Ascites-Derived Organoids to Depict Platinum Resistance in Gynaecological Serous Carcinomas.

Gynaecological serous carcinomas (GSCs) constitute a distinctive entity among female tumours characterised by a very poor prognosis. In addition to late-stage diagnosis and a high rate of recurrent disease associated with massive peritoneal carcinomatosis, the systematic acquisition of resistance to first-line chemotherapy based on platinum determines the unfavourable outcome of GSC patients. To explore the molecular mechanisms associated with platinum resistance, we generated patient-derived organoids (PDOs) from liquid biopsies of GSC patients. PDOs are emerging as a relevant preclinical model system to assist in clinical decision making, mainly from tumoural tissue and particularly for personalised therapeutic options. To approach platinum resistance in a GSC context, proficient PDOs were generated from the ascitic fluid of ovarian, primary peritoneal and uterine serous carcinoma patients in platinum-sensitive and platinum-resistant clinical settings from the uterine aspirate of a uterine serous carcinoma patient, and we also induced platinum resistance in vitro in a representative platinum-sensitive PDO. Histological and immunofluorescent characterisation of these ascites-derived organoids showed resemblance to the corresponding original tumours, and assessment of platinum sensitivity in these preclinical models replicated the clinical setting of the corresponding GSC patients. Differential gene expression profiling of a panel of 770 genes representing major canonical cancer pathways, comparing platinum-sensitive and platinum-resistant PDOs, revealed cellular response to DNA damage stimulus as the principal biological process associated with the acquisition of resistance to the first-line therapy for GSC. Additionally, candidate genes involved in regulation of cell adhesion, cell cycles, and transcription emerged from this proof-of-concept study. In conclusion, we describe the generation of PDOs from liquid biopsies in the context of gynaecological serous carcinomas to explore the molecular determinants of platinum resistance.

Noninvasive detection of microsatellite instability in patients with endometrial cancer.

The analysis of mismatch repair proteins in solid tissue is the standard of care (SoC) for the microsatellite instability (MSI) characterization in endometrial cancer (EC). Uterine aspirates (UAs) or circulating-DNA (cfDNA) samples capture the intratumor heterogeneity and provide a more comprehensive and dynamic molecular diagnosis. Thus, MSI analysis by droplet-digital PCR (ddPCR) in UAs and cfDNA can provide a reliable tool to characterize and follow-up the disease. The UAs, paraffin-embedded tumor tissue (FFPE) and longitudinal plasma samples from a cohort of 90 EC patients were analyzed using ddPCR panel and compared to the SoC. A high concordance (96.67%) was obtained between the analysis of MSI markers in UAs and the SoC. Three discordant cases were validated as unstable by ddPCR on FFPE samples. Besides, a good overall concordance (70.27%) was obtained when comparing the performance of the ddPCR assay on UAs and cfDNA in high-risk tumors. Importantly, our results also evidenced the value of MSI analysis to monitor the disease evolution. MSI evaluation in minimally invasive samples shows great accuracy and sensitivity and provides a valuable tool for the molecular characterization and follow-up of endometrial tumors, opening new opportunities for personalized management of EC.

Improving the Management of Endometrial Cancer Patients through the Use of Liquid Biopsy Analyses: A Case Report.

Endometrial cancer (EC) is the 4th most common neoplasm of the female genital tract, with 15-20% of patients being of high risk of recurrence which leads to a significant decrease in patient survival. Current therapeutic options for patients with EC are poor, being the combined therapy of carboplatin and paclitaxel the standard of care, with limited efficacy. Therefore, new therapeutic options and better monitoring tools are needed to improve the management of the disease. In the current case report, we showcase the value of liquid biopsy analyses in a microsatellite instability EC patient with initially good prognosis that however underwent rapid progression disease within 6 months post-surgery; through the study of plasma cfDNA/ctDNA dynamics to assess the tumour evolution during treatment, as well as the study of the uterine aspirate as a valuable sample that captures the intra-tumour heterogeneity that allows a comprehensive genomic profiling of the disease to identify potential therapeutic options. Furthermore, preclinical models were generated at the time of tumour progression to assess the efficacy of the identified targeted therapies.

Intratumor genetic heterogeneity and clonal evolution to decode endometrial cancer progression.

