatment PDXs for exploration for biomarkers as well as for co-clinical trials to validate the utility of rational drug combinations. All models were validated using short tandem repeats (STR) and compared to the H&E and immunohistochemistry stains from the matching patient's tissue. We also examined the expression of B7-H4, a novel member of the B7 family immune checkpoint regulators, in 9 patient-derived xenografts (PDXs) of high-grade ovarian serous carcinoma. Our data showed that B7-H4 proteins were expressed in 8 of 9 PDX models of high-grade serous carcinoma, suggesting that PDXs may be useful for studying immunotherapy. Citation Format: Li Liang, Imelda Mercado-Uribe, Na Niu, Yi Jiang, Wenjun Cheng, Russell Broaddus, Robert Bast, Gordon Mills, Anil K. Sood, Jinsong Liu. Establishment of Patient-Derived Xenograft (PDX) Models of Ovarian Cancer. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B22.

Abstract AS06: Development and characterization of an oviduct-specific model of high-grade serous carcinoma

Abstract Recent studies suggest the most common and lethal type of “ovarian” cancer, high-grade serous carcinoma (HGSC), often arises from fallopian tube epithelium rather than the ovarian surface epithelium. Their intraperitoneal location and microscopic size make it very difficult to screen for, or study the biology of early tubal lesions in humans, as most are discovered incidentally in women with hereditary predisposition to ovarian cancer who elect prophylactic salpingo-oophorectomy for cancer risk-reduction. Although most cases of pelvic HGSC probably originate in the fallopian tube, roughly one-third lack evidence of tubal origin. These cases may arise from ectopic tubal-type epithelium (endosalpingiosis) present in the ovary, peritoneum, or at other sites. Mouse models that closely mimic the genetics and biology of human HGSCs may be useful for clarifying how tubal and non-tubal pelvic HGSCs develop and progress. We have developed transgenic (Ovgp1-iCreERT2) mice allowing conditional (tamoxifen [TAM]-inducible) expression of Cre recombinase exclusively in the oviductal epithelium, using a single transgene. Using double transgenic Ovgp1-iCreERT2;R26LSL-eYFP mice in which TAM treatment activates eYFP reporter protein expression in oviductal epithelial cells, we have shown that tubal-type inclusion glands (endosalpingiosis) expressing both eYFP and OVGP1 are present in a subset of murine ovaries following treatment with TAM. The findings suggest that oviductal epithelium can detach from the oviduct and traffic to the ovaries, where it implants through breaks in the OSE and maintains its tubal-type differentiation. Preliminary data also suggest that the frequency of ovarian endosalpingiosis increases over time and by superovulating the mice. The latter finding raises the possibility that at least some of the protective effects of high parity and oral contraceptive use on human ovarian cancer risk is related to reduced likelihood of acquiring endosalpingiosis from which “ovarian” HGSCs may eventually arise. We have further shown that Ovgp1-iCreERT2;Trp53fl/fl;Brca1fl/fl mice develop oviductal lesions identical to human serous tubal intraepithelial carcinomas (STICs) after TAM treatment and have identified additional genetic alterations that cooperate with Trp53 and Brca1 inactivation in oviductal HGSC pathogenesis in our model system. Murine models of HGSC that recapitulate their human tumor counterparts provide excellent in vivo systems with which to define the cellular and molecular events associated with HGSC development and progression. Citation Format: Yali Zhai, Rong Wu, Tom C. Hu, Eric R. Fearon, Kathleen R. Cho. Development and characterization of an oviduct-specific model of high-grade serous carcinoma [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr AS06.

Abstract POSTER-BIOL-1308: Macrophage infiltration in high-grade serous carcinomas of humans and mice

