Search

Background CD103 is an integrin specifically expressed on the surface of cancer-reactive T cells. CD103 has been linked with better disease-specific survival in patients with ovarian, cervical, and endometrial cancer. The number of CD103+ T cells significantly increases during successful immunotherapy across human malignancies and therefore might be an attractive biomarker for non-invasive immune PET imaging of T cell infiltration. Indeed, we previously demonstrated that zirconium-89 (89Zr) radiolabeled anti-CD103 antibodies could be used for PET imaging of CD103+ T cells at relevant cell densities. However, the long half-life of antibodies precluded repeat imaging of CD103+ T cell dynamics early in therapy, and is associated with a significant radiation burden.Methods Two different anti-human CD103 Fab fragments radiolabeled with 89Zr or 68Ga were developed, namely 89Zr- hCD103 Fab and 68Ga-hCD103 Fab respectively. In vivo evaluation of these tracers was performed in nude mice (BALB/cOlaHsd-Foxn1nu) with established CD103-expressing CHO (CHO.CD103) or CHO wildtype (CHO.K1) xenografts, followed by serial PET imaging and ex vivo bio-distribution.Results In vivo, both 89Zr- and 68Ga- hCD103 Fab tracers showed high target-to-background ratios, high target site selectivity and high sensitivity in human CD103 positive xenografts.Conclusion We conclude that the two novel human CD103 immuno-PET tracers may be used for future non-invasive assessment of cancer reactive T cell infiltration. Consequently, both 89Zr and 68Ga- hCD103 Fab PET tracers should be explored in the clinical setting for stratification of patients who could benefit from immune checkpoint inhibition therapy.

The chemokine CXCL13 mediates recruitment of B cells to tumors and is essential for the formation of tertiary lymphoid structures (TLSs). TLSs are thought to support antitumor immunity and are associated with improved prognosis. However, it remains unknown whether TLSs are formed in response to the general inflammatory character of the tumor microenvironment, or rather, are induced by (neo)antigen-specific adaptive immunity. We here report on the finding that the TGFβ-dependent CD103+CD8+ tumor-infiltrating T-cell (TIL) subpopulation expressed and produced CXCL13. Accordingly, CD8+ T cells from peripheral blood activated in the presence of TGFβ upregulated CD103 and secreted CXCL13. Conversely, inhibition of TGFβ receptor signaling abrogated CXCL13 production. CXCL13+CD103+CD8+ TILs correlated with B-cell recruitment, TLSs, and neoantigen burden in six cohorts of human tumors. Altogether, our findings indicated that TGFβ plays a noncanonical role in coordinating immune responses against human tumors and suggest a potential role for CXCL13+CD103+CD8+ TILs in mediating B-cell recruitment and TLS formation in human tumors.

Supplementary Tables 1-14 and Supplementary Figures 1-4

Optimum risk stratification in early-stage endometrial cancer combines clinicopathologic factors and the molecular endometrial cancer classification defined by The Cancer Genome Atlas (TCGA). It is unclear whether analysis of intratumoral immune infiltrate improves this. We developed a machine-learning, image-based algorithm to quantify density of CD8+ and CD103+ immune cells in tumor epithelium and stroma in 695 stage I endometrioid endometrial cancers from the PORTEC-1 and -2 trials. The relationship between immune cell density and clinicopathologic/molecular factors was analyzed by hierarchical clustering and multiple regression. The prognostic value of immune infiltrate by cell type and location was analyzed by univariable and multivariable Cox regression, incorporating the molecular endometrial cancer classification. Tumor-infiltrating immune cell density varied substantially between cases, and more modestly by immune cell type and location. Clustering revealed three groups with high, intermediate, and low densities, with highly significant variation in the proportion of molecular endometrial cancer subgroups between them. Univariable analysis revealed intraepithelial CD8+ cell density as the strongest predictor of endometrial cancer recurrence; multivariable analysis confirmed this was independent of pathologic factors and molecular subgroup. Exploratory analysis suggested this association was not uniform across molecular subgroups, but greatest in tumors with mutant p53 and absent in DNA mismatch repair–deficient cancers. Thus, this work identified that quantification of intraepithelial CD8+ cells improved upon the prognostic utility of the molecular endometrial cancer classification in early-stage endometrial cancer.

Supplementary Tables 1-6, 8

Supplementary Table 7. Differential expression analysis of CD103+ vs CD103- CD8+ cytotoxic T cells

Purpose: Recent studies have shown that 7% to 12% of endometrial cancers are ultramutated due to somatic mutation in the proofreading exonuclease domain of the DNA replicase POLE. Interestingly, these tumors have an excellent prognosis. In view of the emerging data linking mutation burden, immune response, and clinical outcome in cancer, we investigated whether POLE-mutant endometrial cancers showed evidence of increased immunogenicity.Experimental Design: We examined immune infiltration and activation according to tumor POLE proofreading mutation in a molecularly defined endometrial cancer cohort including 47 POLE-mutant tumors. We sought to confirm our results by analysis of RNAseq data from the TCGA endometrial cancer series and used the same series to examine whether differences in immune infiltration could be explained by an enrichment of immunogenic neoepitopes in POLE-mutant endometrial cancers.Results: Compared with other endometrial cancers, POLE mutants displayed an enhanced cytotoxic T-cell response, evidenced by increased numbers of CD8+ tumor-infiltrating lymphocytes and CD8A expression, enrichment for a tumor-infiltrating T-cell gene signature, and strong upregulation of the T-cell cytotoxic differentiation and effector markers T-bet, Eomes, IFNG, PRF, and granzyme B. This was accompanied by upregulation of T-cell exhaustion markers, consistent with chronic antigen exposure. In silico analysis confirmed that POLE-mutant cancers are predicted to display more antigenic neoepitopes than other endometrial cancers, providing a potential explanation for our findings.Conclusions: Ultramutated POLE proofreading-mutant endometrial cancers are characterized by a robust intratumoral T-cell response, which correlates with, and may be caused by an enrichment of antigenic neopeptides. Our study provides a plausible mechanism for the excellent prognosis of these cancers. Clin Cancer Res; 21(14); 3347–55. ©2015 AACR.

