High-grade Serous Ovarian Tumors Research Articles

Integrin-linked kinase-frizzled 7 interaction maintains cancer stem cells to drive platinum resistance in ovarian cancer

BackgroundPlatinum-based chemotherapy regimens are a mainstay in the management of ovarian cancer (OC), but emergence of chemoresistance poses a significant clinical challenge. The persistence of ovarian cancer stem cells (OCSCs) at the end of primary treatment contributes to disease recurrence. Here, we hypothesized that the extracellular matrix protects CSCs during chemotherapy and supports their tumorigenic functions by activating integrin-linked kinase (ILK), a key enzyme in drug resistance.MethodsTCGA datasets and OC models were investigated using an integrated proteomic and gene expression analysis and examined ILK for correlations with chemoresistance pathways and clinical outcomes. Canonical Wnt pathway components, pro-survival signaling, and stemness were examined using OC models. To investigate the role of ILK in the OCSC-phenotype, a novel pharmacological inhibitor of ILK in combination with carboplatin was utilized in vitro and in vivo OC models.ResultsIn response to increased fibronectin secretion and integrin β1 clustering, aberrant ILK activation supported the OCSC phenotype, contributing to OC spheroid proliferation and reduced response to platinum treatment. Complexes formed by ILK with the Wnt receptor frizzled 7 (Fzd7) were detected in tumors and correlated with metastatic progression. Moreover, TCGA datasets confirmed that combined expression of ILK and Fzd7 in high grade serous ovarian tumors is correlated with reduced response to chemotherapy and poor patient outcomes. Mechanistically, interaction of ILK with Fzd7 increased the response to Wnt ligands, thereby amplifying the stemness-associated Wnt/β-catenin signaling. Notably, preclinical studies showed that the novel ILK inhibitor compound 22 (cpd-22) alone disrupted ILK interaction with Fzd7 and CSC proliferation as spheroids. Furthermore, when combined with carboplatin, this disruption led to sustained AKT inhibition, apoptotic damage in OCSCs and reduced tumorigenicity in mice.ConclusionsThis “outside-in” signaling mechanism is potentially actionable, and combined targeting of ILK-Fzd7 may lead to new therapeutic approaches to eradicate OCSCs and improve patient outcomes.

Abstract 7347: A transcriptome correlation network analysis of the high grade serous ovarian tumor immune microenvironment

Abstract High Grade Serous Ovarian Cancer (HGSOC) affects many women and is the most common and lethal histopathological type of ovarian cancer. The molecular subtypes delineated by TCGA–mesenchymal, proliferative, immunoreactive, and differentiated subtypes, offer a sophisticated and innovative framework for HGSOC classification. These subtypes not only provide a detailed molecular characterization but also contribute to understanding the HGSOC Tumor Immune Microenvironment (TIME), comprised of both immune and tumor cells. The interplay within the TIME significantly influences pro- or anti-tumor responses, creating an important link between molecular landscape and dynamic immune-tumor interactions. Currently, there is a lack of understanding for the key drivers of clinical outcomes, molecular phenotypes, and diagnostic biomarkers for HGSOC. Here, we answer the question, does immune cell type, and proportion, drive clinical and molecular subtyping of primary HGSOC? Primary ovarian tumor samples from The Cancer Genome Atlas, TCGA (n=358) samples were collected and RNA-sequenced. FPKM values were used to construct the Weighted Gene Correlation Network Analysis (WGCNA), Gene Ontology (GO) and Proportion Estimations of Immune and Cancer cells (EPIC). Enrichment of HGSOC network module overlaps using a Fischer Exact Test were used to identify novel components of HGSOC progression that were significantly associated with clusters of highly correlated gene expression profiles, HGSOC stage, grade, and molecular phenotypes. We show genes associated with T cell proliferation (CD84) and B cell regulation (CD37), epithelial to mesenchymal transition (DOCK2), adaptor proteins present in T cells and myeloid cells (LCP2), inflammatory and metabolic signaling modulators (AIF1,CD53, SASH3), immunosuppressors of T lymphocytes and dendritic cells (FGL2), cell proliferation (PI3K) and relevant tertiary lymphoid structure gene (TNFAIP8L2) significantly correlated with HGSOC progression in both estimated tumor and proportions of immune cells. Future studies aim to confirm phosphorylation cascades associated with the aggressive molecular phenotypes of HGSOC and immune cell pathobiology. Citation Format: Kaylin M. Carey, Eric B. Dammer, Corey Young, Rajesh Singh, Ti'ara Griffen, James W. Lillard. A transcriptome correlation network analysis of the high grade serous ovarian tumor immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7347.

