High-grade Serous Ovarian Carcinoma Research Articles

Prediction of homologous recombination deficiency from Oncomine Comprehensive Assay Plus correlating with SOPHiA DDM HRD Solution.

Poly(ADP-ribose) polymerase (PARP) inhibitors are used for targeted therapy for ovarian cancer with homologous recombination deficiency (HRD). In this study, we aimed to develop a homologous recombination deficiency prediction model to predict the genomic integrity (GI) index of the SOPHiA DDM HRD Solution from the Oncomine Comprehensive Assay (OCA) Plus. We also tried to a find cut-off value of the genomic instability metric (GIM) of the OCA Plus that correlates with the GI index of the SOPHiA DDM HRD Solution. We included 87 cases with high-grade ovarian serous carcinoma from five tertiary referral hospitals in Republic of Korea. We developed an HRD prediction model to predict the GI index of the SOPHiA DDM HRD Solution. As predictor variables in the model, we used the HRD score, which included percent loss of heterozygosity (%LOH), percent telomeric allelic imbalance (%TAI), percent large-scale state transitions (%LST), and the genomic instability metric (GIM). To build the model, we employed a penalized logistic regression technique. The final model equation is -21.77 + 0.200 × GIM + 0.102 × %LOH + 0.037 × %TAI + 0.261 × %LST. To improve the performance of the prediction model, we added a borderline result category to the GI results. The accuracy of our HRD status prediction model was 0.958 for the test set. The accuracy of HRD status using GIM with a cut-off value of 16 was 0.911. The Oncomine Comprehensive Assay Plus provides a reliable biomarker for homologous recombination deficiency.

Abstract 2418: Copy number variation status of DNA in extracellular vesicles as novel biomarkers of high-grade serous ovarian carcinoma

Abstract Objective: Ovarian cancer is a gynecologic malignancy with a dismal prognosis due to the lack of accurate methods for early diagnosis methods and effective therapies. High-grade serous ovarian carcinoma (HGSOC) is the most common subtype and copy number variations (CNVs) are the dominant genomic events in HGSOC. Extracellular vesicles (EVs) circulate in body fluids and carry pathological genomic information positively. In this study, we evaluated the significance of EV-DNA as novel biomarkers for HGSOC patients. Methods: A total of 124 samples from HGSOC patients and cell lines were analyzed. EVs were isolated using the ultracentrifuge method. Comprehensive CNV statuses were analyzed by whole genome sequencing (WGS) and SNP array. In reference to the Ovarian Cancer Moon Shot database, 30 dominant CNVs in HGSOC were selected as dominant targets for analysis by droplet digital PCR (ddPCR). For predicting the response to PARP inhibitors, HGSOC patients who were treated with olaparib were collected, and DNA was extracted from FFPE tissue at the time of surgery. Results: NTA assays and cryo-electron microscopic analyses showed that the EVs were successfully isolated from all samples. Both WGS and SNP arrays detected correlating CNVs in cell-DNA and EV-DNA for cell lines, and in tissue-DNA and EV-DNA in ascites for patients. The CNV status was also accurately measured by ddPCR. Copy numbers of RAD51, BRCA1, AKT2, CCNE1, and MSH6 were significantly higher in malignant tissue than in benign tissue (p<0.0001, p<0.0001, p=0.0012, p=0.002. p=0.0328, respectively), and were detectable in EV-DNA from ascites. Additionally, the amount of DNA in malignant ascites was significantly higher than in benign ascites (p=0.016), and increased DNA in ascites itself suggested the presence of malignancy. Furthermore, EV-DNA more accurately reflected the CNV status of tumor DNA than cell-free DNA in ascites. Regarding the prediction of PARP inhibitor response, the CNV status in EV-DNA from patients who were treated with olaparib, a PARP inhibitor, demonstrated a marked difference between responders and non-responders. The equation calculated based on the combination of six genes (RB1, GABRA6, CTNNB1, MYC, RAD51, BARD1) could predict the response to olaparib (AUC: 1.0). The selected genes were validated in vitro using EV-DNA, and responders could be identified using the prediction equation calculated from CNV status of the selected genes in EV-DNA from ascites. Conclusion: CNV status of EV-DNA was correctly evaluated and can serve as novel diagnostic and prognostic biomarkers for HGSOC. Citation Format: Ryosuke Uekusa, Akira Yokoi, Kosuke Yoshida, Juntaro Matsuazki, Yusuke Yamamoto, Hiroaki Kajiyama. Copy number variation status of DNA in extracellular vesicles as novel biomarkers of high-grade serous ovarian carcinoma [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 2418.

Abstract 7287: High-grade serous ovarian carcinoma detected with TP53 mutation panel and SiMSen-Seq

Abstract Background: Ovarian carcinoma (OC) has the highest mortality among gynecologic malignancies due to absent or unspecific symptoms. The high-grade serous ovarian carcinoma (HGSC) accounts for over 60% of the OC associated deaths. The most frequent genetic aberrations in HGSC are mutations in the TP53 gene, detected in approximately 96% of the tumors. Previous studies have proposed potential diagnostic utility of detecting mutations in circulating tumor DNA (ctDNA) from OC patients. However, the need for diagnostic methods capable of detecting asymptomatic HGSC is unmet. Objectives: The primary aims of this study were to design a screening tool for identification of HGSC and to evaluate the suitability of various clinical specimen types for diagnostic applications. Methods: In this study, we designed a highly sensitive and robust screening tool using the simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing (SiMSen-Seq) technique for identification of HGSC (n=11) by TP53 mutation profile in various clinical specimen for diagnostic applications. Specimens sampled included primary tumors, ascites, plasma, vaginal samples, and both liquid and solid samples from cyst-, endocervical-, and endometrial origin (n=94). Results: The in-house designed panel amplified 17 regions, encompassing 619 nucleotide positions within the TP53 gene. Pathogenic variants were identified in 10/11 of the primary tumors, with a VAF ≥ 9% (range: 9-91). Preliminary data indicated that all (100%) paired samples obtained corresponding somatic mutations between two or more of the different compartments from the same patient. In total 10/10 ascites-, 16/16 cyst-, 8/8 plasma-, 5/8 vaginal-, 17/22 endocervical-, and 14/19 endometrial samples showed somatic variants at a consensus read depth of 3 and a minimum VAF ≥ 0.1%. Conclusion: The present study suggests that the TP53 mutation panel can identify somatic variants in a variety of non-invasive liquid biopsies. Additionally, the assessment of the TP53 mutation panel exhibits potential for forthcoming clinical utilization as a diagnostic tool. Citation Format: Amanda Olsson Widjaja, Peter Micallef, Benjamin Ulfenborg, Maria Lycke, Ulf Gyllensten, Therese Carlsson, Tobias Österlund, Anders Ståhlberg, Anna Linder, Karin Sundfeldt. High-grade serous ovarian carcinoma detected with TP53 mutation panel and SiMSen-Seq [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 7287.

