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Introduction/Background* Endometrial carcinoma patient care was based on histopathologic examination for many years. However, conventional pathologic features are known to suffer from high inter-observer variability and may be irreproducible in many cases. TCGA-derived molecular classification was shown to provide clinically meaningful data and was recently introduced to ESGO/ESTRO/ESP endometrial carcinoma consensus guidelines. It was the aim of this study to quantify alterations in risk-assessment if molecular classification is added to conventional prognosticators. Methodology Consecutive primary endometrial carcinoma patients diagnosed in 2016 were identified in participating centres. Original risk classification (‘ORC16’, ESGO/ESMO guidelines 2016) was compared to current molecular-based risk assessment (‘MBR21’, ESGO/ESMO guidelines 2021). Clinical and histopathological data were collected and tumor specimens were retrospectively submitted to PMS2, MSH6 and p53 immunohistochemistry and POLE mutation testing. Result(s)* 226 patients were identified across five major European gynecologic oncology centres. Complete molecular and clinical data were available from 198 cases with a median follow-up time of 52.1 months. Median age was 64.9 years (30.9-90.9), 165 cases (83,3%) were endometrioid histotype. Grade distribution included 85(42.9%) G1, 63(31.8%) G2, and 46(23.3%) G3 tumors. 98(49.5%) patients had stage IA disease, with the remaining stage IB (n=51;25.8%), stage II (n=16;8.0%), and stage III/IV (n=33;16.7%). Molecular classification yielded 43(21.7%) MMR-D, 18(9.1%) POLE, 43(21.7%) p53abn, and 94(47.5%) p53wt tumors. If ORC16 was compared to MBR21, risk was found to be higher in 15(7.6%) cases, whereas 27(13.6%) cases were assigned to a lower risk group. No survival events were observed in the 9 patients where risk was changed from high-intermediate or high to low-risk. Conclusion* We were able to demonstrate clinically relevant alterations of endometrial carcinoma risk assessment in a significant number of patients after adding TCGA-derived molecular data to conventional risk classification. Potential molecular-guided changes in patient management may help to avoid over- and undertreatment and will ultimately give rise to precision medicine strategies in endometrial carcinoma patient care.

Introduction/Background* The TransPORTEC Consortium was established in 2013 by the PIs and translational science representatives of the PORTEC-3 trial groups from the Netherlands, United Kingdom, Australia, Canada and France. Purpose of the collaboration is to improve treatment of endometrial cancer (EC) patients. Here, we evaluate our experience with international collaboration to identify challenges and strengths. Methodology Result(s)*: Since its establishment, TransPORTEC had a strong scientific team of chief investigators, translational leads and core members from participating groups. Twice-yearly TransPORTEC-meetings were organised to build and maintain friendships, share results and discuss new proposals. Over time, the TransPORTEC-biobank has expanded with PORTEC-trial tumour tissues and other cohorts, and is now the world’s largest set of molecularly classified ECs. The research focus has expanded to include molecular cancer immunology and digital pathology. The group’s output include 10 scientific papers and numerous posters and presentations on (inter)national meetings. Their analysis of PORTEC-3 showing differences in chemotherapy effect by molecular group led to initiating an international program with 4 clinical trials on Refining Adjuvant treatment IN endometrial cancer Based On molecular features (RAINBO) to compare personalised to standard treatment in terms of efficacy, toxicity, quality of life and cost-utility (figure 1). Tumour material of all participants will be collected for translational research. To achieve this, the consortium evolved: new talented members were attracted and trial-specific and expertise teams were installed (figure 2). Despite this impacting on group equilibrium, the collaboration is continuously productive. Keys to success were frequent meetings, sharing of draft protocols and experiences with contacting (inter)national research organisations and potential funders. The first of the RAINBO trials is expected to open by the end of 2021 and the program will probably fuel translational research for years to come. Conclusion* International research collaborations are dynamic and demanding. Challenges include: balancing between a stable organisational structure and flexibility to adapt to opportunities; providing all members with a satisfying share; and acquisition of funding for academic-sponsored international trials. Strengths are the profound interaction and trust between members with different expertise and backgrounds and shared ambitions and successes, resulting in unique and innovative academic research projects with leverage.