Abstract 575: Identification of SORL1 as a key regulator of chemoresistance in ovarian cancer

Abstract This study aimed to understand the molecular mechanism of platinum chemoresistance in recurrent ovarian tumors with a focus on the SORL1-regulated pathways. SORL1 is an intracellular sorting receptor that binds to HER2 in breast cancer; this promotes HER2 recycling to the cell surface, which ultimately impacts the progression and treatment of breast cancer. The role of SORL1 in ovarian cancer has not been extensively studied. Method: RNA was extracted from 15 pairs of matched primary and recurrent high grade serous ovarian tumors collected from patients. Human transcriptomic microarray was used to identify the differentially expressed genes. The identified genes were validated by RT-QPCR and western blot in five ovarian cancer cell lines that were treated or untreated with carboplatin. To study the effects of identified target gene SORL1, overexpression and knockdown in ovarian cancer cell lines were established using SORL1 plasmid and shRNA. Proliferation and response to carboplatin were analyzed using celltiter Glo and caspase-3/7 activity assays respectively. EGFR and FGFR4 were identified as the interacting proteins of SORL1 in ovarian cancer cells through FGF1 and EGF1 treatment and co-immunoprecipitation. A proximity ligation assay was performed to validate their interactions. Finally, a xenograft mouse model was used to compare the tumor forming abilities and the response to carboplatin of SORL1-knockdown ovarian cancer cells and the control cancer cells in vivo. Protein expression of SORL1, EGFR, and FGFR4 in the collected tumors was determined via western blot. Result: Transcriptomic analysis of matched human primary and recurrent ovarian tumors identified SORL1 as an upregulated gene in recurrent tumors compared to primary samples. SORL1 expression was also upregulated in ovarian cancer cells surviving carboplatin treatment. Proliferation assays demonstrated that SORL1-overexpressing and knockdown cell lines had increased and inhibited proliferative capabilities respectively. Caspase-3 activity assays showed that SORL1-overexpressing and knockdown cell lines had reduced and increased sensitivity to carboplatin treatment respectively. SORL1 was found to bind to and promote the expression of EGFR and FGFR4. When injected subcutaneously into nude mice, SORL1-knockdown cells had reduced tumor forming capabilities and increased sensitivity to carboplatin. SORL1-knockdown caused the tumors to express lower levels of SORL1, EGFR, and FGFR4 proteins in comparison with the control tumors. Conclusion: This study demonstrates that SORL1 regulates the EGFR and FGF4 pathways to promote the growth and carboplatin resistance of ovarian cancer cells. Targeting this pathway has potential to aid in the treatment of platinum-resistant ovarian cancer. Citation Format: Miranda Mansolf, Ziyan Jian, Fangfang Bi, Tobias M. Hartwich, Yang Yang-Hartwich. Identification of SORL1 as a key regulator of chemoresistance in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 575.

Abstract A017: An analysis of 13 independently performed assays to measure homologous recombination deficiency using 90 freshly extracted high grade serous ovarian tumors: findings from the friends of cancer research hrd harmonization project

Abstract A017: An analysis of 13 independently performed assays to measure homologous recombination deficiency using 90 freshly extracted high grade serous ovarian tumors: findings from the friends of cancer research hrd harmonization project

Abstract IA012: Targeting AXL in platinum-resistant ovarian cancer: from the lab bench to Phase 3 trial

Abstract IA012: Targeting AXL in platinum-resistant ovarian cancer: from the lab bench to Phase 3 trial

Abstract C147: The DNA replication checkpoint inhibitors, ATRN-1051 (WEE1i) and ATRN-119 (ATRi), are potentially well tolerated and effective cancer treatments

