A toolkit for a modern gynecologic oncology tissue bank.

OBJECTIVES: Tissue banking procedures have evolved to keep pace with precision medicine, technology, emerging understanding of racial disparities, and regulatory requirements. However, there is little published guidance regarding strategies to create and maintain a successful biorepository. Our objective is to describe the infrastructure and protocols used by our Gynecologic Oncology Tissue Bank. METHODS: Our Tissue Bank was founded in 1992. In August 2022, internal funding was used to modernize the Tissue Bank. We hired three full-time employees, implemented universal screening of patients treated by gynecologic oncology faculty, updated consenting protocols, and standardized communication with providers. Tumor tissue, blood derivatives, ascites, and pleural fluid were collected from eligible, consenting patients and processed. Patient-derived cell lines and organoids were generated. For quality control purposes, one formalin-fixed, paraffin-embedded (FFPE) sample per tissue site was analyzed by a board-certified pathologist. All samples were labeled and tracked in an OpenSpecimen collection protocol and clinically annotated in a secure database. RESULTS: From August 2022 to October 2023, 227 patients (83% white, 15% Black, 1% Asian) were enrolled and 4249 specimens were collected. Adherent cell lines were generated from 15 patients with ovarian cancer and cell suspensions for organoid generation were collected from 46 patients with ovarian cancer. A recharge center was established to self-sustain the Tissue Bank. Samples have been shared with academic and commercial collaborators. CONCLUSIONS: Our Tissue Bank has enrolled a large number of diverse patients, collected numerous specimen types, and collaborated widely. The procedures described here provide guidance for other institutions establishing similar resources.

A RAD18-UBC13-PALB2-RNF168 axis mediates replication fork recovery in BRCA1-deficient cancer cells.

BRCA1/2 proteins function in genome stability by promoting repair of double-stranded DNA breaks through homologous recombination and by protecting stalled replication forks from nucleolytic degradation. In BRCA1/2-deficient cancer cells, extensively degraded replication forks can be rescued through distinct fork recovery mechanisms that also promote cell survival. Here, we identified a novel pathway mediated by the E3 ubiquitin ligase RAD18, the E2-conjugating enzyme UBC13, the recombination factor PALB2, the E3 ubiquitin ligase RNF168 and PCNA ubiquitination that promotes fork recovery in BRCA1- but not BRCA2-deficient cells. We show that this pathway does not promote fork recovery by preventing replication fork reversal and degradation in BRCA1-deficient cells. We propose a mechanism whereby the RAD18-UBC13-PALB2-RNF168 axis facilitates resumption of DNA synthesis by promoting re-annealing of the complementary single-stranded template strands of the extensively degraded forks, thereby allowing re-establishment of a functional replication fork. We also provide preliminary evidence for the potential clinical relevance of this novel fork recovery pathway in BRCA1-mutated cancers, as RAD18 is over-expressed in BRCA1-deficient cancers, and RAD18 loss compromises cell viability in BRCA1-deficient cancer cells.

Updates in the Use of Targeted Therapies for Gynecologic Cancers.

Targeted therapies have changed the treatment landscape in gynecologic cancer. Studies released over the past year have led to the incorporation of immunotherapy (IO) into the treatment for all patients with endometrial and cervical cancers at some point during their disease course. Poly(ADP-ribose) polymerase (PARP) inhibitors continue to play a role in women with ovarian carcinoma, particularly in homologous repair deficient tumors. Furthermore, the benefit of PARP inhibitors in challenging subgroups continues to be elucidated. Biomarker identification has led to the approval or compendium listing of several antibody-drug conjugates (ADCs). This review will update on IO, ADCs, and PARP inhibition for the treatment of gynecologic cancers.

ROR2/Wnt5a Signaling Regulates Directional Cell Migration and Early Tumor Cell Invasion in Ovarian Cancer.

Adhesion to and clearance of the mesothelial monolayer are key early events in metastatic seeding of ovarian cancer. ROR2 is a receptor tyrosine kinase that interacts with Wnt5a ligand to activate noncanonical Wnt signaling and has been previously shown to be upregulated in ovarian cancer tissue. However, no prior study has evaluated the mechanistic role of ROR2 in ovarian cancer. Through a cellular high-throughput genetic screen, we independently identified ROR2 as a driver of ovarian tumor cell adhesion and invasion. ROR2 expression in ovarian tumor cells serves to drive directed cell migration preferentially toward areas of high Wnt5a ligand, such as the mesothelial lined omentum. In addition, ROR2 promotes ovarian tumor cell adhesion and clearance of a mesothelial monolayer. Depletion of ROR2, in tumor cells, reduces metastatic tumor burden in a syngeneic model of ovarian cancer. These findings support the role of ROR2 in ovarian tumor cells as a critical factor contributing to the early steps of metastasis. Therapeutic targeting of the ROR2/Wnt5a signaling axis could provide a means of improving treatment for patients with advanced ovarian cancer. IMPLICATIONS: This study demonstrates that ROR2 in ovarian cancer cells is important for directed migration to the metastatic niche and provides a potential signaling axis of interest for therapeutic targeting in ovarian cancer.