Introduction/Background* Tumor cell dissemination is associated with a less favorable outcome in breast cancer patients. In contrast, only limited clinical significance was yet reported for other gynecologic malignancies. We have previously reported disseminated tumor cells (DTC) not to be associated with established risk factors, L1CAM immunoreactivity and outcome in endometrial carcinoma. It was the aim of this study to investigate potential associations of TCGA-derived molecular features such as POLE-mutation status, p53 abnormalities or MMR deficiency and the presence of DTC in the bone marrow of endometrial carcinoma patients. Methodology Patients treated for primary endometrial carcinoma at Tuebingen University women’s hospital between 2003 and 2016 with bone marrow aspirates and FFPE tumor specimens were identified. For DTC detection, cytospins were stained for pan-cytokeratin (A45-B/B3 antibody). Molecular classification was performed according to the Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE). Result(s)* A total of 402 patients with a complete set of bone marrow cytology, molecular and clinical data was evaluable. ProMisE molecular classification revealed 40(10.0%) POLEmut, 103(25,6%) MMRd, 52(12,9%) p53-abnormal and 207(51.5%) tumors with no specific molecular profile (NSMP). Overall DTC were detected in 71/402 (17.7%) patients. DTC occurrence was distributed equally among molecular groups (p=0.651). DTC were present in 7/40(17.5%) POLEmut, 21/103(20.4%) MMRd, 32/207(15.5%) NSMP and 11/52(21.2%) p53 abnormal tumors. Conclusion* The scientific community widely agrees that molecular classification will be of key importance in future endometrial carcinoma patient care. In line with our previous findings, tumor cell dissemination is not associated with TCGA-inspired molecular groups in our large cohort of primary endometrial carcinoma patients. While DTC are detectable in a significant number of patients, even including cases with favorable POLEmut subtype, tumor cell dissemination seems not to play a role in disease progression and clinical outcome in endometrial carcinoma.

Introduction/Background* The role of lymph node (LN) assessment in endometrial cancer (EC) has been a subject of debate for decades, with significant variation in use between centres. Molecular classification of EC provides objective, prognostic information and be performed on diagnostic endometrial biopsy specimens. The Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) uses targeted next-generation sequencing to detect pathogenic POLE exonuclease domain mutations (POLEmut) and immunohistochemistry to evaluate for the presence of mismatch repair deficiency (MMRd), TP53 mutations (p53abn) or tumours with no specific molecular profile (NSMP/p53wt). Herein, we assessed the association between EC molecular subtype and LN metastases in a single institutional cohort with a uniform approach to LN assessment. Methodology All ECs treated with primary surgery from a single institution in 2015 underwent ProMisE molecular subtyping and collection of clinicopathologic and outcomes data. Result(s)* Complete pelvic and para-aortic lymphadenectomies were performed in 171 of 172 consecutive cases of EC. The distribution of ProMisE subtypes and clinicopathologic features associated with molecular subtype are outlined in table 1. The p53abn subtype was observed across a range of EC histotypes, including low grade endometrioid endometrial carcinoma. LN metastases were found in 31/171 (18.1%) patients: pelvic only in 83.9% and pelvic plus para-aortic in 16.1%. LN metastases included macrometastases (19/31), micrometastases (5/31), and isolated tumour cells (ITCs) (7/31). Molecular subtype was significantly associated with LN metastases (p=0.004); there was a strong association between LN metastases and p53abn EC (nodal involvement in 44.8% of cases). LN metastases were observed in 14.2% of POLEmut, 14.9% of MMRd, and 10.8% of NSMP EC. By multivariate analysis, molecular subtype and CA 125 >25 kU/L were significantly associated with LN metastases (p=0.021 and p=0.022 respectively) compared to histotype, which showed no significant association with LN status (p=0.24). Conclusion* EC molecular subtype significantly associates with LN metastases and offers objective, reproducible, and prognostic information from diagnostic specimens. Pre-operative knowledge of molecular subtype can guide biologically-informed approaches to LN sampling, particularly for patients with high molecular risk (p53abn) EC.