Abstract Previous studies have demonstrated WEE1i and ATR inhibitors (ATRi and WEE1i) to be promising cancer therapeutics through synthetic lethality with various cancer associated mutations. However, a key limitation to the use of these inhibitors as cancer therapies in prior clinical trials has been the occurrence of adverse hematological effects, including anemia and thrombocytopenia. Herein, we describe two novel inhibitors, ATRN-1051 (WEE1i) and ATRN-119 (ATRi) that are potentially both effective in tumor suppression in animal models and well tolerated. ATRN-1051 was developed to be both a potent WEE1i and selective for WEE1 over other kinases (PLK1, PLK2 and PLK3). ATRN-1051 has an IC50 of 2.2 nM for WEE1 and limits the proliferation of various cancer cell lines in culture in the 100 nM to 200 nM range. Notably, ATRN-1051 is particularly effective in suppressing the growth of Cyclin E overexpressing cells lines, pinpointing Cyclin E as a potential biomarker for ATRN-1051. In addition, ATRN-1051 has potentially favorable pharmacokinetic properties that permits 3-8 times lower dosing than other clinical WEE1 inhibitors to achieve similar exposure (AUC, 0-24) levels (1). Consistent with the increase selectivity of ATRN-1051 fostering increased tolerability, dose-range finding studies indicate that doses potentially expected to cause significant tumor suppression are hematological well tolerated in mice, with red blood cell and platelet counts remaining in a non-pathogenic range. Importantly, daily oral dosing of ATRN-1051 suppresses the growth of CCNE1-amplified high-grade serous ovarian xenografted tumors over the course of 28 days, providing further evidence of the role of CCNE1-overexpression as a biomarker for ATRN-1051 treatment. Based on these data, ATRN-1051 has entered and is now progressing through IND-enabling studies. As a distinct upstream DNA replication checkpoint inhibitor, ATRN-119 is a macrocyclic ATRi that is highly specific for inhibition of ATR over other phosphatidylinositol kinase-related kinases (PIKKs), such as ATM, DNA-PK, and MTOR, implying the potential for increased tolerability. Supporting this implication, daily dosing of ATRN-119 suppresses tumor growth in xenograft mouse models of colon, pancreatic, and prostate cancers and causes no appreciable loss of body weight or hematologic toxicity. Daily dosing of ATRN-119 in combination with PARP inhibition causes significant tumor reduction in a BRCA2-deficient PDX model of high-grade serous ovarian cancer, again with no appreciable loss of body weight. These findings have led to a biomarker-driven Phase 1/2a clinical trial of ATRN-119 with daily dosing (Simpkins, PI). Together, these findings underscore the promise of ATRN-1051 and ATRN-119 as DNA replication checkpoint inhibitors for the treatment of a variety of cancers. (1) Data from study in A-427 NSCLC xenograft model as reported in Zentalis Corporate Overview, March 2022 Citation Format: Joseph Vacca, Stephen Rocca, Molly Hansbarger, Sonia Ritzmann, Justin Frye, Fiona Simpkins, Eric J. Brown, Oren Gilad. The DNA replication checkpoint inhibitors, ATRN-1051 (WEE1i) and ATRN-119 (ATRi), are potentially well tolerated and effective cancer treatments [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C147.

An integrated model of CDCA5 and FOXM1 expression combined with a residual disease that predicts prognosis in ovarian cancer patients.

Ovarian cancer (OC) is the most prevalent type of gynecologic cancer, leading to global death. Unfortunately, less than half of patients diagnosed with this cancer survive for up to five years. The factor forkhead box M1 (FOXM1) is a crucial oncoprotein in ovarian cancer and is currently recognized as a potential therapeutic target. The role of the Cell division cycle-associated 5 (CDCA5) is critical for advancing different types of cancers. However, the significance of CDCA5 in OC from a clinical perspective is not well comprehended. This study aimed to build a risk prognosis model and assess the data supporting the prognostic usefulness of CDCA5 and FOXM1 expression in patients with OC. In OC, we found that CDCA5 and FOXM1 were expressed. To establish the existence of variables that were independently related to PFS and OS, Cox regression, data from clinics, and Kaplan-Meier analysis were used. A risk score model and nomogram were created using the independent prognostic parameters. The accuracy of the model's predictions was then evaluated using decision curve analysis (DCA), calibration curve, and receiver operating characteristic(ROC) analysis. Finally, the patients were separated into groups based on their cut-off value, and then the differences in survival were investigated. Significant correlations were found between OC and CDCA5, and FOXM1 expression levels (P <0.0001). Serous ovarian tumors (P=0.025) and even specific subgroups of high-grade serous ovarian tumors were shown to have elevated CDCA5 expression levels. In our database, FOXM1 expression levels were discovered to be related to intestinal metastases (P=0.014). In OC, the expression of FOXM1 was positively correlated with the overexpression of CDCA5 (rs=0.46, P<0.0001). The results of the multivariate analysis indicated that residual disease (RD) (P=0.005), CDCA5 expression level (P=0.028), and FOXM1 expression level (P<0.0001) were identified as independent prognostic factors for PFS. Additionally, RD (P=0.023) and FOXM1 expression level (P<0.0001) were identified as independent prognostic factors for OS. While the prediction model's performance with RD was poor (AUC=0.645 for PFS, AUC=0.650 for OS), the model's performance with tissue biomarkers was enhanced (AUC=0.797 for PFS, AUC=0.741 for OS). The nomogram and risk score method showed a benefit for prognosis prediction. In summary, poor outcomes are predicted by CDCA5, which is overexpressed in OC patients and has a positive correlation with the level of FOXM1 expression. An aid to prognosis prediction in patients with OC and a resource for therapy planning is a risk prognosis model based on CDCA5 and FOXM1 expression with RD.

Performance of computational algorithms to deconvolve heterogeneous bulk ovarian tumor tissue depends on experimental factors

BackgroundSingle-cell gene expression profiling provides unique opportunities to understand tumor heterogeneity and the tumor microenvironment. Because of cost and feasibility, profiling bulk tumors remains the primary population-scale analytical strategy. Many algorithms can deconvolve these tumors using single-cell profiles to infer their composition. While experimental choices do not change the true underlying composition of the tumor, they can affect the measurements produced by the assay.ResultsWe generated a dataset of high-grade serous ovarian tumors with paired expression profiles from using multiple strategies to examine the extent to which experimental factors impact the results of downstream tumor deconvolution methods. We find that pooling samples for single-cell sequencing and subsequent demultiplexing has a minimal effect. We identify dissociation-induced differences that affect cell composition, leading to changes that may compromise the assumptions underlying some deconvolution algorithms. We also observe differences across mRNA enrichment methods that introduce additional discrepancies between the two data types. We also find that experimental factors change cell composition estimates and that the impact differs by method.ConclusionsPrevious benchmarks of deconvolution methods have largely ignored experimental factors. We find that methods vary in their robustness to experimental factors. We provide recommendations for methods developers seeking to produce the next generation of deconvolution approaches and for scientists designing experiments using deconvolution to study tumor heterogeneity.