DDR2-regulated arginase activity in ovarian cancer-associated fibroblasts promotes collagen production and tumor progression.

Ovarian cancer has poor survival outcomes particularly for advanced stage, metastatic disease. Metastasis is promoted by interactions of stromal cells, such as cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME), with tumor cells. CAFs play a key role in tumor progression by remodeling the TME and extracellular matrix (ECM) to result in a more permissive environment for tumor progression. It has been shown that fibroblasts, in particular myofibroblasts, utilize metabolism to support ECM remodeling. However, the intricate mechanisms by which CAFs support collagen production and tumor progression are poorly understood. In this study, we show that the fibrillar collagen receptor, Discoidin Domain Receptor 2 (DDR2), promotes collagen production in human and mouse omental CAFs through arginase activity. CAFs with high DDR2 or arginase promote tumor colonization in the omentum. In addition, DDR2-depleted CAFs had decreased ornithine levels leading to decreased collagen production and polyamine levels compared to WT control CAFs. Tumor cell invasion was decreased in the presence CAF conditioned media (CM) depleted of DDR2 or arginase-1, and this invasion defect was rescued in the presence of CM from DDR2-depleted CAFs that constitutively overexpressed arginase-1. Similarly, the addition of exogenous polyamines to CM from DDR2-depleted CAFs led to increased tumor cell invasion. We detected SNAI1 protein at the promoter region of the arginase-1 gene, and DDR2-depleted CAFs had decreased levels of SNAI1 protein at the arginase-1 promoter region. Furthermore, high stromal arginase-1 expression correlated with poor survival in ovarian cancer patients. These findings highlight how DDR2 regulates collagen production by CAFs in the tumor microenvironment by controlling the transcription of arginase-1, and CAFs are a major source of arginase activity and L-arginine metabolites in ovarian cancer models.

Epigenetic regulation during cancer transitions across 11 tumour types.

Chromatin accessibility is essential in regulating gene expression and cellular identity, and alterations in accessibility have been implicated in driving cancer initiation, progression and metastasis1-4. Although the genetic contributions to oncogenic transitions have been investigated, epigenetic drivers remain less understood. Here we constructed a pan-cancer epigenetic and transcriptomic atlas using single-nucleus chromatin accessibility data (using single-nucleus assay for transposase-accessible chromatin) from 225 samples and matched single-cell or single-nucleus RNA-sequencing expression data from 206 samples. With over 1 million cells from each platform analysed through the enrichment of accessible chromatin regions, transcription factor motifs and regulons, we identified epigenetic drivers associated with cancer transitions. Some epigenetic drivers appeared in multiple cancers (for example, regulatory regions of ABCC1 and VEGFA; GATA6 and FOX-family motifs), whereas others were cancer specific (for example, regulatory regions of FGF19, ASAP2 and EN1, and the PBX3 motif). Among epigenetically altered pathways, TP53, hypoxia and TNF signalling were linked to cancer initiation, whereas oestrogen response, epithelial-mesenchymal transition and apical junction were tied to metastatic transition. Furthermore, we revealed a marked correlation between enhancer accessibility and gene expression and uncovered cooperation between epigenetic and genetic drivers. This atlas provides a foundation for further investigation of epigenetic dynamics in cancer transitions.

Stromal DDR2 Promotes Ovarian Cancer Metastasis through Regulation of Metabolism and Secretion of Extracellular Matrix Proteins.

