Uncharted Territory: Journey of a Mullerian Tumor to the Brain, A Case Report

Ovarian clear cell carcinoma (OCCC) is a special histological type of epithelial ovarian cancer (EOC) that is not derived from epithelial cells of the ovarian or fallopian tube as the most common type of ovarian cancer, high-grade serous ovarian carcinoma (HGSOC), but is closely related to endometriosis and similar to endometrial clear cell carcinoma (ECCC) at morphologic and phenotypic features. However, limited data was shown in OCCC genomic features and compared with that in OCCC, HGSOC and ECCC. Herein, we utilized next-generation sequencing analysis of a panel of 1,021 genes to profile the mutational alterations in 34 OCCC and compared them to those from HGSOC (402 cases) and ECCC (30 cases). In result, the ARID1A and PIK3CA are high-frequency mutations of OCCC. Clonal architectures showed that all the mutations of genes occur in the later stage in the OCCC progress, whereas KRAS mutation is the earlier event compared with mutation of ARID1A or PIK3CA, which usually occurs in a group of ARID1A or PIK3CA mutations. The mutation frequency of main driver genes is similar between OCCC and ECCC, while TP53 is the main mutation in HGSOC and ECCC. Shared mutational signatures between OCCC and ECCC tissues with commonly observed a C>T change indicated a common carcinogens-exposed between these two carcinomas, but HGSOC and ECCC have common and distinct mutational signatures across cohorts respectively. In addition, we identified some novel CNV gains in NF1, ASXL1, TCF7L2, CREBBP and LRP1B and loss in ATM, FANCM, RB1 and FLT in OCCC. Our study offered a new perspective for OCCC tumorigenesis from two organs, the ovary and uterus, at genomic architectures and revealed novel CNV events for helping to provide theoretical support for OCCC treatment.

Objective: To explore the origin of ovarian high grade serous carcinoma (HGSC) through analysing the expression and significance of PAX8, PAX2, p53 and RAS in the ovary and fallopian tube of different types and grades of serous carcinoma. Methods: A total of 44 cases tissue samples of ovarian tumor including 34 malignant ovarian tumor and 10 normal normal tissue (as control group) were collected from the admitted patients in Affiliated Tumor Hospital of Guangxi Medical University from January 2015 to January 2016. Fallopian tube tissues were segmented in accordance with the fimbria, ampulla, isthmus and the corresponding ovarian tissues were by the side. There were 34 cases of patients with ovarian cancer including 29 cases of epithelial ovarian cancer (27 serous carcinoma, 1 mucinous carcinoma,1 endometrioid adenocarcinoma) and 5 non-epithelial ovarian cancer (sex cord-interstitial tumor). Among 27 cases of patients with ovarian serous cancer, there were 23 HGSC and 4 low-grade ovarian serous cancer (LGSC). One hundred fifty-three cases of samples were diagnosed as ovarian serous cancer by Shandong University Affiliated Qilu Hospital from 2005 to 2013 and these samples were made tissue microarray. (1) To analyze the expression and differences of PAX8, PAX2, p53 and RAS in the above tissues and tissue microarray from ovarian and tubal of HGSC and control women by immunohistochemistry methods. (2) To compare the expression levels of PAX8, PAX2, p53 and RAS in ovarian and fallopian tubes of ovarian cancer patients with different pathological types. (3) To analyze the correlations of tubal and ovarian tissue in PAX8, PAX2, p53 and RAS expression of HGSC. (4) To analyze the factors of the prognosis of ovarian serous cancer in tissue microarray by single factor analysis method. Results: (1) PAX8, PAX2, p53 and RAS expression was negative in normal ovarian epithelium of control group, but the expression of PAX8, PAX2, p53 and RAS were strongly positive brown in secrete cells of normal fallopian tube epithelium. (2) p53 and RAS expression of fallopian tube epithelium in the epithelial ovarian cancer group were significantly higher than those in the non-epithelial ovarian cancer groups (P<0.05), but the expression of PAX8 and PAX2 in fallopian tube and the expression of PAX8, PAX2, p53 and RAS in ovarian tissue was not statistically significant in the groups (P>0.05). PAX8, PAX2 and p53 expression of the ovarian in HGSC group were significantly higher than those in LGSC group (P<0.05), while the expression of RAS was lower in the ovarian of the high-grade group (P<0.05), while the expression of PAX8, PAX2, p53 and RAS in fallopian tube was not statistically significant in the groups (P>0.05). (3) There was a significantly positive correlation between fallopian tube and the corresponding ovary of HGSC in PAX8 and PAX2 expression (r=0.422, P=0.045; r=0.693, P=0.000), but not correlation in p53 and RAS expression (r=0.058, P=0.793; r= -0.190, P=0.384). (4) Univariate survival analysis showed that the progression free survival time in patients with ovarian serous cancer group was significantly correlated with the protein expression of PAX8, PAX2 and RAS (P<0.05), but there were not correlated with age, surgical staging, cell differentiation, lymph node metastasis and preoperative chemotherapy and p53 protein expression (P>0.05). The total survival time in patients with ovarian serous cancer group was significantly correlated with the protein expression of PAX8 (P<0.05), but there were not correlated with age,surgical staging, cell differentiation, lymph node metastasis and preoperative chemotherapy and the protein expression of PAX2, RAS and p53 (P>0.05). Conclusions: PAX8, PAX2, p53, RAS are of great significance for the study of origin of HGSC. HGSC may be derived from fallopian tube, but further investigation would be necessary to confirm this. PAX8, PAX2, p53, RAS could be expected to be used as predictors of survival prognosis in patients with ovarian serous cancer.