Associations between prediagnostic aspirin use and ovarian tumor gene expression.

Aspirin use has been associated with reduced ovarian cancer risk, yet the underlying biological mechanisms are not fully understood. To gain mechanistic insights, we assessed the association between prediagnosis low and regular-dose aspirin use and gene expression profiles in ovarian tumors. RNA sequencing was performed on high-grade serous, poorly differentiated, and high-grade endometrioid ovarian cancer tumors from the Nurses' Health Study (NHS), NHSII, and New England Case-Control Study (n = 92 cases for low, 153 cases for regular-dose aspirin). Linear regression identified differentially expressed genes associated with aspirin use, adjusted for birth decade and cohort. False discovery rates (FDR) were used to account for multiple testing and gene set enrichment analysis was used to identify biological pathways. No individual genes were significantly differentially expressed in ovarian tumors in low or regular-dose aspirin users accounting for multiple comparisons. However, current versus never use of low-dose aspirin was associated with upregulation of immune pathways (e.g., allograft rejection, FDR = 5.8 × 10-10 ; interferon-gamma response, FDR = 2.0 × 10-4 ) and downregulation of estrogen response pathways (e.g., estrogen response late, FDR = 4.9 × 10-8 ). Ovarian tumors from current regular aspirin users versus never users were also associated with upregulation in interferon pathways (FDR <1.5 × 10-4 ) and downregulation of multiple extracellular matrix (ECM) architecture pathways (e.g., ECM organization, 4.7 × 10-8 ). Our results suggest low and regular-dose aspirin may impair ovarian tumorigenesis in part via enhancing adaptive immune response and decreasing metastatic potential supporting the likely differential effects on ovarian carcinogenesis and progression by dose of aspirin.

Genetic characterization of primary and metastatic high-grade serous ovarian cancer tumors reveals distinct features associated with survival

High-grade serous ovarian cancer (HGSC) is the most lethal histotype of ovarian cancer and the majority of cases present with metastasis and late-stage disease. Over the last few decades, the overall survival for patients has not significantly improved, and there are limited targeted treatment options. We aimed to better characterize the distinctions between primary and metastatic tumors based on short- or long-term survival. We characterized 39 matched primary and metastatic tumors by whole exome and RNA sequencing. Of these, 23 were short-term (ST) survivors (overall survival (OS) < 3.5 years) and 16 were long-term (LT) survivors (OS > 5 years). We compared somatic mutations, copy number alterations, mutational burden, differential gene expression, immune cell infiltration, and gene fusion predictions between the primary and metastatic tumors and between ST and LT survivor cohorts. There were few differences in RNA expression between paired primary and metastatic tumors, but significant differences between the transcriptomes of LT and ST survivors in both their primary and metastatic tumors. These findings will improve the understanding of the genetic variation in HGSC that exist between patients with different prognoses and better inform treatments by identifying new targets for drug development.

Abstract 6068: Methylomic, transcriptomic and immune profiles of high grade serous ovarian cancer among Black and non-Hispanic White women