Abstract 5070: Landscape of clonal hematopoiesis prior to PARP inhibitor treatment in patients with ovarian cancer

Abstract Background: Poly (ADP-ribose) polymerase inhibitors (PARPi) are an important tool for treatment of ovarian and other cancers, particularly for those with mutations in BRCA1 or 2 genes or homologous recombination deficiency. The risk of developing secondary hematologic malignancy, particularly myelodysplastic syndrome or acute myeloid leukemia (MDS/AML), is substantially higher (4-12%) after PARPi maintenance or treatment in the second line and beyond in patients with ovarian cancer. This elevated risk necessitates additional investigation into the pathogenesis of PARPi-related secondary malignancy. Clonal hematopoiesis of indeterminate potential (CHIP) has been associated with the development of therapy-related myeloid neoplasms. We aim to understand the patterns of CHIP in patients with high grade serous ovarian carcinoma who receive PARPi therapy. Methods: We performed ultra-high-depth whole exome sequencing of plasma derived cfDNA or white blood cells (WBCs) barcoded with unique molecular identifiers from patients with high grade serous ovarian carcinoma who were treated with multiple lines of chemotherapy (n=6) or with PARPi in addition to chemotherapy (n = 10). Using spike-in mutated DNAs as positive controls, we detected variant alleles as low as 1% variant allele frequency. Gene set enrichment analysis was performed on identified groups. Results: We identified 7162 recurrent variants among our patients. We began by comparing quantities of CHIP between the samples from patients who eventually receive PARPi and those who do not. Samples contained a median of 2030 recurrent CHIP variants and 4300 total variants. No differences in total CHIP quantity or mutation type was discernable among samples from patients who will receive PARPi (BRCA1/2 enriched) vs those who will not. Among our 16 patients, 14 ultimately developed secondary hematologic malignancy (t-MN+ Comparison of t-MN+ (n=14) and t-MN- (n=2) defined a group of 14 variants enriched in the t-MN+ population and a group of 85 variants depleted in the t-MN+ population. The t-MN+ enriched variants we identified were not previously associated with CHIP and had significant overlaps with alkyl transferase activity (FDR q val < 0.01) and nucleotide binding gene sets (FDR q val < 0.01). Conclusions: We used a novel methodology to identify unbiased low variant mutational changes in the hematopoietic system. These results suggest that baseline clonal hematopoiesis variants are similar among patients newly diagnosed with ovarian carcinoma. We have also identified a signature potentially related to future risk of leukemia requiring further validation. Citation Format: Amma Asare, Sara Corvigno, Jun Yao, Li Zhao, Nicole D. Fleming, Joseph Celestino, Richard A. Hajek, Mark S. Kim, Alejandra Flores Legarreta, Karen H. Lu, Koichi Takahashi, P Andrew Futreal, Amir A. Jazaeri, Shannon N. Westin, Anil K. Sood, Sanghoon Lee. Landscape of clonal hematopoiesis prior to PARP inhibitor treatment in patients with 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 5070.

Abstract 7064: BRCA 1/2 haploinsufficiency-associated metabolic dysfunction in benign fallopian tube epithelia

Abstract In 3-17% of women with BRCA1/2 mutations who undergo risk-reducing surgery, pre-neoplastic and neoplastic lesions are found in their fallopian tubes that display differential loss of heterozygosity of BRCA1/2. Precisely how mutations in BRCA1/2 predispose women to develop high grade serous ovarian carcinoma (HGSC) is not completely understood. Although linked to various processes localized in the nucleus, BRCA1/2 may also have essential roles in the cytoplasm, specifically in the regulation of key processes associated with mitochondrial homeostasis and metabolism. Understanding how haploinsufficiency of BRCA1/2 contributes to the potential neoplastic transformation of fallopian tube epithelial cells should reveal new insights into BRCA1/2 function, and ultimately provide novel opportunities for preventative interventions in individuals with this mutation and new treatment opportunities for HGSC associated with BRCA1/2 heterozygosity. We have identified distinct metabolic and mitochondrial deficits in BRCA2 mutation carriers compared to BRCA1 carriers and non-carriers. Fallopian tube epithelial cell lines derived from BRCA2 mutation carriers showed increased levels of mitochondrial respiration and activity followed by BRCA1 mutation carriers and non-carriers. Western blot analysis of mitochondrial proteins regulating glycolysis and oxidative phosphorylation showed differential expression based on mutation status. Transmission electron microscopy of BRCA2 mutation carriers showed increased disrupted mitochondrial networks when compared to wild type, suggesting that mutation status may contribute to increased mitochondrial fission. These observations reveal an entirely new aspect of BRCA1 and BRCA2 biology and further studies will disaggregate the effects of BRCA1 and BRCA2 on mitochondrial function, versus DNA damage repair. Ultimately, this work will provide new insight into the adaptive changes within FT cells which promote neoplastic transformation and inform how high-grade serous carcinoma (HGSC) cells distinctively respond to microenvironmental stimuli. Citation Format: Jovanka Ravix, Iru Paudel, Gauthami Pulivendala, Ramlogan Sowamber, Elizabeth Smith, Matthew Schlumbrecht, David Lombard, Sophia George. BRCA 1/2 haploinsufficiency-associated metabolic dysfunction in benign fallopian tube epithelia [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 7064.