Ovarian cancer is the leading cause of gynecologic cancer-related deaths. The propensity for metastasis within the peritoneal cavity is a driving factor for the poor outcomes associated with this disease, but there is currently no effective therapy targeting metastasis. In this study, we investigate the contribution of stromal cells to ovarian cancer metastasis and identify normal stromal cell expression of the collagen receptor, discoidin domain receptor 2 (DDR2), that acts to facilitate ovarian cancer metastasis. In vivo, global genetic inactivation of Ddr2 impairs the ability of Ddr2-expressing syngeneic ovarian cancer cells to spread throughout the peritoneal cavity. Specifically, DDR2 expression in mesothelial cells lining the peritoneal cavity facilitates tumor cell attachment and clearance. Subsequently, omentum fibroblast expression of DDR2 promotes tumor cell invasion. Mechanistically, we find DDR2-expressing fibroblasts are more energetically active, such that DDR2 regulates glycolysis through AKT/SNAI1 leading to suppressed fructose-1,6-bisphosphatase and increased hexokinase activity, a key glycolytic enzyme. Upon inhibition of DDR2, we find decreased protein synthesis and secretion. Consequently, when DDR2 is inhibited, there is reduction in secreted extracellular matrix proteins important for metastasis. Specifically, we find that fibroblast DDR2 inhibition leads to decreased secretion of the collagen crosslinker, LOXL2. Adding back LOXL2 to DDR2 deficient fibroblasts rescues the ability of tumor cells to invade. Overall, our results suggest that stromal cell expression of DDR2 is an important mediator of ovarian cancer metastasis. IMPLICATIONS: DDR2 is highly expressed by stromal cells in ovarian cancer that can mediate metastasis and is a potential therapeutic target in ovarian cancer.

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

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

Reproductive Scientist Development Program: Bridging the Gap to the Physician Scientist Career.

Career development awards are a successful strategy to facilitate the advancement of physician-scientists trained in obstetrics and gynecology (OBGYN) toward a path of investigative independence. While these funding mechanisms can be effective approaches to developing the career of future OBGYN scientists, optimizing the probability of obtaining these awards requires determining the appropriate career development award for the applicant. There are many details and opportunities that need to be considered when deciding on the appropriate award. Some of the most sought-after awards are those that integrate career development and applied research, such as the K-series awards supported by the National Institutes of Health (NIH). A quintessential example of an NIH-funded mentor-based career development award to support the scientific training of an OBGYN physician-scientist is the Reproductive Scientist Development Program (RSDP). In this study, we provide data on the academic achievements of past and present RSDP scholars and discuss the structure, impact, and future of the RSDP, a federally funded K12 program dedicated to women's health for OBGYN scientific investigators. As healthcare is changing and physician-scientists comprise a unique and valuable part of the biomedical workforce, programs such as the RSDP are critical to maintaining a well-trained pipeline of OBGYN scientists to maintain and challenge the leading edge of medicine, science, and biology.

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays.

Immunofluorescence is one of the most widely used techniques to visualize target antigens with high sensitivity and specificity, allowing for the accurate identification and localization of proteins, glycans, and small molecules. While this technique is well-established in two-dimensional (2D) cell culture, less is known about its use in three-dimensional (3D) cell models. Ovarian cancer organoids are 3D tumor models that recapitulate tumor cell clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Thus, they are superior to cell lines for the evaluation of drug sensitivity and functional biomarkers. Therefore, the ability to utilize immunofluorescence on primary ovarian cancer organoids is extremely beneficial in understanding the biology of this cancer. The current study describes the technique of immunofluorescence to detect DNA damage repair proteins in high-grade serous patient-derived ovarian cancer organoids (PDOs). After exposing the PDOs to ionizing radiation, immunofluorescence is performed on intact organoids to evaluate nuclear proteins as foci. Images are collected using z-stack imaging on confocal microscopy and analyzed using automated foci counting software. The described methods allow for the analysis of temporal and special recruitment of DNA damage repair proteins and colocalization of these proteins with cell-cycle markers.

Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids.

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the discovery of novel therapeutics and predictive biomarkers for ovarian cancer. These models recapitulate clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Additionally, they have been shown to match the primary tumor morphologically, cytologically, immunohistochemically, and genetically. Thus, organoids facilitate research on tumor cells and the tumor microenvironment and are superior to cell lines. The present protocol describes distinct methods to generate patient-derived ovarian cancer organoids from patient tumors, ascites, and pleural fluid samples with a higher than 97% success rate. The patient samples are separated into cellular suspensions by both mechanical and enzymatic digestion. The cells are then plated utilizing a basement membrane extract (BME) and are supported with optimized growth media containing supplements specific to the culturing of high-grade serous ovarian cancer (HGSOC). After forming initial organoids, the PDOs can sustain long-term culture, including passaging for expansion for subsequent experiments.

Identity-related experiences of Asian American trainees in gynecologic oncology.