Abstract Background: Ovarian cancer (OC) is the most lethal gynecological malignancy. There remains a significant difference in outcomes among black women, whose mortality/incidence ratio is 0.68 - the highest among all ethnic groups. Little is known about racially-defined biological key determinants of OC disparity. We recently characterized transcriptomic and methylomic profiles of high grade serous ovarian tumors (HGSOC) from black vs. non-Hispanic white women (NHW) to understand the observed disparity in survival. Methods: Nucleic acids were extracted from treatment-naïve HGSOC specimens (30 black and 26 NHW) and used for DNA methylation (EPIC array) and RNA-sequencing. Differential expression was performed using EBseq; pathway enrichment was performed using Enrichr. TIMER was used to deconvolute immune cell infiltration based on RNA-seq results, and multiplex immunohistochemistry (mIHC) was used for validation. Top differentially expressed genes (DEGs) were validated by qRT-PCR and studied functionally. Results: 191 and 825 genes were up- and down-regulated, respectively, in HGSOC tumors from black vs. NHW women (FDR-adjusted p-value &amp;lt; 0.10). Significantly enriched pathways among these DEGs included multiple classes of GPCRs - Class A, Monoamine GPCRs, and Peptide GPCRs. Among DEGs, calmodulin like-5 gene CALML5, involved in GPCR signaling, was significantly downregulated in tumors from black patients (0.086 fold-change). CALML5 downregulation in tumors from black vs. NHW women was validated by qRT-PCR. CALML5 knockdown by using shRNA disrupted colony and tumor sphere formation (p&amp;lt;0.05). Analysis using TIMER predicted that CD8+ and CD4+ memory-resting T-cells were enriched in tumors from black patients. mIHC validated increased infiltration by CD3+CD4+ and CD4+FOXP3+ lymphocytes in humors from black women, as well as increased expression of PD-L1, supporting an immunosuppressive phenotype. Global, modest hypermethylation was discovered in tumors from black vs. NHW patients: 47 CpGs were hypermethylated (FDR-adjusted p-value &amp;lt; 0.10) and 19 regions were differentially methylated between the two groups. Conclusions: We identified significant transcriptomic and modest methylation differences in HGSOC tumors from black and NHW patients. Pathway enrichment within GPCR-signaling was observed and an immunosuppressive phenotype was identified. Further exploration of the contribution of these differences to clinical outcomes and treatment response in black women is needed. Citation Format: Hao Huang, Russell Keathley, Ping Xie, JianJun Wei, Ernst Lengyel, Bin Zhang, Daniela Matei. Methylomic, transcriptomic and immune profiles of high grade serous ovarian cancer among Black and non-Hispanic White women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6068.

Correlation between CD44 and membrane fluidity-a study on biopsies of high-grade serous ovarian tumor.

Membrane fluidity is highly correlated with cellular malignancy, which is involved in cancer metastasis and drug resistance. Although the use of a simple, rapid, convenient, and label-free strategy based on Raman spectroscopy has been reported extensively in the study of membrane fluidity, the systematic studies in tumor tissues are underexplored. Using confocal Raman microscopy in the present study, we collect Raman spectra on the biopsies of high-grade serous ovarian cancer (HGSOC) and normal ovary epithelium, and analyze their membrane fluidities. A multiple immunofluorescence assay based on HGSOCs is used to examine the expression profiles of tumor biomarkers, CD44, a membrane fluidity-associated protein, which supports the conclusions drawn by Raman spectroscopy analysis in the study.

Impact of automated methods for quantitative evaluation of immunostaining: Towards digital pathology.

IntroductionWe sought to develop a novel method for a fully automated, robust quantification of protein biomarker expression within the epithelial component of high-grade serous ovarian tumors (HGSOC). Rather than defining thresholds for a given biomarker, the objective of this study in a small cohort of patients was to develop a method applicable to the many clinical situations in which immunomarkers need to be quantified. We aimed to quantify biomarker expression by correlating it with the heterogeneity of staining, using a non-subjective choice of scoring thresholds based on classical mathematical approaches. This could lead to a universal method for quantifying other immunohistochemical markers to guide pathologists in therapeutic decision-making.MethodsWe studied a cohort of 25 cases of HGSOC for which three biomarkers predictive of the response observed ex vivo to the BH3 mimetic molecule ABT-737 had been previously validated by a pathologist. We calibrated our algorithms using Stereology analyses performed by two experts to detect immunohistochemical staining and epithelial/stromal compartments. Immunostaining quantification within Stereology grids of hexagons was then performed for each histological slice. To define thresholds from the staining distribution histograms and to classify staining within each hexagon as low, medium, or high, we used the Gaussian Mixture Model (GMM).ResultsStereology analysis of this calibration process produced a good correlation between the experts for both epithelium and immunostaining detection. There was also a good correlation between the experts and image processing. Image processing clearly revealed the respective proportions of low, medium, and high areas in a single tumor and showed that this parameter of heterogeneity could be included in a composite score, thus decreasing the level of discrepancy. Therefore, agreement with the pathologist was increased by taking heterogeneity into account.Conclusion and discussionThis simple, robust, calibrated method using basic tools and known parameters can be used to quantify and characterize the expression of protein biomarkers within the different tumor compartments. It is based on known mathematical thresholds and takes the intratumoral heterogeneity of staining into account. Although some discrepancies need to be diminished, correlation with the pathologist’s classification was satisfactory. The method is replicable and can be used to analyze other biological and medical issues. This non-subjective technique for assessing protein biomarker expression uses a fully automated choice of thresholds (GMM) and defined composite scores that take the intra-tumor heterogeneity of immunostaining into account. It could help to avoid the misclassification of patients and its subsequent negative impact on therapeutic care.

Characterization of the mutational landscape of high-grade serous ovarian tumors before and after neo-adjuvant chemotherapy (267)

Characterization of the mutational landscape of high-grade serous ovarian tumors before and after neo-adjuvant chemotherapy (267)

Neoadjuvant treatment in ovarian cancer: New perspectives, new challenges.