The vast majority of high grade serous ovarian carcinomas (HGSOC) are diagnosed at an advanced stage. Chromobox 2 (CBX2), a polycomb repressor complex subunit, plays an oncogenic role in a variety of cancers. In prostate cancer, CBX2 is a driver of metastatic progression. Little is known about the role of CBX2 in HGSOC. Our hypothesis is that CBX2 upregulation promotes advanced HGSOC by promoting a stem-like transcriptional profile and inhibiting anoikis. Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) were queried to establish the role of CBX2 in HGSOC. In vitro evaluation of CBX2 occurred in OVCAR4, PEO1, and OVCAR8 HGSOC cell lines. PolyHEMA-coated plates forced cells to grow in suspension and simulated anoikis-escape. Quantitative polymerase chain reaction and immunoblots evaluated CBX2 expression. Small hairpin RNAs (shRNAs) knocked CBX2 down for loss of function studies. To mimic HGSOC progression several culture conditions, including 2D, colony formation, and 3D, spheroid, were examined. Secreted Gaussia luciferase (gLuc) activity was utilized as an indicator of proliferation. Stemness was tested with the Aldefluor assay, measuring aldehyde dehydrogenase activity (ALDH). Using patient tumors derived from the Gynecology Tissue and Fluid Bank (GTFB) and a HGSOC tissue microarray (TMA) with matched primary, metastatic, and lymph nodes, a CBX2 expression profile was established. Student's t-test was used to define statistical significance, with a p value of &amp;lt; 0.05. Analysis of GEO databases established CBX2 is upregulated in HGSOC tumors compared to benign tissues. Analysis of TCGA found CBX2 expression conveyed worse disease-free survival (11.7 vs 17.6 months, Log-rank test p-value &amp;lt; 0.005) and overall survival (34 vs. 44.8 months, Log-rank test p-value &amp;lt;0.005). Examination of primary HGSOC tumors confirmed CBX2 was upregulated at the protein level in HGSOC compared to benign tissue. In vitro, OVCAR4, PEO1, and OVCAR8 cells upregulate CBX2 when grown in suspension compared to adherent conditions. CBX2 knockdown led to a significant inhibition of proliferation in 2D, 3D, and in suspension. Forced suspension promoted increased ALDH3A1 expression and ALDH activity. CBX2 knockdown led to a decrease in both ALDH3A1 expression and ALDH activity. HGSOC cells grown in suspension were found to be more chemoresistant compared cells grown under adherent conditions. Similarly, knockdown of CBX2 sensitized HGSOC cells to cisplatin. Examination of primary tissue in a TMA of matched patient samples revealed CBX2 is expressed in primary and metastatic disease. We conclude that CBX2 directly impacts proliferation and is overexpressed in HGSOC. Our work indicates that CBX2 is an important regulator of stem-ness, which could play a role in anoikis escape, HGSOC dissemination, and chemoresistance, suggesting that CBX2 may be associated with more advanced disease. This exploration expands our understanding of molecular drivers of HGSOC progression and potentially identifies a novel therapeutic target. Citation Format: Lindsay J. Wheeler, Zachary L. Watson, Lubna Qamar, Alexandra McMellen, Tomomi Yamamoto, Kian Behbakht, and Benjamin G. Bitler. CBX2 IDENTIFIED AS DRIVER OF ANOIKIS ESCAPE AND DISSEMINATION IN HIGH GRADE SEROUS OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-058.

