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Objective. Universal screening of endometrial carcinoma (EC) for mismatch repair deficiency (MMRd) and Lynch syndrome uses presence of MLH1 methylation to omit common sporadic cases from follow-up germline testing. However, this overlooks rare cases with high-risk constitutional MLH1 methylation (epimutation), a poorly-recognized mechanism that predisposes to Lynch-type cancers with MLH1 methylation. We aimed to de-termine the role and frequency of constitutional MLH1 methylation among EC cases with MMRd, MLH1- methylated tumors.Methods. We screened blood for constitutional MLH1 methylation using pyrosequencing and real-time methylation-specific PCR in patients with MMRd, MLH1-methylated EC ascertained from (i) cancer clinics (n = 4

We would like to thank ‘the National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands’ for their contribution and ongoing support to the EPIC Study., This work was undertaken during the tenure of a doctoral Fellowship supported by INCa (INCa 2016 − 184), by Fondation ARC pour la recherche sur le cancer (ARC 2019-DOC4) and by IARC. The coordination of EPIC is financially supported by International Agency for Research on Cancer (IARC) and by the Department of Epidemiology and Biostatistics, School of Public Health, Imperial College London which has additional infrastructure support provided by the NIHR Imperial Biomedical Research Centre (BRC). The national cohorts are supported by: Danish Cancer Society (Denmark); Ligue Contre le Cancer, Institut Gustave Roussy, Mutuelle Générale de l’Education Nationale, Institut National de la Santé et de la Recherche Médicale (INSERM) (France); German Cancer Aid, German Cancer Research Center (DKFZ), German Institute of Human Nutrition PotsdamRehbruecke (DIfE), Federal Ministry of Education and Research (BMBF) (Germany); Associazione Italiana per la Ricerca sul Cancro-AIRC-Italy, Compagnia di SanPaolo and National Research Council (Italy); Dutch Ministry of Public Health, Welfare and Sports (VWS), Netherlands Cancer Registry (NKR), LK Research Funds, Dutch Prevention Funds, Dutch ZON (Zorg Onderzoek Nederland), World Cancer Research Fund (WCRF), Statistics Netherlands (The Netherlands); Health Research Fund (FIS) - Instituto de Salud Carlos III (ISCIII), Regional Governments of Andalucía, Asturias, Basque Country, Murcia and Navarra, and the Catalan Institute of Oncology - ICO (Spain); Swedish Cancer Society, Swedish Research Council and County Councils of Skåne and Västerbotten (Sweden); Cancer Research UK (C864/A 14136 to EPIC- Norfolk (DOI https://doi.org/10.22025/2019.10.105.00004); C8221/A29017 to EPIC-Oxford), Medical Research Council (MR/N003284/1 and MC_UU_00006/1 to EPIC-Norfolk; MR/M012190/1 to EPIC-Oxford). (United Kingdom)., Data Availability For information on how to submit an application for gaining access to EPIC data and/or biospecimens, please follow the instructions at http://epic.iarc.fr/access/index.php., Background: Diet may impact important risk factors for endometrial cancer such as obesity and inflammation. However, evidence on the role of specific dietary factors is limited. We investigated associations between dietary fatty acids and endometrial cancer risk in the European Prospective Investigation into Cancer and Nutrition (EPIC). Methods: This analysis includes 1,886 incident endometrial cancer cases and 297,432 non-cases. All participants were followed up for a mean of 8.8 years. Multivariable Cox proportional hazard models were used to estimate hazard ratios (HR) and 95% confidence intervals (CI) of endometrial cancer across quintiles of individual fatty acids estimated from various food sources quantified through food frequency questionnaires in the entire EPIC cohort. The false discovery rate (q-values) was computed to control for multiple comparisons. Results: Consumption of n-6 γ-linolenic acid was inversely associated with endometrial cancer risk (HR comparing 5th with 1st quintileQ5-Q1=0.77, 95% CI = 0.64; 0.92, ptrend=0.01, q-value = 0.15). This association was mainly driven by γ-linolenic acid derived from plant sources (HRper unit increment=0.94, 95%CI= (0.90;0.98), p = 0.01) but not from animal sources (HRper unit increment= 1.00, 95%CI = (0.92; 1.07), p = 0.92). In addition, an inverse association was found between consumption of n-3 α-linolenic acid from vegetable sources and endometrial cancer risk (HRper unit increment= 0.93, 95%CI = (0.87; 0.99), p = 0.04). No significant association was found between any other fatty acids (individual or grouped) and endometrial cancer risk. Conclusion: Our results suggest that higher consumption of γ-linolenic acid and α-linoleic acid from plant sources may be associated with lower risk of endometrial cancer., Institut National du Cancer (INCA) France C864/A 14136, Fondation ARC pour la Recherche sur le Cancer ARC 2019-DOC4, IARC, World Health Organization. International Agency for Research on Cancer (IARC), Department of Epidemiology and Biostatistics, School of Public Health, Imperial College London, NIHR Imperial Biomedical Research Centre (BRC), Danish Cancer Society, Institut Gustave Roussy, Mutuelle Generale de l'Education Nationale, Institut National de la Sante et de la Recherche Medicale (Inserm), Deutsche Krebshilfe. German Cancer Aid, Helmholtz Association, German Institute of Human Nutrition PotsdamRehbruecke (DIfE), Federal Ministry of Education & Research (BMBF), Fondazione AIRC per la ricerca sul cancro, Compagnia di San Paolo, Dutch Ministry of Public Health, Welfare and Sports (VWS), Netherlands Cancer Registry (NKR), LK Research Funds, Dutch Prevention Funds, Dutch ZON (Zorg Onderzoek Nederland), World Cancer Research Fund International (WCRF), Netherlands Government, Health Research Fund (FIS) - Instituto de Salud Carlos III (ISCIII), Regional Governments of Andalucia, Asturias, Basque Country, Murcia and Navarra, and the Catalan Institute of Oncology - ICO (Spain), Swedish Cancer Society, Swedish Research Council and County Councils of Skane and Vaesterbotten (Sweden), Cancer Research UK C8221/A29017 MR/N003284/1, UK Research & Innovation (UKRI) Medical Research Council UK (MRC) MC_UU_00006/1 MR/M012190/1 2016 - 184

Introduction Cervical cytology is a well‐stablished cervical cancer screening method. However, due to the anatomical continuity of the genital tract, it can also detect signs of endometrial disease. Our aim was to estimate the sensitivity of cervical cytology in endometrial cancer detection and prognosis in a large population over a 30‐year period in a large academic tertiary hospital in Spain. Methodology We performed a search for women diagnosed with endometrial cancer from 1990 to 2020, who were surgically treated and had a previous cervical cytology result. Information Technologies Department databases from Bellvitge University Hospital and the Screenwide case–control study's database were used. Cervical cytology results were classified as abnormal when squamous lesions, glandular atypia or malignant cells were identified. Results Overall, we evaluated 371 women with endometrial cancer and a documented cervical cytology performed within 3 years previous to surgical treatment. Overall, the sensitivity of cervical cytology for endometrial cancer detection was 25.6%. Several clinico‐pathological characteristics, such as non‐endometrioid histology and a higher stage, were correlated with higher sensitivity. Discussion We observed a low sensitivity of cervical cytology to effectively diagnose endometrial cancer. However, recent technological advances using genomics and epigenomics may offer a promising perspective to detect endometrial cancer with high sensitivity in these cervical specimens., Cervical cytology sensitivity for endometrial cancer was 25.6%. Several clinico‐pathological factors, such as non‐endometrioid histology and a higher stage, were correlated with higher sensitivity.

BackgroundDespite the growing interest in immunotherapeutic interventions for endometrial cancer (EC), the prevalence, phenotype, specificity and prognostic value of tumor infiltrating lymphocytes (TILs) in this tumor type remains unclear.MethodsTo better understand the role of TILs in EC, we analyzed the phenotypic traits of CD8+and CD4+EC-resident T cells from 47 primary tumors by high-dimensional flow cytometry. In addition, CD8+and CD4+TIL subpopulations were isolated based on the differential expression of programmed cell death protein-1 (PD-1) (negative, dim and high) and CD39 (positive or negative) by fluorescence activated cell sorting (FACS), expanded in vitro, and screened for autologous tumor recognition. We further investigated whether phenotypic markers preferentially expressed on CD8+and CD4+tumor-reactive TIL subsets were associated with the four distinct molecular subtypes of EC, tumor mutational burden and patient survival.ResultsWe found that CD8+TILs expressing high levels of PD-1 (PD-1hi) co-expressed CD39, TIM-3, HLA-DR and CXCL13, as compared with TILs lacking or displaying intermediate levels of PD-1 expression (PD-1−and PD-1dim, respectively). Autologous tumor reactivity of sorted and in vitro expanded CD8+ TILs demonstrated that the CD8+PD-1dimCD39+and PD-1hiCD39+T cell subsets both contained tumor-reactive TILs and that a higher level of PD-1 expression was associated with increased CD39 and a superior frequency of tumor reactivity. With respect to CD4+T conventional (Tconv) TILs, co-expression of inhibitory and activation markers was more apparent on PD-1hicompared with PD-1−or PD-1dimT cells, and in fact, it was the CD4+PD-1hisubpopulation that accumulated the antitumor T cells irrespective of CD39 expression. Most importantly, detection of CD8+PD-1hiCD39+ and CD4+PD-1hitumor-reactive T-cell subsets, but also markers specifically expressed by these subpopulations of TILs, that is, PD-1hi, CD39, CXCL13 and CD103 by CD8+TILs and PD-1hiand CXCL13 by CD4+Tconv TILs, correlated with prolonged survival of patients with EC.ConclusionsOur results demonstrate that EC are frequently infiltrated by tumor-reactive TILs, and that expression of PD-1hiand CD39 or PD-1hican be used to select and expand CD8+and CD4+tumor-reactive TILs, respectively. In addition, biomarkers preferentially expressed on tumor-reactive TILs, rather than the frequency of CD3+, CD8+and CD4+lymphocytes, hold prognostic value suggesting their protective role in antitumor immunity.

Clinical management of endometrial cancer (EC) is handicapped by the limited availability of second line treatments and bona fide molecular biomarkers to predict recurrence. These limitations have hampered the treatment of these patients, whose survival rates have not improved over the last four decades. The advent of coordinated studies such as The Cancer Genome Atlas Uterine Corpus Endometrial Carcinoma (TCGA_UCEC) has partially solved this issue, but the lack of proper experimental systems still represents a bottleneck that precludes translational studies from successful clinical testing in EC patients. Within this context, the first study reporting the generation of a collection of endometrioid-EC-patient-derived orthoxenograft (PDOX) mouse models is presented that is believed to overcome these experimental constraints and pave the way toward state-of-the-art precision medicine in EC. The collection of primary tumors and derived PDOXs is characterized through an integrative approach based on transcriptomics, mutational profiles, and morphological analysis; and it is demonstrated that EC tumors engrafted in the mouse uterus retain the main molecular and morphological features from analogous tumor donors. Finally, the molecular properties of these tumors are harnessed to assess the therapeutic potential of trastuzumab, a human epidermal growth factor receptor 2 (HER2) inhibitor with growing interest in EC, using patient-derived organotypic multicellular tumor spheroids and in vivo experiments.

Background: Obesity is a risk factor for endometrial cancer but whether metabolic dysfunction is associated with endometrial cancer independent of body size is not known. Methods: The association of metabolically defined body size phenotypes with endometrial cancer risk was investigated in a nested case–control study (817 cases/ 817 controls) within the European Prospective Investigation into Cancer and Nutrition (EPIC). Concentrations of C-peptide were used to define metabolically healthy (MH, World Health Organization, Department of Epidemiology and Biostatistics, School of Public Health, Imperial College London, Danish Cancer Society, Ligue nationale contre le cancer, Institut Gustave Roussy, Mutuelle Generale de l'Education Nationale, Institut National de la Sante et de la Recherche Medicale (Inserm), Deutsche Krebshilfe, Helmholtz Association, German Institute of Human Nutrition PotsdamRehbruecke (DIfE), Federal Ministry of Education & Research (BMBF), Fondazione AIRC per la ricerca sul cancro, Compagnia di San Paolo, Consiglio Nazionale delle Ricerche (CNR), Dutch Ministry of Public Health, Welfare and Sports (VWS), Netherlands Cancer Registry (NKR), LK Research Funds, Dutch Prevention Funds Netherlands Organization for Scientific Research (NWO), World Cancer Research Fund (WCRF-ERC) 232997, Netherlands Government, Health Research Fund (FIS)-Instituto de Salud Carlos III (ISCIII), Junta de Andalucia, Principality of Asturias, Basque Government, Regional Government of Murcia, Regional Government of Navarra, Catalan Institute of OncologyICO (Spain), Swedish Cancer Society Swedish Research Council, European Commission, County Council of Skane, County Council of Vasterbotten (Sweden), Cancer Research UK 14136 C8221/A29017 C19335/A21351, UK Research & Innovation (UKRI), Medical Research Council UK (MRC), European Commission 1000143 MR/M012190/1

[Purpose]: Germline pathogenic variants in the exonuclease domain (ED) of polymerases POLE and POLD1 predispose to adenomatous polyps, colorectal cancer (CRC), endometrial tumors, and other malignancies, and exhibit increased mutation rate and highly specific associated mutational signatures. The tumor spectrum and prevalence of POLE and POLD1 variants in hereditary cancer are evaluated in this study. [Methods]: POLE and POLD1 were sequenced in 2813 unrelated probands referred for genetic counseling (2309 hereditary cancer patients subjected to a multigene panel, and 504 patients selected based on phenotypic characteristics). Cosegregation and case–control studies, yeast-based functional assays, and tumor mutational analyses were performed for variant interpretation. [Results]: Twelve ED missense variants, 6 loss-of-function, and 23 outside-ED predicted-deleterious missense variants, all with population allele frequencies, This work was funded by the Spanish Ministry of Science and Innovation, cofunded by FEDER funds (SAF2016-80888-R, SAF2015-68016-R, Severo Ochoa SVP-2014-068895 contract [L.M.-P.]); Instituto de Salud Carlos III (PI16/00563, PI16/00588, PI14/00613, PI19/00553, CIBERONC CB16/12/00234, CIBERESP, Sara Borrell contract [P.M.]); Government of Catalonia [AGAUR 2017SGR1282, CERCA Program]; and Fundación Olga Torres. This study was facilitated by COST Action CA17118, supported by COST (European Cooperation in Science and Technology).

