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Supplementary Table 4

All supplemental text and 9 supplementary figures

Supplementary Table 1

Supplementary Table 3

Table S1. Differential expression analysis for CD8 T cells.

Table S5. List of Genes Used to Generate NK Cell Activation Scores.

Table S2. Differential expression analysis for Treg CD4 T cells.

Table S3. Differential expression analysis for non-Treg CD4 T cells.

Immune therapies have had limited efficacy in high-grade serous ovarian cancer (HGSC), as the cellular targets and mechanism(s) of action of these agents in HGSC are unknown. Here we performed immune functional and single-cell RNA sequencing transcriptional profiling on novel HGSC organoid/immune cell co-cultures treated with a unique bispecific anti-programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) antibody compared with monospecific anti-PD-1 or anti-PD-L1 controls. Comparing the functions of these agents across all immune cell types in real time identified key immune checkpoint blockade (ICB) targets that have eluded currently available monospecific therapies. The bispecific antibody induced superior cellular state changes in both T and natural killer (NK) cells. It uniquely induced NK cells to transition from inert to more active and cytotoxic phenotypes, implicating NK cells as a key missing component of the current ICB-induced immune response in HGSC. It also induced a subset of CD8 T cells to transition from naïve to more active and cytotoxic progenitor-exhausted phenotypes post-treatment, revealing the small, previously uncharacterized population of CD8 T cells responding to ICB in HGSC. These state changes were driven partially through bispecific antibody-induced downregulation of the bromodomain-containing protein BRD1. Small-molecule inhibition of BRD1 induced similar state changes in vitro and demonstrated efficacy in vivo, validating the co-culture results. Our results demonstrate that state changes in both NK and a subset of T cells may be critical in inducing an effective anti-tumor immune response and suggest that immune therapies able to induce such cellular state changes, such as BRD1 inhibitors, may have increased efficacy in HGSC.Significance:This study indicates that increased efficacy of immune therapies in ovarian cancer is driven by state changes of NK and small subsets of CD8 T cells into active and cytotoxic states.

Table S4. Differential expression analysis for NK cells.

Supplementary Materials and Methods, Figures, and Figure and Table legends

Endometrial cancer is the most common gynecologic cancer in the U.S., but metastasis to the brain is rare, and diagnosis can be challenging. Traditional tools for determining if a tumor is a primary or metastatic lesion include pan-imaging, histopathologic studies, and immunohistochemistry. Molecular testing with next-generation sequencing has been increasingly used to augment these tests. We present a case of a patient who initially presented with a brain lesion diagnosed as glioblastoma on histology and immunohistochemistry, but whose diagnosis was later changed to metastasis from an endometrial primary based on molecular findings. The two tumors shared a common microsatellite instability signature and 51 DNA variants, including oncogenic driver mutations KRAS p.G13D, PIK3CA p.E545A, and PTEN p.I135V and p.K267Rfs*9. This highlights the power of molecular analysis in making the diagnosis in cases of rare metastases. Key Points Brain metastasis from endometrial primary is rare, and histopathological features may be augmented with molecular analysis to aid in diagnosis. Comparison of the molecular makeup of the primary endometrial lesion with the metastatic lesion may reveal high-risk molecular features that may be indicative of metastatic potential.

The purpose of this study was to determine the Magnetic Resonance (MR) imaging features that best differentiate leiomyosarcoma (LMS) from leiomyoma, and to explore a scoring system to preoperatively identify those at highest risk of having LMS. Our Institutional Review Board approved this retrospective HIPAA-compliant study with a waiver for written informed consent. Institutional Research Patient Data Registry identified patients with histopathologically-proven LMS (n = 19) or leiomyoma (n = 25) and a pelvic MRI within six months prior to surgery. Qualitative differentiating MRI features were selected based on prior publications and clinical experience. Patient and MRI characteristics for leiomyomas versus LMS were compared using Wilcoxon rank-sum tests or Fisher’s exact tests and using a basic classification tree. Hypothesis testing was two-tailed, with a p value

Immune therapies have had limited efficacy in high-grade serous ovarian cancer (HGSC), as the cellular targets and mechanism(s) of action of these agents in HGSC are unknown. Here we performed immune functional and single-cell RNA sequencing transcriptional profiling on novel HGSC organoid/immune cell co-cultures treated with a unique bispecific anti-programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) antibody compared with monospecific anti-PD-1 or anti-PD-L1 controls. Comparing the functions of these agents across all immune cell types in real time identified key immune checkpoint blockade (ICB) targets that have eluded currently available monospecific therapies. The bispecific antibody induced superior cellular state changes in both T and natural killer (NK) cells. It uniquely induced NK cells to transition from inert to more active and cytotoxic phenotypes, implicating NK cells as a key missing component of the current ICB-induced immune response in HGSC. It also induced a subset of CD8 T cells to transition from naïve to more active and cytotoxic progenitor-exhausted phenotypes post-treatment, revealing the small, previously uncharacterized population of CD8 T cells responding to ICB in HGSC. These state changes were driven partially through bispecific antibody-induced downregulation of the bromodomain-containing protein BRD1. Small-molecule inhibition of BRD1 induced similar state changes in vitro and demonstrated efficacy in vivo, validating the co-culture results. Our results demonstrate that state changes in both NK and a subset of T cells may be critical in inducing an effective anti-tumor immune response and suggest that immune therapies able to induce such cellular state changes, such as BRD1 inhibitors, may have increased efficacy in HGSC. Significance: This study indicates that increased efficacy of immune therapies in ovarian cancer is driven by state changes of NK and small subsets of CD8 T cells into active and cytotoxic states.