Disease-specific survival (DSS) analyses by Kaplan Meier method of the primary surgery (PS) cohort for high and low infiltration of CD27+ cells. CD27 infiltration in the tumor epithelium is a prognostic factor in patients in which the surgical cytoreduction resulted in residual tissue of maximum 1 cm. Groups were created on basis of surgical outcome; red line shows patients that had no residual tumor after surgery, the violet lines depict patients that had residual tumors of {less than or equal to}1 cm, blue line represents patients with >1cm of residual tumor.

Package with all supplemental figures and tables

Supplementary Methods

Purpose: Tumor-infiltrating lymphocytes (TIL) are associated with a better prognosis in high-grade serous ovarian cancer (HGSC). However, it is largely unknown how this prognostic benefit of TIL relates to current standard treatment of surgical resection and (neo-)adjuvant chemotherapy. To address this outstanding issue, we compared TIL infiltration in a unique cohort of patients with advanced-stage HGSC primarily treated with either surgery or neoadjuvant chemotherapy.Experimental Design: Tissue microarray slides containing samples of 171 patients were analyzed for CD8+ TIL by IHC. Freshly isolated CD8+ TIL subsets were characterized by flow cytometry based on differentiation, activation, and exhaustion markers. Relevant T-cell subsets (CD27+) were validated using IHC and immunofluorescence.Results: A prognostic benefit for patients with high intratumoral CD8+ TIL was observed if primary surgery had resulted in a complete cytoreduction (no residual tissue). By contrast, optimal (+ TIL. Subsequent analysis of primary TIL by flow cytometry and immunofluorescence identified CD27 as a key marker for a less-differentiated, yet antigen-experienced and potentially tumor-reactive CD8+ TIL subset. In line with this, CD27+ TIL were associated with an improved prognosis even in incompletely cytoreduced patients. Neither CD8+ nor CD27+ cell infiltration was of prognostic benefit in patients treated with neoadjuvant chemotherapy.Conclusions: Our findings indicate that treatment regimen, surgical result, and the differentiation of TIL should all be taken into account when studying immune factors in HGSC or, by extension, selecting patients for immunotherapy trials. Clin Cancer Res; 22(3); 714–24. ©2015 AACR.

Purpose: Therapeutic vaccination with human papillomavirus type 16 (HPV16) E6 and E7 synthetic long peptides (SLP) is effective against HPV16-induced high-grade vulvar and vaginal intraepithelial neoplasia (VIN/VaIN). However, clinical nonresponders displayed weak CD8+ T-cell reactivity. Here, we studied if imiquimod applied at the vaccine site could improve CD8+ T-cell reactivity, clinical efficacy, and safety of HPV16-SLP (ISA101).Experimental Design: A multicenter open-label, randomized controlled trial was conducted in patients with HPV16+ high-grade VIN/VaIN. Patients received ISA101 vaccination with or without application of 5% imiquimod at the vaccine site. The primary objective was the induction of a directly ex vivo detectable HPV16-specific CD8+ T-cell response. The secondary objectives were clinical responses (lesion size, histology, and virology) and their relation with the strength of vaccination-induced immune responses.Results: Forty-three patients were assigned to either ISA101 with imiquimod (n = 21) or ISA101 only (n = 22). Imiquimod did not improve the outcomes of vaccination. However, vaccine-induced clinical responses were observed in 18 of 34 (53%; 95% CI, 35.1–70.2) patients at 3 months and in 15 of 29 (52%; 95% CI, 32.5–70.6) patients, 8 of whom displayed a complete histologic response, at 12 months after the last vaccination. All patients displayed vaccine-induced T-cell responses, which were significantly stronger in patients with complete responses. Importantly, viral clearance occurred in all but one of the patients with complete histologic clearance.Conclusions: This new study confirms that clinical efficacy of ISA101 vaccination is related to the strength of vaccine-induced HPV16-specific T-cell immunity and is an effective therapy for HPV16-induced high-grade VIN/VaIN. Clin Cancer Res; 22(10); 2342–50. ©2016 AACR.See related commentary by Karaki et al., p. 2317

Multivariable analysis on the prognostic role of the clinicopathological characteristics, molecular subgroups, and potential other classifiers in all cases of early-stage endometrial cancer (n=834) and in the subset of EC without substantial LVSI, >10% L1CAM, p53 and POLE mutation (n=620).

supplementary material and methods Table S1. Reasons for exclusion of patients from outcome analysis Table S2. Summary statistics of patient groups according to linear mixed model analysis Table S3. All related systemic adverse events by study group Table S4. Swelling size of injection site at 12 months follow-up (safety population) Table S5. Overview of treatment-related severe adverse events in patients who received at least one ISA101 vaccination with or without imiquimod Table S6. Overview of serious adverse events (safety population) Figure S1. Study Profile Figure S2. Example of detection of HPV16-specific CD8+ T-cell response measured by flow cytometry Figure S3. Example of detection of HPV16-specific CD8+ T-cell response measured by ex-vivo CD8+ T-cell IFNγ-ELISPOT Figure S4. HPV16-specific CD8+ T-cell reactivity of the PP population Figure S5. No differences in immune response to recall antigens upon treatment Figure S6. Strength of the HPV16-specific immunity versus clinical measurements at 12 months follow-up The strength of the immune response prior to vaccination (pre), after 2 vaccinations (2) and after 4 vaccinations (4) is depicted when ITT patients were grouped according to presence (open circle) or absence (closed circle) of VIN lesion (histology), HPV16 DNA (virology),a non-CR (NR/PR; open circles) or CR (closed circles) at 12 months after the last vaccination. Figure S7. Strength of the HPV16-specific immune responses in the PP population Figure S8. Granulomatous dermatitis at ISA101 vaccination site Figure S9. Basophil activation assay