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy, with 75% of women presenting with metastatic disease at the time of diagnosis. High Grade Serous Ovarian Carcinoma (HGSOC) is the most common and aggressive type of epithelial ovarian cancer, with 5-year survival rate of 30% for most patients. Developing of accurate experimental models of HGSOC is necessary to elucidate the disease pathogenesis and to evaluate new treatments. Our study is aimed at determining the roles of the Large Tumor Suppressor 1 and 2 kinases (LATS1/2), and their downstream effector Dual-specificity tyrosine(Y)-Regulated Protein Kinase 1A (DYRK1A), in HGSOC pathogenesis and treatment sensitivity. The Cancer Genome Atlas study revealed copy number losses of the LATS1, LATS2 and DYRK1A genes in 65%, 59% and 38% of HGSOC cases, respectively. Previous studies found that LATS1/2 and DYRK1A mediate a crosstalk between the Hippo tumor suppressor pathway and the G0/G1 cell cycle checkpoint. However, the role of these kinases in HGSOC pathogenesis is not known. We found that loss of both LATS1 and LATS2, or DYRK1A results in increased proliferation of SKOV3 ovarian cancer cells in 2D or 3D cultures, mediated in part by disruption of the DREAM complex that represses cell cycle-regulated genes. Interestingly, we observed upregulation of cyclin D1 and increased phosphorylation of retinoblastoma (RB) family proteins in SKOV3 cells depleted of LATS1/2 or DYRK1A kinases, indicative of aberrant activation of CDK4/6. Similar changes were observed upon depletion of LATS1/2 or DYRK1A using non-transformed human fallopian epithelial cells, suggesting that genetic losses of these kinases could contribute to the HGSOC pathogenesis. Furthermore, we tested whether an aberrant activation of CDK4/6 in SKOV3 cells with low levels of LATS1/2 or DYRK1A could increase their sensitivity to pharmacological CDK4/6 inhibitors such as palbociclib. Using metabolic cell proliferation assays, we found that loss of LATS1/2 or DYRK1A resulted in a significant decrease of palbociclib IC50 in SKOV3 cells. Interestingly, palbociclib treatment increased the DREAM assembly in these cells, resulting in downregulation of DREAM-regulated cell cycle factors such as cyclin A or B-Myb. Our results show that loss of LATS1/2 or DYRK1A could contribute to HGSOC pathogenesis by CDK4/6-mediated disruption of RB family function. Further studies are underway to validate our findings in vivo using orthotopic ovarian tumor xenografts in mice and a bioluminescence imaging approach. In addition, these models will be used for validation of novel therapeutic approaches for HGSOC, such as palbociclib, as well as for the mechanistic studies of the EOC pathogenesis. Citation Format: Fatmata Sesay, Siddharth Saini, Larisa Litovchick. ROLE OF LATS AND DYRK1A KINASES IN THE PATHOGENESIS AND TREATMENT OF HGSOC [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-053.

The high–grade serous ovarian carcinoma (HGSOC), the most aggressive ovarian cancer subtype, accounts for two–thirds of ovarian cancer deaths. It is a poorly understood disease with high therapeutic resistance and extremely poor prognosis. The current study is an attempt to analyze molecular mechanisms underlying HGSOC and to identify new therapeutic targets. The Cancer Genome Atlas (TCGA) analysis of ovarian carcinoma demonstrated that p53 mutations are near universal (&amp;gt;90%) in HGSOC and forkhead box M1 protein (FOXM1) network is significantly altered in about 87% of HGSOC leading to transcriptional upregulation of proliferation–related target genes. Moreover, mutant p53 and FOXM1 have important roles in resistance to chemotherapy. In the present study, we have used various experimental procedures such as RNAi technology, immunoprecipitation, quantitative real–time PCR, immunoblot analysis, proximity ligation assay (PLA) (to determine protein–protein interaction in situ) and assays for cell cycle, apoptosis and proliferation to analyze estrogen receptor beta (ER–beta) – mutant p53 –FOXM1 signaling crosstalk in OVCAR3 (HGSOC) cells. Knocking down ER–beta resulted in increased mut–p53 transcript levels suggesting transcription of mut–p53 is repressed by ER–beta in OVCAR cells. Similarly, mut–p53 represses transcription of ER–beta gene. We show that not only mut–p53 and ER–beta physically interact, but also they mutually regulate each other's expression at the gene transcriptional level leading to changes in expression of FOXM1 and its downstream signaling targets such as . We show that when ER–beta was knocked down, FOXM1 levels were decreased and resistance to cisplatin and carboplatin, major therapeutic agents currently in use for ovarian cancer, was decreased as evidenced by increased apoptosis and decreased colony formation. Surprisingly, ER–beta binds to both p53 and FOXM1. Our data provides new insight into the mechanisms underlying HGSOC by illustrating how signaling crosstalk between ER–beta and mut–p53 leads to increased expression of pro–proliferative and pro–tumorigenic downstream targets such as FOXM1, overexpression of which has been associated with resistance to therapeutic agents.in HGSOC patients. Our study will be the first, to the best of our knowledge, to investigate whether the crosstalk between ER–beta and mut–p53 along with FOXM1 plays an important role in the onset and progression of HGSOC. We have identified ER–beta–mutant p53–FOXM1 signaling axis as a novel therapeutic target in HGSOC. Correlation between ER–beta, mut–p53, and FOXM1 expression, their interaction, and disease characteristics are being analyzed in HGSOC patient tissue microarrays (TMAs). This study was supported by a Pilot Study Award from the Rivkin Center for Ovarian Cancer. Citation Format: Chetan Oturkar and Gokul M. Das. ESTROGEN RECEPTOR BETA – P53 – FOXM1 SIGNALING AXIS AS A NOVEL MOLECULAR TARGET TO OVERCOME THERAPEUTIC RESISTANCE IN HIGH–GRADE SEROUS OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-054.