To determine whether neoadjuvant chemotherapy (NACT) disproportionately benefits obese patients.Data were collected from stage IIIC-IV ovarian cancer patients treated between 01/2010-07/2015. We performed univariate/multivariate logistic regression analyses with post-operative infection, readmission, any postoperative complication, and time to chemotherapy as outcomes. An interaction term was included in models, to determine if the effect of NACT on post-operative complications was influenced by obesity status.Of 507 patients, 115 (22.6%) were obese and 392 (77.3%) were non-obese (obese defined as BMI ≥30). Among obese patients undergoing primary debulking surgery (PDS) vs. NACT, rates of postoperative infection were 42.9% vs. 30.8% (p = 0.12), 30-day readmission 30.2% vs. 11.5% (p 0.02), and any post-operative complication were 44.4% vs 30.8% (p = 0.133). Among non-obese patients undergoing PDS vs. NACT, rates of post-operative infection were 20.0% vs. 12.9% (p = 0.057), 30-day readmission 16.9% vs. 9.2% (p = 0.02), and any post-operative complication were 19.4% vs 28% (p = 0.044). Obesity was associated with post-operative infection (OR 2.3; 95%CI 1.22-4.33), 30-day readmission/reoperation (OR 2.27; 95%CI 1.08-3.21) and the development of any post-operative complication (OR 2.1; CI 1.13-3.74). However, there was not a significant interaction between obesity and NACT in any of the models predicting post-operative complications.The decision to use NACT should not be predicated on obesity alone, as the reduction in post-operative complications in obese patients is similar to non-obese patients.

IntroductionIn gynecologic patients, few studies describe the accuracy of the American College of Surgeons-National Surgical Quality Improvement Project (ACS-NSQIP) pre-operative risk calculator for women undergoing surgery for ovarian cancer.ObjectiveTo determine whether the ACS-NSQIP risk calculator accurately predicts post-operative complications and length of stay in patients undergoing interval debulking surgery for advanced stage epithelial ovarian cancer.MethodsFor this multi-institutional retrospective cohort study, pre-operative risk factors, post-operative complication rates, and Current Procedural Terminology codes were abstracted from records of patients with ovarian cancer managed with open interval debulking surgery from January 2010 to July 2015. A power calculation was done to estimate the minimum number of complications needed to evaluate the accuracy of the ACS-NSQIP risk calculator. Predicted risk compared with observed risk was calculated using logistic regression. The predictive accuracy of the ACS-NSQIP risk calculator in estimating post-operative complications or length of stay was assessed using c-statistics and Briar scores. Complications with a c-statistic of >0.70 and Brier score of ResultsA total of 261 patients underwent interval debulking surgery, encompassing 21 unique Current Procedural Terminology codes. Readmission (n=25), surgical site infection (n=35), urinary tract infection (n=12), and serious post-operative complications (n=57) met the minimum event threshold (n>10). All predicted complication rates fell within the IQR of the observed incidence rates. However, the ACS-NSQIP calculator demonstrated neither discriminative ability nor accuracy for any post-operative complications based on c-statistics and Brier scores. The calculator accurately predicted length of stay within 1 day for only 32% of patients and could not accurately predict which patients were likely to have a prolonged length of stay (c-statistic=0.65).ConclusionAmong patients undergoing interval debulking surgery, the ACS-NSQIP did not accurately discriminate which patients were at increased risk of complications or extended length of stay. The risk calculator should be considered to have limited utility in informing pre-operative counseling or surgical planning.

Uterine carcinoma (UC) is the most common gynecologic malignancy in the United States. TP53 mutant UCs cause a disproportionate number of deaths due to limited therapies for these tumors and the lack of mechanistic understanding of their fundamental vulnerabilities. Here we sought to understand the functional and therapeutic relevance of TP53 mutations in UC. We functionally profiled targetable TP53 dependent DNA damage repair and cell cycle control pathways in a panel of TP53 mutant UC cell lines and patient-derived organoids. There were no consistent defects in DNA damage repair pathways. Rather, most models demonstrated dependence on defective G2/M cell cycle checkpoints and subsequent upregulation of Aurora kinase-LKB1-p53-AKT signaling in the setting of baseline mitotic defects. This combination makes them sensitive to Aurora kinase inhibition. Resistant lines demonstrated an intact G2/M checkpoint, and combining Aurora kinase and WEE1 inhibitors, which then push these cells through mitosis with Aurora kinase inhibitor-induced spindle defects, led to apoptosis in these cases. Overall, this work presents Aurora kinase inhibitors alone or in combination with WEE1 inhibitors as relevant mechanism driven therapies for TP53 mutant UCs. Context specific functional assessment of the G2/M checkpoint may serve as a biomarker in identifying Aurora kinase inhibitor sensitive tumors.

Objective The objective of this study was to determine the proportion of patients with a pre-invasive endometrial lesion who meet Mayo criteria for lymph node dissection on final pathology to determine if the use of sentinel lymph node biopsy in patients with pre-invasive lesions would be warranted. Methods All women who underwent hysterectomy for a pre-invasive endometrial lesion (atypical hyperplasia or endometrial intra-epithelial neoplasia) between 2009 and 2019 were included for analysis. Relevant statistical tests were utilized to test the associations between patient, operative, and pathologic characteristics. Results 141 patients met inclusion criteria. 51 patients (36%) had a final diagnosis of cancer, the majority (96%) of which were Stage IA grade 1 endometrioid carcinomas. Seven patients (5%) met Mayo criteria on final pathology (one grade 3, seven size >2 cm, one >50% myoinvasive). Three of these seven patients had lymph nodes assessed of which 0% had metastases. Six of these patients had frozen section performed, and 2 met (33%) Mayo criteria intraoperatively. Of the seven patients in the overall cohort that had lymph node sampling, six had a final diagnosis of cancer and none had positive lymph nodes. Of the 51 patients with cancer, only 10 had cancer diagnosed using frozen section, and only two met intra-operative Mayo criteria. Age > 55 was predictive of meeting Mayo criteria on final pathology (p = 0.007). No patients experienced a cancer recurrence across a median follow up of 24.3 months. Conclusions Atypical hyperplasia and endometrial intra-epithelial neoplasia portend low risk disease and universal nodal assessment is of limited value.