Ovarian cancer remains the leading cause of death from gynecological cancer. Survival is significantly related to the stage of the disease at diagnosis. Of quite importance is primary cytoreductive surgery, having as a goal to remove all visible tumor tissue, and is the standard primary treatment in combination with platinum-based chemotherapy for patients with advanced ovarian carcinoma.Neo-adjuvant chemotherapy (NACT) has been implemented mostly in treating advanced disease, with studies performed having numerous limitations. Data extrapolated from these studies have not shown inferiority survival of NACT, compared to primary debulking surgery. The role of NACT is of particular interest because of the intrinsic mechanisms that are involved in the process, which can be proven as therapeutic approaches with enormous potential. NACT increases immune infiltration and programmed death ligand-1 (PDL-1) expression, induces local immune activation, and can potentiate the immunogenicity of immune-exclude high grade serous ovarian tumors, while the combination of NACT with bevacizumab, PARP inhibitors or immunotherapy remains to be evaluated. This article summarizes all available data on studies implementing NACT in the treatment of ovarian cancer, focusing on clinical outcomes and study limitations. High mortality rates observed among ovarian cancer patients necessitates the identification of more effective treatments, along with biomarkers that will aid treatment individualization.

Abstract 2845: Synthetic fusion cytokine, GIFT4, transduced autologous peritoneal immune cells - a novel cell therapy for the treatment of ovarian cancer

Abstract Ovarian Cancer is the 5th leading cause of mortality among women. Even with the development of modern chemo and biologic therapies, there has not been a significant increase in the progression-free and overall survival of patients with high grade serous ovarian tumors. The goal of this study is to develop an individualized cell therapy approach for immunologic targeting of this cancer. Our proposed approach is based on harvesting and subsequently arming natural immune cells present in the peritoneal fluid (ascitic fluid) that accumulates in most ovarian cancer patients and is removed by paracentesis and discarded to alleviate discomfort. Using multiparameter flow cytometry with three 16-18 component antibody panels we have carefully characterized the T cells, dendritic cells and innate lymphoid cells present in the peritoneal fluid of 15 ovarian cancer patients. Analysis of the data using tSNE identified multiple clusters of immune cell subsets. These data indicated that in each patient, a substantial percentage of adaptive and innate lymphoid cells are present that under appropriate conditions can be reactivated to recognize and target autologous tumors (Vazquez, Jessica, et al. "Identification of unique clusters of T, dendritic, and innate lymphoid cells in the peritoneal fluid of ovarian cancer patients." American Journal of Reproductive Immunology 84.3 (2020): e13284). Our goal now is to use fusokines, chimeras of two distinct cytokines, to weaponized the ascitic immune cells and employ them as autologous cell therapy against ovarian cancer. The first candidate under investigation is GIFT4, a fusokine composed of GM-CSF and IL-4 that has previously been shown to eradicate Melanoma in mouse model. Using Vector Builder, we designed and produced lentiviral vectors to express GIFT4 in primary immune cells. The efficiency of human GIFT4 expression in primary immune cells was optimized by testing different promoter constructs. The efficiency of transduction was determined by monitoring the primary immune cells for secretion of GIFT4 using IL-4 and GM-CSF ELISA. The effect of GIFT4 expression on the phenotype of the primary immune cells is under investigation using multiparameter flow cytometry. The tumor targeting potential of the GIFT4 transduced peritoneal immune cells is being investigated in an ID8 mouse model of ovarian cancer. Effects of GIFT4 on the activation of specific subtypes of ascetic immunocytes and their in vivo effects in neutralizing ID8 cells in immunocompetent C57BL/6 mice will be instrumental for developing a novel fusokine-based individualized immune cell therapy for ovarian cancer. Citation Format: Sejal Tarun Sharma, Pradyut Paul, Rahul Das, Shala Yuan, Lisa Barroilhet, Jessica Vazquez, Aleksandar K. Stanic, Jacques Galipeau, Manish Patankar. Synthetic fusion cytokine, GIFT4, transduced autologous peritoneal immune cells - a novel cell therapy for the treatment of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2845.

Abstract 975: Obesity-induced changes in the tumor microenvironment impact the response to chemotherapy and overall ovarian cancer metastatic success

