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Introduction: The clinical role of the molecular endometrial cancer (EC) classification has not been fully explored in patients staged with lymphadenectomy or without adjuvant treatment, conditions that could potentially moderate the prognostic value of the classification. We aimed to evaluate the clinical outcome of the molecular subgroups in patients with high-grade EC staged by lymphadenectomy and those without adjuvant treatment.Methods: DNA-sequencing for the detection of pathogenic POLE-exonuclease domain mutations and immunohistochemistry for mismatch repair (MMR) proteins and p53 expression were performed on 412 high-grade EC from the Danish Gynaecological Cancer Database (2005-2012) to classify them as POLE-ultramutated (POLEmut), MMR-deficient (MMRd), p53-mutant (p53abn), or no specific molecular profile (NSMP). Patients with stage IV or residual disease after surgery were excluded. Kaplan-Meier method, log-rank test and Cox proportional hazard models were used for analysis.Results: Molecular analysis was successful in 367 EC; 251 patients had undergone lymphadenectomy. Five-year recurrence rates in this subgroup of patients was 36.7% for women with p53abn EC, 0.0% for POLEmut EC, 13.4% for MMRd EC and 42.9% for NSMP EC (p < 0.001). Similar results were observed among stage IA-IB patients. Among patients without adjuvant treatment (n = 264), none with POLEmut EC (n = 26) had a recurrence.Conclusion: The molecular EC classification has strong prognostic value, independent of clinicopathological factors, also among high-grade EC patients staged by lymphadenectomy and those without adjuvant treatment. The unfavourable prognosis of early-stage p53abn EC is not due to undetected lymph node metastasis, and the indolent behaviour of POLEmut EC is independent of adjuvant treatment.

Background Risk-assessment of endometrial cancer (EC) is based on clinicopathological factors and molecular subgroup. It is unclear whether adding hormone receptor expression, L1CAM expression or CTNNB1 status yields prognostic refinement. Methods Paraffin-embedded tumour samples of women with high-risk EC (HR-EC) from the PORTEC-3 trial (n = 424), and a Dutch prospective clinical cohort called MST (n = 256), were used. All cases were molecularly classified. Expression of L1CAM, ER and PR were analysed by whole-slide immunohistochemistry and CTNNB1 mutations were assessed with a next-generation sequencing. Kaplan–Meier method, log-rank tests and Cox’s proportional hazard models were used for survival analysis. Results In total, 648 HR-EC were included. No independent prognostic value of ER, PR, L1CAM, and CTNNB1 was found, while age, stage, and adjuvant chemotherapy had an independent impact on risk of recurrence. Subgroup-analysis showed that only in NSMP HR-EC, ER-positivity was independently associated with a reduced risk of recurrence (HR 0.33, 95%CI 0.15–0.75). Conclusions We confirmed the prognostic impact of the molecular classification, age, stage, and adjuvant CTRT in a large cohort of high-risk EC. ER-positivity is a strong favourable prognostic factor in NSMP HR-EC and identifies a homogeneous subgroup of NSMP tumours. Assessment of ER status in high-risk NSMP EC is feasible in clinical practice and could improve risk stratification and treatment.