Background This article reports a unique case of cesarean scar pregnancy, demonstrating importance of early management and diagnosis. Case presentation A 30-year-old pregnant woman with prior history of two cesarean sections found to have cesarean scar pregnancy at approximately 13 weeks’ gestation and underwent a gravid hysterectomy. Conclusions While rare, cesarean scar pregnancies should be considered on the differential diagnosis of any pregnant patient with history of cesarean section who presents in early pregnancy with vaginal bleeding and/or cramping. Given the increased rates of cesarean sections in the times of COVID-19, one may anticipate seeing more cases of cesarean scar pregnancies.

Simple Summary Cancers arising from the lining of the uterus, endometrial cancers, are the most common gynecologic malignancy in the United States. Once endometrial cancer escapes the uterus and grows in distant locations, there are limited therapeutic options. The most aggressive and lethal endometrial cancers carry alterations in the protein p53, which is a critical guardian of many cellular functions. The role of these p53 alterations in endometrial cancer is not well understood. The goal of this work was to use p53 altered models of endometrial cancer to understand which, if any, therapeutically targetable vulnerabilities these p53 alterations may confer in endometrial cancer. Here we show that many of these p53 altered cells have problems with cell division which can be targeted with novel single and combination therapies. These discoveries may lead to relevant new therapies for difficult to treat advanced stage endometrial cancers. Abstract Uterine carcinoma (UC) is the most common gynecologic malignancy in the United States. TP53 mutant UCs cause a disproportionate number of deaths due to limited therapies for these tumors and the lack of mechanistic understanding of their fundamental vulnerabilities. Here we sought to understand the functional and therapeutic relevance of TP53 mutations in UC. We functionally profiled targetable TP53 dependent DNA damage repair and cell cycle control pathways in a panel of TP53 mutant UC cell lines and patient-derived organoids. There were no consistent defects in DNA damage repair pathways. Rather, most models demonstrated dependence on defective G2/M cell cycle checkpoints and subsequent upregulation of Aurora kinase-LKB1-p53-AKT signaling in the setting of baseline mitotic defects. This combination makes them sensitive to Aurora kinase inhibition. Resistant lines demonstrated an intact G2/M checkpoint, and combining Aurora kinase and WEE1 inhibitors, which then push these cells through mitosis with Aurora kinase inhibitor-induced spindle defects, led to apoptosis in these cases. Overall, this work presents Aurora kinase inhibitors alone or in combination with WEE1 inhibitors as relevant mechanism driven therapies for TP53 mutant UCs. Context specific functional assessment of the G2/M checkpoint may serve as a biomarker in identifying Aurora kinase inhibitor sensitive tumors.

PROBLEM Cervical cancer screening strategies in the United States include cotesting (human papillomavirus (HPV) with cytology), primary HPV with genotyping and reflex cytology, and cytology alone. An ongoing challenge is the appropriate triage of patients to colposcopy to those at highest risk. We investigated whether incorporation of p16INK4a immunodetection by enzyme-linked immunosorbent assay (ELISA) on fresh cervical samples obtained at the time of screening could improve appropriate referral to colposcopy. METHOD OF STUDY A derivation group comprised of cervical swabs collected from subjects with high-grade dysplasia or cancer (positive control) and from subjects with negative screening history (negative control). Samples collected from colposcopy were used to evaluate the existing screening strategies individually and with incorporation of p16INK4a ELISA. Histology was used as the gold standard. RESULTS Among 163 subjects recruited, 138 were included. In the derivation group, mean p16INK4a level was 2.86 ng/mL (n = 31) and 0.58 ng/mL (n = 20) among positive and negative controls respectively (p = 0.002) with an area under the receiver operator characteristic curve of 0.79 (p

The purpose of this study was to determine the Magnetic Resonance (MR) imaging features that best differentiate leiomyosarcoma (LMS) from leiomyoma, and to explore a scoring system to preoperatively identify those at highest risk of having LMS.Our Institutional Review Board approved this retrospective HIPAA-compliant study with a waiver for written informed consent. Institutional Research Patient Data Registry identified patients with histopathologically-proven LMS (n = 19) or leiomyoma (n = 25) and a pelvic MRI within six months prior to surgery. Qualitative differentiating MRI features were selected based on prior publications and clinical experience. Patient and MRI characteristics for leiomyomas versus LMS were compared using Wilcoxon rank-sum tests or Fisher's exact tests and using a basic classification tree. Hypothesis testing was two-tailed, with a p value 0.001 used to determine inclusion of variables into an MR imaging predictive (MRP) score. Diagnostic performance [sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV)] of the MRP in diagnosis of LMS used all possible scores as cutoffs.Seven out of 15 MRI features were found to have an association with LMS. The final MRP scores ranged from 0 to 7: a score of 0-3 was associated with 100% NPV for LMS, and a MRP score of 6-7 with 100% PPV for LMS.Seven qualitative MR imaging features, extracted from a standard MR imaging protocol, allow differentiation of LMS from leiomyoma. An exploratory risk stratification MRP score can be used to determine the likelihood of LMS being present.

Highlights • FATWOs are rare gynecologic neoplasms of low malignant potential derived from mesonephric (Wolffian) duct remnants. • FATWOs have diverse presentations from vague abdominal symptoms to incidental diagnosis. • In general, FATWOs require no additional management beyond initial surgical intervention.

Background Heterotopic pregnancies implanted in a rudimentary uterine horn account for 1 in 2–3 million gestations, and confer significant risk of morbidity due to uterine rupture and hemorrhage. Case presentation A 34-year-old nullipara presented with acute pelvic pain at 17 weeks of gestation with dichorionic-diamniotic twins, one in each horn of an anomalous uterus first diagnosed in pregnancy as bicornuate. Three-dimensional ultrasound and MRI revealed myometrial disruption in the left rudimentary uterine horn, and the patient underwent an uncomplicated abdominal hemi-hysterectomy. Fourteen days later, an uncomplicated dilation and curettage was performed for a fetal anomaly in the remaining twin in the right unicornuate uterus. Conclusion This case demonstrates the utility of magnetic resonance imaging and three-dimensional ultrasound in the assessment of myometrial integrity in a gravid patient with a heterotopic pregnancy and ruptured rudimentary uterine horn. This case demonstrates the importance of pre-pregnancy diagnosis and management of mullerian anomalies.