Purpose: Recommendations for adjuvant treatment for women with early-stage endometrial carcinoma are based on clinicopathologic features. Comprehensive genomic characterization defined four subgroups: p53-mutant, microsatellite instability (MSI), POLE-mutant, and no specific molecular profile (NSMP). We aimed to confirm the prognostic capacity of these subgroups in large randomized trial populations, investigate potential other prognostic classifiers, and integrate these into an integrated molecular risk assessment guiding adjuvant therapy.Experimental Design: Analysis of MSI, hotspot mutations in 14 genes including POLE, protein expression of p53, ARID1a, β-catenin, L1CAM, PTEN, ER, and PR was undertaken on 947 available early-stage endometrioid endometrial carcinomas from the PORTEC-1 and -2 trials, mostly high-intermediate risk (n = 614). Prognostic value was determined using univariable and multivariable Cox proportional hazard models. AUCs of different risk stratification models were compared.Results: Molecular analyses were feasible in >96% of the patients and confirmed the four molecular subgroups: p53-mutant (9%), MSI (26%), POLE-mutant (6%), and NSMP (59%). Integration of prognostic molecular alterations with established clinicopathologic factors resulted in a stronger model with improved risk prognostication. Approximately 15% of high-intermediate risk patients had unfavorable features (substantial lymphovascular space invasion, p53-mutant, and/or >10% L1CAM), 50% favorable features (POLE-mutant, NSMP being microsatellite stable, and CTNNB1 wild-type), and 35% intermediate features (MSI or CTNNB1-mutant).Conclusions: Integrating clinicopathologic and molecular factors improves the risk assessment of patients with early-stage endometrial carcinoma. Assessment of this integrated risk profile is feasible in daily practice, and holds promise to reduce both overtreatment and undertreatment. Clin Cancer Res; 22(16); 4215–24. ©2016 AACR.

Supplementary table 1. Demographic and clinicopathological characteristics of cases by EC molecular subtype

Clinicopathological characteristics of the PORTEC-1 and -2 trial populations: comparison of cases included in the current analysis and those excluded for lack of material (n=164), non-endometrioid histology (n=30) or failed molecular analysis (n=86).

Purpose:Endometrioid ovarian carcinoma (ENOC) is generally associated with a more favorable prognosis compared with other ovarian carcinomas. Nonetheless, current patient treatment continues to follow a “one-size-fits-all” approach. Even though tumor staging offers stratification, personalized treatments remain elusive. As ENOC shares many clinical and molecular features with its endometrial counterpart, we sought to investigate The Cancer Genome Atlas–inspired endometrial carcinoma (EC) molecular subtyping in a cohort of ENOC.Experimental Design:IHC and mutation biomarkers were used to segregate 511 ENOC tumors into four EC-inspired molecular subtypes: low-risk POLE mutant (POLEmut), moderate-risk mismatch repair deficient (MMRd), high-risk p53 abnormal (p53abn), and moderate-risk with no specific molecular profile (NSMP). Survival analysis with established clinicopathologic and subtype-specific features was performed.Results:A total of 3.5% of cases were POLEmut, 13.7% MMRd, 9.6% p53abn, and 73.2% NSMP, each showing distinct outcomes (P < 0.001) and survival similar to observations in EC. Median OS was 18.1 years in NSMP, 12.3 years in MMRd, 4.7 years in p53abn, and not reached for POLEmut cases. Subtypes were independent of stage, grade, and residual disease in multivariate analysis.Conclusions:EC-inspired molecular classification provides independent prognostic information in ENOC. Our findings support investigating molecular subtype–specific management recommendations for patients with ENOC; for example, subtypes may provide guidance when fertility-sparing treatment is desired. Similarities between ENOC and EC suggest that patients with ENOC may benefit from management strategies applied to EC and the opportunity to study those in umbrella trials.

Supplementary table 2. Antigen presentation pathway mutations in TCGA POLE proofreading-mutant ECs

Disease-specific survival (DSS) analyses by Kaplan Meier method of cohort treated with primary surgery (PS) and cohort treated with neo-adjuvant chemotherapy (NACT). A) DSS depicted for patients in PS cohort split on surgical outcome. B) Age is a predictor for survival in the PS cohort. C) Prognostic effect of surgical outcome in the NACT cohort. D) Age is not a predictor of survival in the NACT cohort.

Supplementary figure 1. Tumor infiltrating lymphocytes and Crohn's like reaction in POLE- mutant endometrioid endometrial cancers

Supplementary figure 4. CD20+ and FOXP3+ tumor infiltrate according to EC molecular subtype

Legends to supplementary figures and tables

Supplementary figure 2. CD3+ tumor infiltrate according to EC molecular subtype

Hotspot mutation frequency according to the four molecular subgroups in early-stage endometrial cancer (n=834).

Clinicopathological characteristics, additional mutations and protein expression alterations in tumours with multiple classifying alterations.

Supplementary figure 3. Number of TIA-1+ cytolytic T cells according to molecular subtype

Reactivation of tumor infiltrating T lymphocytes (TILs) with immune checkpoint inhibitors or co-stimulators has proven to be an effective anti-cancer strategy for a broad range of malignancies. However, epithelial ovarian cancer (EOC) remains largely refractory to current T cell-targeting immunotherapeutics. Therefore, identification of novel immune checkpoint targets and biomarkers with prognostic value for EOC is warranted. Combining multicolor immunofluorescent staining’s with single cell RNA-sequencing analysis, we here identified a TIM-3/CXCL13-positive tissue-resident memory (CD8/CD103-positive) T cell (Trm) population in EOC. Analysis of a cohort of ~175 patients with high-grade serous EOC revealed TIM-3-positive Trm were significantly associated with improved patient survival. As CXCL13-positive CD8-positive T cells have been strongly linked to patient response to anti-PD1 immune checkpoint blockade, combinatorial TIM-3 and PD-1 blockade therapy may be of interest for the (re)activation of anti-cancer immunity in EOC.

Tumour-infiltrating lymphocytes (TILs) are considered crucial in anti-tumour immunity. Accordingly, the presence of TILs contains prognostic and predictive value. In 2011, we performed a systematic review and meta-analysis on the prognostic value of TILs across cancer types. Since then, the advent of immune checkpoint blockade (ICB) has renewed interest in the analysis of TILs. In this review, we first describe how our understanding of the prognostic value of TIL has changed over the last decade. New insights on novel TIL subsets are discussed and give a broader view on the prognostic effect of TILs in cancer. Apart from prognostic value, evidence on the predictive significance of TILs in the immune therapy era are discussed, as well as new techniques, such as machine learning that strive to incorporate these predictive capacities within clinical trials.

CD103, an integrin specifically expressed on the surface of cancer-reactive T cells, is significantly increased during successful immunotherapy across human malignancies. In this study, we describe the generation and zirconium-89 (First, to determine the feasibility of anti-CD103 immuno-PET to visualize CD103-positive cells at physiologically and clinically relevant target densities, we developed anCD103 immuno-PET tracers visualize CD103 T cells at relevant densities and are suitable for future non-invasive assessment of cancer reactive T cell infiltration.