Abstract 4538: Brigatinib induces synergy with PARP inhibitors through dual inhibition of FAK and EphA2 in high-grade serous ovarian carcinoma

Abstract PARP inhibitors (PARPis) have now become a standard of care for high-grade serous ovarian cancer (HGSOC), especially for those with defects in homologous recombination (HR) DNA repair. However, the innate and acquired PARPi resistance in HGSOC poses a significant concern, necessitating the exploration of novel treatment options and strategies. Here we investigated the potential of combining brigatinib—a second-generation anaplastic lymphoma kinase (ALK) inhibitor approved for treating lung cancer—with PARPis as way to enhance PARPi activity in HGSOC. Clonogenic cell survival assays revealed that brigatinib synergizes with PARPis in HR-proficient and -deficient HGSOC cell lines. In subsequent proteomics approaches and cell-based confirmatory studies, we found that brigatnib suppressed tyrosine kinases FAK and EphA2 with reduction in downstream signaling. Follow-up pharmacologic and gene-silencing techniques demonstrated that dual FAK and EphA2 inhibition additively reduces PI3K/Akt and MAPK/ERK cell survival signaling to levels conferring PARPi sensitivity. Additionally, the efficacy of this therapeutic approach on tumor size reduction was assessed in HGSOC patient-derived xenograft (PDX) models, with combination treatment inducing tumor regression more effectively than either agent alone. Overall, our studies unveil a beneficial ALK-independent effect of brigatinib that may offer new avenues for considering its repurposing as a HGSOC therapy. Citation Format: Julie R. Duffield, Xiaonan Hou, Iman K. McKeon-Makki, Amelia M. Huehls, Benjamin W. Wilson, Anjali Prasad, Xinyan Wu, Scott H. Kaufmann, Larry M. Karnitz, John J. Weroha, Arun Kanakkanthara. Brigatinib induces synergy with PARP inhibitors through dual inhibition of FAK and EphA2 in high-grade serous ovarian carcinoma [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 4538.

Abstract 4940: Unraveling ctDNA features in high-grade serous ovarian carcinoma and insights from genomic and transcriptomic integration

Abstract High-grade serous carcinoma (HGSC) is often diagnosed at advanced stages with less than 40% of patients surviving for five years after diagnosis. Circulating tumor DNA (ctDNA) is a minimally invasive technology that can provide crucial insights into tumor biology. Unique features from ctDNA, fragment patterns and nucleosome footprints at transcription start sites (TSS), complement genomic variants. Our study explored ctDNA features in HGSC, integrating with tumor biopsies' genomic and transcriptomic profiles. We focus on two aspects in HGSC: understanding how ctDNA features assist genomic profiling and track dynamic changes during treatment and exploring the biological factors impacting ctDNA release. We used 500+ longitudinal plasma and 400+ pretreatment tissue samples from HGSC patients in the DECIDER study (NCT04846933). Shallow whole-genome sequencing (sWGS) with 0.4x average coverage was conducted on plasma. Integration of DNA and RNA pretreatment tissue data was performed for 98 and 91 patients with available plasma before treatment, respectively. Plasma samples underwent analysis for fragment patterns and TSS signals. Somatic mutations and mutational signatures were retrieved for patients with available WGS tissue data. Pathway enrichment analysis was applied to diverse gene expression profiles. WGS data from 29 healthy individuals (Zhu, G. et al. (2023)) served as controls. DNA fragment profiles and TSS signals distinguish ctDNA from healthy samples. The ability to cluster sample phases based on ctDNA features facilitates tracking longitudinal changes. Integrating tissue DNA and RNA data reveals a positive correlation between high ctDNA release and mutational burden, single-base substitution signature SBS1 as a marker for cell division/mitotic clock and doublet-base substitution signatures linked to late replication. These mechanisms help explain why high ctDNA release group (n=40) is associated with a shorter progression-free survival. Additionally, transcriptomics data indicates associations with proliferation pathways in cancer cells and ctDNA release. The concordance in results from DNA and RNA points to a higher proliferation rate in patients with high ctDNA release, suggesting the need for alternative treatment compared to those with low ctDNA release. In conclusion, our results provide a multifaceted perspective on ctDNA in HGSC. The identification of longitudinal ctDNA features signifies the capability to stratify sample states and show the potential findings through fragment profiles and the discernment of key TSS regions. The proposed aetiologies and enriched pathways associated with ctDNA release, mutation burden and proliferation, shed light on potential treatment avenues. These findings contribute to our understanding of ctDNA in HGSC and offer insights for personalized treatment strategies and monitoring approaches. Citation Format: Mai TN Nguyen, Jaana Oikkonen, Yilin Li, Daria Afentave, Johanna Hynninen, Sampsa K. Hautaniemi. Unraveling ctDNA features in high-grade serous ovarian carcinoma and insights from genomic and transcriptomic integration [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 4940.

Abstract 565: PDE7 modulates EMT, cells morphology and mitochondrial cristae in high grade serous ovarian cancer

Abstract Background: Due to the acquisition of chemoresistant phenotype by epithelial ovarian cancer (EOC) cells and, consequently, disease relapse, the identification of novel therapeutic strategies is urgent. Herein, we targeted phosphodiesterase 7A (PDE7A) in EOC cells, based on data derived from RNA-sequencing assays comparing high-grade serous ovarian carcinoma (HGSOC) and fallopian tube samples. Methods: To understand the role of PDE inhibition, we evaluated the effects of its inhibitor BRL50481 alone or in combination with paclitaxel (PTX) on untreated A2780 EOC cells and multi-resistant high grade serous ovarian cancer (HGSOC) OVCAR3 cells. To do so, we pursued with the: validation of data obtained by high throughput RNA sequencing assays using qRT-PCR; evaluation of PDE7 expression in normal and EOC cells, as well as in normal Fallopian tube cells by qRT-PCR; immunoblotting; metabolic cell viability (MCV) by MTT assays; cytokine dosage by qRT-PCR and ELISA; scanning electron microscopy; transmission electron microscopy; statistical analysis (GraphPad software). Results: Drug-sensitive A2780 cells, but not OVCAR3 cells, exhibited lower MCV when treated with the PDE7 inhibitor; however, the combinatory therapy decreased MCV in both cell lines. Of clinical relevance, pre-treatment of A2780 and OVCAR3 with the PDE7 inhibitor BRL50481 followed by PTX lowered its IC50 by 103- and 625-fold, respectively. While the phosphatidylinositol 3-kinase (PI3K)/protein kinase B(AKT)/mammalian target of rapamycin (mTOR) pathway was inhibited in both cell lines, the pro-apoptotic protein Bcl-2 Associated X-protein (BAX) was overexpressed in A2780 cells only. Analysis of mRNA and protein expression in OVCAR3 pretreated with BRL50481 showed a 2-fold increase in IL-6 cytokine expression. Pre-treatment of OVCAR3 with BRL50481 followed by the combination of BRL50481 and PTX inhibited the expression of vimentin and Octamer-binding transcription factor 4 (OCT4) by 2-fold. BRL50481 promoted changes in EOC cells morphology and mitochondrial cristae. Conclusions: Altogether, our data led us to postulate that inhibition of PDE7 in EOC cells, specifically in those presenting the chemoresistant phenotype, is a promising strategy to fight this yet highly lethal disease. Citation Format: Leticia B. A. Rangel, Nayara Gusmão Tessarollo, Isabella dos Santos Guimarães, Diandra Zipinotti dos Santos, Taciane Barbosa Henriques, Marcele Lorentz de Souza, Leide Laura Figueiredo Maciel, João Carlos Aquino Almeida, Tatiana Massariol Pimenta, Josiany Carlos de Souza, Ranna Batista Wanzeler, Barbara Silva Martins, Solenny Maria Silva Butzene, Jose Matheus Simões Padilha. PDE7 modulates EMT, cells morphology and mitochondrial cristae in high grade serous 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 565.