The lack of consensus on clinically relevant molecular subtypes by gene expression in high-grade serous ovarian carcinoma (HGSOC) creates a barrier to subtype-based clinical investigation for the development of targeted therapies. We previously discovered aberrant expression of noncoding repeat RNAs across epithelial cancers including ovarian (Ting, Science 2011), which can invoke innate immune responses in tumors (Chiappinelli, Cell 2015). Epigenetic alterations and loss of tumor suppressor function, especially p53, can derepress endogenous repetitive elements in cancer. Further, BRCA1 deficiency induces satellite repeat RNAs, which promote genomic instability via interacting with the BRCA1 complex (Zhu, Mol Cell 2018), highlighting their potential significance in HGSOC. Here we aim to comprehensively define the as yet uncharacterized “repeatome” in HGSOC to refine molecular subtypes and identify novel biomarkers and therapeutic targets. We developed a Total RNASeq platform and novel computational pipelines to quantify the repeatome (Solovyov, Cell Rep 2018). Results from RNASeq are validated in cell lines and human tumors with RNA in situ hybridization (RNA-ISH) using probes to specific repeat RNAs, and combined IHC is performed to quantify intratumoral immune subpopulations. Whole-exome sequencing is performed to link the repeatome with somatic mutations. To obtain a global landscape of the ovarian cancer repeatome, Total RNASeq was performed on 32 patient-derived ovarian cancer cell lines, 11 HGSOC PDX, and 11 additional ovarian cancer cell lines. We detect abundant repeat RNA expression from all major subclasses including retrotransposons, endogenous retroviruses, and satellites. HGSOC are enriched for satellite repeats compared with other cancers, most notably in BRCA-mutant HGSOC. Human satellite II (HSATII), a cancer-specific satellite, is strongly upregulated in HGSOC compared with fallopian tube epithelial cells and displays highly variable expression across different models. RNA-ISH for HSATII on human ovarian cancer tissue microarrays confirms the abundance and variation of HSATII. Additionally, the repeatome is altered in HGSOC following exposure to in vitro chemotherapy and epigenetic agents with distinct patterns of expression linked to specific agents. Additional RNAseq analysis using hierarchical clustering and principal component analysis are being used to understand the relationship of repeatome expression patterns with coding gene expression, somatic mutations, in vitro drug sensitivity, and clinical outcomes. Digital image analysis of repeat RNA-ISH expression and immune cell infiltrate quantitation will determine the link between tumor cell repeat RNA levels and the responding immune tumor microenvironment. Overall, these studies will define the undiscovered repeatome in HGSOC and provide a foundation for discovery of novel biomarkers and potential therapeutic strategies, particularly those to increase efficacy of immunotherapies. Citation Format: Rebecca L. Porter, Anna Szabolcs, Niyati Desai, Vishal Thapar, Raghav Mohan, Alexander Solovyov, David Pepin, Joyce Liu, Benjamin Greenbaum, David T. Ting. Repeatome profiling in high-grade serous ovarian cancer reveals abundant repeat noncoding RNA expression [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A66.

Introduction: Ovarian cancer (OC) is one of the deadliest cancers, with 314,000 new cases and 207,000 deaths globally in 2020. Serum CA125 has been explored as an OC biomarker for the past 40 years, but lacks sensitivity for early stage OC and is not recommended for screening average-risk, asymptomatic women. We hypothesize that co-localization of biomarkers on the surface of individual extracellular vesicles (EVs), which are shed into the circulation by cancer cells, may lead to development of a blood test for early stage OC. We evaluated the potential of our approach in detecting early stage OC in clinical samples. Methods: We isolated EVs using size-exclusion chromatography and immunoaffinity capture, and detected biomarkers co-localized on the surface of individual EVs with proximity ligation qPCR. Using this approach, we evaluated 49 antibody combinations recognizing 2 or more biomarkers. Each combination consisted of 1 capture antibody and 2 oligonucleotide-tagged detection antibodies. We tested plasma samples from women with early stage I/II high-grade serous ovarian carcinoma (HGSOC)(n=18; 48-80 yr, med 57) and late stage HGSOC (n=24; 37-80 yr, med 54). HGSOC samples were sourced from 2 commercial vendors. Controls comprised samples from women with benign ovarian masses (n=26; 23-76 yr, med 39.5) sourced from a single vendor, and samples prospectively collected by Mercy from healthy women with no cancer history (n=24; 22-72 yr, med 52.5). PCR cycle threshold (Ct) values were measured for each of 49 combinations and data was evaluated using univariate analysis. Performance was compared to plasma CA125 measured at Mercy by commercial ELISA. Results: 8 of 49 combinations distinguished all stages of HGSOC relative to benign and healthy controls with AUCs ranging from 0.86 (95% CI 0.78-0.94) to 0.95 (95% CI 0.90-1.00), comparable to CA125 with an AUC of 0.87 (95% CI 0.79-0.95). One of the most effective combinations (STn, BST2, MUC1) had a sensitivity of 0.78 (95% CI 0.52-0.94) at a specificity of 0.96 (95% CI 0.87-0.99) in detecting early stage HGSOC. This combination also detected HGSOC in 6 of 11 women (3 early stage, 3 late stage) with normal CA125 (&amp;lt; 25 U/mL) and correctly classified 7 of 8 women with benign masses and high CA125 (&amp;gt; 25 U/mL). Conclusions: These preliminary data suggest that co-localization of surface biomarkers in single EVs may provide an effective means to identify women with early stage HGSOC, including those with normal CA125, while avoiding false positives in women with benign masses and high CA125. Despite the inherent challenges associated with commercial samples, our finding that several combinations detected early stage HGSOC is promising. Statistically powered studies with curated repository specimens are underway to refine combinations and independently validate our assay for early stage OC detection. Citation Format: Laura T. Bortolin, Daniel P. Salem, Sanchari Banerjee, Kelly M. Biette, Delaney M. Byrne, Anthony D. Couvillon, Peter A. Duff, Jonian Grosha, MacKenzie Sadie King, Christopher R. Sedlak, Ibukunoluwapo O. Zabroski, Claire Alexander, Karen Copeland, Daniel Gusenleitner, Emily S. Winn-Deen, Eric K. Huang, Bo R. Rueda, Joseph Charles Sedlak. Preliminary results for a novel single extracellular vesicle assay for early stage ovarian cancer: The power of co-localized detection of surface biomarkers [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 3390.