Accurate risk prediction of distant recurrence (DR) is crucial for personalized adjuvant systemic therapy of endometrial cancer (EC) stage I-III patients, as DR is associated with a 5-year overall survival of 10-20%. Risk stratification and treatment recommendation are currently based on histopathological and molecular markers, which is challenging due to high inter-observer variability and testing costs respectively. Deep Learning (DL) models can predict prognosis by identifying relevant visual features from H&E whole slide images (WSIs) at different resolutions without prior assumptions. Here, we developed and tested the first interpretable state-of-the-art DL model for WSI-based risk prediction of DR of stage I-III EC (DeREC) from the randomized PORTEC-1/-2/-3 trials and three clinical cohorts with long-term follow-up data. We used one representative H&E WSI each from 1761 EC patients, excluding those who received adjuvant chemotherapy as it lowers the risk of DR. We randomly sampled 20% as a held-out internal test set (N=353 with 62 events; 8.45 year median follow-up) and performed a 5-fold cross-validation on the training set (N=1408). WSIs were partitioned into 360 micron patches at 40x magnification. DeREC combined self-supervised representation learning of patches using a multi-resolution vision transformer and a WSI-level graph attention-based time-to-event prediction model. The model performance of correctly ranking patients by predicted risk scores and true time to DR was measured with the concordance-index and compared with a Cox’ Proportional Hazards (CPH) model fitted on histopathological variables (histotype, grade, lymphovascular space invasion, stage). Discriminative quality of the predicted risk groups was investigated with Kaplan-Meier analysis and the log-rank test. Most predictive patches by predicted risk groups were reviewed by an expert gynecopathologist for identification of prognostic morphological features. DeREC achieved a concordance-index of 0.764 [95%CI 0.754-0.773] on 5-fold cross validation and 0.757 on the test set, as compared to 0.704 [95%CI 0.662-0.746] with CPH. Predicted risk groups around quartiles 1 and 3 accurately stratified patients between low (N=89), intermediate (N=175), high (N=89) risk of DR (p Citation Format: Sarah Fremond, Sonali Andani, Jurriaan Barkey Wolf, Gitte Ørtoft, Estrid Høgdall, Jouke Dijkstra, Jan J. Jobsen, Ina M. Jürgenliemk-Schulz, Ludy CHW Lutgens, Melanie E. Powell, Naveena Singh, Linda R. Mileshkin, Helen J. Mackay, Alexandra Leary, Dionyssios Katsaros, Hans W. Nijman, Stephanie M. de Boer, Remi A. Nout, Vincent T.H.B.M Smit, Carien L. Creutzberg, Nanda Horeweg, Viktor H. Koelzer, Tjalling Bosse. Deep learning risk prediction model of distant recurrence from H&E endometrial cancer slides. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5695.

DNA‐sensing receptor Cyclic GMP–AMP Synthase (cGAS) and its downstream signaling effector STimulator of INterferon Genes (STING) have gained significant interest in the field of tumor immunology, as a dysfunctional cGAS‐STING pathway is associated with poor prognosis and worse response to immunotherapy. However, studies so far have not taken into account the polymorphic nature of the STING‐encoding STING1 gene. We hypothesized that the presence of allelic variance in STING1 would cause variation between individuals as to their susceptibility to cancer development, cancer progression, and potential response to (immuno)therapy. To start to address this, we defined the genetic landscapes of STING1 in cervical scrapings and investigated their corresponding clinical characteristics across a unique cohort of cervical cancer patients and compared them with independent control cohorts. Although we did not observe an enrichment of particular STING1 allelic variants in cervical cancer patients, we did find that the occurrence of homozygous variants HAQ/HAQ and R232H/R232H of STING1 were associated with both younger age of diagnosis and higher recurrence rate. These findings were accompanied by worse survival, despite comparable mRNA and protein levels of STING and numbers of infiltrated CD8+ T cells. Our findings suggest that patients with HAQ/HAQ and R232H/R232H genotypes may have a dysfunctional cGAS‐STING pathway that fails to promote efficient anticancer immunity. Interestingly, the occurrence of these genotypes coincided with homozygous presence of the V48V variant, which was found to be individually associated with worse outcome. Therefore, we propose V48V to be further evaluated as a novel prognostic marker for cervical cancer., Homozygous variants of the STING1 gene are associated with poor outcome in human cervical cancer. Moreover, patients with homozygous variants of STING1 are diagnosed at an earlier age and have a higher recurrence rate. Likely, these patients have a dysfunctional cGAS‐STING pathway that fails to promote efficient anticancer immunity.

The clinical success of cancer immune checkpoint blockade (ICB) has refocused attention on tumor-infiltrating lymphocytes (TILs) across cancer types. The outcome of immune checkpoint inhibitor therapy in cancer patients has been linked to the quality and magnitude of T cell, NK cell, and more recently, B cell responses within the tumor microenvironment. State-of-the-art single-cell analysis of TIL gene expression profiles and clonality has revealed a remarkable degree of cellular heterogeneity and distinct patterns of immune activation and exhaustion. Many of these states are conserved across tumor types, in line with the broad responses observed clinically. Despite this homology, not all cancer types with similar TIL landscapes respond similarly to immunotherapy, highlighting the complexity of the underlying tumor-immune interactions. This observation is further confounded by the strong prognostic benefit of TILs observed for tumor types that have so far respond poorly to immunotherapy. Thus, while a holistic view of lymphocyte infiltration and dysfunction on a single-cell level is emerging, the search for response and prognostic biomarkers is just beginning. Within this review, we discuss recent advances in the understanding of TIL biology, their prognostic benefit, and their predictive value for therapy.