Abstract 6320: CAR-T cells targeting LGR5: An effective treatment for chemotherapy resistant ovarian cancer

Abstract Ovarian cancer is the second leading cause of cancer-related mortality among gynaecological cancers in the Western world. CAR-T cell therapy is an adoptive immunotherapy used to treat some blood cancers. This study has investigated whether leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a suitable target for CAR-T cell therapy for ovarian cancer. We assessed the cytotoxic effects of LGR5-targeting CAR-T cells on a range of ovarian cancer cell lines and primary serous ovarian cancer cells derived from patient ascites (at diagnosis and following relapse with chemotherapy resistant disease) in monolayer culture, 3D spheroid assays and patient-derived explant assays. OVCAR3 and a chemoresistant patient-derived xenograft (PDX) models have been used to assess whether LGR5-targeting CAR-T cells can inhibit growth and metastasis in vivo. We found that LGR5 is highly expressed in high-grade serous ovarian carcinoma (HGSOC) cancer cell lines and LGR5 expression was increased in chemotherapy resistant primary ovarian cancer cells. We demonstrated that LGR5-targeting CAR-T cells were cytotoxic and significantly inhibited the survival of ovarian cancer cell lines and chemotherapy-resistant primary ovarian cancer cells with high LGR5 expression levels in both monolayer culture and 3D spheroids compared to un-transduced CD3+ T cells. In patient-derived explant assays, we observed increased apoptosis in HGSOC tissues expressing high LGR5 following treatment with LGR5-targeting CAR-T cells. In vivo, LGR5-targeting CAR-T cell treatment decreased the tumour burden in mice with OVCAR3 xenografts and a chemotherapy-resistant PDX model with high LGR5 expression, compared to CD3+ T cells. In summary, LGR5-targeting CAR-T cells have great potential to be developed as a novel immunotherapy for ovarian cancer. Citation Format: Wanqi (Jady) Wang, Veronika Bandara, Noor A. Lokman, Zoe K K. Price, Tannith M. Noye, Silvana Napoli, Batjargal Gundsambuu, Martin K. Oehler, Simon C. Barry, Carmela Ricciardelli. CAR-T cells targeting LGR5: An effective treatment for chemotherapy resistant 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 6320.

Abstract 1047: Expression of TYMS in cancer: A tissue microarray study involving 6,613 cancers from 127 tumor entities

Abstract Thymidylate synthase (TS, TYMS) is an enzyme that catalyzes the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) which is one of the nucleotides forming the DNA. TYMS is thus essential for DNA synthesis and represents an important target for several chemotherapeutic drugs including 5-fluorouracil (5-FU) and methotrexate. It has been suggested that tumors with low-levels of TYMS may show a better response to 5-FU than those with high-level expression. Literature data on TYMS expression in different cancer types is controversial, however. To better comprehend the role of TYMS expression in cancer, TYMS was analyzed by immunohistochemistry (IHC) on tissue microarrays (TMAs) containing 9,015 samples from 127 different tumor types as well as 8 samples each of 6 different normal tissue types. TYMS immunostaining was always cytoplasmic and/or nuclear. It was only detectable in a minority of normal cell types and was strongest in a subset of lymphoid cells, especially in germinal centers and in the thymus as well as in hematopoetic cells of the bone marrow. Detectable TYMS staining occurred in 3,749 (46.7%) of the 8,030 analyzable tumors, including 2,958 (36.8%) with weak, 609 (7.6%) with moderate, and 182 (2.3%) with strong positivity. A total of 114 of 127 tumor categories showed TYMS expression in at least one case, 64 tumor categories showed TYMS staining ≥50% of cases, and 47 tumor categories included at least one case with strong TYMS positivity. The frequency of TYMS positivity was highest in various subtypes of B- and T-cell lymphoma (80-100%), sarcomas, carcinosarcomas and sarcomatoid carcinomas (33.3-100%), primary and metastatic malignant melanoma (70.5-90.7%), adenocarcinoma of the uterine cervix (78.3%), squamous cell carcinomas of various sites of origin (57.1-77.9%), and testicular germ cell tumors (19.4-67.4%). In all four tumor entities for which clinico-pathological data could be assessed, high TYMS expression was associated with features of aggressive disease. High TYMS expression was linked to high histologic grade (p<0.0001) in endometrioid endometrium carcinoma, advanced pT stage (p=0.0061) in high-grade serous ovarian carcinoma, poor histologic grade (p<0.0001) in squamous cell carcinomas of 11 different organs of origin, and to advanced stage (p<0.0001), tumor invasion in blood and lymph vessels (p≤0.0064) as well as microsatellite instability (p<0.0001) in colon cancers. TYMS is often overexpressed as compared to normal tissues in a broad range of different cancer entities. Our data suggest that high-level of TYMS expression is a feature of cancer aggressiveness in many different cancer types. Citation Format: Florian Lutz, Nicolai Leege, Katharina Möller, Florian Viehweger, Ria Schlichter, Andreas M. Luebke, Martina Kluth, Claudia Hube-Magg, Andrea Hinsch, Christian Bernreuther, Guido Sauter, David Dum, Andreas H. Marx, Ronald Simon, Till Krech, Till S. Clauditz, Frank Jacobsen, Eike Burandt, Stefan Steurer, Patrick Lebok, Christoph Fraune, Sarah Minner, Natalia Gorbokon, Maximilian Lennartz. Expression of TYMS in cancer: A tissue microarray study involving 6,613 cancers from 127 tumor entities [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 1047.