El càncer d'endometri (CE) és el segon càncer ginecològic més freqüent a tot el món. En general, la majoria de les pacients es diagnostiquen en les primeres etapes de la malaltia amb una taxa de supervivència global a 5 anys del 95%. Tanmateix, aquest percentatge disminueix dràsticament quan les pacients són diagnosticades amb metàstasis regionals fins al 69%, o amb metàstasis distants fins el 17% de supervivència. Encara que la cirurgia com a tractament primari és molt eficaç en aquells casos en els que el tumor està localitzat, és ineficaç quan el tumor es troba fora de l'úter. A més, els tractaments adjuvants actuals no són específics pel CE i no funcionen bé. El maneig de les pacients amb CE es decideix en base a les característiques clinicopatològiques i moleculars del tumor, que són la base del sistema d'estratificació del risc. Tot i això, aquesta classificació no és perfecte i, conseqüentment, hi ha un percentatge de casos que pateixen recurrència independentment del grup de risc identificat al pacient. En conjunt, hi ha un desconeixement clínic en la identificació de pacients que patiran una recurrència després del tractament primari i adjuvant, i també hi ha una falta en la identificació de nous tractaments per aquells casos amb risc elevat on la quimioteràpia, la teràpia més agressiva, no és capaç d'inhibir l'evolució del tumor. Aquesta tesi s'ha dividit en dues parts. D'una banda, hem identificat biomarcadors proteics en el teixit primari per predir la recurrència en pacients de CE amb risc intermig i elevat. En la fase de descobriment hem identificat 439 proteïnes en la histologia endometrioide i 56 proteïnes en la histologia serosa per predir la recurrència a través de proteòmica no dirigida. L'anàlisi in silico mitjançant bases de dades online ens ha confirmat l'ús potencial de 2 d'aquests biomarcadors per a predir la recurrència. Entre les proteïnes identificades, hem generat una llista de 58 proteïnes que hem analitzat en el teixit primari en una cohort independent de 129 pacients a través de proteòmica dirigida. En total, hem verificat l'ús de 4 proteïnes com a biomarcadors potencials en CE per a predir la recurrència. D'altra banda, hem realitzat un estudi preclínic utilitzant una cohort de 15 xenografts derivats de pacients (PDX) de CE per avaluar la toxicitat i l'eficàcia de SYD985 i niraparib, en monoteràpia i en combinació, com a nous tractaments per a pacients de CE amb risc elevat. El tractament dirigit contra HER2 (SYD985) ha estat capaç de reduir el creixement tumoral en un 33% dels PDX analitzats, mentre que la combinació amb la via PPAR (niraparib) ha resultat en una excel·lent combinació per les pacients de CE amb risc elevat, sobretot les pacients que expressen HER2 al tumor. Específicament, un 60% de les pacients ha aconseguit una resposta completa. En conclusió, s'espera que els resultats d'aquesta tesis millorin la detecció de les pacients amb CE amb elevat risc de recurrència i que proporcionin noves alternatives pel tractament d'aquestes pacients. A més, també s'espera que la millora en el maneig de les pacients amb CE de risc elevat, redueixi la mortalitat i la morbiditat associades a aquesta malaltia. El cáncer de endometrio (CE) es el segundo cáncer ginecológico más frecuente a nivel mundial. En general, la mayoría de las pacientes son diagnosticadas en etapas tempranas de la enfermedad con una tasa de supervivencia a los 5 años del 95 %. Sin embargo, este porcentaje desciende drásticamente cuando las pacientes son diagnosticadas con metástasis regional hasta el 69%, o con metástasis a distancia hasta un 17% de supervivencia. Aunque la cirugía, el tratamiento primario, es muy eficaz en aquellos casos en los que el tumor está localizado, es ineficaz cuando el tumor se ha diseminado por el útero. Además, los tratamientos adyuvantes actuales no son específicos para el CE y no funcionan bien. El manejo de las pacientes con CE se decide en base a las características clínico-patológicas y moleculares del tumor, que son la base del sistema de estratificación de riesgo. Sin embargo, esta clasificación no es perfecta y, consecuentemente, hay un porcentaje de casos que recurren independientemente del grupo de riesgo que se identifica para la paciente. En general, existe un desajuste clínico en la identificación de pacientes que tendrán una recurrencia después del tratamiento primario y adyuvante, y también hay una falta en la identificación de nuevos tratamientos para aquellos casos de riesgo elevado donde la quimioterapia, la terapia más agresiva, no es capaz de inhibir la evolución tumoral. Esta tesis se ha dividido en dos partes. Por un lado, hemos identificado biomarcadores proteicos en el tejido primario para predecir la recurrencia en pacientes con CE con riesgo intermedio y elevado. En la fase de descubrimiento hemos identificado 439 proteínas en la histología endometrioide y 56 proteínas en la histología serosa para predecir la recurrencia utilizando proteómica no dirigida. El análisis in silico utilizando bases de datos online confirmó el potencial uso de dos de estos biomarcadores para predecir la recurrencia. Entre las proteínas identificadas, generamos un listado de 58 proteínas que fueron analizas en el tejido primaria de una cohorte independiente formada por 129 pacientes usando proteómica dirigida. En total, verificamos el uso de 4 proteínas como biomarcadores potenciales en CE para predecir la recurrencia. Por otro lado, hemos realizado un estudio preclínico utilizando una cohorte de 15 xenoinjertos derivados de pacientes (PDX) con CE para evaluar la toxicidad y eficacia de SYD985 y niraparib, en monoterapia y en combinación, como nuevos tratamientos para pacientes de CE con riesgo elevado. El tratamiento dirigido contra HER2 (SYD985) fue capaz de reducir el crecimiento tumoral en un 33% de los PDX analizados, mientras que la combinación con la vía PPAR (niraparib) resultó en una excelente combinación para las pacientes de CE con riesgo elevado, sobre todo para aquellas pacientes con tumores que expresan HER2. Específicamente, un 60% de las pacientes consiguió una respuesta completa. En conclusión, se espera que los resultados de esta tesis mejoren la detección de pacientes con CE con elevado riesgo de recurrencia y proporcionen nuevas alternativas para el tratamiento de estas pacientes. Además, la mejora en el manejo de pacientes con CE de riesgo elevado, se espera que disminuya la mortalidad y la morbilidad asociadas a esta enfermedad. Endometrial cancer (EC) is the second most frequent gynecological cancer worldwide. In general, most patients are diagnosed at early stages of the disease with an overall 5-years survival rate of 95%. However, this percentage drastically decreases when patients are diagnosed with regional or distant metastases to 69% and 17%, respectively. Although surgery as a primary treatment is very effective in those cases of localized tumor, it is inefficient when the tumor has spread the uterus. Additionally, the current adjuvant treatments are not specific for EC and are not well working. Management of EC patients is decided based on the clinicopathological and molecular features of the tumor, which are the bases of the risk stratification system. However, this classification is not perfect, and consequently, there is a percentage of cases that recur independently of the risk group that was identified to the patient. Overall, there is a clinical unmet in the identification of patients that will recur after primary and adjuvant treatment, and in the identification of new treatments for those high-risk cases where chemotherapy, the most aggressive therapy, is not able to inhibit tumor evolution. This thesis has been divided in two parts. On the one hand, we have elucidated protein biomarkers in primary tissues to predict recurrence for intermediate to high-risk EC. In a discovery phase, we identified 439 proteins in endometrioid EC and 56 proteins in serous EC to potentially predict recurrence using non-targeted proteomics. In silico analysis using online databases confirmed the potential use of two of those biomarkers predicting recurrence. Among the identified proteins, we prioritized a list of 58 proteins that were analyzed in primary tissues from an independent cohort of 129 patients using targeted proteomics. Altogether, we verified the use of 4 proteins as potential EC biomarkers predicting recurrence. On the other hand, we have performed a preclinical study using a comprehensive cohort of 15 patient-derived xenograft (PDX) EC models to assess the toxicity and efficacy of SYD985 and niraparib, in monotherapy and in combination, as new treatments for high-risk ECs. Targeted-treatment against HER2 (SYD985) was able to reduce tumor growth in 33% of the PDX models analyzed, whilst its combination with PPAR pathway (niraparib) resulted in an excellent combination for high-risk EC patients, specifically for patients bearing HER2-expressing tumors. Especially, 60% of patients achieved a complete response. The results of this thesis are expected to improve the detection of high-risk of recurrence EC patients and provide a new alternative for the treatment of these patients. The improved management of high-risk EC patients is expected to decrease mortality and morbidity associated with this disease.

El càncer d’endometri (CE) és el sisè tumor més comú en dones a tot el món, amb 417.367 casos nous i 97.370 morts el 2020. El diagnòstic precoç del CE s’associa amb el 95% de la taxa de supervivència als 5 anys, mentre que el diagnòstic avançat la redueix al 17%. Actualment, no existeix cap prova de cribratge per a la detecció precoç del CE. Només les dones que presenten els símptomes clàssics del CE, principalment un sagnat uterí anormal (SUA), inicien el llarg procés de diagnòstic del CE, basat en l’obtenció invasiva de biòpsies endometrials. Això suposa una gran càrrega per a la salut femenina, ja que el SUA és un símptoma molt poc específic. Per tant, el desenvolupament i la implementació d’una prova no invasiva per distingir les condicions benignes dels CE urgeix. A més del diagnòstic, les biòpsies endometrials haurien de proporcionar informació sobre la histologia i grau tumorals per avaluar del risc preoperatori en pacients amb CE, utilitzat per guiar el tractament quirúrgic. Malauradament, el limitat material contingut en les biòpsies i l’elevada variabilitat en la interpretació patològica entre especialistes resulta en un 11% i 27% de discordances entre el diagnòstic preoperatori i el final en la determinació del tipus i grau histològic, respectivament. Per tant, la mesura objectiva dels factors pronòstics i/o la identificació de nous biomarcadors pronòstics també són urgents per guiar un tractament quirúrgic òptim en pacients amb CE. L’objectiu d’aquesta tesi és posicionar els fluids ginecològics, com les biòpsies líquides uterines i els fluids cervicals, com a font de biomarcadors de CE altament sensibles i específics. En aquesta tesi, hem volgut identificar biomarcadors altament sensibles, específics i reproduïbles que millorin el diagnòstic i l’avaluació del risc preoperatori dels tumors endometrials en fluids ginecològics, seguint dos enfocs diferents. El primer va ser investigar el fluid de les biòpsies pipelle, és a dir, els fluids uterins, com a font de biomarcadors de tipus i grau histològic, així com la predicció de recurrència. Per aconseguir-ho, vam realitzar una extensa revisió literària identificant 255 proteïnes, 30 de les quals es van validar en un anàlisi in-silico. Paral·lelament, vam generar una biblioteca espectral amb el proteoma dels fluids uterins (n= 42 CE) que posteriorment es va utilitzar per realitzar un estudi clínic retrospectiu en fluids uterins de 149 pacients amb CE quantificats per espectrometria de masses (EM) d’adquisició independent de les dades. L’estudi va permetre definir panells de 2 proteïnes que permeten el diagnòstic del tipus histològic amb sensibilitats que oscil·len entre el 90,9-100%, grau histològic amb una sensibilitat del 85,3%, i predir recurrència amb sensibilitats entre el 85,7-100%. Per avançar en el diagnòstic del CE, el segon enfocament es va dirigir a la identificació de biomarcadors proteics en fluids cervicals pel diagnòstic precís i no invasiu del CE. Vam realitzar dos estudis clínics retrospectius utilitzant els fluids cervicals, incloent un estudi de descobriment amb 59 pacients i un estudi de verificació amb 241 pacients mitjançant EM. Com a resultat, vam identificar biomarcadors de diagnòstic de CE i vam desenvolupar panells proteics que diagnostiquen el CE amb una sensibilitat del 95,4%. Adicionalment, vam identificar panells proteics per determinar el tipus i el grau histològic amb AUC de 0,91 i 0,97, respectivament. S’espera que els resultats d’aquesta tesi generin un canvi de paradigma en el maneig de les dones que pateixen SUA i millorin la detecció precoç del CE. Hem identificat panells de proteïnes que permeten una avaluació objectiva i més precisa del risc preoperatori per a pacients amb CE en fluids uterins i que permeten desenvolupar un diagnòstic de CE precís, objectiu i no invasiu basat en fluids cervicals. El cáncer de endometrio (CE) es el sexto tumor más común en mujeres a nivel mundial, con 417.367 nuevos casos y 97.370 muertes en 2020. El diagnóstico temprano del CE se asocia con el 95% de la tasa de supervivencia a los 5 años, mientras que el diagnóstico avanzado la reduce hasta el 17%. Actualmente no existe ninguna prueba de cribado para la detección precoz del CE. Solo las mujeres que presentan los síntomas clásicos del CE, principalmente un sangrado uterino anormal (SUA), inician el largo proceso de diagnóstico del CE, basado en la obtención invasiva de biopsias endometriales. Esto representa una gran carga para la salud de la mujer, ya que el SUA es un síntoma muy inespecífico. Por lo tanto, urge el desarrollo y la implementación de una prueba no invasiva para distinguir las patologías benignas de los CE. Además del diagnóstico, las biopsias endometriales deben proporcionar información sobre la histología y grado tumorales para evaluar el riesgo preoperatorio en pacientes con CE, utilizado para guiar el tratamiento quirúrgico. Desafortunadamente, el limitado material contenido en las biopsias y la alta variabilidad en la interpretación patológica entre especialistas resulta en un 11% y 27% de discordancias entre el diagnóstico preoperatorio y el final en la determinación del tipo histológico y grado, respectivamente. Por lo tanto, la medición objetiva de los factores pronósticos y/o la identificación de nuevos biomarcadores pronósticos también se necesita con urgencia para guiar un tratamiento quirúrgico óptimo en pacientes con CE. El objetivo de esta tesis es posicionar los fluidos ginecológicos, como las biopsias líquidas uterinas y los fluidos cervicales, como fuente de biomarcadores de CE altamente sensibles y específicos. En esta tesis, nuestro objetivo fue identificar biomarcadores altamente sensibles, específicos y reproducibles que mejoraran el diagnóstico y la evaluación del riesgo preoperatorio de tumores endometriales en fluidos ginecológicos, siguiendo dos enfoques diferentes. El primero fue investigar el fluido de las biopsias pipelle, es decir, los fluidos uterinos, como fuente de biomarcadores de tipo y grado histológico, y predicción de recurrencia. Para lograrlo, realizamos una extensa revisión literaria identificando 255 proteínas, 30 de las cuales fueron validadas en un análisis in-silico. Paralelamente, generamos una biblioteca espectral con el proteoma de fluidos uterinos (n=42 CE) que posteriormente se utilizó para realizar un estudio clínico retrospectivo en fluidos uterinos de 149 pacientes con CE cuantificados mediante espectrometría de masas (EM) de adquisición independiente de datos. El estudio permitió definir paneles de 2 proteínas que permiten el diagnóstico de tipo histológico con sensibilidades entre el 90,9-100%, grado histológico con sensibilidad del 85,3%, y predicen recurrencia con sensibilidades del 85,7-100%. Para avanzar en el diagnóstico del CE, el segundo enfoque se dirigió a identificar biomarcadores proteicos en los fluidos cervicales para diagnosticar el CE de manera precisa y no invasiva. Realizamos dos estudios clínicos retrospectivos sobre fluidos cervicales, incluyendo un estudio de descubrimiento de 59 pacientes y un estudio de verificación de 241 pacientes mediante EM. Como resultado, identificamos biomarcadores de diagnóstico de CE y desarrollamos paneles de proteínas que diagnostican el CE llegando a una sensibilidad del 95,4%. Además, también identificamos paneles de proteínas para determinar el tipo histológico y el grado con AUCs de 0,91 y 0,97, respectivamente. Se espera que los resultados de esta tesis generen un cambio de paradigma en el manejo de las mujeres que sufren SUA y mejoren la detección temprana del CE. Identificamos paneles de proteínas que permiten una evaluación objetiva y más precisa del riesgo preoperatorio para pacientes con CE en fluidos uterinos, y que permiten el desarrollo de un diagnóstico preciso, objetivo y no invasivo del CE basado en fluidos cervicales. Endometrial cancer (EC) is the sixth most common tumor in women worldwide, with 417,367 new cases and 97,370 deaths in 2020. Early diagnosis of EC is associated with 95% of 5-year survival rate, while advanced diagnosis drops the survival rate down to 17%. Today, there is no screening test for the early detection of EC. Only women presenting the classic symptoms of EC, i.e., mainly an abnormal uterine bleeding (AUB), start the multistep process of EC diagnosis, which relies on the invasive acquisition of endometrial biopsies. This accounts for a huge burden for women’s health since AUB is a highly unspecific symptom. Therefore, the development and implementation of a non-invasive test to distinguish benign from malignant conditions is urgently needed. In addition to diagnosis, endometrial biopsies should provide information regarding tumor histology and tumor grade to provide the pre-operative risk assessment in EC patients, which is used to guide the surgical treatment. Unfortunately, the limited material contained in endometrial biopsies and the high inter-observer variability in the pathological interpretation, results in 11% and 27% of discordances in the determination of EC histological type and grade, respectively, between preoperative and final diagnosis. Thus, the objective measurement of prognostic factors and/or the identification of novel prognostic biomarkers is as well urgently needed to guide an optimal EC surgical treatment. This thesis was performed aiming to position gynecological fluids, such as uterine liquid biopsies and cervical fluids, as a source of highly sensitive and specific EC biomarkers. In this thesis, we aimed to identify highly sensitive, specific, and reproducible biomarkers that improve the diagnosis and pre-operative risk assessment of EC tumors in gynecological fluids, following two different approaches. The first was to investigate the fluid of pipelle biopsies i.e., uterine fluids, as a source of biomarkers of histological type and grade, and recurrence prediction. To achieve this, we performed an extensive literature review identifying 255 proteins, 30 of which were further validated in an in-silico analysis. In parallel, we generated a spectral library with the proteome of uterine fluids (n=42 EC) that was subsequently used to conduct a clinical retrospective study in uterine fluids of 149 EC patients quantified by data-independent acquisition mass spectrometry (MS). The study allowed us to define panels of 2 proteins that allow diagnosis of histological type with sensitivities ranging from 90.9-100%, histological grade with sensitivity of 85.3%, and predict recurrence with sensitivities ranging from 85.7-100%. As a step towards advancing on EC diagnosis, the second approach was directed to investigate on the identification of protein biomarkers in cervical fluids to accurately and non-invasively diagnose EC. We conducted two clinical retrospective studies on cervical fluids, including a discovery study of 59 patients and a verification study of 241 patients by MS approaches. As a result, we identified EC diagnostic biomarkers and developed protein signatures that accurately diagnose EC with a sensitivity of 95.4%. Additionally, we also identify protein signatures to determine the histological type and grade with AUCs of 0.91 and 0.97, respectively. The results of this thesis are expected to generate a change in the paradigm on how clinicians manage women suffering from AUB, and to improve early detection of EC. We identified protein signatures that permit an objective and more accurate preoperative risk assessment for EC patients in uterine fluids, and that allow the development of a non-invasive, accurate and objective EC diagnosis based on cervical fluids. Universitat Autònoma de Barcelona. Programa de Doctorat en Bioquímica, Biologia Molecular i Biomedicina

Aquest treball es basa en dues hipòtesis: Algunes dietes poden augmentar els nivells d'estrògens en sang, i alguns patrons dietètics relacionats amb els estrògens poden augmentar el risc de càncer d'endometri. Per a comprovar aquestes hipòtesis, hem creat un patró dietètic correlacionat amb els nivells d'estrògens mitjançant el mètode del Reduced rank regression (RRR). A posteriori, hem estudiat l'associació entre aquest patró i el risc de càncer d'endometri utilitzant Cox models. Hem vist que una dieta rica en llegums, làctics i cervesa, i baixa en cereals i llegums, està associada a menors nivells de estrògens. També hem observat una lleu associació entre el nostre patró i els risc de càncer d'endometri.