Objectives: To determine whether the American College of Surgery-National Surgery Quality Improvement Program (ACS-NSQIP) risk calculator accurately predicts postoperative complications and length of stay in patients undergoing interval debulking surgery (IDS) for advanced stage epithelial ovarian cancer. Methods: Pre-operative risk factors, post-operative complication rates, and Current Procedural Terminology codes were abstracted from records of ovarian cancer patients managed with IDS from 01/2010-07/2015. A power calculation was done to estimate the minimum number of complications needed to evaluate accuracy of the ACS-NSQIP risk calculator. Predicted risk compared with observed risk was calculated using logistic regression. The predictive accuracy of the ACS-NSQIP risk calculator in estimating post-operative complications or length of stay (LOS) was assessed using c-statistics and Briar scores. Complications with a c-statistic of >0.70 and Brier score of Results: 261 patients underwent IDS, encompassing 21 unique CPT codes. Readmission(n=25), surgical site infection(n=35), urinary tract infection(n=35), and serious postoperative complications(n=57) met the minimum event threshold (n>10). All predicted complication rates fell within the interquartile range of the observed incidence rates, however the ACS-NSQIP calculator demonstrated neither discriminative ability nor accuracy for any postoperative complications based on c-statistics and Brier scores. The calculator accurately predicted LOS within 1 day for only 32% of patients and could not accurately predict patients likely to have a prolonged LOS (c-statistic=0.65). Download : Download high-res image (99KB) Download : Download full-size image Conclusions: Among patients undergoing IDS, the ACS-NSQIP did not accurately discriminate which patients were at increased risk of complications or extended LOS. The risk calculator should be considered to have limited utility in informing preoperative counseling or surgical planning.

Objective The purpose of this study was to determine the prevalence of incidentally discovered serous tubal intraepithelial carcinoma in women without a genetic risk for or history of high grade serous carcinoma (HGSC) in the gynecologic tract. Methods All pathology reports at our institution that included bilateral salpingectomies from January 2006–December 2011 were examined in women >50years old in which the entire tube or the distal one-third was examined histologically with the complete (proximal and distal fallopian tube) or modified (distal one third of the tube) SEE-FIM protocol. Cases were divided into: Group 1, a history of or known risk factors (BRCA1 or BRCA2 mutations) for HGSC and Group 2, those without these attributes for whom a STIC would be unexpected (incidental). Women undergoing unspecified "risk-reducing" procedures were included in Group 1. Results Of 4051 identified total, 2268 had complete examination of the distal fallopian tube and were age 50 or above. Of these, 1747 were in group 2. Two STICs were identified (0.1%), one associated with a grade 2 endometrial endometrioid adenocarcinoma and one with a low-grade ovarian serous carcinoma in the setting of a serous borderline tumor. Conclusions Incidental STICs in women over age 50 are uncommon. However, the significance of lesser tubal atypias (0.3% in this study), risk of STIC in women with no epithelial pathology and the risk imposed by coexisting endometrioid neoplasia are unclear and require further study.

Although consensus has yet to be reached on universal mismatch-repair (MMR) protein immunohistochemical (IHC) screening for Lynch syndrome (LS) in endometrial cancer (EC), an increasing number of institutions have adopted universal screening protocols similar to those used for colorectal carcinoma. Here we describe our institution's experience with a prospective universal screening protocol in which all ECs resected over a period of 19 months (n=242) were screened for MLH1, PMS2, MSH2, and MSH6 deficiencies using IHC, followed by MLH1 promoter methylation testing when appropriate. When consent was obtained, tumor samples underwent next-generation sequencing. A total of 11 unmethylated MMR-deficient cases (4.5% of cohort) were identified through IHC screening. Germline testing was performed in 10 cases and confirmed LS in 4 patients (1.7% of cohort). Of our 4 confirmed LS cases, 1 did not meet traditional LS screening criteria (eg, age below 50 y, Revised Bethesda criteria). In addition, universal screening identified 6 germline-negative MMR-deficient nonmethylated cases, 4 of which occurred in women older than 50. Although our next-generation sequencing data suggest somatic mutations in 4 of these cases, it is possible that these cases may represent cases of "Lynch-like syndrome." We conclude that a subset of LS cases could be missed using traditional screening guidelines. The value of screening for Lynch-like syndrome has yet to be determined. Although the cost-effectiveness of universal screening in EC has yet to be elucidated, we conclude that universal IHC screening is currently a reasonable, and arguably superior, approach to screening for LS.

It is now well known that the distal fallopian tube is a site of origin for many high-grade serous carcinomas (HGSCs). HGSCs are thought to arise via intra-peritoneal spread of tumor cells from serous tubal intra-epithelial carcinomas (STICs) that share identical TP53 mutations. However, a dualistic model of pathogenesis might be indicated because many HGSCs are not associated with STICs and their site(s) of origin is unclear. This fact has important implications for both screening and cancer preventive strategies. In addition to STICs, the tube also hosts TP53 mutation-positive early serous proliferations (ESPs) that comprise a morphologic spectrum ranging from p53 signatures to serous tubal intraepithelial lesions (STILs). ESPs are more common than STICs and are not considered malignant; however, they are often the only abnormality found in fallopian tubes of women with HGSCs. The purpose of this study was to determine if a genetic relationship exists between isolated ESPs and concurrent HGSCs in the absence of STIC. Fallopian tubes from 32 HGSCs without a co-existing STIC on initial review were exhaustively and serially sectioned. The presence of either occult STIC or ESP in serial sections was documented and DNAs from tissues containing ESPs, cancers and normal control epithelia were interrogated for TP53 mutations by targeted amplicon-based sequencing with average coverage reads >4000 across DNA replicate samples. Serial sectioning unearthed a STIC in 3 of 32 (9.3%) and ESPs in 12 (37.5%). Four of 12 ESPs (33.0%) shared an identical TP53 mutation with the concurrent cancer at an allele frequency ≥5%. An additional 5 ESPs (41.7%) also shared identical TP53 mutations with concurrent cancers at a lower ( Citation Format: Thing Rinda Soong, MD, PhD, MPH, Brooke E. Howitt, MD, Alexander Miron, PhD, Neil Horowitz, MD, Frank Campbell, PhD, Colleen M. Feltmate, MD, Michael G. Muto, MD, Ross S. Berkowitz, MD, Marisa R. Nucci, MD, Wa Xian, PhD, Christopher P. Crum, MD. LINEAGE CONTINUITY BETWEEN EARLY TUBAL SEROUS PROLIFERATIONS (ESPS/STILS) AND DISSEMINATED HIGH-GRADE SEROUS CARCINOMAS: A MODEL FOR “PRECURSOR ESCAPE” [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 AP01.