SummaryBackground Metformin use is associated with reduced cancer risk in epidemiological studies and has preclinical anti-cancer activity in ovarian cancer models. The primary objective of this phase I study was to determine the recommended phase II dose (RP2D) of metformin in combination with carboplatin/paclitaxel in patients with ovarian cancer. Secondary objectives were to describe safety and pharmacokinetics. Methods In this single-center trial the RP2D of metformin in combination with carboplatin area under the concentration-time curve (AUC) 6 and paclitaxel 175 mg/m2 every 3 weeks (q3w) in patients with advanced epithelial ovarian cancer was determined using a 3 + 3 escalation rule at three fixed dose levels: 500 mg three times daily (tds), 850 mg tds and 1000 mg tds. Metformin was commenced on day 3 of cycle 1 and continued until 3 weeks after the last chemotherapy administration. The RP2D was defined as the dose level at which 0 of 3 or ≤ 1 of 6 evaluable subjects experienced a metformin-related dose-limiting toxicity (DLT). Safety was assessed according to CTCAE v4.0. Plasma and serum samples for pharmacokinetic (PK) analyses were collected during treatment cycles 1 and 2. Results Fifteen patients with epithelial ovarian cancer and an indication for neo-adjuvant (n = 5) or palliative (n = 10) treatment were included. No DLTs were observed. Three patients discontinued study treatment during cycle 1 for other reasons than DLT. Six patients were treated at the RP2D of metformin 1000 mg tds. The most frequent low-grade toxicities were anemia, hypomagnesemia and diarrhea. Grade 3 adverse events (AEs) occurred in ten patients, most common were leucopenia (n = 4), thrombocytopenia (n = 3) and increased GGT (n = 3). There were no grade 4 AEs. Metformin increased the platinum (Pt) AUC (Δ22%, p = 0.013) and decreased the Pt clearance (Δ-28%, p = 0.013). Metformin plasma levels were all within the therapeutic range for diabetic patients (0.1–4 mg/L). Conclusion The RP2D of metformin in combination with carboplatin and paclitaxel in advanced ovarian cancer is 1000 mg tds. This is higher than the RP2D reported for combination with targeted agents. A potential PK interaction of metformin with carboplatin was identified.

Introduction Centralization has, among other aspects, been argued to have an impact on quality of care in terms of surgical morbidity. Next, monitoring quality of care is essential in identifying areas of improvement. This nationwide cohort study was conducted to determine the rate of short-term surgical complications and to evaluate its possible predictors in women with early-stage cervical cancer.Material and methods Women diagnosed with early-stage cervical cancer, 2009 FIGO stages IB1 and IIA1, between 2015 and 2017 who underwent radical hysterectomy with pelvic lymphadenectomy in 1 of the 9 specialized medical centers in the Netherlands, were identified from the Netherlands Cancer Registry. Women were excluded if primary treatment consisted of hysterectomy without parametrial dissection or radical trachelectomy. Women in whom radical hysterectomy was aborted during the procedure, were also excluded. Occurrence of intraoperative and postoperative complications and type of complications, developing within 30 days after surgery, were prospectively registered. Multivariable logistic regression analysis was used to identify predictors of surgical complications.Results A total of 472 women were selected, of whom 166 (35%) developed surgical complications within 30 days after radical hysterectomy. The most frequent complications were urinary retention with catheterization in 73 women (15%) and excessive perioperative blood loss >1000 mL in 50 women (11%). Open surgery (odds ratio [OR] 3.42; 95% CI 1.73-6.76), chronic pulmonary disease (OR 3.14; 95% CI 1.45-6.79), vascular disease (OR 1.90; 95% CI 1.07-3.38), and medical center (OR 2.83; 95% CI 1.18-6.77) emerged as independent predictors of the occurrence of complications. Body mass index (OR 0.94; 95% CI 0.89-1.00) was found as a negative predictor of urinary retention. Open surgery (OR 36.65; 95% CI 7.10-189.12) and body mass index (OR 1.15; 95% CI 1.08-1.22) were found to be independent predictors of excessive perioperative blood loss.Conclusions Short-term surgical complications developed in 35% of the women after radical hysterectomy for early-stage cervical cancer in the Netherlands, a nation with centralized surgical care. Comorbidities predict surgical complications, and open surgery is associated with excessive perioperative blood loss.

Contains fulltext : 220978.pdf (Publisher’s version ) (Open Access) AIM: Recently, the safety of laparoscopic radical hysterectomy (LRH) has been called into question in early-stage cervical cancer. This study aimed to evaluate overall survival (OS) and disease-free survival (DFS) in patients treated with abdominal radical hysterectomy (ARH) and LRH for early-stage cervical cancer and to provide a literature review. METHODS: Patients diagnosed between 2010 and 2017 with International Federation of Gynaecology and Obstetrics (2009) stage IA2 with lymphovascular space invasion, IB1 and IIA1, were identified from the Netherlands Cancer Registry. Cox regression with propensity score, based on inverse probability treatment weighting, was applied to examine the effect of surgical approach on 5-year survival and calculate hazard ratios (HR) and 95% confidence intervals (CIs). Literature review included observational studies with (i) analysis on tumours /=30 months (iii) >/=5 events per predictor parameter in multivariable analysis or a propensity score. RESULTS: Of the 1109 patients, LRH was performed in 33%. Higher mortality (9.4% vs. 4.6%) and recurrence (13.1% vs. 7.3%) were observed in ARH than LRH. However, adjusted analyses showed similar DFS (89.4% vs. 90.2%), HR 0.92 [95% CI: 0.52-1.60]) and OS (95.2% vs. 95.5%), HR 0.94 [95% CI: 0.43-2.04]). Analyses on tumour size (/=2 cm) also gave similar survival rates. Review of nine studies showed no distinct advantage of ARH, especially in tumours

ObjectivesCRISPR/Cas9-mediated gene editing has been leveraged for the modification of human and mouse T cells. However, limited experience is available on the application of CRISPR/Cas9 electroporation in cryopreserved T cells collected during e.g. clinical trials.MethodsPBMCs from healthy donors were used to generate knockout T cell models for interferon-γ (IFNγ), Cbl Proto-Oncogene B (CBLB), Fas cell surface death receptor (Fas) and T cell receptor (TCRαβb) genes. The effect of CRISPR-cas9-mediated gene editing on T cells was evaluated using apoptosis assays, cytokine bead arrays and ex vivo and in vitro stimulation assays.ResultsOur results demonstrate that CRISPR/Cas9-mediated gene editing of ex vivo T cells is efficient and does not overtly affect T cell viability. Cytokine release and T cell proliferation were not affected in gene edited T cells. Interestingly, memory T cells were more susceptible to CRISPR/Cas9 gene editing than naïve T cells. Ex vivo and in vitro stimulation with antigens resulted in equivalent antigen-specific T cell responses in gene-edited and untouched control cells; making CRISPR/Cas9-mediated gene editing compatible with clinical antigen-specific T cell activation and expansion assays.ConclusionHere, we report an optimized protocol for rapid, viable and highly efficient genetic modification in ex vivo human antigen specific T cells, for subsequent functional evaluation and/or expansion. Our platform extends CRISPR/Cas9-mediated gene editing for use in gold-standard clinically-used immune-monitoring pipelines and serves as a starting point for development of analogous approaches such as those including transcriptional activators and or epigenetic modifiers.