Background: Anti-Asian violence increased during the COVID-19 pandemic. Asian American/Pacific Islanders (AAPI) represent a diverse population experiencing a long history of stereotyping and exclusionism; however, this group is often left out of diversity/inclusion conversations. In academic medicine, AAPI are under-represented in leadership. We characterized the personal/professional experiences of AAPI gynecologic oncology trainees and assessed the impact of a virtual panel discussion with leaders in the field. Methods: An anonymous survey was disseminated online to trainees in/interested in gynecologic oncology fellowship who identified as AAPI, using modified snowball sampling. A virtual session with AAPI leaders in gynecologic oncology discussed themes emerging from survey responses. Session attendees completed an anonymous follow-up survey. Results were assessed quantitatively and qualitatively. Results: 44/59 (75%) respondents participated in the pre-survey; 23 (39%) participated in the virtual session. All session participants (23/23, 100%) completed the post-session survey. Participants reported increased identity-related thoughts with the COVID-19 pandemic (88% during, 61% prior). Sixty-eight percent reported that identity-related thoughts/awareness changed during the pandemic. Presence of AAPI colleagues was associated with higher perceived identity-related support from their department. Of those without AAPI coworkers, none (0%) felt 'moderately' or 'extremely well supported.' Qualitative analysis demonstrated that the panel discussion created a sense of community and encouragement, combating previously reported isolation and self-consciousness. Participants reported more connection with their heritage and identified more personal/professional topics that might be related to their cultural backgrounds. Discussion: This intervention demonstrates the opportunity to provide a supportive network for mentorship and professional development in a culturally inclusive way.

State of the Biomarker Science in Ovarian Cancer: A National Cancer Institute Clinical Trials Planning Meeting Report.

PURPOSE: Despite therapeutic advances in the treatment of ovarian cancer (OC), 5-year survival remains low, and patients eventually die from recurrent, chemotherapy-resistant disease. The National Cancer Gynecologic Cancer Steering Committee identified the integration of scientifically defined subgroups as a top strategic priority in clinical trial planning. METHODS: A group of experts was convened to review the scientific literature in OC to identify validated predictive biomarkers that could inform patient selection and treatment stratification. Here, we report on these findings and their potential for use in future clinical trial design on the basis of hierarchal evidence grading. RESULTS: The biomarkers were classified on the basis of mechanistic targeting, including DNA repair and replication stress, immunotherapy and tumor microenvironment, oncogenic signaling, and angiogenesis. Currently, BRCA mutations and homologous recombination deficiency to predict poly (ADP-ribose) polymerase inhibitor response are supported in OC by the highest level of evidence. Additional biomarkers of response to agents targeting the pathways above have been identified but require prospective validation. CONCLUSION: Although a number of biomarkers of response to various agents in OC have been described in the literature, high-level evidence for the majority is lacking. This report highlights the unmet need for identification and validation of predictive biomarkers to guide therapy and future trial design in OC.

Community access to primary care is an important geographic disparity among ovarian cancer patients undergoing cytoreductive surgery.

Objective: Given the importance of understanding neighborhood context and geographic access to care on individual health outcomes, we sought to investigate the association of community primary care (PC) access on postoperative outcomes and survival in ovarian cancer patients. Methods: This was a retrospective cohort study of Stage III-IV ovarian cancer patients who underwent surgery at a single academic, tertiary care hospital between 2012 and 2015. PC access was determined using a Health Resources and Services Administration designation. Outcomes included 30-day surgical and medical complications, extended hospital stay, ICU admission, hospital readmission, progression-free and overall survival. Descriptive statistics and chi-squared analyses were used to analyze differences between patients from PC-shortage vs not PC-shortage areas. Results: Among 217 ovarian cancer patients, 54.4 % lived in PC-shortage areas. They were more likely to have Medicaid or no insurance and live in rural areas with higher poverty rates, significantly further from the treating cancer center and its affiliated hospital. Nevertheless, 49.2 % of patients from PC-shortage areas lived in urban communities. Residing in a PC-shortage area was not associated with increased surgical or medical complications, ICU admission, or hospital readmission, but was linked to more frequent prolonged hospitalization (26.3 % vs 14.1 %, p = 0.04). PC-shortage did not impact progression-free or overall survival. Conclusions: Patients from PC-shortage areas may require longer inpatient perioperative care in order to achieve the same 30-day postoperative outcomes as patients who live in non-PC shortage areas. Community access to PC is a critical factor to better understanding and reducing disparities among ovarian cancer patients.

High visceral fat to subcutaneous fat ratios portend a poor prognosis in patients with advanced endometrial cancer.