A chemotherapy response score (CRS) system was recently described to assess the histopathologic response and prognosis of patients with tubo-ovarian high-grade serous carcinoma (HGSC) receiving neoadjuvant chemotherapy. The current study was performed as an independent assessment of this CRS system. We retrospectively identified advanced stage HGSC patients who received neoadjuvant chemotherapy and underwent interval debulking. If available, a hemotoxylin and eosin slide from the omentum and the adnexa was selected for the study. Slides were independently scored by 13 pathologists using the 3-tiered CRS system. Reviewers then received web-based training and rescored the slides. Overall survival and progression-free survival were estimated using the Kaplan-Meier method and compared using the log-rank test. A total of 68 patients with omental (n=65) and/or adnexal (n=59) slides were included in the study. Interobserver reproducibility was moderate for omentum (κ, 0.48) and poor for adnexa (κ, 0.40), which improved for omentum (κ, 0.62) but not for adnexa (κ, 0.38) after online training. For omental slides, a consensus CRS of 1/2 was associated with a shorter median progression-free survival (10.9 mo; 95% confidence interval, 9-14) than a CRS of 3 (18.9 mo; 95% CI, 18-24; P=0.020). In summary, a 3-tiered CRS system of hemotoxylin and eosin-stained omental deposits can yield prognostic information for HGSC patients receiving neoadjuvant chemotherapy, and web-based training improved reproducibility but did not alter determination of clinical outcomes. The CRS system may allow oncologists to identify potential nonresponders and triage HGSC patients for heightened observation and/or clinical trials.

Background The development of intrinsic and acquired resistance to antineoplastic agents is a major obstacle to successful chemotherapy in ovarian cancers. Identification and characterisation of chemoresponse-associated biomarkers are of paramount importance for novel therapeutic development. Methods Global RNA expression profiles were obtained by high-throughput microarray analysis. Cell cycle, proliferation rate, and paclitaxel sensitivity of ovarian cancer cells harbouring cyclin A1-inducible expression construct were compared with and without tetracycline induction, as well as when the cyclin A1 expression was suppressed by short inhibiting RNA (siRNA). Cellular senescence was evaluated by β-galactosidase activity staining. Results Global RNA expression profiling and subsequent correlation studies of gene expression level and drug response has identified that elevated expression of cyclin A1 (CCNA1) was significantly associated with cellular resistance to paclitaxel, doxorubicin and 5-fluorouracil. The role of cyclin A1 in paclitaxel resistance was confirmed in ovarian cancer cells that harbour an inducible cyclin A1 expression construct, which showed reduced paclitaxel-mediated growth inhibition and apoptosis when cyclin A1 expression was induced, whereas downregulation of cyclin A1 expression in the same cell lines using cyclin A1-specific siRNAs sensitised the cells to paclitaxel toxicity. However, ovarian cancer cells with ectopic expression of cyclin A1 demonstrated slowdown of proliferation and senescence-associated β-galactosidase activity. Conclusions Our profiling and correlation studies have identified cyclin A1 as one chemoresistance-associated biomarker in ovarian cancer. The results of the characterisation studies suggest that cyclin A1 functions as an oncogene that controls proliferative and survival activities in tumourigenesis and chemoresistance of ovarian cancer.

// Yanli Zhang 1 , Jamie Sui-Lam Kwok 2 , Pui-Wah Choi 1 , Minghua Liu 2 , Junzheng Yang 1 , Margit Singh 1 , Shu-Kay Ng 4 , William R. Welch 5 , Michael G. Muto 1 , Stephen KW Tsui 2 , Stephen P. Sugrue 3 , Ross S. Berkowitz 1 , Shu-Wing Ng 1 1 Laboratory of Gynecologic Oncology, Division of Gynecologic Oncology, Department of Obstetrics, Gynecology and Reproductive Biology, Harvard Medical School, Boston, MA, USA 2 School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong 3 Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL, USA 4 School of Medicine and Menzies Health Institute Queensland, Griffith University, Meadowbrook, Australia 5 Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA Correspondence to: Shu-Wing Ng, e-mail: sng@partners.org Keywords: ovarian cancer, tumorigenesis, RNA metabolism, cell adhesion, RNA sequencing Received: October 21, 2015 Accepted: January 24, 2016 Published: February 08, 2016 ABSTRACT Unlike many other human solid tumors, ovarian tumors express many epithelial markers at a high level for cell growth and local invasion. The phosphoprotein Pinin plays a key role in epithelial cell identity. We showed that clinical ovarian tumors and ovarian cancer cell lines express a high level of Pinin when compared with normal ovarian tissues and immortalized normal ovarian surface epithelial cell lines. Pinin co-localized and physically interacted with transcriptional corepressor C-terminal binding proteins, CtBP1 and CtBP2, in the nuclei of cancer cells. Knockdown of Pinin in ovarian cancer cells resulted in specific reduction of CtBP1 protein expression, cell adhesion, anchorage-independent growth, and increased drug sensitivity. Whole transcriptomic comparison of next-generation RNA sequencing data between control ovarian cancer cell lines and cancer cell lines with respective knockdown of Pinin, CtBP1, and CtBP2 expression also showed reduced expression of CtBP1 mRNA in the Pinin knockdown cell lines. The Pinin knockdown cell lines shared significant overlap of differentially expressed genes and RNA splicing aberrations with CtBP1 knockdown and in a lesser degree with CtBP2 knockdown cancer cells. Hence, Pinin and CtBP are oncotargets that closely interact with each other to regulate transcription and pre-mRNA alternative splicing and promote cell adhesion and other epithelial characteristics of ovarian cancer cells.