BackgroundThe endometrial cancer molecular classification has been integrated into the 2020 World Health Organization (WHO) diagnostic classification and European treatment guidelines, and provides direction towards more effective and less toxic adjuvant treatment strategies for women with endometrial cancer.Primary Objective(s)The RAINBO program of clinical trials will investigate four molecular class-directed adjuvant treatment strategies following surgical resection to either increase cure rates through the addition of novel targeted therapies or safely reduce toxicity and improve quality of life through treatment de-escalation.Study HypothesisMolecular-directed adjuvant treatment strategies will improve clinical outcomes and reduce toxicity of unwarranted therapies in women with endometrial cancer. The overarching and translational research RAINBO program will advance knowledge of predictive and prognostic (bio)markers that will improve prognostication and treatment allocation.Trial DesignThe RAINBO program is a platform of four international clinical trials and an overarching research program. The randomized phase III p53abn-RED trial for women with invasive stage I–III p53abn endometrial cancer compares adjuvant chemoradiation followed by olaparib for 2 years with adjuvant chemoradiation alone. The randomized phase III MMRd-GREEN trial for women with stage II (with lymphovascular space invasion (LVSI)) or stage III mismatch repair-deficient (MMRd) endometrial cancer compares adjuvant radiotherapy with concurrent and adjuvant durvalumab for 1 year to radiotherapy alone. The randomized phase III NSMP-ORANGE trial is a treatment de-escalation trial for women with estrogen receptor positive stage II (with LVSI) or stage III no specific molecular profile (NSMP) endometrial cancer comparing radiotherapy followed by progestin for 2 years to adjuvant chemoradiation. ThePOLEmut-BLUE trial is a phase II trial in which the safety of de-escalation of adjuvant therapy is investigated for women with stage I–IIIPOLEmut endometrial cancer: no adjuvant therapy for lower-risk disease and no adjuvant therapy or radiotherapy alone for higher-risk disease. The overarching RAINBO program will combine data and tumor material of all participants to perform translational research and evaluate molecular class-based adjuvant therapy in terms of efficacy, toxicity, quality of life, and cost-utility.Major Inclusion/Exclusion CriteriaInclusion criteria include a histologically confirmed diagnosis of endometrial cancer treated by hysterectomy and bilateral salpingo-oophorectomy with or without lymphadenectomy or sentinel lymph node biopsy, with no macroscopic residual disease after surgery and no distant metastases, and molecular classification according to the WHO 2020 algorithm.Primary Endpoint(s)Recurrence-free survival at 3 years in the p53abn-RED, MMRd-GREEN, and NSMP-ORANGE trials and pelvic recurrence at 3 years in thePOLEmut-BLUE trial.Sample SizeThe p53abn-RED trial will include 554 patients, the MMRd-GREEN trial 316, the NSMP-ORANGE trial 600, and thePOLEmut-BLUE trial 145 (120 for lower-risk disease and approximately 25 for higher-risk disease). The overarching research program will pool the four sub-trials resulting in a total sample size of around 1600.Estimated Dates for Completing Accrual and Presenting ResultsThe four clinical trials will have different completion dates; main results are expected from 2028.Trial Registration NumberThe RAINBO program is registered at clinicaltrials.gov (NCT05255653).

Vulvar cancer is a rare gynecological malignancy with a tremendous disease burden⁠. Vulvar squamous cell carcinoma (VSCC) is the most common histologic type of vulvar cancer and constitutes 80–90% of all vulvar cancers⁠. The cornerstone of treatment for VSCC consists of surgery with or without radiochemotherapy⁠. In addition, precursors of VSCC often require surgical or medical intervention as well⁠. Treatment of vulvar (pre)malignancies is a challenging balance act, as on the one hand clearance of all lesions is desired, while on the other hand normal vulvar anatomy and function must be preserved as best as possible⁠. Utilizing the current treatment interventions, recurrence occurs in up to 40% of VSCC patients and no major improvements in 5-year survival rates were observed in the last decades⁠. This is partly caused by the difficulty to recognize vulvar (pre)malignant lesionsfor the medical specialist, either macroscopically or pathologically⁠.This highlights the high unmet medical need for preferably non-invasive and accurate diagnostics for vulvar (pre)malignancies⁠. Further, effective therapies are needed with a favorable safety profile that encompass complete reduction of the affected tissue⁠.Therefore, the aim of this thesis has been to search for disease-specific biomarkers to improve the clinical management of vulvar (pre)malignancies⁠. In the present thesis, we have attempted to test several promising innovative instruments in a search for disease-specific biomarkers for vulvar (pre)cancers⁠. A multimodal profiling approach guided this research, based on systems profiling of disease and drug effects in dermatology⁠. The different investigated aspects include imaging, cellular and molecular characteristics and was complemented by patient reported outcomes and physician-based input⁠. Overall we focused on three important cornerstones within this multimodal approach:1 Application of novel non-invasive imaging instruments as dermatoscopy, optical coherence tomography and reflectance confocal microscopy on healthy and diseases vulvar skin to improve diagnostics;2 Establishment of in vitro healthy and VSCC 3D models aimed for future anticancer drug evaluation studies;3 Investigation of promising targets for tumor-specific molecular real time imaging of vulvar (pre)cancers, to assist complete surgical excision⁠.