Abstract 5727: ISM9682A, a novel and potent KIF18A inhibitor, shows robust antitumor effects against chromosomally unstable cancers

Abstract KIF18A belongs to the kinesin-8 subfamily and is a plus-end-directed motor, which regulates kinetochore microtubules dynamics to control mitotic chromosome alignment and spindle tension. Studies have shown that genetic depletion or pharmacological inhibition of KIF18A is a promising therapeutic approach for cancers, exhibiting high chromosomal instability (CIN-high), without having a significant impact on the normal division of euploid cells. Here, we report the discovery and preclinical evaluation of ISM9682A, a novel KIF18A inhibitor with excellent potency and high selectivity. ISM9682A is a potent KIF18A inhibitor with a single-digit nanomolar IC50 value and exhibits high selectivity over other kinesin family members. In addition, ISM9682A demonstrated excellent monotherapy anti-proliferative activity, with single-digit nanomolar IC50 values, in high-grade serous ovarian carcinoma (HGSOC) as well as in triple-negative breast cancer (TNBC) aneuploid cell lines with p53 mutation. Notably, ISM9682A exhibited a prolonged drug-target residence time indicated by both enzymatic and cellular assays. Consistent with the in vitro findings, ISM9682A displayed significant anti-tumor efficacy with a robust pharmacodynamic response (pH3, mitotic marker; γH2AX, DNA damage marker) in OVCAR3 and HCC1806 CDX models in a dose-dependent manner. In addition to its robust biological potency and high selectivity, ISM9682A exhibits favorable drug-like properties, including desirable in vitro ADMET characteristics and excellent in vivo exposure, clearance, as well as good oral bioavailability across multiple preclinical species. Collectively, these findings highlight the potential of ISM9682A as a novel and highly potent KIF18A inhibitor for the treatment of CIN-high cancers. Citation Format: Jie Zhang, Feng Gao, Wei Zhu, Chenxi Xu, Xiaoyu Ding, Jing Shang, Junwen Qiao, Shan Chen, Xin Cai, Xiao Ding, Supriya Bavadekar, Sujata Rao, Man Zhang, Feng Ren, Alex Zhavoronkov. ISM9682A, a novel and potent KIF18A inhibitor, shows robust antitumor effects against chromosomally unstable cancers [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 5727.

Abstract 1564: NEU4-mediated desialylation ignites the oncogenic receptors for the dissemination of ovarian carcinoma

Abstract Background: Glycosylation profoundly influences the interactions between cancer cells and microenvironmental stromal cells during the peritoneal disseminated metastasis of ovarian carcinoma (OC), which is the major cause of cancer-related death. Although the characteristic cancer glycoconjugates are widely used as biomarkers for cancer diagnosis, our knowledge about cancer glycome remains quite fragmented due to the technique limitations in analyzing glycan chains with tremendous structural and functional heterogeneity. Method: Given the dysregulated cancer glycome is defined by the altered glycosylation machinery, here we performed a systematic loss-of-function screen on 498 genes involved in glycosylation for key regulators of OC dissemination. We identified neuraminidase 4 (NEU4), an enzyme capable of hydrolyzing terminal sialic acid from glycoconjugates, as a vital peritoneal dissemination-promoting modifier of OC glycome. What’s more, we also identified membrane proteins whose sialylation was able to be modified by NEU4 in OC cells by pull down and mass spectrum to investigate the functional significance of NEU4-mediated desialylation. Results: In human patients with high-grade serous OC (HGSOC), increased NEU4 was detected in the disseminated OC cells when compared with that in the primary tumor cells, which significantly correlated with the worse survival. Among three alternative splice-generated isoforms of human NEU4, we revealed that only the plasma membrane-localized NEU4 isoform 2 (NEU4-iso2) and intracellular isoform 3 promoted the peritoneal dissemination of OC by enhancing the cell motility and epithelial-mesenchymal transition. We also identified NEU4-iso2-regulated cell surface glycoproteome and found that NEU4-iso2 desialylated the epithelial growth factor receptor (EGFR), in particular at N196 residue, for the hyperactivation of EGFR and its downstream tumor-promoting signaling cascades. Conclusion: Our results provide new insights into how the OC glycome is dysregulated by NEU4 during OC progression and reveals a functionally important glycosite on EGFR for its abnormal activation in cancer. Citation Format: Jie Shi, Rui Zhou, Shuo Wang, Yuxin Liu, Baorui Tian, Yanhua Liu, YaNan Chen, Taoyu Hu, Yuhao Mu, Shufan Wang, Xintao Shao, Jie Yan, Pengpeng Qu, Wei Ding, Shuang Yang, Yi Shi, Jia Li, Longlong Wang. NEU4-mediated desialylation ignites the oncogenic receptors for the dissemination of ovarian carcinoma [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 1564.

Abstract 7014: Integrative DNA methylation cancer profiling reveals patterns associated with tumor progression and treatment response

Abstract DNA methylation plays a critical role in tumor progression and response to chemotherapy. Frequently dysregulated during tumor development, it is closely intertwined with mixed effects from patient epigenetic background, tissue context, and treatment pressure. Here, we leveraged methylomic and transcriptomic profiling of 400 whole-genome sequencing samples from 125 patients with ovarian high-grade serous carcinoma (HGSC), belonging to the prospective, longitudinal, multi-region DECIDER trial (NCT04846933). This cohort included 120 samples from tubo-ovarian tumors, 209 samples from intra-abdominal metastases, and 71 ascites, representing all major sites of HGSC metastases. We profiled cancer methylome across tissues, patient groups, and various anticancer treatments using decomposition method, which allows separating the effects of tumor microenvironment and treatment, tissue, and patient specific changes, thus, facilitating integration of the heterogeneous collection of samples. First, we systematically examined the tissue-specific carcinogenesis and characterized methylomes of all types of metastases and excess fluid accumulations in the abdominal cavity. In our analyses, the cancer spread from tubo-ovarian sites to metastatic deposits involved gradual loss of DNA methylation and increase in transcriptomic levels, possibly indicating stemness. Specifically, several members of the ATP-binding-cassette (ABC) family of transporters, as well as receptor tyrosine kinases were frequently hypomethylated in bowel and omentum. The methylome of ascites showed most commonality to the tubo-ovarian tumors and peritoneum. Our results confirmed that several well-known tumor-associated genes, such as PTEN, GSK3B, ESR1, FGFR2, TNC, BCL6, HIF1A, NOTCH2 are regulated epigenetically, and this dysregulation stems early from the sites of origin. Next, we built methylomic profiles of patients with various treatment responses. Direct comparisons between good and poor responders revealed pronounced methylation differences, with refractory disease being associated with severely aberrated methylome prior to chemotherapy treatment. Neoadujuvant chemotherapy resulted in very few changes to these epigenetic aberrations and no acquired mechanisms, suggesting that intrinsic methylomes of poor responders accommodate multiple drivers for EMT, hypoxia, and chronic inflammation to resist chemotherapy-induced cell death already prior to treatment. In conclusion, using a large whole-genome sequencing DNA methylation dataset we elucidated the major underlying epigenetic mechanisms of tumorigenesis and platinum resistance, which provide possible drug targets for improving platinum sensitivity. Our multi-omics analyses enabled the identification of aberrated patterns that drive transcriptional changes, shedding light on epigenetic landscape of HGSC. Citation Format: Alexandra Lahtinen, Giovanni Marchi, Daria Afenteva, Kari Lavikka, Susanna Holmström, Elsi Pöllänen, Giulia Micoli, Ilari Maarala, Yilin Li, Taru A. Muranen, Jaana Oikkonen, Sakari Hietanen, Anni Virtanen, Johanna Hynninen, Antti Häkkinen, Sampsa Hautaniemi. Integrative DNA methylation cancer profiling reveals patterns associated with tumor progression and treatment response [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 7014.