Genomic analyses indicate that 50% of high-grade serous ovarian cancers (HGSC) harbor a genomic alteration in a DNA damage repair gene that may lead to functional defects. Using functional assays on patient-derived HGSC organoid cultures to test the capacity of the tumor cells to repair double-strand DNA breaks and to protect stalled replication forks, we have found that many HGSCs have stalled fork protection defects regardless of the genomic background of the tumor and that these defects correlate with sensitivity to replication stress inducing therapeutic agents. We hypothesized that gaining a better understanding of the mechanisms of replication fork instability and stability in HGSC organoid cultures would help to better understand the mechanisms of therapeutic sensitivity of the tumor cells. The purpose of this work is to understand how replication fork stabilization either in the primary tumor or through selection post-treatment leads to alterations in tumor cell biology, including therapeutic sensitivity and interaction of the tumor cells with the surrounding microenvironment. We utilized bulk RNA sequencing analysis of HGSC organoid cultures with varied replication fork protection capacity, some matched pairs of untreated and post-neoadjuvant tumors, to stratify differences in functional profiles in fork stable versus unstable tumors; we then used basic molecular biology techniques to understand the mechanisms of fork stabilization and how this stabilization affects the therapeutic sensitivity of the cells. We also developed and utilized multiple functional assays to assess the interaction of HGSC organoids of varying fork protection capacity with their immune microenvironment in different drug exposure settings. We identified multiple proteins that through either up- or downregulation lead to stabilization of replication forks in the tumor cells and found that the mechanisms of stabilization can occur at both the level of the replication fork and the overall transcriptional level of the cell and can alter the therapeutic sensitivity of the cells. We have determined that replication fork stability leads to increased mesenchymal characteristics in tumors and to decreased activation of the antitumor immune response within the cultures after treatment with DNA damage repair and immuno-oncologic (IO) agents. Overall, these results indicate that replication fork stabilization in HGSC through multiple different mechanisms can lead to altered interactions of the tumor cells with their microenvironment and altered therapeutic sensitivity. This abstract is also being presented as Poster B10. Citation Format: Sarah J. Hill, Patrick Lizotte, Nikolas Kesten, Neil S. Horowitz, Michael G. Muto, Michael J. Worley, Colleen M. Feltmate, Ross S. Berkowitz, Henry Long, Ursula A. Matulonis, Christopher P. Crum, Myles Brown, Alan D. D'Andrea. Dissecting mechanisms of replication fork stabilization in patient-derived high-grade serous organoid cultures and their impact on therapeutic sensitivity and the immune-tumor interaction [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 PR06.

Objectives To determine whether perioperative red blood cell transfusion (PRBCT) affects infection, thrombosis, or survival rates in epithelial ovarian cancer (EOC) patients undergoing neoadjuvant chemotherapy (NACT) and interval debulking surgery (IDS). Methods Demographics, operative characteristics, and outcome data were abstracted from records of stage IIIC-IV EOC patients managed with NACT-IDS from 01/2010–07/2015. Associations of PRBCT with morbidity and oncologic outcomes were evaluated. Results Of 270 patients, 136 (50.4%) received PRBCT. Patients with preoperative anemia and higher estimated blood loss (EBL) were more likely to undergo PRBCT (OR,95%CI 1.80, 1.02–3.17) and (OR,95%CI 1.00, 1.002–1.004), respectively. There were no significant differences in PRBCT based on patient age, Charlson Comorbidity Index, or stage. When compared to low complexity operations, patients with moderate and high complexity surgeries were more likely to receive PRBCT (OR,95%CI 1.81, 1.05–3.09) and (OR,95%CI 2.25, 1.13–4.50), respectively. On univariate analysis, PRBCT was associated with intraabdominal infection (OR,95%CI 8.31, 1.03–67.41), but not wound complications (OR,95%CI 1.57, 0.76–3.23) or venous thromboembolism/pulmonary embolism (VTE/PE) (OR,95%CI 2.02, 0.49–8.23). After adjusting for surgical complexity and preoperative anemia, PRBCT was not independently associated with intraabdominal infection (OR,95%CI 7.66, 0.92–63.66), wound complications (OR,95%CI 1.70, 0.80–3.64), or VTE/PE (OR,95%CI 2.15, 0.51–9.09). When comparing patients undergoing PRBCT versus those who did not, there were no significant differences in median progression-free survival (PFS) or median overall survival (OS) on univariate analysis after adjusting for age, stage and residual disease. Conclusions Among patients undergoing NACT-IDS, intraabdominal infection, wound complication and VTE/PE rates are similar, regardless of PRBCT. PRBCT does not impact PFS or OS.