El càncer d'endometri és la neoplàsia ginecològica més freqüent a la nostra població. La majoria de pacients són diagnosticades en estadis inicials i tenen pronòstics favorables. Tot i així, un percentatge de pacients situat entre un 10% i un 20%, poden presentar recidives o diagnosticar-se en fases més avançades de la neoplàsia, per tant, la investigació en aquesta patologia ha centrar-se en desenvolupar noves estratègies per precisar el pronòstic i millorar les opcions terapèutiques. Les catecolamines, que intervenen com a neurotransmissors en resposta a l'estrès físic o emocional, s'han perfilat com un dels principals mediadors de la relació entre l'estrès crònic i la progressió del càncer. El treball que es planteja en aquesta tesi doctoral sorgeix de la investigació amb mostres de pacients, amb models in vivo i amb línies cel·lulars de càncer d'endometri per tal de caracteritzar el paper dels receptors catecolaminèrgics en la progressió d'aquest tipus tumoral. L'anàlisi dels nivells d'expressió a nivell de ARNm per als diferents subtipus de receptors catecolaminèrgics, associat a la revisió de resultats de la literatura, ha permès escollir ADRβ2, DRD1 i DRD2 com a principals dianes a ser avaluades mitjançant la tècnica immunohistoquímica. Les nostres dades suggereixen que el receptor adrenèrgic ADRβ2 no determina el pronòstic de les pacients avaluades, a diferència del que s'ha descrit en altres neoplàsies. Per la seva banda DRD1 presenta una expressió reduïda al tumor respecte al teixit endometrial sa. L'anàlisi de l'associació del receptor amb les variables clinico-patològiques suggereix que la seva expressió podria tenir una relació inversa en referència a la mida del tumor i a l'estadi tumoral. Finalment, l'anàlisi de DRD2 s'associa significativament al tipus histològic de càncer d'endometri no endometrioide, a tumors d'alt grau histològic i també a una pitjor supervivència lliure de malaltia i global en la cohort de pacients estudiada. A més, hem establert dos models xenògrafs derivats de mostres de pacient implantats a nivell ortotòpic en ratolí. Aquests models presenten una major rellevància traslacional i clínica. El model generat a partir del tumor END 17 presenta una progressió més ràpida i un pes mitjà tumoral final superior al model END 19, alhora que una supervivència menor; definint-se per tant com un potencial model de CE agressiu. A més, s'ha confirmat en tots dos models la conservació de les característiques del tumor original a partir del perfil immunohistoquímic i del patró d'expressió dels receptors catecolaminèrgics al tumor generat. D'altra banda, l'experimentació in vitro ens ha permès avaluar l'efecte de la neuromodulació de les vies de senyalització adrenèrgica i dopaminèrgica en línies cel·lulars representatives de CE i la seva potencial utilització en teràpia antitumoral. La combinació de l'agonisme de DRD1 i de l'antagonisme de DRD2 ha induït una reducció significativament superior en la capacitat de proliferació cel·lular de la línia AN3CA, comparat amb el seu tractament en monoteràpia, representant una nova estratègia terapèutica. La informació obtinguda en aquest projecte aporta a la comunitat científica nou coneixement sobre el paper dels receptors catecolaminèrgics en càncer d'endometri. La seva aplicació a nivell pronòstic i/o terapèutic podria contribuir a millorar el maneig clínic de les pacients que el pateixen. A partir d'aquest punt, es poden definir dues vies d'actuació. En primer lloc, prosseguir l'experimentació per caracteritzar més detalladament, la contribució nerviosa al microentorn tumoral i els mecanismes implicats en aquesta contribució. En segon lloc, avaluar l'efectivitat terapèutica de la combinació d'agonistes i antagonistes a nivell preclínic, i valorar el reposicionament d'alguns fàrmacs ja aprovats i que podrien tenir noves aplicacions a nivell oncològic. El cáncer de endometrio (CE) es la neoplasia ginecológica más frecuente en nuestra población. La mayoría de pacientes son diagnosticadas en estadios tempranos y tienen pronósticos favorables. Aun así, un porcentaje de pacientes situado entre un 10% y un 20%, pueden presentar recidivas o diagnosticarse en fases más avanzadas de la neoplasia, por lo que la investigación en esta patología debe colaborar en el desarrollo de nuevas estrategias para precisar el pronóstico y las opciones terapéuticas. Las catecolaminas, que intervienen como neurotransmisores en respuesta al estrés físico o emocional, se han perfilado como uno de los principales mediadores de la relación entre el estrés crónico y la progresión del cáncer. El trabajo que se plantea en esta tesis doctoral surge de la investigación con muestras de pacientes, modelos in vivo y líneas celulares de cáncer de endometrio con el objetivo de caracterizar el papel de los receptores catecolaminérgicos en la progresión de este tipo tumoral. El análisis de los niveles de expresión a nivel de ARNm para los distintos subtipos de receptores catecolaminérgicos, asociado a la revisión de resultados de la literatura, ha permitido escoger ADRβ2, DRD1 y DRD2 como principales dianas a evaluar por la técnica inmunohistoquímica. Nuestros datos sugieren el receptor adrenérgico ADRβ2 no determina el pronóstico de las pacientes evaluadas, a diferencia de lo que se ha descrito en otras neoplasias. Por su parte DRD1 presenta una expresión reducida en el tumor con respecto al tejido endometrial sano. El análisis de la asociación del receptor con las variables clínico-patológicas sugiere que su expresión podría tener una relación inversa en referencia al tamaño y al estadio tumoral. Finalmente, el análisis de DRD2, se asocia significativamente al tipo histológico de cáncer de endometrio no endometrioide, a tumores de alto grado histológico y también a una peor supervivencia libre de enfermedad y global en la cohorte de pacientes estudiada. Además, hemos establecido dos modelos xenógrafos derivados de muestras de paciente implantados a nivel ortotópico en ratón. Estos modelos presentan una mayor relevancia traslacional y clínica. El modelo generado a partir del tumor END 17 presenta una progresión más rápida y un peso medio tumoral final superior al modelo END 19, a la vez que una supervivencia menor; definiéndose como potencial modelo de CE agresivo. Además, se ha confirmado en ambos modelos la conservación de las características del tumor original a partir del perfil inmunohistoquímico y del patrón de expresión de los receptores catecolaminérgicos en el tumor generado. Por otra parte, la experimentación in vitro, nos ha permitido evaluar el efecto de la neuromodulación de las vías de señalización adrenérgica y dopaminérgica en líneas celulares representativas de CE, y su potencial utilización en terapia antitumoral. La combinación del agonismo de DRD1 y del antagonismo de DRD2 ha inducido una reducción significativamente mayor en la capacidad de proliferación celular de la línea AN3CA, comparado con su tratamiento en monoterapia, representando una novedosa estrategia terapéutica. La información obtenida de este proyecto aporta a la comunidad científica nuevo conocimiento sobre el papel de los receptores catecolaminérgicos en cáncer de endometrio. Su aplicación a nivel pronóstico y/o terapéutico, podría contribuir a mejorar el manejo clínico de las pacientes. A partir de este punto, se pueden definir dos vías de actuación. En primer lugar, proseguir la experimentación para caracterizar más en detalle, la contribución nerviosa al microentorno tumoral y los mecanismos implicados en dicha contribución. En segundo lugar, evaluar la efectividad terapéutica de la combinación de agonistas y antagonistas a nivel preclínico, y valorar el reposicionamiento de algunos fármacos ya aprobados que podrían tener nuevas aplicaciones a nivel oncológico. Endometrial cancer is the most common gynecologic malignancy in our population. Most patients are diagnosed in early stages and have favorable prognoses. However, somewhere between 10% and 20% of those patients may present recurrences or be diagnosed in more advanced stages. Therefore, researchers must make a concerted effort to develop new strategies to optimize the prognosis and offer new therapeutic options for this pathology. Catecholamines, which act as neurotransmitters in response to physical or emotional stress, have emerged as one of the main mediators of the relationship between chronic stress and cancer progression. There are data suggesting that tumor cells use neurotransmitters and their neuroreceptors to activate the mechanisms of tumor growth and metastasis via nerve fibers that are part of the so-called tumor microenvironment. The project proposed in this doctoral thesis arises from research based on patient samples, in vivo models and established cell lines of endometrial cancer to characterize the role of catecholaminergic receptors in the progression of this tumor. The analysis of the mRNA expression levels for the different subtypes of catecholaminergic receptors along with the results seen in a review of the literature, has made it possible to choose ADRβ2, DRD1 and DRD2 as the main targets to be evaluated by means of the immunohistochemical technique. Our data suggest that ADRβ2 does not determine the prognosis of the patients evaluated, unlike what has been described in other tumor types. On the other hand, DRD1 has a reduced expression in the tumor tissue compared to healthy endometrial tissue. The analysis of the association of the receptor with clinicopathological variables suggests that its expression may have an inverse relationship with tumor size and stage. Finally, DRD2 expression analysis is significantly associated with non-endometrioid endometrial cancer, high grade FIGO classification tumors and worse disease-free and overall survival in the cohort of patients studied. In addition, we have established two patient-derived xenograft models from patient samples implanted orthotopically in mice. These models have greater translational and clinical relevance. Initially, its viability as well as its capacity for tumor implantation and dissemination have been verified. The model generated from the END 17 tumor shows a more rapid progression and a greater tumor weight as well as shorter survival than the END 19 model, thereby defining itself as a potential model of aggressive endometrial cancer. In addition, the conservation of the characteristics of the original tumor has been confirmed in both models based on the immunohistochemical profile and the expression pattern of the catecholaminergic receptors in the generated tumor. In the end, in vitro experimentation has made it possible to evaluate the effect of neuromodulation of adrenergic and dopaminergic signaling pathways in cell lines representative of EC and its potential use in antitumor therapy. The combination of DRD1 agonism and DRD2 antagonism induce a significantly greater reduction in the cell proliferation capacity of the AN3CA cell line, compared to its monotherapy treatment, representing a new therapeutic strategy. The information obtained from this project provides new knowledge to the scientific community on the role of catecholaminergic receptors play in endometrial cancer. Its application as a prognostic and/or therapeutic tools may well contribute to improving the clinical management of affected patients. From this point, two courses of action can be defined. First, continued experimentation to further characterize the nervous contribution to the tumor microenvironment and the mechanisms involved in endometrial cancer is called for. Second, there is a need to evaluate the therapeutic effectiveness of the combination of agonists and antagonists at a preclinical level and to assess the repositioning of some drugs already approved that might have new applications for oncological treatment.

Endometrial cancer (EC) is the fourth most common cancer in women in developed countries. Although it is usually diagnosed in postmenopausal women, its incidence has increased in young women, as well in recent decades, with an estimated rate of 4% in those under 40 years of age. Factors involved in this increase, particularly in resource-rich countries, include delayed childbearing and the rise in obesity. The new molecular classification of EC should help to personalize treatment, through appropriate candidate selection. With the currently available evidence, the use of oral progestin either alone or in combination with other drugs such as metformin, levonorgestrel-releasing intrauterine devices and hysteroscopic resection, seems to be feasible and safe in women with early-stage EC limited to the endometrium. However, there is a lack of high-quality evidence of the efficacy and safety of conservative management in EC. Randomized clinical trials in younger women and obese patients are currently underway.

Simple Summary PTEN is a protein highly altered in endometrial cancer. PTEN mutation or deficiency leads to the activation of other downstream proteins that are important to the development of cancers. In this study, we have identified the SMAD2/3 proteins as targets of PTEN deficiency. We have found that loss of PTEN in endometrial cells leads to SMAD2/3 activation. To investigate the role of SMAD2/3 activation downstream of PTEN deficiency, we have used endometrial cells lacking both PTEN and SMAD2/3 proteins. These cells display even more tumorigenic potential than cells lacking only PTEN. These results suggest that SMAD2/3 acts as an obstacle for cancer development triggered by PTEN loss. Abstract TGF-β has a dichotomous function, acting as tumor suppressor in premalignant cells but as a tumor promoter for cancerous cells. These contradictory functions of TGF-β are caused by different cellular contexts, including both intracellular and environmental determinants. The TGF-β/SMAD and the PI3K/PTEN/AKT signal transduction pathways have an important role in the regulation of epithelial cell homeostasis and perturbations in either of these two pathways’ contributions to endometrial carcinogenesis. We have previously demonstrated that both PTEN and SMAD2/3 display tumor-suppressive functions in the endometrium, and genetic ablation of either gene results in sustained activation of PI3K/AKT signaling that suppresses TGF-β-induced apoptosis and enhances cell proliferation of mouse endometrial cells. However, the molecular and cellular effects of PTEN deficiency on TGF-β/SMAD2/3 signaling remain controversial. Here, using an in vitro and in vivo model of endometrial carcinogenesis, we have demonstrated that loss of PTEN leads to a constitutive SMAD2/3 nuclear translocation. To ascertain the function of nuclear SMAD2/3 downstream of PTEN deficiency, we analyzed the effects of double deletion PTEN and SMAD2/3 in mouse endometrial organoids. Double PTEN/SMAD2/3 ablation results in a further increase of cell proliferation and enlarged endometrial organoids compared to those harboring single PTEN, suggesting that nuclear translocation of SMAD2/3 constrains tumorigenesis induced by PTEN deficiency.

This article provides practical recommendations developed from the International Society of Gynecological Pathologists Endometrial Carcinoma Project to address 4 issues that may arise in the diagnosis of uterine corpus low-grade endometrioid carcinoma: (1) The distinction between atypical hyperplasia and low-grade endometrioid carcinoma. (2) The distinction between low-grade endometrioid carcinoma and serous carcinoma. (3) The distinction between corded and hyalinized or spindle cell variants of low-grade endometrioid carcinoma and carcinosarcoma. (4) The diagnostic criteria for mixed endometrial carcinomas, a rare entity that should be diagnosed only after exclusion of a spectrum of tumors including morphologic variants of endometrioid carcinoma, dedifferentiated endometrial carcinoma, carcinosarcoma, and endometrial carcinomas with ambiguous morphology.

Advanced endometrial cancer (EC) lacks therapy, thus, there is a need for novel treatment targets. CXCR4 overexpression is associated with a poor prognosis in several cancers, whereas its inhibition prevents metastases. We assessed CXCR4 expression in EC in women by using IHC. Orthotopic models were generated with transendometrial implantation of CXCR4-transduced EC cells. After in vitro evaluation of the CXCR4-targeted T22-GFP-H6 nanocarrier, subcutaneous EC models were used to study its uptake in tumor and normal organs. Of the women, 91% overexpressed CXCR4, making them candidates for CXCR4-targeted therapies. Thus, we developed CXCR4+ EC mouse models to improve metastagenesis compared to current models and to use them to develop novel CXCR4-targeted therapies for unresponsive EC. It showed enhanced dissemination, especially in the lungs and liver, and displayed 100% metastasis penetrance at all clinically relevant sites with anti-hVimentin IHC, improving detection sensitivity. Regarding the CXCR4-targeted nanocarrier, 60% accumulated in the SC tumor; therefore, selectively targeting CXCR4+ cancer cells, without toxicity in non-tumor organs. Our CXCR4+ EC models will allow testing of novel CXCR4-targeted drugs and development of nanomedicines derived from T22-GFP-H6 to deliver drugs to CXCR4+ cells in advanced EC. This novel approach provides a therapeutic option for women with metastatic, high risk or recurrent EC that have a dismal prognosis and lack effective therapies.

Purpose: This study aimed to report the uptake of hysterectomy and/or bilateral salpingo-oophorectomy (BSO) to prevent gynaecological cancers (risk-reducing surgery [RRS]) in carriers of pathogenic MMR (path_MMR) variants.Methods: The Prospective Lynch Syndrome Database (PLSD) was used to investigate RRS by a cross-sectional study in 2292 female path_MMR carriers aged 30-69 years.Results: Overall, 144, 79, and 517 carriers underwent risk-reducing hysterectomy, BSO, or both combined, respectively. Two-thirds of procedures before 50 years of age were combined hysterectomy and BSO, and 81% of all procedures included BSO. Risk-reducing hysterectomy was performed before age 50 years in 28%, 25%, 15%, and 9%, and BSO in 26%, 25%, 14% and 13% of path_MLH1, path_MSH2, path_MSH6, and path_PMS2 carriers, respectively. Before 50 years of age, 107 of 188 (57%) BSO and 126 of 204 (62%) hysterectomies were performed in women without any prior cancer, and only 5% (20/392) were performed simultaneously with colorectal cancer (CRC) surgery.Conclusion: Uptake of RRS before 50 years of age was low, and RRS was rarely undertaken in association with surgical treatment of CRC. Uptake of RRS aligned poorly with gene-and age-associated risk estimates for endometrial or ovarian cancer that were published recently from PLSD and did not correspond well with current clinical guidelines. The reasons should be clarified. Decision-making on opting for or against RRS and its timing should be better aligned with predicted risk and mortality for endometrial and ovarian cancer in Lynch syn-drome to improve outcomes. (C) 2021 The Author(s). Published by Elsevier Ltd.

Ectonucleoside triphosphate diphosphohydrolase-2 (NTPDase2/CD39L1) has been described in human non-pathological endometrium in both epithelial and stromal components without changes along the cycle. It was identified as a stromal marker of basalis. In the present study, we aimed to evaluate NTPDase2 distribution, using immunolabeling and in situ enzyme activity approaches, in endometrial carcinoma (EC) at different tumor grades. NTPDase2 was present in tumor epithelial EC cells, as in the non-pathological endometria, but the expression underwent changes in subcellular distribution and also tended to decrease with the tumor grade. In stroma, NTPDase2 was identified exclusively at the tumor-myometrial junction but this expression was lost in tumors of invasive phenotype. We have also identified in EC samples the presence of the perivascular population of endometrial mesenchymal stem cells (eMSCs) positive for sushi domain containing 2 (SUSD2) and for NTPDase2, already described in non-tumoral endometrium. Our results point to NTPDase2 as a histopathological marker of tumor invasion in EC, with diagnostic relevance especially in cases of EC coexisting with other endometrial disorders, such as adenomyosis, which occasionally hampers the assessment of tumor invasion parameters.

A European consensus conference on endometrial carcinoma was held in 2014 to produce multidisciplinary evidence-based guidelines on selected questions. Given the large body of literature on the management of endometrial carcinoma published since 2014, the European Society of Gynaecological Oncology (ESGO), the European SocieTy for Radiotherapy & Oncology (ESTRO) and the European Society of Pathology (ESP) jointly decided to update these evidence-based guidelines and to cover new topics in order to improve the quality of care for women with endometrial carcinoma across Europe and worldwide. ESGO/ESTRO/ESP nominated an international multidisciplinary development group consisting of practicing clinicians and researchers who have demonstrated leadership and expertise in the care and research of endometrial carcinoma (27 experts across Europe). To ensure that the guidelines are evidence-based, the literature published since 2014, identified from a systematic search was reviewed and critically appraised. In the absence of any clear scientific evidence, judgment was based on the professional experience and consensus of the development group. The guidelines are thus based on the best available evidence and expert agreement. Prior to publication, the guidelines were reviewed by 191 independent international practitioners in cancer care delivery and patient representatives. The guidelines comprehensively cover endometrial carcinoma staging, definition of prognostic risk groups integrating molecular markers, pre- and intra-operative work-up, fertility preservation, management for early, advanced, metastatic, and recurrent disease and palliative treatment. Principles of radiotherapy and pathological evaluation are also defined.