Abstract 1617: Tumor evolution during therapy in ovarian cancer

Abstract The evolution of tumors from diagnosis to relapse in ovarian high-grade serous carcinoma (HGSC) patients is poorly understood. Consequently, it is unknown whether diagnostic samples are representative enough to guide treatment directions at relapse, or whether acquired changes during therapy provide targets in patients with relapses. To answer these questions and characterize tumor evolution during treatment, we used data from >700 tissue and plasma samples from 200 HGSC patients belonging to the observational, longitudinal DECIDER trial (NCT04846933). Mutations from whole-genome sequencing data from tissue samples (2-12 per patient) were used to construct phylogenetic trees. The trees were corrected by the copy-number alterations and curated to integrate circulating tumor DNA (ctDNA) exome- and targeted-sequencing data from plasma samples. Evolutionary states presenting within and between sample heterogeneity were evaluated through clonal complexity and divergence. Additionally, genetic differences between persistent and non-persistent subclones were studied to identify features affecting treatment resistance. Our results indicate that the majority of subclones survive chemotherapy treatment and major subclonal replacement takes place in only a few patients. Within-sample heterogeneity was higher in plasma samples than in ascites, highlighting the importance of ctDNA samples to estimate persistent subclones. Additionally, our recently reported evolutionary states (Lahtinen et al. Cancer Cell 2023) were often altered by the treatment induced selection pressure. At late relapses, subclone compositions typical of maintaining evolutionary state tumors were less common, and a higher number of acquired subclones were detected, suggesting further evolution of the persisting clones. In summary, chemotherapy does not suppress heterogeneity in HGSC tumors. Interestingly, patients with the worst prognosis have a stable composition of multiple clones in their tumors, and tumors with low heterogeneity responded better to primary treatment. Relapse samples show lower within-sample heterogeneity and further evolution. Furthermore, we detected several evolutionary trajectories from stable, persistent cancer subclone populations to highly dynamic subclone structures. This renders diagnostic samples suitable to guide relapse treatment decisions in a subset of patients with HGSC but not for all. Our characterization of the dynamics of tumor clonal heterogeneity during treatment allows to define better therapeutic interventions for HGSC patients with relapsed disease. Citation Format: Sampsa Kalervo Hautaniemi, Jaana Oikkonen, Alexandra Lahtinen, Mai T.N. Nguyen, Giovanni Marchi, Kari Lavikka, Anna Rajavuori, Sakari Hietanen, Anni Virtanen, Johanna Hynninen. Tumor evolution during therapy 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 1617.

Abstract 5169: Circulating levels of ANGPTL3, PCSK9, apoCIII and lipoprotein (a) in high-grade serous ovarian cancer

Abstract Background: Ovarian cancers require abundant amounts of lipids for their growth. Recent years have highlighted the role of ANGPTL3, PCSK9, ApoCIII and Lp(a) as key players in lipid metabolism. But circulating levels of these factors in ovarian cancers have not been thoroughly investigated to date. Objective: We assessed the profiles of plasma ANGPTL3, PCSK9, ApoCIII and Lp(a) in ovarian high-grade serous carcinoma (HGSC). Methods: Plasma samples collected from women diagnosed with an HGSC (n=31) or a benign ovarian lesion (BOL, n=40) were analyzed for plasma lipid profile (ApoB, total cholesterol, HDL, triglycerides, Lp(a) levels) on a clinical modular platform (Roche). ApoCIII, ANGPTL3 and PCSK9 levels were measured with commercially-available ELISA kits. Differences between HGSC and BOL were assessed by two-group comparisons. Pairwise correlation strength between variables was evaluated with Spearman’s rank order tests. Logistic regression modelling was used to further examine the associations between selected variables and HGSC diagnoses. Results: ANGPTL3 levels increased in HGSC (84.1 ng/ml, SD=29.2ng/ml, n=31) compared to BOL (66.9ng/ml, SD=30.7 ng/ml, n=40; HGSC vs BOL p=0.02). Receiver operating characteristic (ROC) curve suggested that plasma ANGPTL3 levels were moderately associated with HGSC (AUC=67.7%, p=0.005 1) and did not increase predictive performance of ovarian tumor biomarkers CA125 and HE4 in multivariable model. Associations between ANGPTL3 levels and cholesterol (HDL, non-HDL, LDL and total) could be observed in the control group but not in the HGSC group. The same dissociation was observed for the inverse correlation between HDL and triglycerides in HGSC. Furthermore, moderate associations were observed between PCSK9 & CA19-9 in the entire cohort (rho=0.34, n=53, p=0.013) and Lp(a) & CA125 in HGSC cases (rho=0.51, p= 0.017). Conclusion: In this cohort of 71 women, we report increased levels of ANGPTL3 in HGSC with an effect size (Cohen’s D) of 0.57. Confirmation of this result in larger cohort studies (minimal n>50 in each group) would warrant further investigation into the roles of circulating ANGPTL3. Its inhibition may allow to tackle deregulated lipid metabolic pathways contributing to ovarian cancer progression. Citation Format: Emilie Wong Chong, France-Hélène Joncas, Pierre Douville, Dimcho Bachvarov, Frédéric Calon, Nabil Seidah, Caroline Diorio, Anne Gangloff. Circulating levels of ANGPTL3, PCSK9, apoCIII and lipoprotein (a) in high-grade serous 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 5169.