The ability of ovarian steroids to modulate ovarian cancer (OC) risk remains controversial. High grade serous ovarian carcinoma (HGSC), the most aggressive OC subtype, contains abundant estrogen (ER; 76%) and progesterone (PR; 35%) receptors. Progesterone is thought to be a primarily protective factor that reduces OC risk but the relevance of PR actions in HGSC is unknown. Our IHC analyses of human clinical tissues showed robust expression of total and activated phospho-S294 PR within serous tubal intraepithelial carcinomas (STIC), precursor HGSC lesions of fallopian tube epithelia (FTE), suggesting a role for PR signaling in early disease. To explore the molecular mechanisms of PR isoform actions, p53-dominant negative mutant FTE lines expressing the PR-A or PR-B isoform were created. Progestin treatment (R5020 or progesterone) of these models induced spheroid formation and reaggregation, transwell migration and collagen invasion. RNAseq analyses revealed enrichment of cell cycle progression pathways; in particular, the gene targets of the repressive DREAM complex that drives quiescent cell fate. Progestins induced inhibition of proliferation (decreased Ki67; decreased BrdU; increased B-galactosidase) and accumulation of cells in G0/G1 that was reversed following growth factor stimulation. Subsequent immunoprecipitation of DREAM (G0) and B-MYB/MMB complexes (G1/S) confirmed that PR-driven changes in cell fate occur via regulation of the DREAM complex. PR activation enhanced DREAM and suppressed B-MYB/MMB complex formation. PR also induced transcriptional regulation of DREAM/B-MYB/MMB genes (LIN9, MYBL2, DYRK1A, FOXM1) via promoter recruitment and mRNA regulation. Interestingly, DREAM complexes contained PR and PR was co-recruited to DREAM binding sites within DREAM target promoters, along with the DREAM complex proteins p130 and E2F4. The relevance of the quiescent state to early OC phenotypes was examined through the disruption of DREAM/DYRK1s by pharmacological inhibition (harmine) , HPV E6/E7 expression or the depletion of DYRK1A/B kinases that promote DREAM complex formation. These manipulations blocked progestin-induced cell cycle arrest and attenuated PR-driven gene expression and associated OC phenotypes. Our results reveal that activated PRs support quiescence and pro-survival/pro-dissemination cell behaviors that may contribute to early HGSC progression. Taken together, our data suggest an alternative perspective on the tenant that progesterone always confers protection against OC. STICs can reside undetected for decades prior to invasive disease. Our studies reveal clinical opportunities to prevent the ultimate development of HGSC by targeting activated PRs, DREAM complexes, and/or DYRKs (Supported by the Minnesota Ovarian Cancer Alliance to LJM and CAL). Citation Format: Laura J. Mauro, Megan I. Seibel, Caroline H. Diep, Angela Spartz, Carlos Perez Kerkvliet, Hari Singhal, Elizabeth M. Swisher, Lauren E. Schwartz, Ronny Drapkin, Siddharth Saini, Fatmata Seesay, Larisa Litovchick, Carol A. Lange. Progesterone receptors promote quiescence &amp; ovarian cancer cell phenotypes via modulation of DREAM in p53-mutant fallopian tube models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 744.