The nuclease MRE11A is often included in genetic test panels for hereditary breast and ovarian cancer (HBOC) due to its BRCA1-related molecular function in the DNA repair pathway. However, whether MRE11A is a true predisposition gene for HBOC is still questionable. We determined to investigate this notion by dissecting the molecular genetics of the c.1516G > T;p.E506* truncating MRE11A variant, that we pinpointed in two unrelated French-Canadian (FC) HBOC patients. We performed a case–control study for the variant in ~ 2500 breast, ovarian, and endometrial cancer patients from the founder FC population of Quebec. Furthermore, we looked for the presence of second somatic alterations in the MRE11A gene in the tumors of the carriers. In summary, these investigations suggested that the identified variant is not associated with an increased risk of developing breast or ovarian cancer. We finally performed a systematic review for all the previously reported MRE11A variants in breast and ovarian cancer. We found that MRE11A germline variants annotated as pathogenic on ClinVar often lacked evidence for such classification, hence misleading the clinical management for affected patients. In summary, our report suggests the lack of clinical utility of MRE11A testing in HBOC, at least in the White/Caucasian populations.

Colorectal (CRC) and endometrial cancer (EC) are the most common types of cancer in Lynch syndrome (LS). Risk reducing surgeries (RRS) might impact cancer incidence and mortality. Our objectives were to evaluate cumulative incidences of CRC, gynecological cancer and all-cause mortality after RRS in LS individuals.Retrospective analysis of 976 LS carriers from a single-institution registry. Primary endpoints were cumulative incidence at 75 years of cancer (metachronous CRC in 425 individuals; EC and ovarian cancer (OC) in 531 individuals) and all-cause mortality cumulative incidence, comparing extended (ES) vs. segmental surgery (SS) in the CRC cohort and risk reducing gynecological surgery (RRGS) vs. surveillance in the gynecological cohort.Cumulative incidence at 75 years of metachronous CRC was 12.5% vs. 44.7% (RRS in LS reduces the incidence of metachronous CRC and gynecological neoplasms, also indicating a reduction in all-cause mortality cumulative incidence in females undergoing RRGS.

El càncer d’endometri (CE) és el més freqüent dels tumors infiltrants del tracte genital femení, i el tercer càncer més prevalent entre les dones europees. La supervivència a cinc anys és del 56% quan hi ha metàstasis regionals, i del 20% quan són distals. Cal desenvolupar noves estratègies terapèutiques que puguin millorar el tractament actual aconseguint una major inhibició de la disseminació metastàtica, així com una reducció significativa de la seva toxicitat sistèmica associada a efectes adversos. En col·laboració amb el grup de Nanobiotecnologia de la Universitat Autònoma de Barcelona, el nostre grup ha desenvolupat nanopartícules proteiques autoensamblables, basades en el disseny i la estructura T22-GFP-H6, dirigides específicament a cèl·lules que expressen el receptor de quimiocines CXCR4 (cèl·lules CXCR4+). Aquest receptor està sobreexpressat, en el teixit tumoral en CE. El seu anàlisis per immunohistoquímica de biòpsies tumorals d’una cohort de 79 pacients afectades per CE, va demostrar la seva sobreexpressió en més del 90% de les pacients, presentant un patró d’expressió en membrana. Això suggereix que es tracta d’una bona diana terapèutica també en aquest tipus tumoral, fent-lo susceptible d’esser tractat amb nanopartícules terapèutiques dirigides a cèl·lules tumorals CXCR4+. Per l’avaluació preclínica de les nostres nanopartícules, vam generar noves línies cel·lulars de CE amb sobreexpressió de CXCR4 i luciferasa. D’entre les línies que vam transduir, vam seleccionar la línia AN3CA CXCR4+ pel desenvolupament de models xenògrafs d’implantació subcutània i ortotòpica. Amb aquest últim vam aconseguir imitar les característiques clinicopatològiques del CE en estadis avançats, amb una tassa d’implantació de tumor primari i desenvolupament de metàstasis distals del 100% dels animals. A més, vam demostrar que la sobreexpressió de CXCR4 va exercir un gran efecte respecte a l’augment de la disseminació metastàtica. El nanoportador T22-GFP-H6 va demostrar una gran eficiència d’internalització depenent de CXCR4 sense citotoxicitat in vitro. En el model subcutani, va ser captat principalment pel teixit tumoral, CXCR4+, amb un pic de fluorescència a les 5 hores després de l’administració, sense alteracions histopatològiques en òrgans no tumorals. Després del cribratge amb diverses nanopartícules terapéutiques, vam observar que les basades en toxines bacterianes, T22-DITOX-H6 i T22-PE24-H6, exercien in vitro, a la línia AN3CA CXCR4+, un major efecte citotòxic, que era depenent de CXCR4 i estava mediat per inducció d’apoptosi. Aquesta via de mort cel·lular va ser confirmada in vivo desprès de l’administració de les nanotoxines en el model subcutani CXCR4+. També vam demostrar que ambdues, quan s’administraven a dosis repetides en el model subcutani, inhibien el creixement tumoral i augmentaven la supervivència dels ratolins en absència de toxicitat sistèmica associada. Per últim, l’administració repetida de T22-DITOX-H6, la nanotoxina que va demostrar un efecte antitumoral més potent, en el model ortotòpic CXCR4+ va inhibir el desenvolupament de metàstasis peritoneals, limfàtiques, hepàtiques i pulmonars, disminuint dràsticament tant el nombre com l’àrea dels focs metastàtics en fetge, així com l’àrea de teixit envaït al pulmó. Aquest potent efecte antimetastàtic va tenir lloc a dosis baixes (5 µg q3d x 14 dosis) i en absència de toxicitat sistèmica. La nostra nanopartícula T22-DITOX-H6 presenta múltiples avantatges respecte d’altres teràpies oncològiques pel que fa al seu direccionament actiu a cèl·lules CXCR4+, la seva mida nanomètrica i la seva naturalesa proteica, així com el seu singular mecanisme d’acció que inhibeix la síntesi de proteïnes. Aquestes propietats contribueixen a aconseguir un gran efecte antineoplàstic sense toxicitat inespecífica, com hem demostrat en els models animals CXCR4+ que hem generat en aquesta tesi. Creiem que aquesta nanopartícula pot ser una excel·lent alternativa terapèutica, i promet contribuir a solucionar la manca de tractament efectiu per a les pacients de CE CXCR4+ avançat, recurrent o amb alt risc de recaiguda. El cáncer de endometrio (CE) es el más frecuente de los tumores infiltrantes del tracto genital femenino, y el tercer cáncer más prevalente entre las mujeres europeas. La supervivencia a cinco años es del 56% si existen metástasis regionales, y de solo el 20% si las metástasis son distales. Es necesario desarrollar nuevas estrategias terapéuticas que mejoren el tratamiento actual para conseguir una mayor inhibición de la diseminación metastática, así como una reducción significativa de su toxicidad sistémica asociada. En colaboración con el grupo de Nanobiotecnología de la Universidad Autónoma de Barcelona, nuestro grupo ha desarrollado nanopartículas proteicas autoensamblables, basadas en el diseño y la estructura T22-GFP-H6, dirigidas específicamente a células con expresión del receptor de quimiocinas CXCR4 (células CXCR4+). Este receptor está sobreexpresado en tejido tumoral de CE. El análisis por inmunohistoquímica de biopsias tumorales de una cohorte de 79 pacientes afectadas de CE demostró sobreexpresión de CXCR4 en más del 90% de los casos, con un patrón predominantemente membranal. Esto sugiere que podría ser una buena diana terapéutica en este tipo tumoral, siendo susceptible de ser tratado con nanopartículas terapéuticas dirigidas a células tumorales CXCR4+. Para la evaluación preclínica de las nanopartículas, generamos nuevas líneas de CE con sobreexpresión de CXCR4 y de luciferasa. De entre ellas, seleccionamos la línea AN3CA CXCR4+ para el desarrollo de modelos xenógrafos de implantación subcutánea, en ratones Swiss nude, y ortotópica, en NSG. Este último modelo replicó las características clinicopatológicas del CE en estadios avanzados, con una tasa de implantación de tumor primario y desarrollo de metástasis distales en el 100% de los animales. También demostramos que la sobreexpresión de CXCR4 ejerció un gran efecto en el aumento de la diseminación metastática. El nanotransportador T22-GFP-H6 demostró una gran eficiencia de internalización y dependencia de CXCR4 en ausencia de citotoxicidad in vitro. En el modelo subcutáneo, se biodistribuyó principalmente en el tejido tumoral, CXCR4+, con un pico de fluorescencia a las 5 horas tras la administración, sin observarse acumulación o alteraciones histopatológicas en órganos no tumorales. Por otro lado, tras un cribaje de diversas nanopartículas portadoras de distintos agentes terapéuticos, observamos que las que incorporaban toxinas bacterianas, T22-DITOX-H6 y T22-PE24-H6, ejercían en la línea AN3CA CXCR4+ un gran efecto citotóxico, dependiente de CXCR4 y mediado por inducción de apoptosis. Esta vía de muerte celular fue confirmada en el modelo subcutáneo CXCR4+ tras la administración de las nanotoxinas. También demostramos que ambas, administradas a dosis repetidas en el modelo subcutáneo, inhibían el crecimiento tumoral, aumentando la supervivencia de los ratones, en ausencia de toxicidad sistémica y de acumulación en órganos no tumorales. Por último, T22-DITOX-H6, con mayor efecto antitumoral en el modelo subcutáneo, fue administrada a dosis repetidas también en el modelo ortotópico CXCR4+, demostrando inhibir el desarrollo de metástasis peritoneales, linfáticas y hematógenas. Disminuyó drásticamente tanto el número como el área de los focos metastáticos en hígado, así como el porcentaje de pulmón invadido por células tumorales. Este potente efecto antimetastático tuvo lugar a una dosificación baja y en ausencia de toxicidad sistémica. Nuestra nanopartícula T22-DITOX-H6 muestra múltiples ventajas respecto a otras terapias oncológicas, en cuanto a su direccionamiento activo a células CXCR4+, su tamaño nanométrico, y su naturaleza proteica, así como su singular mecanismo de acción. Todo ello conlleva un elevado efecto antineoplásico en ausencia de toxicidad sistémica. Creemos que esta nanopartícula puede ser un excelente abordaje terapéutico,tras el pertinente desarrollo preclínico, contribuyendo a solucionar la falta de tratamiento efectivo para las pacientes de CE CXCR4+ avanzado, recurrente o con alto riesgo de recaída. Endometrial cancer (EC) is the most common cancer in the female genital tract, and the third leading cancer in European women. Its five-year survival is of a 56% or 20% when locoregional or distal metastasis occur, respectively. Thus, there’s an unmet medical need calling for developing new therapeutic approaches that improve current treatment by achieving a higher inhibition of metastatic dissemination, as well as a significant reduction of its associated systemic toxicity. In collaboration with the Nanobiotechnology group at Universitat Autònoma de Barcelona, our group has developed self-assembling protein nanoparticles, based on T22-GFP-H6 design and structure, to specifically target cells which express chemokine receptor CXCR4 (CXCR4+ cells). This receptor is overexpressed in EC tumor tissue. Immunohistochemical analysis of tumor biopsies from 79 EC patients showed its overexpression in more than 90% of the cases, with predominantly membrane localization. This suggests CXCR4 could be a good therapeutic target in this tumor type, which could be treated using therapeutic nanoparticles targeted to CXCR4+ cells. For the preclinical development of our nanoparticles, we generated new CXCR4+ EC cell lines, which also overexpressed luciferase to allow cell tracking. Among them, we selected the CXCR4+ AN3CA cell line to develop xenograft models of subcutaneous and orthotopic implantation, in Swiss nude or NSG mice, respectively. Our orthotopic models mimicked the clinicopathological features of EC in advanced stages, having a rate of tumor implantation and metastasis development of 100%. Moreover, CXCR4 overexpression showed a large effect on metastatic dissemination. Our nanocarrier, T22-GFP-H6, showed a high and CXCR4-dependent internalization, especially in the AN3CA cell line, without associated cytotoxicity. Moreover, in our subcutaneous model, it was mainly uptaken by CXCR4+ cells in the tumor, reaching its peak after 5 hours from administration. Non-tumor organs showed no histopathological alterations or nanoparticle accumulation. Besides, a screening of multiple nanoparticles carrying different therapeutic agents identified those incorporating bacterial toxins, T22-DITOX-H6 and T22-PE24-H6, as the ones exerting a higher cytotoxic effect in CXCR4+ AN3CA cell line. This effect was CXCR4-dependent and mediated by apoptosis induction, a cell death mechanism that was also confirmed in our subcutaneous model after the intravenous administration of the nanotoxins. In addition, the repeated administration of both nanotoxins in the same model, achieved inhibition of tumor growth, leading to increased mice survival in the absence of side toxicity or nanoparticle accumulation in non-tumor organs. Finally, we used T22-DITOX-H6, which showed the highest antitumor effect, on a repeated dose schedule, to assess its antimetastatic effect on our orthotopic CXCR4+ model. This nanotoxin inhibited the development of peritoneal, lymph node, liver and lung metastasis. Regarding hematogenous metastasis, it dramatically reduced both the number and the area of metastatic foci in liver, as well as the percentage of the area occupied by metastatic cells in the lung. This powerful antitumor effect was exerted at a low dosage, without systemic toxicity. To sum up, our nanoparticle T22-DITOX-H6 shows many advantages over other oncological therapies, including its active targeting to CXCR4+ cells, its nanometric size, and its proteic nature, as well as its unique mechanism of action, inhibiting protein synthesis. All of these nanoparticle properties lead to a high antineoplastic effect in the absence of systemic toxicity. We believe this nanoparticle could represent an excellent therapeutic approach for CXCR4+ EC patients with an advance stage, high risk of progression or recurrent tumors, once after its appropriate preclinical development, since no effective treatments are currently available. Universitat Autònoma de Barcelona. Programa de Doctorat en Biologia Cel·lular

Purpose To evaluate the preliminary results of the use of 68 Gy EQD2(α/β=3 Gy) as a dose limit to the lowest dose in the most exposed 2 cm3 of the vagina in order to reduce G2 late vaginal problems in postoperative endometrial carcinoma (EC). Methods From November 2016 to October 2019, 69 postoperative EC patients receiving vaginal brachytherapy (VBT) ± external beam radiotherapy (EBRT) were prospectively analyzed. The median EBRT dose was 45 Gy (range: 44–50.4 Gy), 1.8−2 Gy/day, 5 fractions(Fr)/week. VBT was administered with the following schedule: 1Fr of 7 Gy after EBRT and 2 daily Fr × 7.5 Gy in exclusive VBT. The dose was prescribed at 0.5 cm from the applicator surface with an active length of 2.5 cm; 56 patients were treated with vaginal cylinders (49–3.5 cm, 6–3 cm, and 1–2.5 cm) and 13 with the colpostat technique. The overall VBT dose was adjusted to meet the vaginal restriction of (α/β=3 Gy) at 2 cm3. Late toxicity was prospectively assessed using RTOG scores for bladder and rectum, and the objective LENT-SOMA criteria for vagina. Results With a median follow-up of 31.0 months, no vaginal-cuff recurrences were found. Late toxicity: only 1G1(1.4%) rectal toxicity; 21G1(30.4%) and 3G2(4.3%) vaginal complications. Only one (1.4%) of 3 G2 manifested as vaginal shortening. Conclusions In postoperative EC patients treated with VBT, only one developed G2 vaginal stenosis with the use of 68 Gy EQD2(α/β=3 Gy) as a dose constraint. These preliminary results seem to indicate the value of this dose limit for reducing G2 vaginal stenosis. Nonetheless, these findings should be confirmed in a larger number of patients with longer follow-up.

Objective. Endometrial cancers have historically been classified by histomorphologic appearance, which is subject to interobserver disagreement. As molecular and biomarker testing has become increasingly available, the prognostic significance and accuracy of histomorphologic diagnoses have been questioned. To address these issues for a large, prospective cohort study, we provide the results of a centralized pathology review and biomarker analysis of all incidental endometrial carcinomas occurring between 1976 and 2012 in the Nurses’ Health Study. Methods. Routine histology of all ( n = 360 ) cases was reviewed for histomorphologic diagnosis. Cases were subsequently planted in a tissue microarray to explore expression of a variety of biomarkers (e.g., ER, PR, p53, PTEN, PAX2, AMACR, HNF1β, Napsin A, p16, PAX8, and GATA3). Results. Histologic subtypes included endometrioid (87.2%), serous (5.6%), carcinosarcoma (3.9%), clear cell (1.7%), and mixed type (1.7%). Biomarker results within histologic subtypes were consistent with existing literature: abnormal p53 was frequent in serous cases (74%), and HNF1β (67%), Napsin A (67%), and AMACR (83%) expression was frequent in clear cell carcinomas. Our dataset also allowed for examination of biomarker expression across non-preselected histologies. The results demonstrated that (1) HNF1β was not specific for clear cell carcinoma, (2) TP53 mutations occurred across many histologies, and (3) GATA3 was expressed across multiple histotypes, with 75% of positive cases demonstrating high-grade features. Conclusions. Our findings establish the subtypes of endometrial cancer occurring in the Nurses’ Health Study, corroborate the sensitivity of certain well-established biomarkers, and call into question previously identified associations between certain biomarkers (e.g., HNF1B) and particular histotypes.