Abstract 6127: Characterization of germline and somatic homologous recombination deficiency features in high-grade serous ovarian cancer among Black individuals

Abstract Background: Approximately 50% of high-grade serous ovarian carcinomas (HGSOC) are homologous recombination deficient (HRD). HRD is associated with increased T-cell infiltration and improved survival. This study aimed to describe the distribution of HRD features and cytotoxic T-cell infiltration by gene expression subtype among Black HGSOC cases. Methods: We included Black HGSOC cases from the African American Cancer Epidemiology Study and the North Carolina Ovarian Cancer Study. We used RNASeq data to assign gene expression subtypes (n=160), and whole exome sequencing data from matched blood and tumor formalin-fixed paraffin-embedded specimens (n=206) to characterize HRD features. We defined HRD features as (1) germline variants and somatic mutations in HRD genes, and (2) HRD-associated signatures (SBS3 and ID6) that we obtained from de novo mutational signature analysis for single-base pair substitution, insertion, and deletion. We estimated the proportion of intratumoral cytotoxic T cells using multiplex immunofluorescence (n=125). Results: The subtype distribution in our study was 39% immunoreactive, 29% mesenchymal 27% proliferative, and 4% differentiated. Germline variants and somatic mutations in any HRD gene were observed in 27% and 23% of cases, respectively, and at least one of these was observed in 42% of cases. Pathogenic germline variants and somatic mutations were observed in 7% and 3% of cases, respectively. The prevalence of germline or somatic variants by gene was 16% for BRCA1, 14% for BRCA2, 11% for ATM, 5% for RAD51, and 2% each for BRIP1, PALB2, and BARD1. We observed a COSMIC HRD signature in 43% of cases, with 25% SBS3 only, 5% ID6 only, and 13% with both signatures. The prevalence of any HRD gene variant or signature was higher in immunoreactive cases (52% and 59%, respectively) than in mesenchymal (28% and 43%) or proliferative (44% and 28%) cases. Germline HRD variants were more common in the proliferative subtype (44%) compared with immunoreactive (25%) and mesenchymal (17%). In contrast, somatic HRD mutations were more common in the immunoreactive subtype (33%), compared with proliferative (16%) and mesenchymal (15%). This was due to a higher occurrence of BRCA1 somatic mutations in the immunoreactive subtype (25%) compared with proliferative (9%) and mesenchymal (8%). A higher proportion of cytotoxic T-cells (≥1% vs <1%) was also observed in the immunoreactive subtype (44%) compared with mesenchymal (31%) and proliferative (11%). Conclusion: We observed differences in the distribution of HRD features and cytotoxic T-cells by gene expression subtype. In particular, in the immunoreactive subtype, the most common gene expression subtype in Black HGSOC cases, we observed a higher occurrence of any HRD feature, somatic BRCA1 mutations, and cytotoxic T cell infiltration than in any of the other gene expression subtypes. Citation Format: Katherine Lawson-Michod, Mollie E. Barnard, Natalie Davidson, Lindsay J. Collin, Courtney Johnson, Lucas A. Salas, Casey Greene, Jeffrey R. Marks, Lauren Peres, Joellen M. Schildkraut, Jennifer A. Doherty. Characterization of germline and somatic homologous recombination deficiency features in high-grade serous ovarian cancer among Black individuals [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 6127.

Abstract 1066: Colocalization of ovarian cancer-associated biomarkers in tumors, cancer cells and extracellular vesicles

Abstract Introduction: We have previously shown that ovarian cancer can be detected with high specificity and sensitivity by interrogating colocalized membrane-associated cell-surface biomarkers on single tumor-associated extracellular vesicles (EVs). Our blood based Ovarian Cancer Test (OC Test) is composed of 5 cancer-associated biomarkers (BST-2, FOLR1, MUC-1, MUC-16, and sialylated Thomsen-nouveau antigen (sTn)) known to be overexpressed in ovarian cancer relative to healthy tissues. The test employs these 5 biomarkers in 3 combinations to capture and detect EVs from human plasma to distinguish high-grade serous ovarian carcinoma (HGSC) from both benign ovarian tumors and normal samples. Using super-resolution microscopy on ovarian cancer cell lines and EVs and multiplex immunohistochemistry of ovarian benign and tumor tissue, our study aimed to show that the 5 ovarian cancer biomarkers show 1) colocalization on ovarian cancer cells, 2) colocalization on the surface of EVs, and 3) colocalization on cancer cells within ovarian tumor biopsies. Methods: Super-resolution microscopy was employed to visualize stained biomarkers on the cell surface of a collection of ovarian cancer cell lines as well as on the surface of extracellular vesicles released by these cell lines. 58 HGSC (17 Stage I, 30 Stage II, and 10 Stage III) and 17 benign ovarian tumor K2EDTA plasma samples and paired FFPE tissue were sourced from the Ovarian Cancer Research Program (OVCARE, Vancouver BC, Canada). A tumor microarray from identical tissue samples was stained with an Opal multiplex-IHC (mIHC) assay comprised of antibodies to BST2, MUC1, sTn, and pan-cytokeratin and a DAPI counterstain. Machine learning based image analysis was utilized to determine colocalization of the biomarkers at the cellular level within benign and tumor tissue. RNA sequencing was carried out to assess expression levels of the biomarkers in each FFPE tissue sample. OC Tests were run on each plasma sample. Results: Super-resolution microscopy showed colocalization of OC Test combination biomarkers on ovarian cancer cells and EVs derived therefrom. Machine learning-based image analysis of tumor and benign tissue microarrays stained for BST2/MUC1/sTn using mIHC, showed strong evidence for colocalization of these biomarkers on single cells within ovarian tumor tissue, particularly in early-stage tumors, but not on benign tissue. RNA sequencing analysis showed good correlation of biomarker RNA expression levels and with the OC Test results. Conclusions: The results presented here demonstrate that the OC Test biomarkers are colocalized on tumor tissue, ovarian cancer cells, and EVs from ovarian cancer cell lines. RNA and protein expression levels show good correlation with OC Test results. These results provide further evidence that colocalized biomarkers on EVs can be utilized for early detection of cancer. Citation Format: Anthony D. Couvillon, Emily S. Winn-Deen, Shelby Thornton, Shuhong Liu, Michael DeRan, Maciej Pacula, Sanchari Banerjee, Daniel Gusenleitner, Laura T. Bortolin, Jonian Grosha, Timothy Santos-Heiman, Gabrielle Sangiuliano, Kelly M. Biette, Christopher R. Sedlak, Bilal Hamzeh, Delaney M. Byrne, Peter A. Duff, Julie Ho, Dongxia Gao, Amy Jamieson, Lauren T. Cuoco, MacKenzie S. King, Dawn R. Mattoon, Toumy Guettouche, Jessica N. McAlpine, David Huntsman. Colocalization of ovarian cancer-associated biomarkers in tumors, cancer cells and extracellular vesicles [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 1066.