Chromosomal instability is a hallmark of cancer, but also an instigator of aneuploidy-induced stress, reducing cellular fitness. To better understand how cells with CIN adjust to aneuploidy and adopt a malignant fate in vivo, we performed a genome-wide mutagenesis screen in mice. We find that specifically aneuploid tumors inactivate Stat1 signaling in combination with increased Myc activity. By contrast, loss of p53 is common, but not enriched in CIN tumors. Validation in another tissue type confirmed that CIN promotes immune cell infiltration, which is alleviated by Stat1 loss combined with Myc activation, but not with p53 inactivation, or Myc activation alone. Importantly, we find that this mechanism is preserved in human aneuploid cancers. We conclude that aneuploid cancers inactivate Stat1 signaling to circumvent immune surveillance.

Introduction/Background* On the 11th of March 2020, the novel severe acute respiratory syndrome corona virus 2 (SARS-COV-2) was declared a pandemic. We studied the incidence and missed diagnoses in gynaecological oncology as a result of the impact of the COVID-19 pandemic and consequent lockdown and overcrowded hospitals in the Netherlands. Methodology We performed a retrospective cohort study using data from the Netherlands Cancer Registry (NCR) on women of 18 years and older diagnosed with invasive endometrial, ovarian, cervical or vulvar cancer in the period 2017-2020. The incidence was calculated for the period before the COVID-19 pandemic (mean number of the period 2017-2019) and compared with the incidence during the COVID-19 pandemic (2020) for each type of gynaecological cancer. The number of missed diagnoses was calculated as the difference in incidence between the period before and during the pandemic. Analyses were stratified for age, socioeconomical status (SES) and region. Result(s)* The incidence rate of gynaecological cancer was 57/100.000 (n = 4921) before and 53/100.000 (n = 4682) during the pandemic. Comparing the incidence of the two periods for the four types of cancer showed no significant difference (chi2 p=0.23). During the first wave of COVID-19 (March-June 2020), a clear decrease in incidence was visible for all four types of gynaecological cancer (figure 1). Subsequently, large increases in incidence were visible. A total of 299 diagnoses were missed during the pandemic (6.2% of the incidence of the period before COVID-19). The largest number of missed diagnoses was observed in ovarian cancer (n = 157, 10.9%), followed by cervical cancer (8.3%, n = 76), vulvar cancer (7.1%, n=31) and endometrial cancer (1.7%, n = 35). No significant differences in incidence were found for different age groups, SES and between regions. Conclusion* In the Netherlands, a clear drop in incidence was visible for all four types of gynaecological cancers during the first wave, which was caught up in the second part of 2020 hence no significant difference with the period before COVID-19. The difference in incidence was not caught up completely, for 299 women walk around with missed diagnoses, which could result in higher stage disease or even worse.

Introduction/Background* Ovarian cancer (OC) causes high mortality due to late diagnosis and high rate of relapse following initial therapy. Immunotherapy in combination with standard treatment modalities forms a promising new treatment approach. DCP-001 is an intradermally applied cancer relapse vaccine derived from the human leukaemic cell line DCOne® and is currently tested in acute myeloid leukaemia patients. To obtain the highly immunogenic DCP-001 vaccine, DCOne cells are shifted towards a mature dendritic cell phenotype. Since DCOne cells express multiple common tumour associated antigens such as WT-1, RHAMM, PRAME and MUC-1, which are also documented as target antigens in OC, DCP-001 vaccination may also be efficacious in OC. To support this hypothesis, the capacity of DCP-001 to induce immune responses against OC was studied in human peripheral blood mononuclear cells (PBMCs) from OC patients and a humanized mouse model for OC. Methodology The effect of DCP-001 on T cells was evaluated after a 3 week culture of PBMCs with or without DCP-001. Cytotoxic activity was analysed by IFNγ production and CD107a expression when these cells were subsequently cultured with OC cell line SKOV3. The effect of DCP-001 vaccination in vivo was evaluated in humanised NCG mouse subcutaneously engrafted with SKOV3 OC cells. Mice received two intra-peritoneal (i.p.) vaccinations with DCP-001 either after or prior to SKOV3 engraftment and tumour size was measured to evaluate the efficacy of DCP-001. Result(s)* In vitro, DCP-001 was shown to activate both CD4+ as well as CD8+ T cells and to induce formation of memory T cells. Importantly, DCP-001-stimulated CD8+ T cells from OC patients were shown to exert a HLA class I dependent, immune response to OC cells. In vivo, in an ovarian tumour mouse model, significant reduction of tumour growth rate and partial or even complete tumour regressions were observed in mice vaccinated with DCP-001, particularly when administered as relapse vaccine (prior to tumour engraftment), as compared to PBS treated mice. Conclusion* These pre-clinical in vitro and in vivo results support the potential use of DCP-001 as a cancer relapse vaccine in ovarian cancer, with the aim to reduce disease recurrence following initial standard of care therapy.