Abstract Background The majority of women with epithelial ovarian cancer (OvCa) are diagnosed with metastatic disease, resulting in a poor 5-year survival of 31%. Obesity is a recognized epidemic that increases OvCa incidence, enhances metastatic success and reduces survival. We have previously demonstrated a link between obesity and OvCa metastatic success in a diet-induced obesity (DIO) mouse model where a significantly enhanced tumor burden was associated with a decreased M1/M2 tumor-associated macrophage (TAM) ratio (Liu Y et al. Can, Res. 2015; 75:5046-57). TAMs are the most abundant immune cell type in the ovarian tumor microenvironment and are generally categorized as M1-polarized (cytotoxic to tumor cells), or M2-polarized (growth promoting). For this study we sought to examine if a similar correlation between body mass index (BMI) and TAMs were also true for human ovarian cancer patients. Furthermore, we used the DIO mouse model to examine any potential effect of obesity on response to chemotherapy. Methods We analyzed high grade serous ovarian tumors from 6 normal weight patients (BMI 20-25), and 10 obese patients (BMI ≥35) for an in-depth analysis of immune cells using MultiOmyx™, an immunofluorescence (IF) multiplexing assay. After multiplexing FFPE sections with a custom panel of 13 immuno-oncology biomarkers, images were analyzed by applying the deep-learning based cell classification platform NeoLYTX.For the analysis of response to chemotherapy we used a DIO mouse model in which mice were fed a low-fat diet (LFD) vs high-fat diet (HFD) and then injected with ID8-Trp53-/- cells to establish tumor burden. Mice bearing equivalent tumor burden were treated with weight-adjusted standard of care chemotherapy. After sacrifice, remaining tumor burden was evaluated by quantitative fluorescence imaging. Results We found that the mean M1/M2 ratio in obese OvCa patients was nearly half (ratio=0.16) compared to patients who were normal weight (ratio=0.29), consistent with our previously published mouse data. Quantitation of residual tumor burden in the mouse model showed significantly reduced efficacy of chemotherapy (i.e., greater remaining tumor burden) in HFD mice (n=11/cohort). The M1/M2 TAM ratio in these mouse tumors is now under evaluation. Conclusions Our observations suggest that the negative impact of obesity on OvCa survival may be due in part to an increased M1/M2 TAM ratio and reduced response to chemotherapy. These data will be key to a more detailed understanding of the contribution of host obesity to ovarian tumor progression. Citation Format: Anna Juncker-Jensen, Yueying Liu, Tyvette S. Hilliard, Nicholas Stavrou, M Sharon Stack. Obesity-induced changes in the tumor microenvironment impact the response to chemotherapy and overall ovarian cancer metastatic success [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 975.

Abstract 2707: Factoring expression data of high-grade serous ovarian cancer tumors for unbiased longitudinal analysis

Abstract We address challenges in longitudinal RNA-seq analysis of patient tumors, which can reveal powerful insights on cancer evolution at personalized level, proposing methods for unbiased analysis and integration Experimental procedures: 214 whole-genome bulk RNA-seq samples from 61 high-grade serous ovarian cancer (HGSC) patients were collected before and after chemotherapy, along with single-cell RNA-seq data from &amp;gt;93,000 cells in 11 patients. 308 TCGA treatment-naive bulk RNA-seq samples from HGSC, 474 from melanoma (SKCM), and 581 from endometrial carcinoma (UCEC) were used for validation. Results: We show that in a longitudinal analysis, cancer progression and chemotherapy exert both microenvironmental and phenotypic changes. We developed PRISM [1] to factor the expression data at individual bulk level, and show the adjustment improves the association between expression profiles and patient survival. Our findings extend to pathway and expression-derived tumor subtype levels in HGSC and SKCM. Technical batch effects pose challenges for multi-institute or long-running sample collections. Unbiased correction requires replicates, which are typically unavailable for patient data. We developed POIBM [submitted] to simultaneously infer a suitable reference and factor out the batch effects. We show that POIBM effectively discovers true replicates, batch effects plague many cancer types in TCGA data, and batch correction allows more meaningful expression subtyping in UCEC. Distinct genomic backgrounds of the patients hinders stratification and discovering shared functional states. We developed PRIMUS [submitted] to simultaneously factor the patient background (or other confounders) and a sample clustering, which is necessary when the underlying stratification is uneven or shared by patient subsets. Among the commonly aberrant cell states, such as EMT, PRIMUS analysis on HGSC scRNA identified a novel stress signature, enriched by chemotherapy and indicating poor survival, which were validated in PRISM-factored TCGA bulk ovarian data. Conclusions: Our methodology facilitates unbiased longitudinal expression analysis and integration. The more accurate phenotypic changes at gene, pathway, and expression subtype level may confer sensitivity/resistance chemotherapy, and, as shown, allow enhance patient survival prediction and discovery of novel chemotherapy related subtypes. Consequently, our work aids ranking putative intervention strategies for overcoming ovarian cancer chemoresistance for future validation experiments. [1]: Hakkinen et al., Bioinformatics 37: 2882 (2021) Citation Format: Antti Häkkinen, Kaiyang Zhang, Susanna Holmström, Sanaz Jamaldazeh, Johanna Hynninen, Sakari Hietanen, Kaisa Huhtinen, Sampsa Hautaniemi. Factoring expression data of high-grade serous ovarian cancer tumors for unbiased longitudinal analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2707.