Analyzing different tumor regions by next generation sequencing allows the assessment of intratumor genetic heterogeneity (ITGH), a phenomenon that has been studied widely in some tumor types but has been less well explored in endometrial carcinoma (EC). In this study, we sought to characterize the spatial and temporal heterogeneity of 9 different ECs using whole-exome sequencing, and by performing targeted sequencing validation of the 42 primary tumor regions and 30 metastatic samples analyzed. In addition, copy number alterations of serous carcinomas were assessed by comparative genomic hybridization arrays. From the somatic mutations, identified by whole-exome sequencing, 532 were validated by targeted sequencing. Based on these data, the phylogenetic tree reconstructed for each case allowed us to establish the tumors' evolution and correlate this to tumor progression, prognosis, and the presence of recurrent disease. Moreover, we studied the genetic landscape of an ambiguous EC and the molecular profile obtained was used to guide the selection of a potential personalized therapy for this patient, which was subsequently validated by preclinical testing in patient-derived xenograft models. Overall, our study reveals the impact of analyzing different tumor regions to decipher the ITGH in ECs, which could help make the best treatment decision.

Haemodynamic-dependent arrest of circulating tumour cells at large blood vessel bifurcations as new model for metastasis.

Homing of circulating tumour cells (CTC) at distant sites represents a critical event in metastasis dissemination. In addition to physical entrapment, probably responsible of the majority of the homing events, the vascular system provides with geometrical factors that govern the flow biomechanics and impact on the fate of the CTC. Here we mathematically explored the distribution of velocities and the corresponding streamlines at the bifurcations of large blood vessel and characterized an area of low-velocity at the carina of bifurcation that favours the residence of CTC. In addition to this fluid physics effect, the adhesive capabilities of the CTC provide with a biological competitive advantage resulting in a marginal but systematic arrest as evidenced by dynamic in vitro recirculation in Y-microchannels and by perfusion in in vivo mice models. Our results also demonstrate that viscosity, as a main determinant of the Reynolds number that define flow biomechanics, may be modulated to limit or impair CTC accumulation at the bifurcation of blood vessels, in agreement with the apparent positive effect observed in the clinical setting by anticoagulants in advanced oncology disease.

Genomic Profiling of Uterine Aspirates and cfDNA as an Integrative Liquid Biopsy Strategy in Endometrial Cancer.

The incidence and mortality of endometrial cancer (EC) have risen in recent years, hence more precise management is needed. Therefore, we combined different types of liquid biopsies to better characterize the genetic landscape of EC in a non-invasive and dynamic manner. Uterine aspirates (UAs) from 60 patients with EC were obtained during surgery and analyzed by next-generation sequencing (NGS). Blood samples, collected at surgery, were used for cell-free DNA (cfDNA) and circulating tumor cell (CTC) analyses. Finally, personalized therapies were tested in patient-derived xenografts (PDXs) generated from the UAs. NGS analyses revealed the presence of genetic alterations in 93% of the tumors. Circulating tumor DNA (ctDNA) was present in 41.2% of cases, mainly in patients with high-risk tumors, thus indicating a clear association with a more aggressive disease. Accordingly, the results obtained during the post-surgery follow-up indicated the presence of ctDNA in three patients with progressive disease. Moreover, 38.9% of patients were positive for CTCs at surgery. Finally, the efficacy of targeted therapies based on the UA-specific mutational landscape was demonstrated in PDX models. Our study indicates the potential clinical applicability of a personalized strategy based on a combination of different liquid biopsies to characterize and monitor tumor evolution, and to identify targeted therapies.

Endometrial Tumour Microenvironment.

Endometrial cancer (EC) is the most common gynaecological tumour in developed countries, and its incidence is increasing in part due to the prevalence of obesity and its related hormone dysregulation. As described in this chapter, the tumour microenvironment plays a principal role in unopposed oestrogen stimulation promoting tumour cell proliferation. Factors and cytokines secreted by the different cell types defining the reactive tumour stroma also determine the invasive abilities of the tumour cells. Cancer-associated fibroblasts and tumour-associated macrophages actively participate through SDF-1, TGF-b or HGF to promote epithelial-to-mesenchymal transition or to generate an appropriate tumour niche. Likewise, endothelial cells facilitate lymph node and vascular infiltration through VEGF. Finally, the possibility to balance the immunosuppressive phenotypes in advanced endometrial cancer through the tumour microenvironment will probably represent a main therapeutic strategy in the near future.

Extracellular Vesicles-Based Biomarkers Represent a Promising Liquid Biopsy in Endometrial Cancer.