Background: The causal link between elevated systemic inflammation biomarkers and poor survival has been demonstrated in cancer patients. However, the evidence for this correlation in endometrial cancer (EC) is too weak to influence current criteria of risk assessment. Here, we examined the role of inflammatory indicators as a tool to identify EC patients at higher risk of death in a retrospective observational study. Methods: A total of 155 patients surgically diagnosed with EC stage I-III FIGO 2009 and treated with postoperative External Beam Radiotherapy (EBRT) &plusmn, brachytherapy and chemotherapy according to ESMO-ESTRO-ESGO recommendation for patients at high risk of recurrence at the Gustave Roussy Institut, France, and Hospital Cl&iacute, nic, Spain, between 2008 and 2017 were evaluated. The impact of pre-treatment Neutrophil-to-Lymphocyte Ratio (NLR &ge, 2.2), Monocyte-to-Lymphocyte Ratio (MLR &ge, 0.18), Systemic Immune-Inflammatory Index (SII &ge, 1100) and lymphopenia (&lt, 1.0&times, 109/L) on overall survival (OS), cancer-specific survival and progression-free survival was evaluated. Subsequently, a cohort of 142 patients within high-advanced risk groups according to ESMO-ESGO-ESTRO classification was evaluated. Results: On univariate analysis, NLR (HR = 2.2, IC 95% 1.1&ndash, 4.7), SII (HR = 2.2, IC 95% 1.1&ndash, 4.6), MLR (HR = 5.0, IC 95% 1.1&ndash, 20.8) and lymphopenia (HR = 3.8, IC 95% 1.6&ndash, 9.0) were associated with decreased OS. On multivariate analysis, NLR, MLR, SII and lymphopenia proved to be independent unfavorable prognostic factors. Conclusions: lymphopenia and lymphocytes-related ratio are associated with poorer outcome in surgically staged I-III FIGO EC patients classified as high risk and treated with adjuvant EBRT and could be considered at cancer diagnosis. External validation in an independent cohort is required before implementation for patients&rsquo, stratification.

Endometrial cancer (EC) incidence rates vary ~10‐fold worldwide, in part due to variation in EC risk factor profiles. Using an EC risk model previously developed in the European EPIC cohort, we evaluated the prevention potential of modified EC risk factor patterns and whether differences in EC incidence between a European population and low‐risk countries can be explained by differences in these patterns. Predicted EC incidence rates were estimated over 10 years of follow‐up for the cohort before and after modifying risk factor profiles. Risk factors considered were: body mass index (BMI, kg/m2), use of postmenopausal hormone therapy (HT) and oral contraceptives (OC) (potentially modifiable); and, parity, ages at first birth, menarche and menopause (environmentally conditioned, but not readily modifiable). Modeled alterations in BMI (to all ≤23 kg/m2) and HT use (to all non‐HT users) profiles resulted in a 30% reduction in predicted EC incidence rates; individually, longer duration of OC use (to all ≥10 years) resulted in a 42.5% reduction. Modeled changes in not readily modifiable exposures (i.e., those not contributing to prevention potential) resulted in ≤24.6% reduction in predicted EC incidence. Women in the lowest decile of a risk score based on the evaluated exposures had risk similar to a low risk countries; however, this was driven by relatively long use of OCs (median = 23 years). Our findings support avoidance of overweight BMI and of HT use as prevention strategies for EC in a European population; OC use must be considered in the context of benefits and risks., What's new? Endometrial cancer rates vary considerably around the world, with incidence rates higher in Europe and North America than in parts of Asia and Africa. Here, the authors investigated how much of the risk disparity arises from modifiable factors, and how much modifying these factors could reduce cancer incidence. The 10% of European women with lowest risk had similar incidence to women in low‐risk countries, they found. Their model predicted that in European women, maintaining BMI below 23 kg/m2 and avoiding postmenopausal hormone use could reduce risk by 30%. Long‐term use of oral contraceptives could reduce risk by 42.5%.

Els dos subtipus més freqüents de càncer d’endometri (CE) són el carcinoma endometrioide (CEE) i el carcinoma serós (CSE). El diagnòstic diferencial entre aquestes dues tipologies no sempre és fàcil, hi ha casos que presenten característiques histològiques molt dubtoses i ambigües que dificulten el correcte diagnòstic final. Aquests dos tipus tumorals presenten diferents perfils moleculars i un pronòstic diferencial, fent que el CSE sigui el subtipus més agressiu i amb el pronòstic més desfavorable. Actualment s’està posant en coneixement la importància que pot jugar l’heterogeneïtat intratumoral (HIT) en els comportaments més agressius dels tumors i en la resistència als tractaments, com és el cas del CSE. En els últims anys, la irrupció de les tecnologies d’alt rendiment com la NGS (Next Generation Sequencing) i la MLPA (Multiple Ligase-dependent Probe Amplification) han ofert l’opció d’ampliar el coneixement molecular del càncer d’una manera més precisa i resolutiva. Per aquests motius, els dos primers objectius d’aquesta tesi s’han centrat en analitzar, en els dos tipus més freqüents de càncer d’endometri, diverses alteracions moleculars que ens permetin obtenir uns perfils genètics específics, i classificar d’una manera més precisa i objectiva aquests dos tipus tumorals. Per realitzar les anàlisis de les alteracions genètiques s’han utilitzat, l’aplicació de targeted sequencing (NGS) amb un panell personalitzat de 40 gens per determinar els gens alterats respecte a variants somàtiques, i la tècnica MLPA per determinar les alteracions somàtiques en el número de còpies (ASNC) de 106 gens. Finalment, el tercer objectiu s’ha focalitzat en conèixer el paper que juga l’heterogeneïtat intratumoral en el CSE. L’efecte de l’HIT ha estat estudiat a dos nivells d’alteració molecular, a nivell de l’alteració dels gens determinant variants somàtiques i de l’alteració en el número de còpies dels gens, mitjançant les dues tècniques anteriors. Els resultats del primer objectiu d’aquesta tesi han demostrat la idoneïtat del nostre estudi personalitzat de NGS com a eina molecular addicional per confirmar la classificació histològica del CE. Aquesta estratègia sembla interessant com una eina per classificar tumors amb troballes microscòpiques inusuals i ambigües. Amb els resultats del segon objectiu s’ha descrit que de manera general el CSE presenta moltes més ASNC que el CEE. Tot i això, també s’ha mostrat que dins el CSE hi ha un percentatge de casos (42%) que presenten unes característiques genètiques que no es corresponen amb el fenotip, i que per tant, en aquests casos pot ser de gran ajuda l’ús del perfil genètic d’ASNC per realitzar una classificació més correcta. A més a més, s’ha suggerit l’ús de la combinació de la determinació de p53 per immunohistoquímica i del número de còpies de la CCNE1 per classificar els casos dins el grup serous-like. Per finalitzar, els resultats del tercer objectiu han mostrat que el CSE, per una banda, presenta unes alteracions moleculars clonals com les variants somàtiques al gen TP53 i guanys en els gens CCNE1 i PIK3CA, i per altra banda, que la seva heterogeneïtat intratumoral està caracteritzada principalment per les ASNC dels gens. A més a més, s’ha remarcat que un dels gens principalment afectats per aquesta heterogeneïtat és el gen ERBB2, que és una diana terapèutica àmpliament utilitzada. Los dos subtipos más frecuentes de cáncer de endometrio (CE) son el carcinoma endometrioide (CEE) y el carcinoma seroso (CSE). El diagnóstico diferencial entre estas dos tipologías no siempre es fácil, hay casos que presentan características histológicas muy dudosas y ambiguas que dificultan su correcto diagnóstico final. Estos dos tipos tumorales presentan perfiles moleculares y pronósticos diferenciales, haciendo que el CSE sea el subtipo más agresivo y con el pronóstico más desfavorable. Actualmente se está poniendo en conocimiento la importancia que puede jugar la heterogeneidad intratumoral (HIT) en los comportamientos más agresivos de los tumores y en la resistencia a los tratamientos, como es el caso del CSE. En los últimos años, la irrupción de las tecnologías de alto rendimiento como la NGS (Next Generation Sequencing) y la MLPA (Multiple Ligase-dependiente Probe Amplification) han ofrecido la opción de ampliar el conocimiento molecular del cáncer de una manera más precisa y resolutiva. Por estos motivos, los dos primeros objetivos de esta tesis se han centrado en analizar en los dos tipos más frecuentes de cáncer de endometrio varias alteraciones moleculares que nos permitan obtener unos perfiles genéticos específicos, y clasificar de una manera más precisa y objetiva estos dos tipos tumorales. Para realizar los análisis de las alteraciones genéticas se han utilizado, la aplicación de targeted sequencing (NGS) con un panel personalizado de 40 genes para determinar los genes alterados respecto a variantes somáticas, y la técnica MLPA para determinar las alteraciones somáticas en el número de copias (ASNC) de 106 genes. Finalmente, el tercer objetivo se ha focalizado en conocer el papel que juega la heterogeneidad intratumoral en el CSE. El efecto de la HIT ha sido estudiado a dos niveles de alteración molecular, a nivel de la alteración de los genes analizando las variantes somáticas y la alteración en el número de copias de los genes, mediante las dos técnicas anteriores. Los resultados del primer objetivo de esta tesis han demostrado la idoneidad de nuestro estudio personalizado de NGS como herramienta molecular adicional para confirmar la clasificación histológica del CE. Esta estrategia parece interesante como una herramienta para clasificar tumores con hallazgos microscópicos inusuales y ambiguos. Con los resultados del segundo objetivo se ha descrito que de manera general el CSE presenta muchas más ASNC que el CEE. Sin embargo, también se ha mostrado que dentro el CSE hay un porcentaje de casos (42%) que presentan unas características genéticas que no corresponden con el fenotipo, por lo que en estos casos puede ser de gran ayuda el uso del perfil genético de ASNC para realizar una clasificación más correcta. Además, se ha sugerido el uso de la combinación de la determinación de p53 por inmunohistoquímica y del número de copias de la CCNE1 para clasificar los casos dentro del grupo serous-like. Para finalizar, los resultados del tercer objetivo han mostrado que el CSE, por una parte, presenta unas alteraciones moleculares clonales como las variantes somáticas en el gen TP53 y ganancias en los genes CCNE1 y PIK3CA, y por otra parte, que su heterogeneidad intratumoral está caracterizada principalmente por las ASNC de los genes. Además, se ha remarcado que uno de los genes principalmente afectados por esta heterogeneidad es el gen ERBB2, que es una diana terapéutica ampliamente utilizada. The two most common subtypes of endometrial cancer (CE) are endometrioid carcinoma (CEE) and serous carcinoma (CSE). Differential diagnosis between these two types is not always easy, there are cases with very doubtful and ambiguous histological features that make it difficult their final diagnostic. These two tumor types have different molecular profiles and differential prognosis, making the CSE the most aggressive subtype and with the most unfavorable prognosis. At present, the importance of intratumoral heterogeneity (HIT) can be played out in the most aggressive behaviors of tumors and in resistance to treatment, as in the case of CSE. In recent years, the advent of high-performance technologies such as NGS (Next Generation Sequencing) and MLPA (Multiple Ligase-dependent Probe Amplification) have offered the option of extending molecular knowledge of cancer in a way more accurate and resolute. For these reasons, the first two objectives of this dissertation have been to analyze in the two most common types of endometrial cancer, various molecular alterations that allow us to obtain specific genetic profiles, and to classify more precisely and objective these tumor types. To preform genetic alteration analyzes have been used the application of targeted sequencing (NGS) with a personalized panel of 40 genes to determine the altered genes with respect to somatic variants, and the MLPA technique to determine somatic alterations in the number of copies (ASNC) of 106 genes. Finally, the third objective was focused on understanding the role that intratumoral heterogeneity plays in CSE. The effect of HIT has been studied at two levels of molecular alteration, at the level of the genes determining somatic variants and the number of copies of the genes, using the two previous techniques. The results of the first objective of this thesis have shown the suitability of our personalized NGS study as an additional molecular tool to confirm the histological classification of CE. This strategy seems interesting as a tool for classifying tumors with unusual and ambiguous microscopic findings. The results of the second objective have described that in general the CSE has many more ASNCs than the EEC. However, it has also been shown that in the CSE there is a percentage of cases (42%) that have genetic characteristics that do not correspond to the phenotype, and therefore in these cases the use may be very helpful of the ASNC gene profile for more accurate classification. In addition, the combination of p53 determination by immunohistochemistry and the number of CCNE1 copies has been suggested to classify cases within the group serous-like. Finally, the results of the third objective have shown that CSE, on the one hand, has clonal molecular alterations such as the somatic variants in the TP53 gene and gains in the CCNE1 and PIK3CA genes, and on the other hand, that its intratumoral heterogeneity is characterized mainly by the ASNC of the genes. It has also been emphasized that one of the genes that is most affected by this heterogeneity is the ERBB2 gene, which is a widely used therapeutic target.

Background: To evaluate whether EQD2(&alpha, /&beta, = 3Gy) at 2 cm3 of the most exposed area of the vagina is related to late vaginal toxicity in postoperative endometrial cancer (PEC) patients (p) treated with exclusive brachytherapy (BT). Methods: From 2014 to 2017, 43p were included in this study. BT was administered: 3-fractions of 6Gy in 37p and 2-fractions of 7.5Gy in 6p. The dose was prescribed at a depth of 5 mm from the applicator surface with dose-point optimization based on distance. The active treatment length was 2.5 cm. CTV-D90 and the dose to the most exposed 2 cm3 of the vagina was calculated for each patient. Late toxicity of the bladder and rectum was assessed using Radiation Therapy Oncology Group (RTOG) criteria, and vaginal toxicity by objective Late Effects Normal Tissue Task Force (LENT)-Subjective, Objective, Management, Analytic (SOMA) (LENT-SOMA) criteria. Statistics: frequency tables, mean, median, range, standard deviation, and box plot. Results: The median follow-up was 51 months (12&ndash, 68). 20 p (46.5%) and 2 p (4.7%) developed G1 and G2 vaginal complications, respectively. Only 1/2 p-G2 receiving EQD2(&alpha, = 3Gy) at 2 cm3 &gt, 68Gy presented vaginal shortening and 18/20 p-G1 received doses &lt, 68Gy. Conclusions: PECp receiving exclusive brachytherapy with doses &lt, 68Gy EQD2(&alpha, = 3Gy) at 2 cm2 of the vagina presented only G0&ndash, G1 vaginal toxicity, except for one with bleeding telangiectasias. Larger prospective studies are necessary to confirm the present results.

This work was supported by Cancer Research UK (CRUK) (grant number C19335/A21351, to MJG and HK) . The metabolomics infrastructure in the Division of Cancer, Imperial College London is supported by the Imperial College Experimental Can-cer Medicine Centre, the This work was supported by Cancer Research UK (CRUK) (grant number C19335/A21351, to MJG and HK) . The metabolomics infrastructure in the Division of Cancer, Imperial College London is supported by the Imperial College Experimental Can-cer Medicine Centre, the Imperial College Cancer Research UK Centre and the NIHR Imperial Biomedical Research Centre (APS & HK) . The co-ordination of EPIC is financially supported by International Agency for Research on Cancer (IARC) and also by the Department of Epidemiology and Biostatistics, School of Public Health, Imperial College London which has additional infrastructure support provided by the NIHR Im-perial Biomedical Research Centre (BRC) . The national cohorts are sup-ported by: Danish Cancer Society (Denmark) ; Ligue Contre le Cancer, Institut Gustave Roussy, Mutuelle Generale de l'Education Nationale, Institut National de la Sante et de la Recherche Medicale (INSERM) (France) ; German Cancer Aid, German Cancer Research Center (DKFZ) , German Institute of Human Nutrition PotsdamRehbruecke (DIfE) , Federal Ministry of Education and Research (BMBF) (Germany) ; Associazione Italiana per la Ricerca sul CancroAIRCItaly, Compagnia di SanPaolo and National Research Council (Italy) ; Dutch Ministry of Public Health, Welfare and Sports (VWS) , Netherlands Can-cer Registry (NKR) , LK Research Funds, Dutch Prevention Funds, Dutch ZON (Zorg Onderzoek Nederland) , World Cancer Research Fund (WCRF) , Statistics Netherlands (The Netherlands) ; Health Research Fund (FIS) -Instituto de Salud Carlos III (ISCIII) , Regional Governments of Andalucia, Asturias, Basque Country, Murcia and Navarra, and the Catalan Institute of Oncology-ICO (Spain) ; Swedish Cancer Society, Swedish Research Council and County Councils of Skkne and Vasterbotten (Sweden) ; Cancer Research UK (14136 to EPICNorfolk; C8221/A29017 to EPICOxford) , Medical Research Council (1000143 to EPICNorfolk; MR/M012190/1 to EPICOxford) . (United Kingdom) ., Background. Endometrial cancer is strongly associated with obesity and dysregulation of metabolic factors such as estrogen and insulin signaling are causal risk factors for this malignancy. To identify additional novel metabolic pathways associated with endometrial cancer we performed metabolomic analyses on pre-diagnostic plasma samples from853 case-control pairs fromthe European Prospective Investigation into Cancer and Nutrition (EPIC). Methods. A total of 129metabolites (acylcarnitines, amino acids, biogenic amines, glycerophospholipids, hexoses, and sphingolipids) were measured by liquid chromatography-mass spectrometry. Conditional logistic regression estimated the associations of metabolites with endometrial cancer risk. An analysis focusing on clusters of metabolites using the bootstrap lasso method was also employed. Results. After adjustment for body mass index, sphingomyelin [SM] C18:0was positively (OR1SD: 1.18,95%CI: 1.05–1.33), and glycine, serine, and free carnitine (C0) were inversely (OR1SD: 0.89, 95% CI: 0.80–0.99; OR1SD: 0.89, 95% CI: 0.79–1.00 and OR1SD: 0.91, 95% CI: 0.81–1.00, respectively) associated with endometrial cancer risk. Serine, C0 and two sphingomyelins were selected by the lasso method in >90% of the bootstrap samples. The ratio of esterified to free carnitine (OR1SD: 1.14, 95% CI: 1.02–1.28) and that of short chain to free acylcarnitines (OR1SD: 1.12, 95% CI: 1.00–1.25) were positively associated with endometrial cancer risk. Further adjustment for C-peptide or other endometrial cancer risk factors only minimally altered the results. Conclusion. These findings suggest that variation in levels of glycine, serine, SMC18:0 and free carnitine may represent specific pathways linked to endometrial cancer development. If causal, these pathways may offer novel targets for endometrial cancer prevention., Cancer Research UK C19335/A21351, Imperial College Experimental Cancer Medicine Centre Imperial College Cancer Research UK Centre, NIHR Imperial Biomedical Research Centre, World Health Organization, Department of Epidemiology and Biostatistics, School of Public Health, Imperial College London, Danish Cancer Society, Ligue Contre le Cancer (France) Institut Gustave Roussy (France) Mutuelle Generale de l'Education Nationale (France), Institut National de la Sante et de la Recherche Medicale (Inserm), Deutsche Krebshilfe, German Cancer Research Center (DKFZ) (Germany) German Institute of Human Nutrition PotsdamRehbruecke (DIfE) (Germany), Federal Ministry of Education & Research (BMBF), Fondazione AIRC per la ricerca sul cancro Compagnia di San Paolo Consiglio Nazionale delle Ricerche (CNR), Netherlands Government Netherlands Government, World Cancer Research Fund International (WCRF), Health Research Fund (FIS) - Instituto de Salud Carlos III (ISCIII) (Spain), Junta de Andalucia, Regional Government of Asturias (Spain), Regional Government of Basque Country (Spain), Regional Government of Murcia (Spain), Regional Government of Navarra (Spain), Catalan Institute of Oncology-ICO (Spain), Swedish Cancer Society, Swedish Research Council, County Council of Skkne (Sweden), County Council of Vasterbotten (Sweden), Cancer Research UK 14136 C8221/A29017, UK Research & Innovation (UKRI), Medical Research Council UK (MRC) 1000143 MR/M012190/1

There have been significant advances in our understanding of the biology and classification of endometrial carcinoma, over the last few years, and the new prediction models proposed for prognostication. To accurately diagnose and stage tumors and apply these prediction models, it is necessary that there be standardized processing of specimens, and a common understanding and usage of the diagnostic terminology of endometrial carcinoma. The International Society of Gynecological Pathologists embarked on an ambitious project to achieve this goal in 2015. An early step in the process was to collect baseline information on existing practices with regard to the processing, diagnosis, and reporting of endometrial carcinomas among the members of the society. This was carried out using a web-based survey comprising 112 questions. The results are presented herein and reveal areas of uniformity but also areas of substantial variation among pathologists. The results of the survey assisted in developing the subsequent recommendations that follow as separate articles in this issue of the journal with regard to processing, diagnosis, and reporting of endometrial carcinomas.