Introduction/Background* Vulvar squamous cell carcinoma (VSCC) is subclassified into three prognostically relevant groups: i) HPV-Associated (HPV-A), ii) HPV-Independent TP53 mutated (HPV-I p53abn), and iii) HPV-Independent TP53 wild type (HPV-I p53wt). Immunohistochemistry (IHC) for p16 and p53 are used as surrogate markers for HPV viral integration and TP53 mutational status, respectively. We assessed the reproducibility of subclassification based on p16 and p53 IHC and evaluated the prognostic significance of VSCC molecular subgroups in a patient cohort treated by vulvar field resection (VFR) surgery. Methodology VSCC formalin-fixed paraffin-embedded tumour tissue and patient clinicopathologic data from 68 cases treated by vulvar field resection (VFR) were collected from the Leipzig School of Radical Pelvic Surgery. Immunohistochemistry for p16 and p53 was performed at three different institutions and evaluated independently by seven pathologists and two pathology research fellows. Tumors were classified into one of four groups: HPV-A, HPV-I p53wt, HPV-I p53abn, and Indeterminate (requiring additional investigation). Selected cases were further interrogated by HPV RNA in situ hybridisation (ISH) and TP53 sequencing. Result(s)* Final VSCC subclassification yielded 22 tumors (32.4%) were HPV-A, 41 (60.3%) were HPV-I p53abn, and 5 (7.3%) were HPV-I p53wt; the clinicopathologic data is summarised in table 1. Interobserver agreement (overall Fleiss’ kappa statistic) for the four-category classification was 0.74. No statistically significant differences in clinical outcomes between HPV-A and HPV-I VSCC were observed (figure 1). Conclusion* Interobserver reproducibility of VSCC subclassification based on p16 and p53 IHC is very good, supporting their routine use in clinical practice. VSCCs treated by VFR surgery showed no significant difference in clinical outcomes when stratified based on HPV status. This contrasts with the significant prognostic differences between HPV-A and HPV-I VSCC demonstrated in cohorts treated by conventional conservative surgical approaches and suggests that a change in surgical approach can improve outcomes in patients with HPV-I VSCC.

Introduction The benefit of systematic lymphadenectomy (LNE) in low-stage, low-grade ovarian carcinoma is unknown. However, most guidelines still recommend LNE in these patients. Prior studies examining the benefit of this invasive procedure have been hampered small numbers, and large-scale studies that consider modern classification are needed. Methods A cohort of 666 pathology-reviewed and immunohistochemistry-validated endometrioid ovarian carcinomas has recently been evaluated using endometrial carcinoma-inspired molecular subtyping. This molecularly characterized series is now being used to assess the value of LNE. Contributing centers are performing detailed chart reviews, so that surgical procedures and lymph node status can be correlated with molecular subtype and outcomes. Results 349 stage I, 181 stage II, 85 stage III, and 22 stage IV cases with a median OS follow-up of 6.11 years (RevKM) were collected from 17 centres across Canada and Europe. Analysis of the first 70/666 cases revealed positive nodes in only a single presumed low stage patient after systematic pelvic and paraaortic LNE (n=1/44). LNE was not performed in 3/44 and restricted to pelvic nodes in 6/44 low-stage cases, all of which were pN0. Tumor spread beyond the Uterus and/or Adnexa was associated with positive nodes in 33%. Conclusion Preliminary results indicate that abandonment of LNE in low-stage, low-grade endometrioid ovarian carcinoma may reduce morbidity without worsening prognosis for these patients. Completion and expansion of our international team initiative stands to provide a powerful statement on the value of LNE, and influence of molecular subtype on disease spread, possibly improving precision care for ovarian carcinoma patients.