Tumor-derived extracellular vesicles (EVs) are secreted in large amounts into biological fluids of cancer patients. The analysis of EVs cargoes has been associated with patient´s outcome and response to therapy. However, current technologies for EVs isolation are tedious and low cost-efficient for routine clinical implementation. To explore the clinical value of circulating EVs analysis we attempted a proof-of-concept in endometrial cancer (EC) with ExoGAG, an easy to use and highly efficient new technology to enrich EVs. Technical performance was first evaluated using EVs secreted by Hec1A cells. Then, the clinical value of this strategy was questioned by analyzing the levels of two well-known tissue biomarkers in EC, L1 cell adhesion molecule (L1CAM) and Annexin A2 (ANXA2), in EVs purified from plasma in a cohort of 41 EC patients and 20 healthy controls. The results demonstrated the specific content of ANXA2 in the purified EVs fraction, with an accurate sensitivity and specificity for EC diagnosis. Importantly, high ANXA2 levels in circulating EVs were associated with high risk of recurrence and non-endometrioid histology suggesting a potential value as a prognostic biomarker in EC. These results also confirmed ExoGAG technology as a robust technique for the clinical implementation of circulating EVs analyses.

Liquid Biopsy in Endometrial Cancer: New Opportunities for Personalized Oncology.

The identification of new molecular targets and biomarkers associated with high risk of recurrence and response to therapy represents one of the main clinical challenges in the management of advanced disease in endometrial cancer. In this sense, the field of liquid biopsy has emerged as a great revolution in oncology and is considered "the way" to reach personalised medicine. In this review, we discuss the promising but already relatively limited advances of liquid biopsy in endometrial cancer compared to other types of tumours like breast, colorectal or prostate cancer. We present recent data analysing circulating tumour material in minimally-invasive blood samples, but also in alternative forms of liquid biopsy like uterine aspirates. Proteomic and genomic studies focused on liquid-based uterine samples are resulting not only in optimal diagnostic tools but also in reliable approaches to address tumour heterogeneity. Likewise, circulating tumour cells (CTCs) and circulating tumour DNA (ctDNA) represent an opportunity for the correct stratification of patients, for the assessment of early recurrent disease or for the real-time monitoring of therapy responses. Appropriately designed studies and implementation in clinical trials will determine the value of liquid biopsy for precision oncology in endometrial cancer.

Retraction and triangulation with neodymium magnetic forceps for single-port laparoscopic cholecystectomy.

INTRODUCTION: There have been attempts to minimize the invasiveness of laparoscopic cholecystectomy by reducing the size and/or the number of the operating ports and instruments. These attempts create technical challenges related principally to retraction and triangulation necessary to expose the surgical field for a safe surgery. A new technique based on retraction and triangulation with magnetic instruments for single port laparoscopic surgery is presented. METHODS: Between March 2007 and December 2008, 40 laparoscopic cholecystectomies were performed with single-port laparoscopic surgery with the assistance of magnetic forceps (IMANLAP project). The surgical technique is described, and the intraoperative and postoperative course of the patients is assessed. RESULTS: There were no intraoperative complications, no need to convert to open surgery, and no need to add a second port. Depending on the patient's anatomy, a 1-mm needle was added in some cases. There were no interactions observed between the magnetic devices and the anesthetic monitoring and the rest of the devices of the operation room. CONCLUSIONS: This new procedure is feasible and safe. The main goal is control of the magnetic field, allowing enough controlled strength for retraction and sufficient triangulation for adequate exposure of the surgical field. This allows for the use of a single port through which an optic device with a working channel can perform the operation with safety. Finally, the procedure can be performed in a manner similar to the traditional laparoscopic cholecystectomy, and it also appears to be simple to learn.

Tratamiento laparoscópico de la enfermedad de reflujo gastro-esofágico (ERGE) / Laparoscopic treatment of gastro-esophagic reflux disease

Antecedentes: El aumento de la frecuencia de presentación de la ERGE, y el avance de los conocimientos de su fisiopatología otorgan a la cirugía antirreflujo un rol importante en su tratamiento. Desde 1991 se incorpora la vía laparoscópica, con la que se lograrían resultados similares con menor tiempo de internación. Material y métodos: Entre junio de 1995 y febrero de 1997 se operaron 37 pacientes (21 mujeres) de ERGE por vía laparoscópica, la edad promedio fue de 59,1 años. El tiempo promedio de evolución de la enfermedad fue de 10,8 años, período en el cual recibieron tratamiento médico, 30 enfermos presentaban hernia hiatal y 7 tenían esclerodermia. Todos fueron estudiados con SEGD, VEDA y manometría esofágica que mostró un EEI hipotensivo y en 7 ausencia de peristaltismo distal, característico de la esclerodermia. A 21 (57 por ciento) se les realizó pHmetría de 24 hs. Se realizaron 8 operaciones de Nissen-Rossetti, 22 de Nissen y 7 de Toupet. Se procedió al cierre sistemático del hiato esofágico en todos aquellos en que se encontraba dilatado. Resultados: tiempo operatorio y de internación: 160 min y 55 hs de promedio respectivamente. En 4 casos (10,8 por ciento) se convirtió a abdomen abierto. A 9,3 meses (2-23 m) de seguimiento, 29 pacientes (88 por ciento) presentan muy buena evolución, 4 pac. (12 por ciento) refirieron pirosis ocasional (todos con esclerodermia). Se realizó manometría postoperatoria en 22 pac. (59 por ciento), la que mostró recuperación de la presión del EEI. Conclusiones: Se puede reproducir la cirugía antirreflujo por vía laparoscópica con resultados iniciales similares a los obtenidos por laparotomía, con menor tiempo de internación. Se requiere experiencia en cirugía laparoscópica de avanzada y conocimientos actualizados de la fisiopatología de la ERGE (AU)