Abstract 2854: Logic gates controlled by priming receptors increase specificity and potency of CAR T cells

Abstract CAR T cell therapies for solid tumors are limited by a paucity of tumor-specific single target antigens and insufficient potency due to limitations of CAR T biology. Logic gated (LG) CARs increase control of cell therapy activation and target cell killing by requiring recognition of two or more antigens through AND or AND-NOT Boolean logic. They represent an attractive strategy for cell therapy to improve the therapeutic index by increasing specificity for tumor antigens. We created an AND logic gate cassette deliverable by CRISPR-mediated, non-viral, site-specific integration into human T cells. This logic gate activates expression of a MSLN-targeting CAR upon ALPP/G binding of a priming receptor (PrimeR) within high-grade serous carcinoma ovarian tumors. The PrimeR triggers proteolytic release of a chimeric, fully human transcription factor, which then translocates to the nucleus to induce expression of a minigene encoding a CAR. The PrimeR is built only from human protein sequences, reducing theoretical risk of cell therapy immunogenicity and rejection within a patient. In order to test the specificity of our LG CARs, we integrated LG cassettes into a defined site in the T cell genome, and then co-cultured T cells with K562 cells engineered with only one target antigen (K562-ALPG or K562-MSLN), both target antigens (K562-ALPG/MSLN), or neither target antigen (K562). LG CAR T cells only killed K562-ALPG/MSLN cells. The specificity of this LG was also demonstrated in vivo in a dual-flank K562 model, in which only the ALPG/MSLN tumor was inhibited by LG CAR T cells. CAR expression was not observed on LG CAR T cells recovered outside the ALPG/MSLN tumor, and no growth inhibition was observed in K562-MSLN tumors, unlike a constitutive CAR T control. To model priming antigen heterogeneity expected in tumors, we mixed K562-MSLN cells with varying proportions of K562-ALPG/MSLN cells. We found that the presence of only 5-15% ALPG/MSLN cells was sufficient to effect killing of all target cells by the induced MSLN CAR. The ALPG/MSLN LG CAR will be tested as a component of AB-1015, a novel therapy for indications including ovarian, fallopian tube, or primary peritoneal cancer. Citation Format: Jasper Williams, Kevin Martinez, Angela Boroughs, Laura Lim, Marian Sandoval, Cate Sue, Matthew Drever, Anzhi Yao, Joseph Choe, Maxim Sidorov, Sophia Phillips, Dina Polyak, Suchismita Mohanty, Stephen Santoro, Aaron Cooper, W. Nicholas Haining. Logic gates controlled by priming receptors increase specificity and potency of CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2854.

Abstract 2840: Identification of a safe harbor CRISPR-Cas9 integration site for improved cell therapy safety and potency

Abstract T cell therapies for cancer have shown striking clinical efficacy in different tumor settings. Integration of a transgene cassette expressing an antigen receptor into the genome enables T cells to recognize specific tumor antigens. Viral vectors and transposons can introduce transgenes into primary human T cells. However these approaches cannot control genomic location and have limited control over the copy number of cassette insertions. Here, we used epigenetic analysis, transcriptional profiling, high-throughput gene-editing and T cell functional experiments to identify the optimal genomic locus amenable to non-viral genome targeting. First we analyzed the epigenetic landscape of primary human T cells to identify regions (“gene deserts”) that are accessible but devoid of annotated regulatory elements or coding sequences. Candidate loci were validated using CRISPR/Cas9 genome editing to directly cut and a marker gene cassette was inserted into these sites. Knock-in efficiency and transgene expression stability in primary human T cells were evaluated for all loci. Evaluation of 40 loci demonstrated substantial variability (10 fold) in transgene expression and confirmed that not all sites were equivalent in their ability to sustain transgene expression. Next, we evaluated 8 most promising candidate loci for compatibility with complex T cell programs embodied by integrated circuits, containing a priming receptor (PrimeR) that triggers expression of a CAR in response to a priming antigen. We identified one integration site (termed “GS94”) that supported: 1) stable and high PrimeR expression; 2) high and inducible CAR expression; and 3) a superior T cell cytotoxic and cytokine secretion profile. Finally, we assessed off-target cutting mediated by the CRISPR sgRNA targeting GS94. The sgRNA is the most specific out of the candidates evaluated with iGUIDE and targeted PCR. We were unable to detect any measurable off-target activity by targeted PCR on predicted sites. The GS94 site is a component of the integrated circuit T cell (ICT) therapy, AB-1015, which is now under development for the treatment of high-grade serous carcinoma ovarian tumors. AB-1015 includes an inserted cassette that contains a logic gate and two shRNAs that enhance the potency and solid tumor microenvironment resistance of the AB-1015 ICT cells. These computational and experimental approaches can be generalized to identify new safe harbor sites for different cell types, expanding the genome engineering toolkit for diverse cell therapy applications. Citation Format: Grace X.Y. Zheng, Somya Khare, Brendan Galvin, Robby Moot, Aaron Cooper, Michelle Nguyen, Michelle Tan, Shan Sabri, Audre May, Jun Fung, Anzhi Yao, Andrea Liu, Matt Drever, Steve Santoro, W Nicholas Haining, Tarjei Mikkelsen. Identification of a safe harbor CRISPR-Cas9 integration site for improved cell therapy safety and potency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2840.