Objectives In preparation for provincial implementation of ProMisE molecular classification, two KT objectives were identified: 1) build awareness of ProMisE with first line knowledge users (KUs); and 2) elucidate barriers and facilitators to KUs use of ProMisE. Methods KUs were defined as pathologists, and clinicians (general practitioners, gynecologists, and oncologists) from rural and urban centres. The KT intervention was an explanatory video reviewing the rationale and relevance of molecular classification via three segments: ProMisE overview, clinician-focused, and pathologist-focused. Dissemination of the video to KUs occurred using a mixed-methods approach in two phases: I: KUs were exposed to the video and survey through institutional listservs; II: KUs were engaged for in-person viewing and focus group. Results The survey received 41 partial and 37 complete responses with representation from all targeted KUs: gynecologists(62.2%), oncologists(rad+gyn-oncs+other 32.4%), pathologists(5.4%) and all provincial health authorities. 95.1% of KUs watched the ProMisE overview, with 80.5% and 68.3% completing the clinician and pathology segments, respectively. Over 90% of respondents reported it having contained enough information to understand the advantages, and 64% felt ProMisE would be useful to guide management. KUs across specialties participated in a focus group (n=19;7 rural) to identify barriers and facilitators to implementation. Thematic analysis of survey and focus group illuminated implementation concerns related to cost, availability, accessibility, process, training, and interpretation of the test. Conclusions Motivation and engagement for EC molecular classification was recognized by KUs with implementation concerns. Addressing cross-provincial access, process, and timely results will ensure rapid uptake and fidelity.

Introduction/Background Four pragmatic molecular subtypes of endometrial cancer (EC) has been established and these seem promising approach in risk stratification. The mismatch repair deficient (MMRd) and p53 wildtype subtypes are the most common and given their molecular subgroups with intermediate prognosis additional biomarkers to further stratify these groups are needed. Fibroblast growth factor receptor (FGFR2) has two major splice isoforms, FGFR2b and FGFR2c. FGFR2c is an oncogen and has been associated with aggressive tumour behaviour in several cancers. The objective was to investigate the role of FGFR2c in progression and risk stratification within the MMRd and p53 wt subtypes. Methodology We have developed, optimized and validated a novel RNA in situ hybridization assay to detect FGFR2c. Expression was determined in screening cohort of ECs (n=78) and the clinically annotated Canadian cohort (n=465). Kaplan Meier curve and Cox regression model analyses were performed to assess the prognostic value of FGFR2c. Results There was a significant difference in mRNA FGFR2c expression between endometrioid EC and non-endometrioid EC, 50% versus 23%, p Conclusion FGFR2c is an independent prognostic biomarker in EC and its expression further discerns the outcome of EC patients with molecularly classified MMRd and p53 wildtype tumours. Integration of FGFR2c into the new molecular subtyping can further refine risk stratification of endometrioid EC. Disclosure Nothing to disclose.

Introduction/Background Endometrioid ovarian carcinoma (ENOC) is generally associated with a more favorable prognosis compared to other ovarian carcinoma histotypes. Nonetheless, patients are still treated according to a ‘one size fits all’ approach. While tumor staging offers some stratification, the development of personalized treatment concepts remain elusive. Our group has recently shown that the Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE), also provides additional prognostic information in it’s ovarian endometrioid counterpart. The aim of this study was to investigate ProMisE in a large cohort of low stage (FIGO I-II) ovarian endometrioid carcinoma. Methodology After establishment of prognostic groups in >500 ENOC we examined a subset of n=379 FIGO stage I-II cases. Cases were aligned into low risk POLE mutant (POLE); moderate risk mismatch repair deficient (MMRd); high-risk p53 abnormal (p53abn); and a final moderate risk category lacking these biomarkers (p53wt). Kaplan-Meier survival analysis was performed. Results 4% of cases were POLE, 15% MMRd, 73% p53wt and 8% p53abn. Groups showed distinct progression-free and overall survival (p Conclusion ProMisE risk classification provides additional prognostic information in a large cohort of low stage ENOC. In the context of low-stage ENOC, and especially younger women wishing to preserve fertility, the introduction of ProMisE-stratified treatment algorithms may serve not only to improve patient care overall but to reduce chemotherapy burden and highlight cases where fertility-sparing surgeries pose little or no risk. Disclosure Nothing to disclose.