Tratamiento laparoscópico de la enfermedad de reflujo gastro-esofágico (ERGE) / Laparoscopic treatment of gastro-esophagic reflux disease

Antecedentes: El aumento de la frecuencia de presentación de la ERGE, y el avance de los conocimientos de su fisiopatología otorgan a la cirugía antirreflujo un rol importante en su tratamiento. Desde 1991 se incorpora la vía laparoscópica, con la que se lograrían resultados similares con menor tiempo de internación. Material y métodos: Entre junio de 1995 y febrero de 1997 se operaron 37 pacientes (21 mujeres) de ERGE por vía laparoscópica, la edad promedio fue de 59,1 años. El tiempo promedio de evolución de la enfermedad fue de 10,8 años, período en el cual recibieron tratamiento médico, 30 enfermos presentaban hernia hiatal y 7 tenían esclerodermia. Todos fueron estudiados con SEGD, VEDA y manometría esofágica que mostró un EEI hipotensivo y en 7 ausencia de peristaltismo distal, característico de la esclerodermia. A 21 (57 por ciento) se les realizó pHmetría de 24 hs. Se realizaron 8 operaciones de Nissen-Rossetti, 22 de Nissen y 7 de Toupet. Se procedió al cierre sistemático del hiato esofágico en todos aquellos en que se encontraba dilatado. Resultados: tiempo operatorio y de internación: 160 min y 55 hs de promedio respectivamente. En 4 casos (10,8 por ciento) se convirtió a abdomen abierto. A 9,3 meses (2-23 m) de seguimiento, 29 pacientes (88 por ciento) presentan muy buena evolución, 4 pac. (12 por ciento) refirieron pirosis ocasional (todos con esclerodermia). Se realizó manometría postoperatoria en 22 pac. (59 por ciento), la que mostró recuperación de la presión del EEI. Conclusiones: Se puede reproducir la cirugía antirreflujo por vía laparoscópica con resultados iniciales similares a los obtenidos por laparotomía, con menor tiempo de internación. Se requiere experiencia en cirugía laparoscópica de avanzada y conocimientos actualizados de la fisiopatología de la ERGE

La evolución de los cálculos abandonados en el abdomen durante las colecistectomías laparoscópicas / Biliary stones spilled into the abdomen

En 2604 colecistectomías laparoscópicas se produjo la efracción de la vesícula en 573 (22 por ciento) y en 194 (7,5 por ciento) cayeron cálculos en la cavidad abdominal. Los autores analizan la evolución de 32 cálculos abandonados en la cavidad abdominal o en los trayectos de los trócares. En 8 de ellas se produjeron complicaciones. De los pacientes con 4 cálculos en la cavidad peritoneal 2 curaron con punción de una colección serosa sin necesidad de retirarlos. Los otros 2 desarrollaron abscesos intraperitoneales debiendo ser reoperados. Los 4 pacientes con cálculos en trayectos fistulosos peritoneales curaron con la extracción de los mismos. Los autores consideran que deben extremarse las medidas para no dejar cálculos abandonados luego de la colecistectomía pero no consideran imperativa la conversión salvo en cálculos grandes (AU)

La evolución de los cálculos abandonados en el abdomen durante las colecistectomías laparoscópicas / Biliary stones spilled into the abdomen