The high-grade serous ovarian carcinoma (HGSOC), the most aggressive ovarian cancer subtype, constitutes 90% of serous carcinomas and it accounts for two-thirds of ovarian cancer deaths. It is a poorly understood disease with high therapeutic resistance and extremely poor prognosis. The current study is an attempt to analyze molecular mechanisms underlying HGSOC and to identify new therapeutic targets. The Cancer Genome Atlas (TCGA) analyses of ovarian carcinoma demonstrated that p53 mutations are near universal (90%) in HGSOC and forkhead box M1 protein (FOXM1) network is significantly altered in 87% of HGSOC leading to transcriptional upregulation of proliferation-related target genes. Moreover, Mutant p53 and FOXM1 have important roles in resistance to chemotherapy. In the present study, we have used various experimental procedures such as RNAi technology, quantitative real-time PCR, immunoblot analysis, proximity ligation assay (PLA) (to determine protein-protein interaction in situ) and assays for cell cycle, apoptosis and proliferation to analyze signaling crosstalk in OVCAR3 (HGSOC) cells. Our experiments have identified estrogen receptor-beta (ER-beta) as a novel member of the mutant p53-FOXM1 axis. We show that not only mut-p53 and ER-beta physically interact, but also they mutually regulate each other's expression at the gene transcriptional level leading to changes in expression of downstream signaling targets such as FOXM1. Importantly, the effect of knocking down ER-beta on transcription of FOXM1 was similar to that of knocking down endogenous mut-p53, suggesting that both mutant p53 and ER-beta are transcriptional activators of FOXM1. Knocking down ER-beta results in decreased mut-p53 transcript levels suggesting transcription of mut-p53 is activated by ER-beta in OVCAR cells. On the other hand, mut-p53 represses transcription of ER-beta gene. Thus, our experiments have revealed a feedback loop between mut-p53 and ER–beta at the transcriptional level.. We show that when ER-beta was knocked down, FOXM1 levels were decreased and apoptosis was increased. Of note, knocking down ER-beta decreased resistance to cisplatin and carboplatin, major therapeutic agents currently in use for ovarian cancer. Our data provides new insight into the mechanisms underlying HGSOC by illustrating how signaling crosstalk between ER-beta and mut-p53 leads to increased expression of pro-proliferative and pro-tumorigenic downstream targets such as FOXM1, overexpression of which has been associated with resistance to therapeutic agents.in HGSOC patients. Our study will be the first, to the best of our knowledge, to investigate whether the crosstalk between ER-beta and mut-p53 play an important role in the onset and progression of HGSOC. Thus, we have identified mutant p53-ER beta signaling axis as a novel therapeutic target in HGSOC. Citation Format: Chetan C. Oturkar, Gokul M. Das. Estrogen receptor-beta and mutant p53 signaling crosstalk as a novel molecular target to overcome therapeutic resistance in high-grade serous ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2135.

The field of personalized disease prognosis, risk prediction and disease management has always attracted the attention of scientists and clinicians worldwide. Translating current clinical research and emerging genome-scale molecular biomarkers of ovarian and other cancers is yielding promising new insights into predictive signatures and potential clinical targets, moving treatment in favor of more personalized therapeutic approaches. Integrative genomics, big data analysis and the disease prediction modelling have become the major driving factors in understanding how to reproducibly stop a disease for individual patients - a goal of precision medicine. In clinical practice, prognostic and predictive factors could be measured as continuous or discrete variables. Here, we proposed a novel methodology to predict disease/treatment outcome via analysis of the similarities between any pair of patients who are each characterized by a certain set of pre-defined biological variables (biomarkers or clinical features) represented initially as a prognostic binary variable vector (PBVV) and subsequently transformed to a prognostic signature vector (PSV). High-grade serous ovarian carcinoma (HGSC) is the most prevalent ovarian cancer, and is one of the most lethal gynecological diseases in the world today. Many molecular signatures for prognostic and disease prediction of HGSC have been proposed, but no been implemented in clinical practice yet. In our previous study, the highly-confidence prognostic classifier, comprising 36 mRNAs that can stratify the HGSC patients into low, intermediate or high-risk subgroups with significantly distinct overall survival outcomes and sensitivity to post-surgery chemotherapy, has been proposed [1]. In this work, we used this classifier as the discriminative model of prognosis of HGSC patients. We implemented our methods to personalized prognosis of HGSC patients of TCGA database based on our molecular predictor [1]. Our results revealed that patient's age, when converted into discrete binary values, was positively correlated with the overall risk of succumbing to the disease and can be used as independent prognostic factor. The inclusion of age into the molecular survival predictor provided more robust personalized prognosis of overall survival when applied to an independent testing metadata composing 359 HGSC patients. In summary, we propose that our personalized prognosis methods and results after including age as prognostic factor could provide clinical benefits in the context of personalized prognosis and therapeutic assignment of HGSC patients. Our method can be generalized and implemented in many other diseases to accurately predict personalized patients' outcomes. [1]. Tang Z, Ow GS, Thiery JP, Ivshina AV and Kuznetsov VA. Int J Cancer. 2013; 134(2):306-318. Citation Format: Ghim Siong Ow, Zhiqun Tang, Anna Vladimirovna Ivshina, Vladimir Andreevich Kuznetsov. Big data and computational biology method for personalized prognosis of high-grade serous ovarian carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A75.