Introduction/Background Mismatch repair (MMR) deficiency is found in 20 to 40% of endometrial cancers (ECs) and was recently identified as a discerning feature of one of the four prognostic subgroups identified by The Cancer Genome Atlas. There is accumulating evidence that MMR proteins are involved in the DNA repair processes following radiotherapy. We investigated the predictive value of MMR status for response to adjuvant radiotherapy in patients with stage IB/II, grade 3 endometrioid endometrial cancer (EEC). Methodology A retrospective multicenter cohort study was performed to compare patients with histopathologically confirmed stage IB/II grade 3 EEC with and without adjuvant radiotherapy. Patients were classified according to the Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) identifying ECs as either MMR-deficient, POLE, p53abn or p53wt. Multivariable Cox regression analysis explored associations between adjuvant treatment and outcome. Results A total of 128 patients were analyzed, including 57 patients (43.0%) with MMR-deficient EECs. Baseline characteristics were comparable, except a higher proportion of MMR-deficient EECs were stage II (36.8% vs. 15.5%, p=0.006). Eighty-two patients (64.1%) received adjuvant radiotherapy (external beam [n=55], vaginal brachytherapy [n=27]). In multivariable analysis, adjuvant radiotherapy was associated with improved disease-specific survival in patients with MMR-deficient EECs (hazard ratio 0.19, 95%-CI 0.05–0.77), but not in patients with MMR-proficient EECs (hazard ratio 0.92, 95%-CI 0.37–2.31). Conclusion Adjuvant radiotherapy improved survival in patients with MMR-deficient EECs. MMR status could be used as a predictive biomarker to select patients that benefit most from adjuvant radiotherapy. Disclosure Nothing to disclose.

Introduction/Background Mismatch repair deficiency (MMRd) is observed in 25–30% of all endometrial cancers. This can be detected by the absence of MMR protein staining on immunohistochemistry (IHC). Only 10% of women with MMRd have Lynch syndrome, but MMRd may still have prognostic significance. The objective of this study was to compare clinical outcomes between MMR deficient and proficient low-risk endometrioid endometrial cancers (stage IA, grade 1/2). Methodology This was a retrospective population-based cohort study of all low-risk endometrial cancers from Vancouver Coastal Health authority region from 2011 to 2016 that were assessed for MMR deficiency. Primary outcome measures were recurrence rates expressed per person-years (py), progression free survival (PFS) and overall survival (OS) calculated using Kaplan-Meier method and log-rank tests. Cox proportional hazards model estimated the association between MMRd and recurrence/death after adjustment for covariates. Results There were 477 low-risk patients, including 132 MMRd (27.7%) and 345 MMRp (proficient) patients. Women with MMRd tumors had higher recurrence rates (3.53p100py vs 1.21p100py) and worse PFS (p=0.0086) compared to women with MMRp tumors. After adjustment for age, LVSI status, adjuvant therapy, and post-operative grade, MMRd status remained associated with a higher risk of recurrence (HR 2.99, 95% CI 1.27–7.04). There was no significant difference in OS between MMR groups (HR 1.38, 95% CI 0.57–3.33). Conclusion In low-risk stage IA grade 1 or 2 endometrioid endometrial cancers, MMR deficiency is associated with a higher recurrence rate than in MMR proficient cases, after adjustment for covariates, implying that MMR deficiency reflects a different biology in endometrial cancer. Disclosure Nothing to disclose.