Based on genomic analysis, 50% of high-grade serous ovarian cancers (HGSC) are predicted to have DNA repair defects. Whether this substantial subset of HGSCs actually have functional repair defects remains unknown. Here, we devise a platform for functional profiling of DNA repair in short-term patient-derived HGSC organoids. We tested 33 organoid cultures derived from 22 patients with HGSC for defects in homologous recombination (HR) and replication fork protection. Regardless of DNA repair gene mutational status, a functional defect in HR in the organoids correlated with PARP inhibitor sensitivity. A functional defect in replication fork protection correlated with carboplatin and CHK1 and ATR inhibitor sensitivity. Our results indicate that a combination of genomic analysis and functional testing of organoids allows for the identification of targetable DNA damage repair defects. Larger numbers of patient-derived organoids must be analyzed to determine whether these assays can reproducibly predict patient response in the clinic. Significance: Patient-derived ovarian tumor organoids grow rapidly and match the tumors from which they are derived, both genetically and functionally. These organoids can be used for DNA repair profiling and therapeutic sensitivity testing and provide a rapid means of assessing targetable defects in the parent tumor, offering more suitable treatment options. Cancer Discov; 8(11); 1404–21. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1333

The distal Fallopian tube is a site of origin for many 'ovarian' high-grade serous carcinomas (HGSCs) with intraepithelial carcinomas (STICs) that share identical TP53 mutations with metastatic tumors. TP53 mutation-positive early serous proliferations (ESPs) comprise a spectrum including p53 signatures and serous tubal intraepithelial lesions (STILs) and are not considered malignant; however, ESPs are often the only abnormality found in Fallopian tubes of women with metastatic HGSC. The purpose of this study was to determine if a relationship exists between isolated ESPs and concurrent metastatic HGSCs in the absence of STIC. Fallopian tubes from 32 HGSCs without a co-existing STIC/HGSC in the endosalpinx were exhaustively sectioned. The presence of either STIC/HGSC or ESP in the endosalpinx was documented and DNA from tissues containing ESPs, concurrent HGSC, and control epithelia were interrogated for TP53 mutations by targeted amplicon-based sequencing with average coverage reads >4000 across DNA replicate samples. Serial sectioning revealed a previously unrecognized STIC/HGSC in 3 of 32 (9.3%) and ESPs in 13 (40.6%). Twelve contained TP53 mutations. Nine (75%) shared identical TP53 mutations with concurrent HGSCs, four at high (≥ 5%) and five at low (< 5%) allele frequency. All control epithelia were TP53 mutation-negative. This study, for the first time, indicates lineage identity between ESPs in the distal tube and some metastatic HGSCs via a shared site-specific TP53 mutation. It supports a novel serous carcinogenic sequence in which an ESP could eventually culminate in a metastatic serous cancer via 'precursor escape' and would explain the apparent sudden onset of cancers without co-existing STICs. This paradigm for serous cancer development underscores the likelihood that multiple precursor types in the Fallopian tube contribute to serous cancer development with implications for the evolution, pathologic classification, and prevention of this lethal malignancy. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Objective To evaluate the impact of insurance status on the stage of cervical cancer diagnosed and treated at a tertiary care center in Massachusetts and review the preceding screening history. Methods An IRB approved retrospective cohort study was conducted of patients with a diagnosis of cervical cancer treated at Brigham and Women's Hospital (BWH) between January 2011 and June 2016. Clinical and demographic data was extracted from the longitudinal medical record. Statistical analysis was performed using SAS. Results 117 cases of cervical cancer met the inclusion criteria during the study period. Most patients (76%) were diagnosed with stage I disease. On univariate analysis, compared to patients with private insurance, patients with public insurance or no documented insurance presented at older ages, were more likely to be non-white races, and present with advanced stage disease. In an adjusted model, the risk of being diagnosed with advanced stage disease persisted among women with public or no documented insurance, adjusted odds ratio (aOR) 4.13 (1.37–12.45). There was no difference in screening history among women with private vs. public insurance, p = 0.30. Conclusions Despite access to insurance, patients with public issued insurance had an increased risk of presenting with advanced stage cervical cancer in this cohort. These data suggest that additional barriers to screening and prevention may exist and are important for future investigation.

To examine the relationship between volume of residual disease and oncologic outcomes among patients with advanced-stage epithelial ovarian/fallopian tube/primary peritoneal carcinoma undergoing primary debulking surgery (PDS). For patients that did not undergo a complete surgical resection (CSR), a surrogate for volume of residual disease was used to assess oncologic outcomes.Medical records of patients with FIGO stage IIIC and IV epithelial ovarian/fallopian tube/primary peritoneal carcinoma undergoing PDS between January 2010 and November 2014 were reviewed. Patient demographics, operative characteristics, residual disease, anatomic site of residual disease and outcome data were collected. Among patients who did not undergo CSR, but had ≤1 cm of residual disease, the number of anatomic sites (single location vs. multiple locations) with residual disease was utilized as a surrogate for volume of residual disease. The effect of residual disease volume on progression-free survival (PFS) and overall survival (OS) was evaluated.Of 240 patients undergoing PDS, 94 (39.2%) had CSR, 41 (17.1%) had ≤1 cm of residual disease confined to a single anatomic location (≤1 cm-SL), 67 (27.9%) had ≤1 cm of residual disease in multiple anatomic locations (≤1 cm-ML) and 38 (15.8%) were sub-optimally (SO) debulked. Median PFS for CSR, ≤1 cm-SL, ≤1 cm-ML and SO-debulked were: 23, 19, 13 and 10 months, respectively (p 0.001). Median OS for CSR, ≤1 cm-SL, ≤1 cm-ML and SO-debulked were: Not yet reached, 64, 50 and 49 months, respectively (p = 0.001).Following PDS, CSR and ≤ 1 cm-SL patients have the best prognosis. In contrast, despite being considered "optimally debulked", ≤1 cm-ML patients have survival similar to those SO-debulked.