Simple SummaryOvarian cancer is the most lethal gynecological malignancy. The overall survival of patients this disease had not substantially changed for several decades, mainly due to the lack of early diagnosis. Type II OC is the most common and most aggressive form of OC, which mainly includes high-grade serous ovarian carcinoma (HGSOC). Our study aimed to pilot whether the detection of TP53 mutation in uterine cavity lavage can be used as a diagnostic method for type II OC. Uterine lavage technique was successfully applied to all patients, also ovarian tissue biopsy was taken. All 136 samples (90 uterine cavity lavages and 46 tissues) were sequenced using six gene panel that included genes commonly associated with ovarian and endometrial cancers (TP53, BRCA1, BRCA2, PIK3CA, KRAS, and PTEN). Our pilot study proved that ctDNA from ovarian neoplasms can be collected from uterine lavage for diagnostic needs. We revealed precise detection of TP53, BRCA1, BRCA2 in uterine lavage from HGSOC by means of NGS. However, for improved sensitivity of such test, additional disease-specific biomarkers have to be discovered.Background: Type II ovarian cancer (OC) is generally diagnosed at an advanced stage, translating into a poor survival rate. Current screening methods for OC have failed to demonstrate a reduction in mortality. The uterine lavage technique has been used to detect tumor-specific TP53 mutations from cells presumably shed from high-grade serous ovarian cancer (HGSOC). We aimed to pilot whether the detection of TP53 mutation in uterine cavity lavage can be used as a diagnostic method for HGSOC using an expanded gene panel. Methods: In this study 90, uterine lavage and 46 paired biopsy samples were analyzed using next-generation sequencing (NGS) targeting TP53 as well as five additional OC-related genes: BRCA1, BRCA2, PI3KCA, PTEN, and KRAS. Results: Uterine lavage was successfully applied to all patients, and 56 mutations were detected overall. TP53 mutations were detected in 27% (10/37) of cases of type HGSOC; BRCA1 and BRCA2 mutations were also frequent in this group (46%; 17/37). Overall concordance between tissue and liquid biopsy samples was 65.2%. Conclusion: Uterine lavage TP53 mutations in combination with other biomarkers could be a useful tool for the detection of lowly invasive HGSOC.

Evaluation of TP53 mutations in Pap test and blood DNA as novel biomarkers of ovarian cancer risk

Extensive utilization of organoids from high-grade serous ovarian carcinoma (HGSOC), the most common and lethal ovarian cancer, has been hampered by low success rates of long-term culture and scarcity of fresh tumor material. Here we present the development of a novel method for efficient generation, expansion and use of HGSOC organoids from cryopreserved tumor material. First, we assessed commonly used organoid media components and found that supplements such as FGF-2, R-Spondin1, Wnt or Noggin had negative impact on the HGSOC organoid derivation. But further, we found that supplementation with FGF-4, which has not been used in cancer organoid culture before, is beneficial for HGSOC organoid growth. Through extensive testing of various supplements and their combinations, we designed two novel HGSOC organoid media formulations - Medium 1 (M1) and Medium 2 (M2). Using M1 and M2 enabled generation and long-term expansion of living HGSOC organoid biobank with markedly improved success rate than in previous reports (55% vs. 23-38%). The organoids were established from cryopreserved tumor material, demonstrating the feasibility of using frozen tissue biobanks for HGSOC organoid derivation. Overall, we generated a collection of 18 expandable HGSOC organoid lines from 11 patients, encompassing samples from different tissue sites and disease progression stages. We validated the organoids using whole-genome sequencing, immunohistochemistry and single-cell RNA sequencing and demonstrated that they are genetically and phenotypically representative of original patient samples over long-term culture. Based on available patient consents, we deposited 3 organoid lines in a publicly accessible biobank. Finally, we investigated whether organoid drug responses correlate to those observed earlier in the clinic in the corresponding patients. Organoid-based drug-response profiling of clinically used HGSOC drug collection was performed in 384-well microplate format. To explore whether growth conditions impact correlation between organoid drug responses and clinical response, we compared the organoid drug responses in the nutrient-rich M1/M2 growth media to the ones observed in human plasma-like medium (HPLM), supplemented with relevant niche factors from M1/M2. Organoid drug responses correlated with clinical treatment outcomes, but only for organoids maintained in HPLM (Spearman r = 0.987, p=0.007 in HPLM vs 0.607, p=0.167 in growth medium, n=7), highlighting the importance of culture conditions in organoid-based functional assays. Taken together, we introduce a resource for efficient development and use of HGSOC organoids from cryopreserved material in ovarian cancer research. Citation Format: Wojciech Senkowski, Laura Gall Mas, Matias M. Falco, Yilin Li, Kari Lavikka, Mette C. Kriegbaum, Jaana Oikkonen, Daria Bulanova, Elin J. Pietras, Karolin Voßgröne, Erdogan P. Erkan, Terese K. Høj, Mia K. Høg, Tarja Lamminen, Katja Kaipio, Anni Virtanen, Lars H. Engelholm, Pernille Christiansen, Eric Santoni-Rugiu, Kaisa Huhtinen, Olli Carpén, Johanna Hynninen, Sampsa Hautaniemi, Anna Vähärautio, Krister Wennerberg. Efficient establishment and utilization of a high-grade serous ovarian cancer organoid biobank [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 3069.