Objectives Approximately 15% of ECs are diagnosed in women before the natural age of menopause. Fertility-sparing conservative management options are increasingly utilized, however biomarkers to inform prognosis or direct therapy are lacking. We sought to determine the value of additional immunohistochemical biomarkers in young women with EC in the context of modern TCGA-based molecular classification. Methods Allred scores for estrogen/progesterone/androgen receptor and Ki67 in addition to immune characterization measuring stromal and epithelial expression of CD3/CD8/CD79a/CD138/PD1 and TILhigh vs. TILlow clusters was performed in a cohort of previously characterized(n=257) young women ( Results Young women had a high proportion of immune-rich ECs: 80% TILhigh compared with 60% TILhigh in non-age stratified cohorts. Expression of all immune biomarkers was enriched within POLE and MMRd subtypes. Within MMRd and p53wt ECs TILhigh immune cluster was associated with improved overall-(OS)and disease-specific survival (DSS)(p Conclusions Molecular classification with additional selective biomarker testing can provide additional prognostic information and may help stratify young women with ECs for targeted therapies.

Objectives We sought to determine the feasibility and characterize the extinction kinetics of circulating cell-free tumor DNA (cfDNA) testing in endometrial and ovarian carcinomas (ECs, OCs) using a clinically-approved commercially-available assay. Methods Women with suspected EC/OC undergoing surgery were consented for tissue and plasma sampling including pre-operative and serial post-operative draws. Tumour tissue and patient-matched buffy coat was extracted for DNA and sequenced for somatic mutations using FINDIT™ panel assay. Plasma samples were extracted for cfDNA and sequenced using FOLLOWIT™, Illumina platform, and analyzed using Contextual Genomics’s QUALITY NEXUS analysis pipelines. Low-frequency variants were confirmed by digital droplet PCR. Results 44 individuals had sufficient tissue and follow-up for inclusion; 24 ECs (13 endometrioid, 10 high-grade serous (HGS), 1 clear cell(CC)), 18 OCs (17 HGS 1, CC), and 2 synchronous endometrial and ovarian carcinomas. Eight ECs and 15 OC cases were advanced stage (II-IV) with residual disease in 2 ECs and 5 OCs, 8 recurrence events and 3 deaths recorded. Compliance with plasma sampling was high(>95%) when requested in hospital or at routine surveillance visits but dropped to 68% for ‘extra’ study-associated visits. Analysis to date reveals cfDNA was detectable in pre-operative samples of 19 individuals (9 ECs, 10 OCs including 4 early stage) and 6/10 tested post-operatively. Normalization of conventional tumour markers post-operatively took a median of 3mo in contrast to rapid loss of detectable cfDNA. Conclusions cfDNA testing is feasible and may enhance surveillance of endometrial and ovarian carcinomas by reflecting i) volume of disease pre-/post-operatively, ii) response to therapy, and/or iii) recurrence.

Objectives Endometrioid ovarian carcinoma (ENOC) is generally associated with a more favorable prognosis compared to other ovarian carcinoma histotypes. Nonetheless, patients are still treated according to a “one size fits all” approach. While tumor staging offers some stratification, the development of personalized treatment concepts remain elusive. Our group has recently validated the Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE), to distinguish clinically relevant prognostic groups. ENOC shares risk factors, genomics, and histology with it’s endometrial counterpart. The aim of our study was to apply and investigate ProMisE in ovarian endometrioid carcinoma. Methods ProMisE was applied to n=509 ENOC after biomarker-assisted review of endometrioid histotype. Cases were aligned into four groups: low risk POLE mutant (POLE); moderate risk mismatch repair deficient (MMRd); high-risk p53 abnormal (p53abn); and a final moderate risk category lacking these biomarkers (p53wt). Kaplan-Meier and multivariable survival analyses were performed. Results 4% of cases were POLE, 16% MMRd, 10% p53abn and 71% p53wt. Groups showed distinct progression-free and overall survival (p Conclusions ProMisE risk classification provides additional prognostic information in a large cohort of ENOC. Our findings support the introduction of ProMisE-stratified treatment algorithms to ultimately improve endometrioid ovarian carcinoma patient care. Further, ENOC may benefit from parallel efforts under investigation in endometrial carcinoma.