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* The molecular endometrial cancer (EC) classification has proven prognostic value and can direct adjuvant treatment decisions. Despite this, a relatively large group of EC is still molecularly unclassified (NSMP-EC). In this study we aimed to identify biomarkers among high-risk NSMP-EC patients with prognostic and/or predictive relevance. Methodology Paraffin-embedded tumour material (n=423) from the PORTEC-3 HREC trial were available for analysis. All patients with NSMP-EC were selected, hence those without pathogenic POLE mutations, mismatch repair deficiency and p53-abnormal immunohistochemistry (IHC). Protein expression of L1CAM, ER and PR (ongoing) were analysed by IHC using a 10% threshold for positivity. Tumour DNA was analysed for pathogenic somatic mutations using a next generation sequencing (NGS) cancer hotspot panel. Time to recurrence was analysed using the Kaplan-Meier method, log-rank tests and Cox’s proportional hazard models. Result(s)* In total, 126 NSMP-EC were identified in PORTEC-3, the majority were hormone receptor positive (ER n=106/121, 87.6%, PR will be presented at ESGO2021). L1CAM overexpression was observed in 11.2% (n=14/125) and mutations in CTNNB1-exon-3 were identified in 34.3% (n=36/105). Clustering showed that ER-positive NSMP-EC were predominantly endometrioid EC (n=99, 93.4%), low grade (n=88, 83.0%) and L1CAM-negative (n=102, 97.1%) (figure 1). PIK3CA and KRAS mutations were present in 27.3% (n=24) and 19.3% (n=17), respectively. ER-negative NSMP-EC were frequently non-endometrioid (n=11, 73.3%), L1CAM-positive (n=11, 73.3%) and rarely harboured PTEN and CTNNB1 mutations (n=1, 7.1% and 0%, respectively). ER-positivity was associated with lower risk of recurrence (HR 0.32 [95%CI 0.14-0.70]; figure 2A), while L1CAM-overexpression and CTNNB1-exon-3 mutations were not (HR 2.25 [95%CI 0.93-5.43] and HR 1.20 [95%CI 0.57-2.54], respectively). Multivariable analysis confirmed independent favourable prognostic impact of ER-positivity and LVSI. Figure 2B shows impact of LVSI on time to recurrence among patients with ER-positive NSMP-EC. Conclusion* The vast majority of NSMP-HREC are ER-positive (87.6%) and are likely sensitive to hormonal therapy. Other treatment targets might be found in this subgroup too as 27.3% had PIK3CA and 19.3% had KRAS mutations. NSMP-EC with loss of ER-expression were often of non-endometrioid histology and had a high risk of recurrence. Future studies should investigate whether this subgroup would benefit from other systemic therapies.

Introduction/Background* CD103-positive tissue resident memory-like CD8+ T cells (CD8CD103 TRM) are associated with improved prognosis across malignancies, including high-grade serous ovarian cancer (HGSOC). We investigated whether quantification of CD8, CD103 or both is required to improve existing survival prediction and whether all HGSOC patients or only specific subgroups of patients benefit from infiltration. Methodology We applied image-based quantification of CD8 and CD103 multiplex immunohistochemistry in the intratumoral and stromal compartments of 268 advanced-stage HGSOC patients from two independent clinical institutions. Result(s)* Infiltration denisty of CD8CD103 TRM was independent of clinicopathological factors and primary treatment strategy. A survival benefit of CD8CD103 TRM infiltration was observed in patients treated with primary cytoreductive surgery. Moreover, survival benefit in this group was limited to patients with no macroscopic tumor lesions after surgery (high epithelial CD8CD103 TRM infiltration 5 year survival 83% versus 52%, p=0.03; high stromal CD8CD103 TRM 5 year survival 77% versus 54%, p=0.01). No effect of CD8CD103 TRM infiltration on overall survival was observed in patients treated with neo-adjuvant chemotherapy, with or without macroscopic tumor lesions after surgery (high epithelial CD8CD103 TRM infiltration, p=0.77; high stromal CD8CD103 TRM infiltration, p=0.32). Conclusion* Our results suggest CD8CD103 TRM quantification as a superior method for prognostication compared to single CD8 or CD103 quantification, and supports the further exploration of image-based quantification of CD8CD103 TRM in HGSOC. This approach provides novel insights into prognostic stratification of HGSOC patients and may contribute to personalized treatment strategies in the future.

B-cells play a key role in cancer suppression, particularly when aggregated in tertiary lymphoid structures (TLS). Here, we investigated the role of B-cells and TLS in endometrial cancer (EC). Single cell RNA-sequencing of B-cells showed presence of activated/memory B-cells, cycling/germinal center B-cells and antibody-secreting cells. Differential gene expression analysis showed association of TLS with L1CAM overexpression. Immunohistochemistry and co-immunofluorescence showed L1CAM expression in mature TLS localized in the myometrial wall or at the tumor invasive border, independent of L1CAM expression the tumor. Using L1CAM as a marker, 378 of the 411 molecularly classified ECs from the PORTEC-3 biobank were evaluated. TLS were found in 19%, predominantly in mismatch-repair deficient and polymerase-epsilon mutant EC. Multivariable Cox regression analysis showed strong favorable prognostic impact of TLS, independent of clinicopathological and molecular factors. Our data suggests a pivotal role of TLS in outcome of EC patients, and establishes L1CAM as a simple biomarker.Statement of significance Tertiary lymphoid structures have a pivotal role in the immune response against endometrial cancer. Presence of mature tertiary lymphoid structures can be easily assessed using L1CAM immunohistochemistry and has independent favorable predictive value for recurrence and endometrial cancer-specific survival.

The chemokine CXCL13 mediates recruitment of B cells to tumors and is essential for the formation of tertiary lymphoid structures (TLSs). TLSs are thought to support antitumor immunity and are associated with improved prognosis. However, it remains unknown whether TLSs are formed in response to the general inflammatory character of the tumor microenvironment, or rather, are induced by (neo)antigen-specific adaptive immunity. We here report on the finding that the TGFβ-dependent CD103+CD8+ tumor-infiltrating T-cell (TIL) subpopulation expressed and produced CXCL13. Accordingly, CD8+ T cells from peripheral blood activated in the presence of TGFβ upregulated CD103 and secreted CXCL13. Conversely, inhibition of TGFβ receptor signaling abrogated CXCL13 production. CXCL13+CD103+CD8+ TILs correlated with B-cell recruitment, TLSs, and neoantigen burden in six cohorts of human tumors. Altogether, our findings indicated that TGFβ plays a noncanonical role in coordinating immune responses against human tumors and suggest a potential role for CXCL13+CD103+CD8+ TILs in mediating B-cell recruitment and TLS formation in human tumors.