The Wilms Tumor protein 1 (WT1) transcription factor (TF) is a ubiquitous biomarker of high-grade serous ovarian carcinoma (HGSOC), which is the most common and aggressive subtype of epithelial ovarian cancer (EOC). This TF interacts with a host of cellular factors including the tumor suppressor P53, which is mutated in virtually all HGSOC cases. Studies have suggested a role for WT1 in HGSOC development, but little is known about the underlying mechanism. We have generated unique catalogues of epigenomic landscapes (specifically H3K27ac) in primary HGSOC using chromatin immunoprecipitation sequencing (ChIP-seq). Using Bayesian analyses of H3K27ac profiling in HGSOCs, we identified significant tissue specific enrichment of active chromatin at the WT1 locus in HGSOC when compared to all other EOC tumor histotypes. Furthermore, this locus contains a superenhancer in HGSOCs and in normal fallopian tube secretory epithelial cells (FTSECs). This locus features a bidirectional promoter that is shared by WT1-AS lncRNA and that contains a WT1 binding site. LncRNAs are essential in many cellular processes, and have been shown to play pivotal roles in tumorigenicity. The WT1-AS lncRNA is implicated in other diseases but little is known of its function in EOC. We have investigated the mechanisms of interaction between WT1 and WT1-AS. We observed that both protein and transcript expression for WT1 and WT1-AS are positively correlated in HGSOC cell lines. Analysis of data for &amp;gt;400 primary HGSOCs from TCGA further supported this correlation. We hypothesized that WT1 is a critical TF in the development of HGSOC, and that WT1-AS plays a key role in its regulatory pathway. We evaluated the regulatory role of WT1 first by analyzing cellular phenotypes and target gene expression changes in HGSOC cell lines after shRNA mediated knockdown (KD) of WT1. HGSOC cells with stable WT1 knockdown had impaired proliferation and concomitant reduction of WT1-AS expression. We also identified a decrease in expression of SHMT1 in WT1 KD cells, which is a known WT1 target gene. Integrated genome-wide analysis of the WT1 transcriptome by RNA-seq and the WT1 cistrome by ChIP-seq, in parallel with ChIP-MS, is being used to establish the architecture of the WT1 regulome, specifically to establish the regulatory mechanisms and relationships between WT1 and WT1-AS. Ultimately, these studies aim to establish the mechanisms of action for the WT1 TF in neoplastic transformation of HGSOC. Citation Format: Norma I. Rodriguez-Malave, Justyna E. Kanska, Kevin C. Vavra, Dennis Hazelett, Rosario I. Corona, Ji-Heui Seo, Matthew Freedman, Simon R. Knott, Sandra Orsulic, Beth Y. Karlan, Kate Lawrenson, Simon A. Gayther. Role of the WT1 transcription factor in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5435.

Purpose: Platinum and PARP inhibitors (PARPi) demonstrate activity in breast and ovarian cancers, but drug resistance ultimately emerges. B7-H4 is over expressed in breast and ovarian cancer compared to normal tissues making it a promising target for therapies, however, to date it has not been evaluated if the expression is maintained in drug resistant cancer. Here we examine B7-H4 expression in high grade serous ovarian carcinoma (HGSOC) tumors at diagnosis and post-platinum or PARPi-resistance. We also evaluate the activity of a novel B7-H4-directed antibody-drug conjugate (ADC) bearing the pyrrolobenzodiazepine-dimer payload tesirine, in preclinical models of breast and ovarian cancer, including those resistant to standard of care therapies. Experimental Design: B7-H4 expression was measured by quantitative flow cytometry and immunohistochemistry. ADC efficacy was tested against multiple cell lines in vitro and patient-derived xenografts (PDX) in vivo. The effect of ADC treatment on cell cycle, DNA damage, and apoptosis was measured using flow cytometry. Results: B7-H4 was over-expressed in 92% of HGSOC tumors at diagnosis (n = 12), persisted in recurrent matched samples after platinum treatment, and was expressed at similar levels at multiple metastatic sites after acquired multi-drug resistance (n = 4 donors). Treatment with the ADC resulted in target-specific growth inhibition in a panel of ten B7-H4 expressing ovarian and breast cancer cell lines (IC50 6.18-273.9 pM, median 48.3 pM). Co-cultures of B7-H4 +/- lines support bystander killing effects leading to further tumor cell death in B7-H4- clones. In platinum- or PARPi-resistant ovarian cancer cells, ADC treatment significantly decreased viability and colony formation (P &amp;lt; 0.001) while increasing S or G2/M phase cell cycle arrest (P &amp;lt; 0.001) and DNA damage (P &amp;lt; 0.0001), ultimately leading to apoptosis (P &amp;lt; 0.01). A single 0.3 mg/kg dose of the ADC resulted in tumor regression in 61% of breast and ovarian PDX models tested (n = 23), where lower activity was identified in B7-H4 low or negative expressing models (P = 0.048). In PARPi- and platinum-resistant HGSOC models (n = 3), continuous B7-H4 ADC treatment (dosed once every 28 days) resulted in sustained anti-tumor activity, leading to complete (7 of 16) or partial responses (3 of 16) and increased survival (P &amp;lt; 0.004). Conclusions: These data support B7-H4 as an attractive ADC target for treatment of drug-resistant HGSOC. Citation Format: Sarah B. Gitto, Margaret Whicker, Gareth Davies, Sushil Kumar, Krista Kinneer, Arthur Lewis, Srinivas Mamidi, Sergey Medvedev, Judith Anderton, Jessica Tang, Benjamin Ferman, Steve Coats, Robert W. Wilkinson, Eric J. Brown, Daniel J. Powell, Fiona Simpkins. A B7-H4 targeting antibody-drug conjugate shows anti-tumor activity in PARPi and platinum resistant cancers with B7-H4 expression [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 1133.