Background Extracellular vesicles (EVs) are considered as a new class of resources for potential biomarkers. We analyzed expression of specific mRNA and miRNA in EVs derived from ovarian cancer ascites and the ideal controls, peritoneal fluids from benign patients for potential early detection and prognostic biomarkers. Methods Fluids were collected from subjects with benign cysts or endometrioma (n = 10), or low/high grade serous ovarian carcinoma (n = 8). EV particles were captured using primarily ExoComplete filterplate or ultracentrifugation and analyzed by nanoparticle tracking analysis, ELISA, and scanning electron microscopy. EV RNAs extracted from two ascites and three peritoneal fluids were submitted for next-generation sequencing. The expression of 34 mRNA and 18 miRNAs in the EVs isolated from patient fluids and cell line media was determined using qPCR. Results EVs isolated from patient samples had concentrations greater than 1010 EV particles/mL and 30% were EpCAM-positive based on ELISA. EV particle sizes averaged 113 ± 11.5 nm. The qPCR studies identified five mRNA (CA11, MEDAG, LAMA4, SPINT2, NANOG) and six miRNA (let-7b, miR23b, miR29a, miR30d, miR205, miR720) that were significantly differentially expressed between cancer ascites and peritoneal fluids. In addition, CA11 mRNA was decreased to 0.5-fold and SPINT2 and NANOG mRNA were significantly increased up to 100-fold in conditioned media of cancer cells compared to immortalized ovarian surface and fallopian tube epithelial cell lines, the hypothesized cells of origin for ovarian cancer development. Conclusions This study indicates that EV mRNA profiles can reflect the disease stage and may provide a potentially novel source for discovery of biomarkers in ovarian cancer. Electronic supplementary material The online version of this article (10.1186/s13048-018-0391-2) contains supplementary material, which is available to authorized users.

Aim To report on the unique, innovative management of a super morbidly obese patient presenting for fertility preservation in the setting of Grade 2 endometrioid endometrial adenocarcinoma. Case report A back-to-back oocyte retrieval and robot-assisted total laparoscopic hysterectomy (RA-TLH) with bilateral salpingo-oophorectomy (BSO) was performed in a 37-year-old nulligravid woman with super morbid obesity (BMI 63 kg/m2) with endometrial cancer. Main outcome measure the successful retrieval of oocytes for fertility preservation and oncologic intervention via RA-TLH with BSO in the same operative and anesthetic episode. Results This combined procedure strategy was successful in our case and is a feasible option for similar high-risk patients in the future. Conclusion Identifying safe and effective approaches for oocyte retrieval in morbidly obese patients represents a pressing need as obesity rates rise among reproductive-aged women. Back-to-back oocyte retrieval and RA-TLH with BSO are not only feasible, but could also decrease significant anesthetic risks for morbidly obese patients.

An 85-yr-old woman was diagnosed with endometrial adenocarcinoma, endometrioid type. Imaging studies showed a large tumor distending the endometrial canal without evidence of local invasion or extrauterine disease. A hysterectomy was performed, followed by microscopic examination of longitudinal tissue sections. Histopathological review showed only focal myometrial invasion, equivocal lymphovascular invasion, and negative bilateral sentinel lymph nodes (FIGO stage IA). A sample of the tumor was submitted for molecular testing (massively parallel sequencing on OncoPanel) and was found to harbor an inversion on Chromosome 2 resulting in an EML4-ALK gene fusion. Confirmatory immunohistochemistry showed ALK overexpression in just a portion of the tumor. Additional genomic characterization on a region of the tumor lacking ALK overexpression by immunohistochemistry was highly congruous with the genomic profile of the ALK-positive portion, showing similar patterns of copy-number variation and mutations in TP53 and KDM5C, with no evidence for an EML4-ALK gene fusion, confirming that EML4-ALK rearrangement had occurred as a subclonal process. EML4-ALK fusions are driver events in 2%–5% of non-small-cell lung cancers; crizotinib is an approved targeted therapy for these patients. EML4-ALK rearrangements have not previously been reported in endometrial cancer.

This chapter updates several important issues pertaining to epithelial malignancies of the ovary. These include: (1) origins, including fallopian tube and updates on the potential significance of and interpretive pitfalls with serous tubal intraepithelial carcinoma (STIC), (2) subdivisions of high-grade serous carcinoma (HGSC) that have clinical and possibly pathogenetic importance, (3) molecular parameters useful in subclassification, (4) recent data on the outcome of stage I noninvasive low-grade serous carcinomas (LGSCs; also known as micropapillary borderline tumors), and (5) the spectrum of borderline tumors of mixed epithelial type. As always mucinous tumors include both endocervical and intestinal type, with an additional gastric variant that must be kept in mind that can be associated with Peutz-Jeghers syndrome.

This chapter addresses several important aspects of ovarian cancer prevention. One pertains to the risk factors and efforts at early detection, critically reviewing the data on screening tests for ovarian cancer. Another addresses the role of the fallopian tube, a topic that has been at the center of efforts to reduce the risk of high-grade serous cancer via both risk reduction salpingo-oophorectomy and opportunistic salpingectomy. The role of the pathologist is first and foremost by more careful scrutinizing of the fallopian tube using the Sectioning and Extensively Examining the FIMbraited End (SEE-FIM) protocol, which has allowed all pathologists to participate in this paradigm shift to the oviduct as a potential source and target of prevention. Both supporting data and caveats are presented on this compelling issue that continues to evolve and the various entities that deserve attention as either potential premalignant lesions or their mimics are illustrated.

Increased inclusion cyst formation in the ovary is associated with ovarian cancer development. We employed in vitro three-dimensional (3D) organotypic models formed by normal human ovarian surface epithelial (OSE) cells and ovarian cancer cells to study the morphologies of normal and cancerous ovarian cortical inclusion cysts and the molecular changes during their transitions into stromal microenvironment. When compared with normal cysts that expressed tenascin, the cancerous cysts expressed high levels of laminin V and demonstrated polarized structures in Matrigel; and the cancer cells migrated collectively when the cyst structures were positioned in a stromal-like collagen I matrix. The molecular markers identified in the in vitro 3D models were verified in clinical samples. Network analysis of gene expression of the 3D structures indicates concurrent downregulation of transforming growth factor beta pathway genes and high levels of E-cadherin and microRNA200 (miR200) expression in the cancerous cysts and the migrating cancer cells. Transient silencing of E-cadherin expression in ovarian cancer cells disrupted cyst structures and inhibited collective cell migration. Taken together, our studies employing 3D models have shown that E-cadherin is crucial for ovarian inclusion cyst formation and collective cancer cell migration.