Focal segmental glomerulosclerosis (FSGS) is a heterogeneous renal histopathological entity that affects the glomerulus. It is characterized by podocyte injury, proteinuria and scarring of the glomerular tuft. In this thesis we investigated various mechanisms that are implicated in the development of FSGS. Firstly, we studied biopsy samples of patients with minimal change disease, a related glomerulopathy with minimal glomerular injury and better prognosis. We showed that loss of nephrin could serve as a biomarker for renal function loss and the progression to FSGS in this patient group. We also investigated renal biopsy material of patients with FSGS to determine the role of complement activation and endothelial-podocyte interaction in the pathogenesis of FSGS. We show that complement activation via the classical pathway and endothelial injury, especially via endothelin-1 signaling, are associated with the development of FSGS in humans. This thesis also describes the study of the genomic architecture of the Munich Wistar Frömter rat model for FSGS, which led to the prioritization of TMEM63c and PTGR2 in the development of podocyte injury and proteinuria in this model. The identification of these mechanisms in the development of FSGS, increases our understanding of the pathophysiology of FSGS and can guide future studies into new, highly needed therapeutic strategies.

Endometrial cancer (EC) is the most common gynaecological cancer in developed countries. Standard treatment consists of surgery (hysterectomy and bilateral salpingo-oophorectomy) followed by either no adjuvant treatment, vaginal brachytherapy (VBT) or external beam radiotherapy (EBRT) with or without chemotherapy. The type of adjuvant treatment is based on clinicopathologic risk factors as age, FIGO-stage, histologic type and grade, myometrial invasion and lymph-vascular space invasion. In the recent years, knowledge has been gained on molecular risk factors in EC and four different molecular subgroups with distinct prognosis have been defined. The implementation of these subgroups into the treatment guidelines is being investigated in the PORTEC-4a trial. In this trial women with high-intermediate risk EC are randomised to either VBT versus an experimental arm in which a molecular-integrated risk profile is used to guide adjuvant treatment. With the improved patient selection women with favourable prognosis can be spared unnecessary treatment, while those with unfavourable prognosis are treated with more intensive treatment (EBRT). Besides the improvement of patient selection, radiotherapy techniques have developed as well. Modern radiotherapy techniques can increasingly spare healthy tissues with comparable outcomes and less toxicity. These developments will lead to better results and higher(er) quality-of-life for women with EC.

A proportion of patients with endometrial carcinoma are currently over- or undertreated due to the lack of reproducibility of some of the traditional factors used to assess risk of recurrence and death due to the cancer, aswell as intrinsic differences in the biological background of tumours within the same risk category. This underlinesthe need for additional biomarkers for the improvement of current risk classification systems and adjuvant treatment selection. In this context, the molecular endometrial carcinoma classification offers an opportunity to categorize tumours according to their molecular background, resulting in more biologically homogeneous groups of patients, with a more precise prognostic and, possibly, predictive value. However, before clinical implementation is possible, information regarding the interpretation of non-hotspot POLE exonuclease domain mutations, aswell as the molecular background and clinical outcome of EC with more than one molecular classifying feature (multiple classifier EC) is needed in order to obtain a reproducible and accurate classification system. Additionally, the integration of the molecular subgroups with clinicopathological features has proven to have a strong prognostic value in intermediate tohigh-risk and unselected cohorts, highlighting its potential to refine prognosis in high-risk patients and perhaps its predictive value. Finally, not all women in the molecularlyprofiled EC cohorts published were staged by lymphadenectomy and most patients had received adjuvant treatment. These features could have influenced the prognostic value of the molecular subgroups. The aims of this thesis were:1) To refine the molecular profiling of endometrial carcinoma by addressing essential remaining questionson the interpretation of POLE variants and characterization of multiple classifier ECs.2) To elucidate the prognostic role of the molecular subgroups in high-risk patients.3) To evaluate the value of the molecular classification to guide adjuvant treatment decisions.4) To investigate the natural behaviour of the molecular EC subgroups among patients staged with lymphadenectomy or not receiving adjuvant treatment.