Ascitic fluid; Biomarkers; Endometrial cancer Líquido ascítico; Biomarcadores; Cáncer de endometrio Ascites; Biomarcadors; Càncer d'endometri Endometrial cancer (EC) is the sixth most common cancer in women worldwide and is responsible for more than 89,000 deaths every year. Mortality is associated with presence of poor prognostic factors at diagnosis, i.e., diagnosis at an advanced stage, with a high grade and/or an aggressive histology. Development of novel approaches that would permit us to improve the clinical management of EC patients is an unmet need. In this study, we investigate a novel approach to identify highly sensitive and specific biomarkers of EC using extracellular vesicles (EVs) isolated from the peritoneal lavage of EC patients. EVs of peritoneal lavages of 25 EC patients were isolated and their miRNA content was compared with miRNAs of EVs isolated from the ascitic fluid of 25 control patients. Expression of the EV-associated miRNAs was measured using the Taqman OpenArray technology that allowed us to detect 371 miRNAs. The analysis showed that 114 miRNAs were significantly dysregulated in EC patients, among which eight miRNAs, miRNA-383-5p, miRNA-10b-5p, miRNA-34c-3p, miRNA-449b-5p, miRNA-34c-5p, miRNA-200b-3p, miRNA-2110, and miRNA-34b-3p, demonstrated a classification performance at area under the receiver operating characteristic curve (AUC) values above 0.9. This finding opens an avenue for the use of EV-associated miRNAs of peritoneal lavages as an untapped source of biomarkers for EC. Funded by Generalitat de Catalunya, grant numbers SLT002/16/00274, 2017SGR1661, and 2017SGR1368; by CIBERONC, grant numbers CB16/12/00328 and CB16/12/00231; by Instituto Salud Carlos III, grant number PIE15/00029 and by Asociación Española Contra el Cáncer (AECC), grant number GCTRA1804MATI

Endometrial carcinoma is the commonest gynecological malignancy in developed countries, and the various aspects of the pathology report are critical for patient management. There are many areas of controversy with regard to the handling of resection specimens and the pathologic reporting of endometrial carcinomas. These controversies include those related to sampling, diagnosis, reporting of parameters important for staging, and the undertaking of ancillary studies. These controversies stimulated the International Society of Gynecological Pathologists (ISGyP) endometrial carcinoma project. The project was devised at the ISGyP Board of Directors meeting in March 2015 under the Presidency of Richard Zaino. An organizing committee was selected from the members of the Board of Directors and the education committee of the ISGyP. The organizing committee (comprising the 5 authors of this editorial), as a first step, devised a comprehensive survey, which was emailed to all members of the ISGyP; the survey covered all aspects of endometrial cancer reporting, including specimen dissection and sampling, diagnosis, staging, prognostic factors, and ancillary studies.

This review of challenging diagnostic issues concerning high-grade endometrial carcinomasisderivedfromtheauthors’ reviewoftheliteraturefollowedbydiscussionsatthe Endometrial Cancer Workshop sponsored by the International Society of Gynecological Pathologists in 2016. Recommendations presented are evidence-based, insofar as this is possible,giventhatthelevelsofevidenceareweakormoderateduetosmallsamplesizesand nonuniform diagnostic criteria used in many studies. High-grade endometrioid carcinomas include FIGO grade 3 endometrioid carcinomas, serous carcinomas, clear cell carcinomas, undifferentiated carcinomas, and carcinosarcomas. FIGO grade 3 endometrioid carcinoma is diagnosed when an endometrioid carcinoma exhibits >50% solid architecture (excluding squamousareas), orwhenan architecturallyFIGOgrade2endometrioid carcinomaexhibits marked cytologic atypia, provided that a glandular variant of serous carcinoma has been excluded. The most useful immunohistochemical studies to make the distinction between these 2 histotypes are p53, p16, DNA mismatch repair proteins, PTEN, and ARID1A. Endometrial clear cell carcinomas must display prototypical architectural and cytologic features for diagnosis. Immunohistochemical stains, including, Napsin A and p504s can be used as ancillary diagnostic tools; p53 expression is aberrant in a minority of clear cell carcinomas. Of note, clear cells are found in all types of high-grade endometrial carcinomas, leading to a tendency to overdiagnose clear cell carcinoma. Undifferentiated carcinoma (which when associated with a component of low-grade endometrioid carcinoma is termed “dedifferentiated carcinoma”) is composed of sheets of monotonous, typically dyscohesive cells, which can have a rhabdoid appearance; they often exhibit limited expression of cytokeratins and epithelial membrane antigen, are usually negative for PAX8 and hormone receptors, lack membranous e-cadherin and commonly demonstrate loss of expression of DNA mismatch repair proteins and SWI-SNF chromatin remodeling proteins. Carcinosarcomas must show unequivocal morphologic evidence of malignant epithelial and mesenchymal differentiation. This work was funded in part through the NIH/NCI Cancer Center Support Grant P30 CA008748.

Background: Colorectal (CRC) and endometrial cancer (EC) are the most common types of cancer in Lynch syndrome (LS). Risk reducing surgeries (RRS) might impact cancer incidence and mortality. Our objectives were to evaluate cumulative incidences of CRC, gynecological cancer and all-cause mortality after RRS in LS individuals. Methods: Retrospective analysis of 976 LS carriers from a single-institution registry. Primary endpoints were cumulative incidence at 75 years of cancer (metachronous CRC in 425 individuals, EC and ovarian cancer (OC) in 531 individuals) and all-cause mortality cumulative incidence, comparing extended (ES) vs. segmental surgery (SS) in the CRC cohort and risk reducing gynecological surgery (RRGS) vs. surveillance in the gynecological cohort. Results: Cumulative incidence at 75 years of metachronous CRC was 12.5% vs. 44.7% (p = 0.04) and all-cause mortality cumulative incidence was 38.6% vs. 55.3% (p = 0.31), for ES and SS, respectively. Cumulative, incidence at 75 years was 11.2% vs. 46.3% for EC (p = 0.001) and 0% vs. 12.7% for OC (p N/A) and all-cause mortality cumulative incidence was 0% vs. 52.7% (p N/A), for RRGS vs. surveillance, respectively. Conclusions: RRS in LS reduces the incidence of metachronous CRC and gynecological neoplasms, also indicating a reduction in all-cause mortality cumulative incidence in females undergoing RRGS.

Endometrial cancer is the most frequent gynecological tumor in development countries. Currently there is a lack of therapeutic options in this type of tumors, which gives rise to a great interest in the search for therapeutic targets that allows the treatment to be customized for the benefit of the patient. The objective of the project was to find out significant differences in the expression of the genes in patients with MSI-H endometrial cancer against MSI-L and MSS, with the objective of finding possible therapeutics options. Methods: To carry out the study, data were obtained from the RNA-seq analysis of samples from patients with TCGA endometrial cancer. The differential expression analysis was carried out using the limma package. Subsequently an analysis of pathway enrichment and gene co-expression was carried out using the ReactomeFIPlugin application through cytoscape version 3.6.1. Finally, the study was completed with a survival analysis of both groups. El cáncer de endometrio es el tumor ginecológico más frecuente en países desarrollados. Actualmente existe una falta de opciones terapéuticas en este tipo de tumores, lo que origina un gran interés en la búsqueda de dianas terapéuticas que permitan personalizar el tratamiento en beneficio del paciente. El objetivo del proyecto fue buscar si había diferencias significativas en la expresión de los genes de pacientes con cáncer de endometrio MSI-H frente a MSI-L y MSS, con la finalidad de encontrar posibles alternativas terapéuticas. Para llevar a cabo el estudio se obtuvieron datos del análisis de RNA-seq de muestras de pacientes con cáncer de endometrio del TCGA. Mediante el paquete limma se realizó el análisis de expresión diferencial. Posteriormente se llevó a cabo un análisis de enriquecimiento de pathways y de co-expresión de genes mediante la aplicación ReactomeFIPlugin a través de Cytoscape versión 3.6.1. Por último, el estudio se completó con un análisis de supervivencia de ambos grupos. El càncer d'endometri és el tumor ginecològic més freqüent en països desenvolupats. Actualment existeix una falta d'opcions terapèutiques en aquest tipus de tumors, la qual cosa origina un gran interès en la cerca de dianes terapèutiques que permetin personalitzar el tractament en benefici del pacient. L'objectiu del projecte va ser buscar si hi havia diferències significatives en l'expressió dels gens de pacients amb càncer d'endometri MSI-H enfront de MSI-L i MSS, amb la finalitat de trobar possibles alternatives terapèutiques. Per dur a terme l'estudi es van obtenir dades de l'anàlisi de RNA-seq de mostres de pacients amb càncer d'endometri del TCGA. Mitjançant el paquet limma es va realitzar l'anàlisi d'expressió diferencial. Posteriorment es va dur a terme una anàlisi d'enriquiment de pathways i de co-expressió de gens mitjançant l'aplicació ReactomeFIPlugin a través de Cytoscape versió 3.6.1. Finalment, l'estudi es va completar amb una anàlisi de supervivència de tots dos grups.

In this issue of Virchows Archiv, there is a comprehensive review article on the most recent advances in our understanding of the pathologic and molecular features of endometrial stromal tumors [1]. The review is written by a team and a senior author, with considerable experience in the field. The manuscript describes the changes made since the first classification based on the number of mitotic figures to the current approach based on the resemblance to normal endometrial stroma of the proliferative phase.

Our aim was to investigate whether molecular classification can be used to refine prognosis in grade 3 endometrial endometrioid carcinomas (EECs). Grade 3 EECs were classified into 4 subgroups: p53 abnormal, based on mutant-like immunostaining (p53abn); MMR deficient, based on loss of mismatch repair protein expression (MMRd); presence of POLE exonuclease domain hotspot mutation (POLE); no specific molecular profile (NSMP), in which none of these aberrations were present. Overall survival (OS) and recurrence-free survival (RFS) rates were compared using the Kaplan-Meier method (Log-rank test) and univariable and multivariable Cox proportional hazard models. In total, 381 patients were included. The median age was 66 years (range, 33 to 96 y). Federation Internationale de Gynecologie et d'Obstetrique stages (2009) were as follows: IA, 171 (44.9%); IB, 120 (31.5%); II, 24 (6.3%); III, 50 (13.1%); IV, 11 (2.9%). There were 49 (12.9%) POLE, 79 (20.7%) p53abn, 115 (30.2%) NSMP, and 138 (36.2%) MMRd tumors. Median follow-up of patients was 6.1 years (range, 0.2 to 17.0 y). Compared to patients with NSMP, patients with POLE mutant grade 3 EEC (OS: hazard ratio [HR], 0.36 [95% confidence interval, 0.18-0.70]; P=0.003; RFS: HR, 0.17 [0.05-0.54]; P=0.003) had a significantly better prognosis; patients with p53abn tumors had a significantly worse RFS (HR, 1.73 [1.09-2.74]; P=0.021); patients with MMRd tumors showed a trend toward better RFS. Estimated 5-year OS rates were as follows: POLE 89%, MMRd 75%, NSMP 69%, p53abn 55% (Log rank P=0.001). Five-year RFS rates were as follows: POLE 96%, MMRd 77%, NSMP 64%, p53abn 47% (P=0.000001), respectively. In a multivariable Cox model that included age and Federation Internationale de Gynecologie et d'Obstetrique stage, POLE and MMRd status remained independent prognostic factors for better RFS; p53 status was an independent prognostic factor for worse RFS. Molecular classification of grade 3 EECs reveals that these tumors are a mixture of molecular subtypes of endometrial carcinoma, rather than a homogeneous group. The addition of molecular markers identifies prognostic subgroups, with potential therapeutic implications. This study was funded in part by the Dutch Cancer Society (KWF-UL2012-5719) (Dr. Bosse, Dr. Nout). This study was funded in part through the NIH/NCI Support Grant P30 CA008748 (Dr. Abu-Rustum, Dr. Levine, Dr. Soslow).

Endometrial cancer (EC) is the most common gynecological cancer in developed countries. Most patients are diagnosed at an early stage with a low risk of relapse. However, there is a group of patients with a high risk of relapse and poor prognosis. Despite the recent publication of randomized trials, the adjuvant treatment of high-risk EC is still to be defined and there are many open questions about the best approach and the right timing. Unfortunately, the survival of metastatic or recurrent EC is short, due to the poor results of chemotherapy and the lack of a second line of treatment. Advances in the knowledge of the molecular abnormalities in EC have permitted the development of promising targeted therapies.

Endometrial cancer (EC) remains the most common malignancy of the genital tract among women in developed countries. Although much research has been performed at genomic, transcriptomic and proteomic level, there is still a significant gap in the metabolomic studies of EC. In order to gain insights into altered metabolic pathways in the onset and progression of EC carcinogenesis, we used high resolution mass spectrometry to characterize the metabolomic and lipidomic profile of 39 human EC and 17 healthy endometrial tissue samples. Several pathways including lipids, Kynurenine pathway, endocannabinoids signaling pathway and the RNA editing pathway were found to be dysregulated in EC. The dysregulation of the RNA editing pathway was further investigated in an independent set of 183 human EC tissues and matched controls, using orthogonal approaches. We found that ADAR2 is overexpressed in EC and that the increase in expression positively correlates with the aggressiveness of the tumor. Furthermore, silencing of ADAR2 in three EC cell lines resulted in a decreased proliferation rate, increased apoptosis, and reduced migration capabilities in vitro. Taken together, our results suggest that ADAR2 functions as an oncogene in endometrial carcinogenesis and could be a potential target for improving EC treatment strategies. This work was supported by the Spanish Ministry of Health (RD12/0036/0035), the Spanish Ministry of Economy and Competitivy (PI14/02043), the AECC (Grupos Estables de Investigacion 2011 - AECC- GCB 110333 REVE), the Fundació La Marató TV3 (2/C/2013), the CIRIT Generalitat de Catalunya (2014 SGR 1330) and the European Commission, 7th Framework Program, IRSES (PROTBIOFLUID –269285) – Belgium. Te Spanish Ministry of Economy and Competitiveness (IJCI-2015-25000) granted Dr. Colás and and the AGAUR Generalitat de Catalunya (2015FI_B00703) granted Tatiana Altadill. Te authors would like to acknowledge the Proteomics and Metabolomics Shared Resource partially supported by Cancer Center Support Grant NIH/NCI grant P30-CA051008. Te Institut de Salud Carlos III (FIS (PI13/01701)) also supported this project. Tissue samples were obtained with the support of “Xarxa Catalana de Bancs de Tumors” and “Plataforma de Biobancos” ISCIII (PT13/0010/0014).