En 2604 colecistectomías laparoscópicas se produjo la efracción de la vesícula en 573 (22 por ciento) y en 194 (7,5 por ciento) cayeron cálculos en la cavidad abdominal. Los autores analizan la evolución de 32 cálculos abandonados en la cavidad abdominal o en los trayectos de los trócares. En 8 de ellas se produjeron complicaciones. De los pacientes con 4 cálculos en la cavidad peritoneal 2 curaron con punción de una colección serosa sin necesidad de retirarlos. Los otros 2 desarrollaron abscesos intraperitoneales debiendo ser reoperados. Los 4 pacientes con cálculos en trayectos fistulosos peritoneales curaron con la extracción de los mismos. Los autores consideran que deben extremarse las medidas para no dejar cálculos abandonados luego de la colecistectomía pero no consideran imperativa la conversión salvo en cálculos grandes

Estudio prospectivo de un año sobre seguridad en el quirófano / A year prospective study on safety in the operating room

En un trabajo prospectivo se registraron todos los hechos no esperados ocurridos durante el acto quirúrgico en un año, en una clínica privada con condiciones edilicias y equipamiento adecuados, personal entrenado y médicos con título de especialistas. En 2.752 operaciones se detectaron 10 (0.36%) problemas que por su poca jerarquía hubieran pasado inadvertidos, pero sirvieron para hacer correcciones y aumentar las medidas de seguridad

Estudio prospectivo de un año sobre seguridad en el quirófano / A year prospective study on safety in the operating room

En un trabajo prospectivo se registraron todos los hechos no esperados ocurridos durante el acto quirúrgico en un año, en una clínica privada con condiciones edilicias y equipamiento adecuados, personal entrenado y médicos con título de especialistas. En 2.752 operaciones se detectaron 10 (0.36%) problemas que por su poca jerarquía hubieran pasado inadvertidos, pero sirvieron para hacer correcciones y aumentar las medidas de seguridad

Necrosis pancreática: aspectos clínicos y terapéuticos / Pancreatic necrosis: clinical and therapeutical aspects

Se analizan retrospectivamente 22 pacientes portadores de necrosis pancreática. De los exámenes complementarios por imágenes, la tomografía computada es la más útil para el diagnóstico y evaluación de la extensión de la necrosis pancreática y peripancreática. Si bien la presente serie favorecería la indicación de necrosectomía y cierre primario del abdómen abierto, el número de pacientes y la imposibilidad ética de realizar estudios "randomizados", impiden conclusiones definitivas. La mortalidad global fue del 54% y las causas más importantes, fallas multiorgánicas y sepsis. Se estima que la alta mortalidad se debe a demora en el diagnóstico, debridamiento incompleto e insuficiente apoyo nutricional.

Necrosis pancreática: aspectos clínicos y terapéuticos / Pancreatic necrosis: clinical and therapeutical aspects

Se analizan retrospectivamente 22 pacientes portadores de necrosis pancreática. De los exámenes complementarios por imágenes, la tomografía computada es la más útil para el diagnóstico y evaluación de la extensión de la necrosis pancreática y peripancreática. Si bien la presente serie favorecería la indicación de necrosectomía y cierre primario del abdómen abierto, el número de pacientes y la imposibilidad ética de realizar estudios "randomizados", impiden conclusiones definitivas. La mortalidad global fue del 54% y las causas más importantes, fallas multiorgánicas y sepsis. Se estima que la alta mortalidad se debe a demora en el diagnóstico, debridamiento incompleto e insuficiente apoyo nutricional. (AU)

Peritonitis difusas graves / Severe diffuse peritonitis

Fueron tratados en un período de 5 años 93 pacientes portadores de peritonitis difusa grave. La mayor mortalidad se registró en los pacientes con peritonitis a punto de partida del intestino delgado y colon (53,8%). La edad constituyó uno de los factores de predicción de la mortalidad, siendo ésta más elevada después de los 60 años. Se analizan los aspectos controvertidos en el tratamiento de la peritonitis, concluyendose de acuerdo con nuestros resultados que la mortalidad sigue siendo alta y que el lavado peritoneal no los mejora. Los factores de predicción de mortalidad en nuestra serie fueron falla orgánica múltiple, etiología de las peritonitis y edad avanzada. (AU)

Peritonitis difusas graves / Severe diffuse peritonitis

Fueron tratados en un período de 5 años 93 pacientes portadores de peritonitis difusa grave. La mayor mortalidad se registró en los pacientes con peritonitis a punto de partida del intestino delgado y colon (53,8%). La edad constituyó uno de los factores de predicción de la mortalidad, siendo ésta más elevada después de los 60 años. Se analizan los aspectos controvertidos en el tratamiento de la peritonitis, concluyendose de acuerdo con nuestros resultados que la mortalidad sigue siendo alta y que el lavado peritoneal no los mejora. Los factores de predicción de mortalidad en nuestra serie fueron falla orgánica múltiple, etiología de las peritonitis y edad avanzada.