Abstract Over the past 10-15 years, focus on the immune system has reinvigorated cancer research and oncology. Both clinical and experimental evidence generated during this time has shown that immune cells and their molecular mediators represent viable targets for therapeutic manipulation. The immune cell composition of high-grade serous carcinoma (HGSC) consists primarily of macrophages and various T cell subsets. However, the functional contribution of each of these cell types is largely unknown. In order to gain insight into whether these cells are pro- or anti-tumorigenic, we generated tissue microarrays containing tumors from 169 patients with pretreated ovarian cancer and quantified the number of CD68+ macrophages in each biopsy. Tumors were divided into high- or low-infiltrated groups based on the median number of macrophages amongst all HGSCs. Interestingly, patients whose tumors were highly infiltrated with macrophages exhibited worsened overall survival, suggesting that macrophages function as pro-tumorigenic cells in these tumors. To investigate the role of macrophages in HGSC progression, we have begun to characterize their abundance and phenotype in tumors of Dicerflox/flox;Ptenflox/flox;Amhr2cre/+ mice, a genetically engineered mouse model of HGSC. Macrophages constituted the predominant stromal and immune cell population in both primary tumors and secondary metastatic lesions. By contrast, macrophages were only found at low frequency in normal oviduct and ovaries. Gene expression analysis of these Dicer/Pten-deficient mouse tumors also revealed the increase in many macrophage-associated genes, as compared with normal oviduct tissue. Taken together, our data from mice and humans strongly indicate a crucial role for macrophages in facilitating ovarian cancer progression. Current efforts are underway to understand how macrophages promote ovarian tumor progression. Citation Format: Tanja Pejovic, Yukie Bean, Jaeyeon Kim, Martin M. Matzuk, Seth B. Coffelt. Macrophage infiltration in high-grade serous carcinomas of humans and mice [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1308.

Evidence for a dualistic model of high-grade serous carcinoma: BRCA mutation status, histology, and tubal intraepithelial carcinoma.

Most early adnexal carcinomas detected in asymptomatic women with germline BRCA mutations (BRCA) present as serous tubal intraepithelial carcinomas (STIC). However, STICs are found in only ∼40% of symptomatic high-grade serous carcinomas (HGSCs) and less frequently in pseudoendometrioid variants of HGSC. Consecutive cases of untreated HGSC from BRCA and BRCA women with detailed fallopian tube examination (SEE-FIM protocol) were compared. STIC status (+/-) was determined, and tumors were classified morphologically as SET ("SET", >50% solid, pseudoendometrioid, or transitional) or classic predominate ("Classic"). SET tumors trended toward a higher frequency in BRCA versus BRCA women (50% vs. 28%, P=0.11), had a significantly younger mean age than those with classic HGSC in BRCA women (mean 56.2 vs. 64.8 y, P=0.04), and displayed a better clinical outcome in both groups combined (P=0.024). STIC was significantly more frequent in tumors from the BRCA cohort (66% vs. 31%, P=0.017) and specifically the BRCA tumors with classic morphology (83%) versus those with SET morphology (22%, P=0.003). Overall, several covariables-histology, BRCA status, age, coexisting STIC, and response to therapy-define 2 categories of HGSC with differences in precursor (STIC) frequency, morphology, and outcome. We introduce a dualistic HGSC model that could shed light on the differences in frequency of STIC between symptomatic and asymptomatic women with HGSC. This model emphasizes the need for further study of HGSC precursors to determine their relevance to the prevention of this lethal malignancy.

Type-specific cell line models for type-specific ovarian cancer research.

BackgroundOvarian carcinomas consist of at least five distinct diseases: high-grade serous, low-grade serous, clear cell, endometrioid, and mucinous. Biomarker and molecular characterization may represent a more biologically relevant basis for grouping and treating this family of tumors, rather than site of origin. Molecular characteristics have become the new standard for clinical pathology, however development of tailored type-specific therapies is hampered by a failure of basic research to recognize that model systems used to study these diseases must also be stratified. Unrelated model systems do offer value for study of biochemical processes but specific cellular context needs to be applied to assess relevant therapeutic strategies.MethodsWe have focused on the identification of clear cell carcinoma cell line models. A panel of 32 “ovarian cancer” cell lines has been classified into histotypes using a combination of mutation profiles, IHC mutation-surrogates, and a validated immunohistochemical model. All cell lines were identity verified using STR analysis.ResultsMany described ovarian clear cell lines have characteristic mutations (including ARID1A and PIK3CA) and an overall molecular/immuno-profile typical of primary tumors. Mutations in TP53 were present in the majority of high-grade serous cell lines. Advanced genomic analysis of bona-fide clear cell carcinoma cell lines also support copy number changes in typical biomarkers such at MET and HNF1B and a lack of any recurrent expressed re-arrangements.Conclusions: As with primary ovarian tumors, mutation status of cancer genes like ARID1A and TP53 and a general immuno-profile serve well for establishing histotype of ovarian cancer cell We describe specific biomarkers and molecular features to re-classify generic “ovarian carcinoma” cell lines into type specific categories. Our data supports the use of prototype clear cell lines, such as TOV21G and JHOC-5, and questions the use of SKOV3 and A2780 as models of high-grade serous carcinoma.