Alrededor del 30% de las pacientes con cáncer de endometrio (CE) son diagnosticadas en un estadio avanzado de la enfermedad, asociado con un drástico descenso de la supervivencia a los 5 años. El diagnóstico se realiza mediante el examen histopatológico de una biopsia endometrial, que se obtiene preferiblemente mediante una aspiración mínimamente invasiva del interior de la cavidad uterina (aspirado uterino). Desafortunadamente, el número limitado de células en estas biopsias está asociado a dos inconvenientes importantes: i) alrededor del 22% de pacientes no reciben un diagnóstico definitivo con esta muestra; y ii) el tipo histológico y grado de los casos de CE son asignados incorrectamente hasta en el 50% de los casos. El objetivo principal de esta tesis es la identificación de paneles proteicos específicos y sensibles que permitan: i) discriminar todos los casos CE y no-CE empleando muestras de aspirado uterino y así reducir el número de biopsias más invasivas; y ii) proporcionar una información preoperatoria del tipo histológico y el grado tumoral de los casos de CE más precisa para mejorar la clasificación de las pacientes en grupos de riesgo. En el Capítulo 1 se realizó una extensa revisión bibliográfica y se generó una lista de 506 proteínas asociadas con CE. El análisis de las características de los estudios incluidos en esta revisión nos permitió definir nuevas aproximaciones que podrían acelerar la identificación de proteínas clínicamente útiles para el diagnóstico del CE. Estas aproximaciones se aplicaron en los pasos descritos en los siguientes capítulos. Así, en el Capítulo 2 se demostró que esas proteínas previamente asociadas con CE, principalmente estudiadas por inmunohistoquímica a nivel de tejido, se pueden medir en muestras de aspirado uterino mediante técnicas de proteómica dirigida. Además, se seleccionó la fracción líquida de estas muestras como la fracción más apropiada para la identificación de biomarcadores. En el Capítulo 3 se describió un flujo de experimentales para reducir la lista inicial de 506 proteínas a los 52 candidatos con mayor potencial, que fueron analizados en la fracción líquida de aspirados uterinos de 20 pacientes con CE y 18 controles por LC-PRM. Se observó la expresión diferencial de 26 proteínas, y entre ellos diez biomarcadores mostraron una alta sensibilidad y especificidad (AUC> 0.9). Demostramos la importancia de los estudios de verificación por espectrometría de masas dirigida, y destacamos los beneficios del LC-PRM para este propósito. En el Capítulo 4 las mismas 52 proteínas fueron validadas en aspirados uterinos de 116 pacientes por LC-PRM. Los niveles de 28 proteínas eran significativamente mayores en las pacientes de CE (n=69) en comparación con los controles (n=47). La combinación de MMP9 y KPYM alcanzaba un 94% de sensibilidad y un 87% de especificidad para detectar casos de CE. Este panel complementa perfectamente el diagnóstico histopatológico estándar, logrando el 100% de diagnósticos correctos en este estudio. Los niveles de nueve proteínas se encontraron significativamente aumentados en los CE de tipo endometrioide (n=49) comparados con los CE de tipo seroso, más agresivos (n=20). La combinación de CTNB1, XPO2 y CAPG alcanzaba una sensibilidad del 95% y una especificidad del 96% en la discriminación de estos subtipos. Con el fin de facilitar la implementación de estos paneles de biomarcadores en la práctica clínica, se evaluó la transferencia de los resultados de LC-PRM a kits de ELISA disponibles comercialmente. Además, en el Capítulo 5 se evaluó la simplificación del ensayo analítico y de la preparación de la muestra. Se espera que estos paneles proteómicos en aspirado uterino mejoren el diagnóstico de CE, reduciendo el número de biopsias invasivas, ayudando en la predicción del tratamiento quirúrgico óptimo, y reduciendo los costes sanitarios asociados a esta enfermedad., About 30% of endometrial cancer (EC) patients are still diagnosed at an advanced stage of the disease associated with a drastic decrease in the 5-year survival rate. Diagnosis is achieved by the histopathological examination of an endometrial biopsy, which is preferably obtained by a minimally invasive aspiration from the uterine cavity (i.e., uterine aspirate). Unfortunately, the limited number of cells available for examination in these biopsies is associated with two important drawbacks: i) around 22% of undiagnosed patients due to histologically inadequate specimens; and ii) up to 50% of incorrectly assigned EC histotype and grade. The main goal of this thesis is the identification of specific and sensitive proteomic signatures able to: i) accurately discriminate between EC cases and non-EC women with suspicion of EC in uterine aspirate samples, and thus to reduce the number of more invasive sampling methods; and ii) provide clinicians more accurate preoperative information regarding EC histological type and tumor grade that could improve the risk stratification of the patients. In Chapter 1 an extensive literature review was performed, and a list of 506 proteins associated with EC was generated. The analysis of the main characteristics of the studies included in this literature review allowed us to outline approaches not yet exploited that might accelerate the identification of clinically useful proteins for EC diagnosis. These approaches were applied in the workflow described in the next chapters. In Chapter 2 we demonstrated that those proteins previously associated with EC, mainly studied by immunohistochemistry at the tissue level, can be measured in uterine aspirate samples by highly multiplexing targeted proteomics. Moreover, we selected the fluid fraction of these uterine aspirates as the most appropriate fraction for biomarker identification. In Chapter 3 we described a stepwise workflow to reduce the initial list of 506 candidate biomarkers generated in Chapter 1 down to 52 proteins that were analyzed in the fluid fraction of uterine aspirates from 20 EC patients and 18 non-EC controls by LC-PRM. The differential expression of 26 proteins was observed, and among them ten biomarkers showed a high sensitivity and specificity (AUC > 0.9). We demonstrated the importance of targeted mass spectrometry-based approaches in biomarker verification studies to prioritize the most promising biomarkers to enter a validation study, and highlighted the benefits of LC-PRM acquisition performed on high-resolution accurate mass spectrometers for this purpose. The same 52 proteins were then validated in the uterine aspirates from 116 patients by LC-PRM, as described in Chapter 4. The levels of 28 proteins were significantly higher in EC patients (n=69) compared to controls (n=47). The combination of MMP9 and KPYM exhibited 94% sensitivity and 87% specificity for detecting EC cases. This panel perfectly complemented the standard histopathological diagnosis, achieving 100% of correct diagnosis in this dataset. Nine proteins were significantly increased in endometrioid EC (n=49) compared to less common but more aggressive serous EC (n=20). The combination of CTNB1, XPO2 and CAPG achieved 95% sensitivity and 96% specificity for the discrimination of these subtypes. In order to facilitate the implementation of these biomarker signatures in the clinical practice, the translation of the LC-PRM results to commercially available ELISA kits was then evaluated. Moreover, simplification of the analytical assay and the sample preparation was assessed in Chapter 5. The two uterine aspirate-based proteomic signatures developed in this thesis are expected to improve EC diagnosis, precluding the use of subsequent invasive sampling methods; and to assist in the prediction of the optimal surgical treatment. In the long term, the implementation of these signatures is expected to improve the management of EC patients and substantially reduce healthcare costs.

El carcinoma endometrial (CE) es el tumor infiltrante más común del tracto genital femenino y el cuarto en la mujer en países desarrollados, siendo la tipología endometrioide (CEE) la más frecuente. El 80% de las pacientes son diagnosticadas cuando el tumor sigue localizado en el útero, presentado una supervivencia a los 10 años que alcanza el 80%. Sin embargo, un número pequeño aunque relevante de estas pacientes recurren. Por ello, es de vital importancia encontrar marcadores para predecir la recidiva en esas pacientes para las cuales no se conoce un factor de riesgo suficientemente robusto. El 20% restante de las pacientes son diagnosticadas en una etapa más avanzada y presentan invasión miometrial, siendo este proceso uno de los factores de riesgo más relevantes y significativamente asociado a la recurrencia. La progresión del cáncer combina múltiples procesos que implican a moléculas de adhesión celular. Su expresión aberrante se relaciona con: pérdida de adhesión célula-célula, disgregación de las células tumorales de la lámina propia y adquisición de un fenotipo invasivo y migratorio. ALCAM, miembro de la superfamilia de las inmunoglobulinas, participa en interacciones homotípicas y heterotípicas entre células adyacentes. Su expresión se ha relacionado con la progresión de distintos tipos de cáncer, aunque su función sigue sin conocerse. Esta tesis se centra en dos objetivos: (i) el estudio de ALCAM como marcador de recurrencia en CEE y su función en la progresión tumoral; (ii) la caracterización de los mecanismos moleculares asociados a ALCAM en el frente superficial e invasivo del tumor y en el proceso de infiltración miometrial. Un estudio multicéntrico retrospectivo en 174 tumores primarios de CEE evidenció que ALCAM es un marcador independiente de recurrencia en los estadios tempranos de la enfermedad. Además, el silenciamiento de ALCAM in vitro e in vivo mostró una disminución en la migración celular, la invasión y una reducción significativa del tamaño tumoral primario así como de las metástasis loco-regionales. Análisis de expresión génica evidenciaron las funciones más alteradas asociadas a la inhibición de ALCAM: la motilidad, la invasión, y las funciones de ensamblaje y organización celular. FLNB, TXNRD1 y LAMC2, se validaron como genes efectores implicados en las funciones mediadas por ALCAM. Por otro lado, se estudió la expresión de ALCAM en el frente superficial e invasivo del tumor en 116 tumores primarios de CEE, así como su relación con marcadores epiteliales y mesenquimales relevantes en el CE. Se utilizó un anticuerpo que reconocía la región extracelular de ALCAM, permitiendo la detección de la proteína no-clivada en la superficie celular. Se observó que mientras en el área superficial, ALCAM-extracelular correlaciona con el complejo E-cadherina/β-catenina, en el invasivo, esta correlación se establece con los marcadores mesenquimales COX-2, MMP-9 y ETV5. La correlación negativa significativa entre ALCAM y MMP-9 se observa en tres cohortes específicas, tumores moderadamente-pobremente diferenciados, tumores con invasión miometrial >50% y tumores ETV5-positivos, todos ellos asociados con un peor pronóstico. Además, en el frente invasivo, la negatividad de ALCAM-extracelular resultó un marcador pronóstico independiente de invasión miometrial. Junto a estos hallazgos a nivel tisular, se confirma en aspirados uterinos el papel de ALCAM-soluble como marcador de infiltración miometrial y una correlación significativa positiva de las expresiones de MMP-9 y ALCAM-soluble para estos tumores no bien diferenciados, indicando la existencia de un clivaje de la proteína. Los modelos in vitro e in vivo demostraron, mediante la recuperación de la expresión de la proteína entera, su participación en la adhesión y la migración celular colectiva, sugiriendo que la invasión tumoral requiere la existencia de un “switch” dinámico y adaptativo entre la expresión de ALCAM en la superficie celular y su clivaje por MMP-9., Endometrial carcinoma (EC) is the most common infiltrative tumour of the female genital tract and the fourth most common women cancer in developed western countries. Around 80% of patients are diagnosed when the tumour is still confined to the uterus, which is usually associated with a good prognosis. In fact, the early stages of endometrioid endometrial cancer (EEC) present a 10-year survival of 80%. However, a small but consistent number of these patients relapse. Therefore, it is of vital importance to find markers to predict recurrence for those patients for whom a sufficiently robust risk factor is still not known. The other 20% of patients, diagnosed at a more advanced stage of the disease, present myometrial invasion that is one of the most relevant risk factors, being strongly correlated with an increase in the rate of recurrence. Cancer progression combines multiple processes, which involve cell adhesion molecules. Aberrant expression of these molecules has been related to loss of cell-cell adhesion, disaggregation of tumour cells from the lamina propria, and acquisition of an invasive and migratory phenotype. ALCAM is a member of the immunoglobulin superfamily, which participates in homotypic and heterotypic interactions between adjacent cells. In the last years, ALCAM expression has been related to the tumorigenesis of many cancers, but its function still remain unclear. Under this scenario, this thesis has focused on two specific objectives: (i) The study of ALCAM as a marker of recurrence in EEC and its function in tumour progression; (ii) The characterization of the molecular mechanisms associated with ALCAM in the superficial and the invasive front of the tumour and its relation to myometrial infiltration. A retrospective multicentre study in 174 EEC primary tumours evidenced that ALCAM is an independent marker of recurrence in early stage EEC. Moreover, ALCAM knockdown in vitro and in vivo experiments showed a decreased in cell migration, cell invasion and a reduced primary tumour size and metastatic local spread. Microarray gene expression analysis of Hec1A ALCAM-depleted cells demonstrated that motility, invasiveness, cellular assembly and organization were the most associated deregulated functions. FLNB, TXNRD1 and LAMC2, were validated as downstream effector genes involved in ALCAM-mediated cell migration. In addition, we focused on the role of ALCAM in two tumour areas (superficial and invasive front) in 116 EEC primary tumours and its correlation with a set of important epithelial and mesenchymal EC markers. For that, we used an antibody that specifically recognised its extracellular region, allowing us to detect the presence of full-ALCAM (non-cleaved protein) at the cell surface. While at the superficial tumour extracellular-ALCAM was correlated with the epithelial E-Cadherin/β-catenin complex, at the invasive tumour ALCAM correlated with the mesenchymal markers COX-2, MMP-9 and ETV5 mesenchymal markers. Interestingly, the inverse correlation between ALCAM and MMP-9 was only significant in the moderately-poorly differentiated tumours, in patients presenting a myometrial invasion >50% and in the ETV5-positive tumours cohorts, all the scenarios related to poor prognosis. Moreover, at the invasive front extracellular ALCAM-negativity was an independent prognostic marker of myometrial invasion. Together with these findings at tissue level, we confirmed that in the moderately-poorly differentiated patients, soluble ALCAM detected in uterine aspirates was significantly increased in tumours presenting myometrial invasion and positively correlated to MMP-9 expression, suggesting that ALCAM decrease at the invasive front of primary tumours is caused by protein shedding. In vitro and in vivo models evidenced that ALCAM participates in cell-cell adhesion and collective cell migration, and suggest that to invade in the frontier between tissues-restricted carcinoma and disseminated tumour cells, a dynamic and adaptive switch between ALCAM expression at the cell surface and ALCAM cleavage by MMP-9 might take place.

Endometrial cancer risk prediction models including lifestyle, anthropometric and reproductive factors have limited discrimination. Adding biomarker data to these models may improve predictive capacity; to our knowledge, this has not been investigated for endometrial cancer. Using a nested case-control study within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort, we investigated the improvement in discrimination gained by adding serum biomarker concentrations to risk estimates derived from an existing risk prediction model based on epidemiologic factors. Serum concentrations of sex steroid hormones, metabolic markers, growth factors, adipokines and cytokines were evaluated in a step-wise backward selection process; biomarkers were retained at p < 0.157 indicating improvement in the Akaike information criterion (AIC). Improvement in discrimination was assessed using the C-statistic for all biomarkers alone, and change in C-statistic from addition of biomarkers to preexisting absolute risk estimates. We used internal validation with bootstrapping (1000-fold) to adjust for over-fitting. Adiponectin, estrone, interleukin-1 receptor antagonist, tumor necrosis factor-alpha and triglycerides were select-ed into the model. After accounting for over-fitting, discrimination was improved by 2.0 percentage points when all evaluated biomarkers were included and 1.7 percentage points in the model including the selected biomarkers. Models including etiologic markers on independent pathways and genetic markers may further improve discrimination.