OBJECTIVES: Visceral adiposity has been established as a predictor of outcomes in various cancers. We aimed to determine the association of radiographic measurements of visceral fat with clinical outcomes in patients with endometrial cancer. METHODS: A retrospective review of patients with stage III-IV endometrial cancer who underwent surgery between 2004 and 2014 was performed. Visceral adipose tissue (VAT), subcutaneous adipose tissue (SAT), and total adipose tissue (TAT;VAT+SAT) were assessed on preoperative computed tomography (CT) scans. Exploratory analysis was performed to establish the optimal cut-off values for VAT, SAT, and TAT to identify patients with poor prognostic body composition. Survival rates were analyzed using Kaplan-Meier analysis, log-rank tests, and cox-regression. RESULTS: Eighty-three patients were included. Forty-two (51%) patients had a low VAT/SAT ratio (<0.45) and 41 (49.4%) had a high VAT/SAT ratio (>0.45). There were no significant differences in demographics between the groups. The mean VAT, SAT, and TAT were 176.3 cm2, 379.3 cm2, and 555.3 cm2 respectively. Compared to patients with low VAT/SAT ratios, patients with high VAT/SAT ratios had a shorter recurrence-free survival (median 29.6 vs 32.3 months, P = 0.01) and shorter overall survival (median 56 vs 93.7 months, P = 0.03). CONCLUSIONS: Visceral fat measurements are predictive of outcomes in patients with advanced stage endometrial cancer. Specifically, VAT to SAT ratios are predictive of overall survival. Future studies should be pursued to identify potential therapeutic targets and biological mechanisms that underlie obesity's relationship with endometrial cancer.

Gynecologic oncology patient perspectives and knowledge on advance care planning: A quality improvement intervention.

Objectives: Assess and improve advance care planning (ACP) awareness and uptake among gynecologic oncology patients. Methods: Using a quality improvement Plan-Do-Check-Act framework, we completed a single institution needs assessment and intervention. The needs assessment was a 26-question survey assessing baseline ACP knowledge and preferences of gynecologic oncology patients. We used this survey to implement an outpatient intervention in which patients were offered ACP resources (pamphlet, discussion with their gynecologic oncologist, and/or social work referral). We conducted a post-intervention survey among patients who had and had not received ACP resource(s) to assess whether our intervention increased ACP knowledge, discussions, or uptake. Results: Among 106 patients surveyed in the needs assessment, 33 % had ACP documents, 26 % had discussed ACP with a physician, and 82 % thought discussing ACP was important. The majority preferred these conversations in the outpatient setting (52 %) with their gynecologic oncologist (80 %) instead of nurses or trainees. In the intervention, 526 patients were offered ACP resources. Compared to women who did not receive resources (n = 324), patients who received ACP resource(s) (n = 202) were more likely to have ACP discussions with their gynecologic oncologist (38 % vs 68 %, P = 0.001) and had greater proficiency regarding how to create ACP documents (median score 5/10 vs 8/10, P = 0.048), although they were no more likely to have ACP documented in their electronic medical record (27 % vs 9 %, p = 0.08). Conclusions: ACP uptake among gynecologic oncology patients is low, but ACP discussions with an oncologist during outpatient visits are important to patients and improve their knowledge regarding completing ACP documents.

DDR2 Expression in Cancer-Associated Fibroblasts Promotes Ovarian Cancer Tumor Invasion and Metastasis through Periostin-ITGB1.

Ovarian cancer has the highest mortality of all gynecologic malignancies. As such, there is a need to identify molecular mechanisms that underlie tumor metastasis in ovarian cancer. Increased expression of receptor tyrosine kinase, DDR2, has been associated with worse patient survival. Identifying downstream targets of DDR2 may allow specific modulation of ovarian cancer metastatic pathways. Additionally, stromal cells play a critical role in metastasis. The crosstalk between tumor and stromal cells can lead to tumor progression. We first identified that tumor cells co-cultured with DDR2-expressing fibroblasts had lower periostin expression when compared to tumor cells co-cultured with DDR2-depleted fibroblasts. We confirmed that DDR2 regulates POSTN expression in ovarian cancer-associated fibroblasts (CAFs). We found that mesothelial cell clearance and invasion by tumor cells were enhanced three-fold when DDR2 and POSTN-expressing CAFs were present compared to DDR2 and POSTN-depleted CAFs. Furthermore, DDR2-depleted and POSTN-overexpressing CAFs co-injected with ovarian tumor cells had increased tumor burden compared to mice injected with tumor cells and DDR2 and POSTN-depleted CAFs. Furthermore, we demonstrated that DDR2 regulates periostin expression through integrin B1 (ITGB1). Stromal DDR2 is highly correlated with stromal POSTN expression in ovarian cancer patient tumors. Thus, DDR2 expression in CAFs regulates the steps of ovarian cancer metastasis through periostin.