Abstract 7154: Ivospemin/doxorubicin combination modulates polyamine metabolism to improve survival in murine ovarian cancer models

Abstract Polyamines are small polycationic alkylamines that are absolutely required for the continual growth and proliferation of cancer cells. As cancer cells maintain elevated polyamine pools through dysregulated polyamine metabolism, its pharmacological modulation is a promising avenue in cancer therapeutics. The polyamine analogue ivospemin has shown efficacy in slowing pancreatic and ovarian tumor progression in vitro and in vivo and has demonstrated encouraging results in pancreatic cancer clinical trials. Considering nearly 75% of late-stage ovarian cancer patients develop resistant to platinum-based chemotherapies, limiting treatment options, the aim of our study is to determine the potential efficacy of ivospemin in combination with doxorubicin, a common chemotherapeutic used in platinum-resistant ovarian tumors. We have previously shown that ivospemin exposure decreases polyamine content in a variety of cancer cell lines through downregulation of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC) and induction of the polyamine catabolic enzyme spermidine/spermine-N1-acetyltransferase (SSAT). Here we examine the potential of combining ivospemin with doxorubicin. Ivospemin treatment reduces cell viability in ovarian adenocarcinoma cell lines and increases the toxicity of doxorubicin regardless of cisplatin sensitivity. Cells treated with the combination exhibit a greater decrease in polyamine levels than cells treated with either single agent. This increased polyamine depletion and decreased survival is accomplished through modulation of polyamine metabolism, predominately through an additive induction of SSAT activity. Using the syngeneic VDID8+ ovarian murine model, we further evaluated the ability of ivospemin to improve response to doxorubicin at clinical and sub-clinical dosing. Ascites fluid was used as a marker for tumor burden and evaluated for polyamine content. We found that the combination treatment increases median survival, delays tumor onset, and decreases overall tumor burden compared to either clinical or subclinical doxorubicin dosing schemes. Combination treatment also decreases overall polyamine content in the ascites by 75%. N1-acetylated spermidine is enriched in ascites from combination-treated mice, consistent with an upregulation of SSAT in response to treatment. Recognizing the non-representative mutational status of the VDID8+ model as a limitation, we are currently evaluating the combination of ivospemin and doxorubicin in genetically defined murine models that better recapitulate human high-grade serous ovarian carcinomas. Ongoing studies will determine influences on the tumor microenvironment and will mechanistically evaluate the cooperativity of ivospemin and doxorubicin on pathways outside of polyamine metabolism. Citation Format: Cassandra E. Holbert, Jackson R. Foley, Ashley C. Nwafor, Ting-Ann Liu, Elizabeth Bruckheimer, Jennifer K. Simpson, Tracy Murray Stewart, Robert A. Casero. Ivospemin/doxorubicin combination modulates polyamine metabolism to improve survival in murine ovarian cancer models [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 7154.

Abstract 3027: p53 aggregation promotes transformation from non-malignant lesions to high-grade serous ovarian carcinomas

Abstract High-Grade Serous Ovarian Cancer (HGSOC) is the most common and aggressive gynecological malignancy. It is typically diagnosed in late stages due to lack of diagnostic tests and current therapies available are not efficacious when the tumor is advanced. HGSOC is characterized by ubiquitous loss of functional p53 tumor suppressor protein function, largely due to point mutations that arise very early in carcinogenesis. P53 mutations are detected in pre-neoplastic cells of the fallopian tube mucosa, called p53 signature, and in early precursor lesions called serous tubal intraepithelial carcinoma (STIC). Some mutations promote the misfolding and aggregation of p53, transforming the protein into a cancer-promoting oncogene. Here, we hypothesize that the transition from folded, soluble to aggregated mutant p53 underlies the transformation from a pre-neoplastic lesion (p53 signature, STILs), to STICs and eventually HGSOC. We first set to determine the proportion of cells carrying soluble vs misfolded mutant p53 in fallopian tube tissues collected from n=10 patients undergoing salpingo-oophorectomy. We performed conformation-sensitive staining and quantification of folded and unfolded p53 cases and demonstrated that p53 misfolding, a mandatory precursor to aggregation, is present in STICs and HGSOCs, but notably absent from p53 signatures and surrounding healthy tissue. To better characterize the role of aggregated p53 in the progression of HGSOC, we took advantage of primary human fallopian tube secretory epithelial cells (FTSEC) that mimic early and more advanced stages of carcinogenesis. We utilize the FTSEC cells line FT282, immortalized by introducing hTERT and the aggregation-prone mutant TP53R175H that is found in p53 signature lesions and STILs, as well as the FT282-CCNE1, derived from FT282 cell line with overexpression of CCNE1, representing a more malignant phenotype.We used these models to investigate p53 aggregation, characterize the mutant p53 interactome and test the efficacy of ReACp53, a p53 aggregation-targeting peptide we developed. Our results demonstrated that p53 aggregation is more severe in the FT282-CCNE1 line, with a higher susceptibility to ReACp53 compared to the FT282 cells. Overall, our results demonstrated that aggregation of mutant p53 is a structural defect that can distinguish between pre-malignant and malignant HGSOC lesions and can potentially be targeted, offering a potential window for early intervention in halting ovarian cancer progression. Citation Format: Sara Sartini, Lexi Omholt, Neda A. Moatamed, Alice Soragni. p53 aggregation promotes transformation from non-malignant lesions to high-grade serous ovarian carcinomas [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 3027.

Abstract 1855: Proteomics driven biomarker discovery for high-grade serous ovarian carcinoma from archived tissue specimens

Abstract High-Grade Serous Ovarian Carcinoma (HGSOC) is a highly aggressive and heterogeneous cancer with variable treatment responses. Current treatment strategies, including surgical resection and chemotherapy, are often ineffective due to a lack of molecular markers to predict drug sensitivity and patient prognosis. Mass spectrometry-based proteomics analysis is a powerful tool for biomarker discovery, but it is restricted by the availability and quality of archived clinical specimens. In this study, we developed a proteomics-based biomarker discovery pipeline that optimizes the extraction and sample preparation from archived tissue specimens of HGSOC patients. Macrodissection of the tumor rich tissue areas of interest ensured sample purity and statistical power for comparative analysis. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we conducted a comprehensive, global proteomics analysis of FF tissue specimens from 25 HGSOC patients to associate proteomic profiles with patient outcomes. Combining high-resolution mass spectrometry proteomics of clinical samples with advanced computational tools for clinical data mining, we were able to identify pathways associated with HGSOC that hold clinical significance. Our unique biomarker discovery platform provides a novel strategy for detecting cancer vulnerabilities from archived tissue samples and identifying proteomic biomarkers linked with treatment response and patient prognosis beyond BRCA mutation status. Citation Format: Anjana Shenoy, Gali Arad, Dimitri Kovalerchik, Amit Manor, Nitzan Simchi, Dina Daitchman, Kirill Pevzner, Eran Seger. Proteomics driven biomarker discovery for high-grade serous ovarian carcinoma from archived tissue specimens [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 1855.