El càncer d’endometri (CE) és la malaltia del tracte genital femení més freqüent als EE.UU i ocupa la quarta posició del rànquing de càncers més comuns en dones. Malauradament, les morts per CE estan augmentant ràpidament i les taxes van incrementar un 2% anual del 2010 al 2014. Tot i que els tumors confinats dins l’úter i diagnosticats precoçment representen un 67% dels casos i presenten un supervivència als 5 anys d’un 95%, resta un 30% dels casos diagnosticats en estadis avançats de la malaltia que presenten una davallada dràstica en la taxa de supervivència a 5 anys (69% i 17% pels casos amb disseminació regional i distant, respectivament). El CE es pot dividir en tipus 1 (el més comú i dependent d’estrògens) i tipus 2 (menys comú però més agressiu i independent d’estrògens). Hi ha una necessitat clara de desenvolupar un test no invasiu capaç de detectar els casos de CE de manera precoç i que pugui discriminar entre els subtipus. El procés diagnòstic actual es tediós i inclou un examen pèlvic i una ultrasonografia transvaginal, seguida de l’examen histopatològic d’una biòpsia endometrial, obtinguda per aspiració o per histerescopia. Per la millora del diagnòstic del CE, l’ús dels aspirats uterins (AUs) reuneix un seguit de característiques que fan d’aquest biofluid una prometedora font de biomarcadors pel cribat, diagnòstic i monitorització de patologies relacionades amb els tracte genital: (i) el mostreig és mínimament invasiu i es poden obtenir entre 50-1000 µl de mostra d’una pacient, material suficient per estudis moleculars; (ii) els AUs estan en contacte directe amb la cavitat uterina i representen el seu ambient complex. La composició dels AUs deriva de secrecions i cèl·lules descamades dels diferents teixits circumdants, de l’epiteli luminal i de les glàndules i de proteïnes transsudades selectivament de la sang; i (iii) els AUs exhibeixen les alteracions moleculars del CE i són útils per estudiar la heterogeneïtat tumoral. Els AUs contenen proteïnes abundants provinents del plasma, que emmascaren la detecció i l’anàlisi de les proteïnes menys abundants, que són generalment els candidats més prometedors. Per solucionar el problema de rang dinàmic i per ampliar la recerca en el camp de nous biomarcadors en CE, els exosomes (exosome-like vesicles, ELVs), que són vesícules de mida nanomètrica alliberades pels cosos multivesiculars i que es fusionen amb la membrana plasmàtica per ser secretats, sorgeixen com un repositori de biomarcadors. Aquest estudi se centra en la identificació de marcadors proteics en ELVs aïllats de AUs. Els extractes proteics es van obtenir de ELVs obtinguts per ultracentrifugació diferencial dels AUs de 3 grups de pacients: control, CE tipus 1 i CE tipus 2 (10 pacients/grup). La qualitat dels ELVs es va comprovar per Nanoparticle tracking Analysis i immunoblots. Per comparar l’abundància de proteïnes entre els 3 grups, vàrem utilitzar la tècnica del Super-SILAC, on els ELVs aïllats de 3 línies de CE cultivades en medi amb marcatge pesat (Lys i Arg) es van combinar amb les proteïnes sense marcatge dels ELVs de cada pacient per tenir un estàndard intern. Les mostres es van separar electroforèticament i les bandes tallades es van digerir en-gel amb tripsina per ser analitzades per LC-MS/MS. Várem generar una llista de 54 candidats que es van analitzar per LC-SRM en una cohort independent de 107 pacients dividits en 3 grups: control(n=41), CE tipus 1 (n=45) i CE tipus 2 (n=21). L’estudi de proteòmica dirigida va confirmar que els ELVs dels AUs contenen proteïnes que poden discriminar entre pacients amb càncer i controls. Cal destacar que es va trobar un panell de 2 proteïnes que incrementa la capacitat de discriminació, i que és independent del tipus de càncer (ROC AUC=0.94). A més a més, també es va trobar un panell amb poder per discriminar entre tipus 1 i tipus 2 (ROC AUC=0.93). Aquest estudi presenta implicacions importants en la detecció primerenca de CE i en l'estratificació dels pacient., Endometrial cancer (EC) is the most common cancer of the female genital tract and the fourth most frequent cancer in women in the United States. Discouraging, EC deaths are increasing rapidly and rates grew about a 2% per year from 2010 to 2014. Even though early-stage diagnosed EC restricted to the uterus represent 67% of the cases with a 5-year survival rate of 95%, it still remains around a 30% of patients diagnosed at advanced stages of the disease, when the 5-year survival rate decreases dramatically to 69% and 17% in case of regional and distant spreading, respectively. EC is divided into the more common estrogen dependent type 1 and the less common but more aggressive estrogen independent type 2. There is an urgent need to develop non-invasive tests that can provide early detection of EC and that can discriminate EC subtypes. The current diagnostic process is laborious and includes a pelvic examination and a transvaginal ultrasonography followed by a confirmatory histopathological evaluation of an endometrial biopsy, which can either be obtained by aspiration or by hysteroscopy. To improve EC diagnosis, the use of uterine aspirates (UAs) meet some features that makes this biofluid a promising source of biomarkers for screening, diagnosis and monitoring of pathologies related to the female genital tract: (i) sampling of UAs is minimally invasive and it is feasible to retrieve between 50-1000 µl of sample from a patient, which is sufficient to extract material for molecular studies; (ii) UAs are in direct contact with the uterine cavity, representing faithfully its complex environment. UA’s composition derives from secretions and cells flaking from a variety of surrounding tissues, from the luminal epithelium and glands, from proteins selectively transudated from blood, and likely contributors from tubal fluid; and (iii) UAs exhibit molecular alterations present in EC and are useful to study the EC tumor heterogeneity. UAs highly express abundant proteins coming from plasma; and those mask the detection and analysis of less abundant proteins, which are generally the most promising candidates for biomarkers discovery. To solve this dynamic-range problem, and to expand research in the field of biomarkers discovery for EC, exosome-like vesicles (ELVs), nanosized-membranous vesicles released by multivesicular bodies fusing with the cell membrane, arise as a promising source of EC biomarkers. This study focuses on the identification of protein markers in ELVs isolated from uterine aspirates. Protein extracts from purified ELVs were obtained following ultracentrifugation of UAs from age-matched groups of control, type1 and type2 EC patients (10 patients/group). The quality of isolated ELVs was monitored by Nanoparticle Tracking Analysis, and immunoblots. To profile protein abundance across different groups, we develop a super-SILAC approach where ELV proteins from 3 different EC cell lines grown in heavy Lys and Arg amino acids were combined with ELV protein extracts of each patient. Proteins were separated by SDS-PAGE and gel-isolated bands were digested with trypsin and analyzed by Mass Spectrometry. We generated a list of 54 protein candidates that was further validated by LC-SRM in an independent cohort of 107 patients including 3 age-matched groups: type 1 EC (n=45) EC, type 2 EC (n=21) and healthy individuals (n=41). Our targeted mass spectrometry approach confirmed that ELVs from UAs contain proteins that can discriminate between cancer and healthy patients. More importantly, a 2-protein signature improves this capacity to discriminate healthy from EC patients (ROC AUC=0.94). This protein signature can detect endometrial cancer independently of the cancer type. In addition, we also report a new protein signature that can differentiate type1 versus type2 EC (ROC AUC=0.93). This study has important implications in early detection of EC and in patient stratification.

Endometrial carcinosarcoma (ECS) represents one of the most extreme examples of tumor heterogeneity among human cancers. ECS is a clinically aggressive, high-grade, metaplastic carcinoma. At the morphological level, intratumor heterogeneity in ECS is due to an admixture of epithelial (carcinoma) and mesenchymal (sarcoma) components that can include heterologous tissues, such as skeletal muscle, cartilage, or bone. Most ECSs belong to the copy-number high serous-like molecular subtype of endometrial carcinoma, characterized by the TP53 mutation and the frequently accompanied by a large number of gene copy-number alterations, including the amplification of important oncogenes, such as CCNE1 and c-MYC. However, a proportion of cases (20%) probably represent the progression of tumors initially belonging to the copy-number low endometrioid-like molecular subtype (characterized by mutations in genes such as PTEN, PI3KCA, or ARID1A), after the acquisition of the TP53 mutations. Only a few ECS belong to the microsatellite-unstable hypermutated molecular type and the POLE-mutated, ultramutated molecular type. A common characteristic of all ECSs is the modulation of genes involved in the epithelial to mesenchymal process. Thus, the acquisition of a mesenchymal phenotype is associated with a switch from E- to N-cadherin, the up-regulation of transcriptional repressors of E-cadherin, such as Snail Family Transcriptional Repressor 1 and 2 (SNAI1 and SNAI2), Zinc Finger E-Box Binding Homeobox 1 and 2 (ZEB1 and ZEB2), and the down-regulation, among others, of members of the miR-200 family involved in the maintenance of an epithelial phenotype. Subsequent differentiation to different types of mesenchymal tissues increases tumor heterogeneity and probably modulates clinical behavior and therapy response., This review was funded by grants from the Instituto de Salud Carlos III (ISCIII) (PIE15/00050 and PI16/00887) and CIBERONC (CB16/12/00316 and CB16/12/00231, CB16/12/00361), co-financed by the European Development Regional Fund. ‘A way to achieve Europe’ (FEDER), and by the Spanish Association Against Cancer Scientific Foundation (grants: AIO-aecc 2016 and Grupos Coordinados Traslacionales aecc 2018).

Undifferentiated and dedifferentiated endometrial carcinomas are rare and highly aggressive subtypes of uterine cancer, not well characterized at a molecular level. To investigate whether dedifferentiated carcinomas carry molecular genetic alterations similar to those of pure undifferentiated carcinomas, and to gain insight into the pathogenesis of these tumours, we selected a cohort of 18 undifferentiated endometrial carcinomas, 8 of them with a well differentiated endometrioid carcinoma component (dedifferentiated endometrioid carcinomas), and studied them by immunohistochemistry and massive parallel and Sanger sequencing. Whole exome sequencing of the endometrioid and undifferentiated components as well as normal myometrium, was also carried out in one case. According to The Cancer Genome Atlas classification, we distributed 95% of the undifferentiated carcinomas in this series as follows: a) hypermutated tumours with loss of any mismatch repair protein expression and microsatellite instability (eight cases, 45%); b) ultramutated carcinomas carrying mutations in the exonuclease domain of POLE (two cases, 11%); c) high copy number alterations (copy-number high) tumours group exhibiting only TP53 mutations and high number of alterations detected by FISH (two cases, 11%) ; and d) low copy number alterations (copy-number low) tumours with molecular alterations typical of endometrioid endometrial carcinomas (five cases, 28%). Two of the latter cases, however, also had TP53 mutations and higher number of alterations detected by FISH and could have progressed to a copy-number high phenotype. Most dedifferentiated carcinomas belonged to the hypermutated group whereas pure undifferentiated carcinomas shared molecular genetic alterations with copy-number low or copy-number high tumours. These results indicate that undifferentiated and dedifferentiated endometrial carcinomas are molecularly heterogeneous tumours, which may have prognostic value.

Acrylamide is an organic chemical that has several important uses in industry, such as a flocculant in public water supplies, and in biomedical research. In 1994, acrylamide was classified by the International Agency for Research on Cancer as ‘probably carcinogenic’ to humans. In 2002, acrylamide was discovered in some starchy foods that are cooked at high temperature (e.g., potato crisps and chips). Acrylamide also is a common component of cigarette smoke. From 2006-2010, epidemiologic studies began to examine and report on the possible association between dietary acrylamide intake and risk of human cancer, including ovarian and endometrial cancers (two of which suggested possible associations). Endometrial and ovarian are the fourth and fifth most common cancers diagnosed in European women. The general aim of this thesis was to assess the risk of endometrial cancer (EC) and epithelial ovarian (EOC) cancer in relation to dietary acrylamide exposure based on questionnaire information and biomarkers of internal exposure. The study population for this project was 368,010 women from the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. Dietary acrylamide intake in the full EPIC cohort was estimated using the most recent European acrylamide-content food databases, country-specific food intake questionnaires, and 24-hour dietary recalls at baseline. Multivariate Cox Proportional hazard models were used to estimate hazard ratios (HR) and 95% confidence intervals (95% CI) for questionnaire-based acrylamide intake and EC or EOC risk. Cohort analyses were conducted in all women and in subgroups. Two nested case-control studies of acrylamide biomarkers and EC or EOC risk were conducted in non-smoking postmenopausal women. Pre-diagnostic biomarkers of acrylamide and its epoxide metabolite, glycidamide, were measured as hemoglobin adducts (HbAA and HbGA, respectively) using HPLC/MS/MS in red blood cells collected at baseline 5-10 years before diagnosis. Unconditional logistic regression models were used to estimate odds ratios (OR) and 95% CI for the relation between acrylamide biomarkers (HbAA, HbGA, HbAA+HbGA, and HbGA/HbAA) and EC or EOC risk. The relation between dietary and demographic determinants of biomarker levels of acrylamide and glycidamide were also examined using simple and multiple linear regression analyses in 801 control women. After a mean follow-up of 11 years, there were 1,382 cases of EC and 1,191 cases of EOC in the EPIC cohort. In general, the results of the dietary acrylamide intake analyses showed no associations with EOC and EC, except for a possible association between higher acrylamide intake and type-I EC in women who were non-smokers and non-users of oral contraceptives (HRQ5vsQ1: 1.97, 95% CI: 1.08-3.62). The main food group determinants of acrylamide and glycidamide biomarker levels were biscuits, crackers and cakes; and alcohol intake and body mass index were important determinants of the ratio of glycidamide to acrylamide. The nested case-control study of acrylamide and glycidamide biomarker levels and EC (383 cases, 171 type-I, and 385 controls) did not find any support for associations between acrylamide or glycidamide and EC risk. The nested case-control study of acrylamide and glycidamide biomarkers and EOC risk (334 cases and 417 controls) found some evidence for associations with the third quintile of HbGA, and the second and third quintiles of HbAA+HbGA. Participants classified in the fifth quintile of HbGA or HbAA+HbGA showed statistically non-significant higher risks of EOC (ORHbGA.Q5vsQ1: 1.63, 95% CI: 0.92-2.86; P-trend: 0.04, and ORHbAA+HbGGA.Q5vsQ1: 1.60, 95% CI: 0.89-2.87; P-trend: 0.14). In conclusion, results in women from the EPIC cohort did not provide convincing support for the hypothesis that higher acrylamide and glycidamide exposures are associated with higher risk for EC. For EOC, inconsistent results were observed in our study. Additional nested case- control studies with larger sample size in different populations, and pooled analysis of existing studies of the relation between acrylamide and glycidamide biomarkers and EOC risk should be performed.

Los carcinomas de ovario y de endometrio son los tumores malignos ginecológicos más frecuentes en nuestro entorno. Se han descrito alteraciones genéticas recurrentes que son características de algunos tipos histológicos y que han permitido definir vías patogenéticas distintas tanto para ovario como para endometrio. Entre estas alteraciones, algunas afectan directamente las vías de señalización celular que regulan el crecimiento y proliferación de la célula. Estas vías representan una de las alteraciones clave implicadas en el desarrollo del cáncer y que han adquirido una trascendencia importante, desde la aplicación en la práctica clínica, de tratamientos dirigidos contra algunas de estas moléculas. Sin embargo, la comprensión de estas vías se complica por la presencia de múltiples interacciones entre ellas, así como redundancias que hacen que el bloqueo de las mismas no siempre produzca los efectos esperados. Dada la complejidad y redundancia de vías que transmiten la señal proliferativa desde la membrana hacia al núcleo, deben existir factores que centralicen la acción oncogénica, de forma que sobre ellas converjan diversas vías y sean representativos del grado de activación de los mismos y por tanto de interés pronóstico y terapéutico. Se seleccionaron 129 neoplasias de ovario que incluyeron 27 tumores benignos, 27 tumores borderline y 75 carcinomas de ovario, y 138 muestras de endometrio que incluyeron 120 carcinomas, 15 endometrios funcionales y atróficos y 3 hiperplasias endometriales. Se construyeron matrices de tejidos (tissue microarrays) a partir de 3 muestras de cada caso y se realizó estudio inmunohistoquímico con los anticuerpos para HER2, EGFR y las formas fosforiladas de AKT, ERK, 4E-BP1, p70S6K y S6. Los resultados se analizaron estadísticamente para valorar de forma descriptiva la expresión de estos marcadores, su relación con las variables clínico-patológicas, la relación entre ellos y con la supervivencia global y libre de enfermedad. De los parámetros estudiados, el estadio según los criterios de la FIGO y el grado de diferenciación fueron las únicas variables clínico-patológicas de valor pronóstico tanto en cáncer de ovario como de endometrio. De los marcadores, HER2 se asoció a peor pronóstico en cáncer de endometrio pero no de ovario, EGFR a mejor pronóstico en cáncer de endometrio. La sobreexpresión de p-4E-BP1 identifica carcinomas de ovario y de endometrio de peor pronóstico y su expresión se relaciona tanto con la activación de AKT como de ERK por lo que apoya su importancia como proteína centralizadora de la señal oncogénica procedente de cualquiera de las 2 vías, representando una buena diana para el tratamiento de estas pacientes., Ovarian and endometrial carcinomas are the most frequent gynecological malignant tumors in our area. Recurrent genetic alterations that are characteristic of some histological types have been described and have allowed the identification of pathogenetic pathways, in both endometrial and ovarian cancers. Among these alterations, some affect cell signaling pathways that regulate cell growth and cell proliferation. These pathways represent one of the hallmarks of cancer, and have become especially important since the appearance of new drugs that target specific molecules involved in their regulation. However, our understanding of these signaling pathways is complicated by the presence of multiple cross-talks between them, as well as multiple redundancies of molecules, both of with are probably responsible for some of the unexplained results that occur when one of the molecules involved is blocked by a drug. Due to the complexity and redundancy of cell signaling pathways that transmit the proliferative signal from the cellular membrane to the nucleus, there should be some factors that centralize the oncogenic action in a way that several stimuli could converge on them. These factors should be representative of the activation level of the oncogenic process, and therefore, should be of prognostic and therapeutic interest. 129 ovarian neoplasms were selected, including 27 benign tumors, 27 borderline tumors, and 75 ovarian carcinomas. Additionally, 138 endometrial samples were selected including 15 atrophic and functional endometria, 3 hyperplastic endometria and 120 endometrial carcinomas. Tissue microarrays were constructed using 3 samples from each case, and an immunohistochemical study with HER2, EGFR, and the phosphorylated forms of AKT, ERK, 4E-BP1, p70S6K and ribosomal protein S6 was performed. The results obtained were statistically analyzed to evaluate the correlation of these markers with clinicopathological features, the relationship between them, and their impact on overall survival and progression-free survival. Surgical stage defined by FIGO, and differentiation grade were the only clinicopathologic variants with prognostic significance in ovarian and endometrial cancer. Among the cell signaling markers evaluated, HER2 was associated with prognosis in endometrial cancer but not in ovarian cancer and EGFR with better survival in endometrial cancer. P-4E-BP1 overexpression identifies a subset of ovarian and endometrial cancers with worse prognosis, and its expression is related to the activation of AKT and ERK pathways, suggesting that it has a central role in cell signal transduction by both pathways, and therefore could be a therapeutic target in these patients.

El càncer constitueix la primera causa de mort als països industrialitzats. El risc de desenvolupar un càncer abans dels 75 anys per a un espanyol és del 25% és a dir un de cada quatre espanyols desenvoluparà un càncer al llarg de la seua vida. En l’actualitat existeix gran evidència científica de que l’exercici físic per si mateix o en combinació amb la dieta i el normo pes, és capaç de disminuir el risc de patir càncer ginecològic. Així mateix, mitjançant l’activitat física podem revertir els efectes perniciosos tant del tractament del càncer com de les seues seqüeles, i es pot arribar a recuperar l’estat de forma previ a la malaltia i millorar així la qualitat de vida de la pacient., Cancer is the first death cause in industrialised countries. The risk to develop cancer before 75 years old for a Spanish citizen is 25%, which means that one out of ten Spaniards will develop cancer throughout their lives. Nowadays, it has been scientifically proven that physical exercise by itself in combination with diet and normal weight is able to diminish the risk of suffering gynaecological cancer. Moreover, the practice of physical exercise can revert the negative effects of both the treatment against cancer and the aftermath of the disease. Consequently, it is even possible for the patient to recover good shape and quality of life., El cáncer constituye la primera causa de muerte en los países industrializados. El riesgo de desarrollar un cáncer antes de los 75 años para un español es del 25%, es decir uno de cada cuatro españoles desarrollará un cáncer a lo largo de su vida. En la actualidad existe una gran evidencia científica de que el ejercicio físico por si solo o en combinación con la dieta y el normo peso, es capaz de disminuir el riesgo de sufrir cáncer ginecológico. Así mismo, mediante la actividad física podemos revertir los efectos perniciosos tanto del tratamiento del cáncer como de las secuelas del mismo, pudiendo llegar a recuperar el estado de forma previo a la enfermedad y mejorar así la calidad de vida de la paciente.