Objectives Discordance between frozen section diagnosis and the definite histopathological diagnosis and the fact that the frozen section result is not always unambiguous, may contribute to differences in clinical practice regarding perioperative treatment and follow-up of borderline ovarian tumours (BOTs) patients amongst gynaecologic oncologists, which may lead to over- and undertreatment. The aim of the study was to map the Dutch gynaecologists' preferred treatment and follow-up strategy in case of BOTs. Methods A questionnaire was sent to all Dutch gynaecologists involved in ovarian surgery with perioperative frozen section analysis, and the outcomes were assessed using descriptive statistics. Results Nearly half of the respondents (41.0%) would not perform a staging procedure in case of a BOT. In case of an ambiguous frozen section diagnosis, tending towards invasive carcinoma, a considerable number (sBOT 56.4%; mBOT 30.8%) would perform a lymph node sampling as part of the staging procedure. A relaparotomy/relaparoscopy, to perform a lymph node sampling in case of a serous or mucinous carcinoma after a BOT frozen section diagnosis, would be performed by 97.4% and 48.7% of the respondents, respectively. Conclusions A considerable number of gynaecologists would perform a staging procedure that is recommended for ovarian cancer in case of an ambiguous BOT frozen section diagnosis, especially for serous tumours. In addition, nearly all gynaecologists would perform a second procedure including a lymph node sampling in case of a serous invasive carcinoma after a BOT frozen section diagnosis, which applies to half of the gynaecologists in case of a mucinous carcinoma., Highlights • In case of an unambiguous frozen section BOT diagnosis, a staging procedure is omitted by nearly half of the gynaecologists. • When the frozen section BOT diagnosis is ambiguous, a full ovarian cancer staging procedure is frequently performed. • Most gynaecologists perform a second procedure to complete staging when definitive diagnosis shows invasive ovarian cancer. • Full staging is performed more frequently in case of serous histopathology, when compared to mucinous tumours.

BACKGROUND:The PORTEC-3 trial investigated the benefit of combined adjuvant chemotherapy and radiotherapy versus pelvic radiotherapy alone for women with high-risk endometrial cancer. We updated the analysis to investigate patterns of recurrence and did a post-hoc survival analysis.METHODS:In the multicentre randomised phase 3 PORTEC-3 trial, women with high-risk endometrial cancer were eligible if they had International Federation of Gynaecology and Obstetrics (FIGO) 2009 stage I, endometrioid grade 3 cancer with deep myometrial invasion or lymphovascular space invasion, or both; stage II or III disease; or stage I-III disease with serous or clear cell histology; were aged 18 years and older; and had a WHO performance status of 0-2. Participants were randomly assigned (1:1) to receive radiotherapy alone (48·6 Gy in 1·8 Gy fractions given on 5 days per week) or chemoradiotherapy (two cycles of cisplatin 50 mg/m2 given intravenously during radiotherapy, followed by four cycles of carboplatin AUC5 and paclitaxel 175 mg/m2 given intravenously), by use of a biased coin minimisation procedure with stratification for participating centre, lymphadenectomy, stage, and histological type. The co-primary endpoints were overall survival and failure-free survival. Secondary endpoints of vaginal, pelvic, and distant recurrence were analysed according to the first site of recurrence. Survival endpoints were analysed by intention-to-treat, and adjusted for stratification factors. Competing risk methods were used for failure-free survival and recurrence. We did a post-hoc analysis to analyse patterns of recurrence with 1 additional year of follow-up. The study was closed on Dec 20, 2013; follow-up is ongoing. This study is registered with ISRCTN, number ISRCTN14387080, and ClinicalTrials.gov, number NCT00411138.FINDINGS:Between Nov 23, 2006, and Dec 20, 2013, 686 women were enrolled, of whom 660 were eligible and evaluable (330 in the chemoradiotherapy group, and 330 in the radiotherapy-alone group). At a median follow-up of 72·6 months (IQR 59·9-85·6), 5-year overall survival was 81·4% (95% CI 77·2-85·8) with chemoradiotherapy versus 76·1% (71·6-80·9) with radiotherapy alone (adjusted hazard ratio [HR] 0·70 [95% CI 0·51-0·97], p=0·034), and 5-year failure-free survival was 76·5% (95% CI 71·5-80·7) versus 69·1% (63·8-73·8; HR 0·70 [0·52-0·94], p=0·016). Distant metastases were the first site of recurrence in most patients with a relapse, occurring in 78 of 330 women (5-year probability 21·4%; 95% CI 17·3-26·3) in the chemoradiotherapy group versus 98 of 330 (5-year probability 29·1%; 24·4-34·3) in the radiotherapy-alone group (HR 0·74 [95% CI 0·55-0·99]; p=0·047). Isolated vaginal recurrence was the first site of recurrence in one patient (0·3%; 95% CI 0·0-2·1) in both groups (HR 0·99 [95% CI 0·06-15·90]; p=0·99), and isolated pelvic recurrence was the first site of recurrence in three women (0·9% [95% CI 0·3-2·8]) in the chemoradiotherapy group versus four (0·9% [95% CI 0·3-2·8]) in the radiotherapy-alone group (HR 0·75 [95% CI 0·17-3·33]; p=0·71). At 5 years, only one grade 4 adverse event (ileus or obstruction) was reported (in the chemoradiotherapy group). At 5 years, reported grade 3 adverse events did not differ significantly between the two groups, occurring in 16 (8%) of 201 women in the chemoradiotherapy group versus ten (5%) of 187 in the radiotherapy-alone group (p=0·24). The most common grade 3 adverse event was hypertension (in four [2%] women in both groups). At 5 years, grade 2 or worse adverse events were reported in 76 (38%) of 201 women in the chemoradiotherapy group versus 43 (23%) of 187 in the radiotherapy-alone group (p=0·002). Sensory neuropathy persisted more often after chemoradiotherapy than after radiotherapy alone, with 5-year rates of grade 2 or worse neuropathy of 6% (13 of 201 women) versus 0% (0 of 187). No treatment-related deaths were reported.INTERPRETATION:This updated analysis shows significantly improved overall survival and failure-free survival with chemoradiotherapy versus radiotherapy alone. This treatment schedule should be discussed and recommended, especially for women with stage III or serous cancers, or both, as part of shared decision making between doctors and patients. Follow-up is ongoing to evaluate long-term survival.FUNDING:Dutch Cancer Society, Cancer Research UK, National Health and Medical Research Council, Project Grant, Cancer Australia Grant, Italian Medicines Agency, and the Canadian Cancer Society Research Institute.