Comparison of Methods for Testing Mismatch Repair Status in Endometrial Cancer.

Approximately 20-30% of endometrial carcinomas (EC) are characterized by mismatch repair (MMR) deficiency (dMMR) or microsatellite instability (MSI), and their testing has become part of the routine diagnosis. The aim of this study was to establish and compare the MMR status using various approaches. Immunohistochemistry (IHC), PCR-based MSI, and the detection of defects in the four key MMR genes (MLH1, PMS2, MSH2, and MSH6) via methylation-specific multiplex ligation-dependent probe amplification (MLPA) and targeted next-generation sequencing (NGS) were performed. MSH3 expression was also evaluated. A set of 126 early-stage EC samples were analyzed, 53.2% of which were dMMR and 46.8% of which were proficient MMR (pMMR) as determined using IHC, whereas 69.3% were classified as microsatellite stable, while 8.8% and 21.9% were classified MSI-low (MSI-L) and MSI-high (MSI-H), respectively. In total, 44.3% of the samples showed genetic or epigenetic alterations in one or more genes; MLH1 promoter methylation was the most common event. Although acceptable concordance was observed, there were overall discrepancies between the three testing approaches, mainly associated with the dMMR group. IHC had a better correlation with MMR genomic status than the MSI status determined using PCR. Further studies are needed to establish solid conclusions regarding the best MMR assessment technique for EC.

The Prognostic Significance of Tumor-Infiltrating Lymphocytes, PD-L1, BRCA Mutation Status and Tumor Mutational Burden in Early-Stage High-Grade Serous Ovarian Carcinoma-A Study by the Spanish Group for Ovarian Cancer Research (GEICO).

Early stages are under-represented in studies on the molecular and immune features of high-grade serous ovarian carcinoma (HGSOC), and specific studies focused on early-stage HGSOC are required for a better prognostic stratification and to personalize chemotherapy. The aim of this study was to determine the prognostic significance of CD8+ and CD4+ tumor-infiltrating lymphocytes (TILs), tumoral cell PD-L1 expression, BRCA mutational status and tumor mutation burden (TMB) in early-stage HGSOC. A retrospective study was performed on stage I and II HGSOC from the Molecular Reclassification of Early Stages of Ovarian Cancer (RECLAMO) cohort from the Spanish Group of Ovarian Cancer Research (GEICO). Centralized histological typing was performed based on morphological and immunohistochemical features. Intraepithelial (i) and stromal (s) CD8+ and CD4+ T cells and PD-L1 were evaluated on tissue microarrays by immunohistochemistry. BRCA1 and BRCA2 mutation status and TMB were analyzed in tumor DNA using next-generation sequencing. The study included 124 tumors. High iCD8+ (>20 TILs/core), low/intermediate CD4+ (<20 TILs/core) and high CD8+/CD4+ ratio (>35/core) were associated with favorable outcomes. Tumor cell PD-L1 expression (TPS ≥ 1) was present in only 8% of tumors. In total, 11 (16%) and 6 (9%) out of 69 HGSOC tested carried pathogenic or likely pathogenic BRCA1 or BRCA2 mutations, respectively. Median TMB of 40 tumors analyzed was 5.04 mutations/Mb and only 6 tumors had 10 or more mutations/Mb. BRCA status and TMB were not associated with TILs or prognosis. When compared with studies on advanced HGSOC, our results suggested that prognostic variables differed according to stage and that more studies focused on early stages of HGSOC are needed to better stratify these tumors.

Spatial distribution of CD3- and CD8-positive lymphocytes as pretest for POLE wild-type in molecular subgroups of endometrial carcinoma.

Introduction: Over the years, the molecular classification of endometrial carcinoma has evolved significantly. Both POLEmut and MMRdef cases share tumor biological similarities like high tumor mutational burden and induce strong lymphatic reactions. While therefore use case scenarios for pretesting with tumor-infiltrating lymphocytes to replace molecular analysis did not show promising results, such testing may be warranted in cases where an inverse prediction, such as that of POLEwt, is being considered. For that reason we used a spatial digital pathology method to quantitatively examine CD3+ and CD8+ immune infiltrates in comparison to conventional histopathological parameters, prognostics and as potential pretest before molecular analysis. Methods: We applied a four-color multiplex immunofluorescence assay for pan-cytokeratin, CD3, CD8, and DAPI on 252 endometrial carcinomas as testing and compared it to further 213 cases as validation cohort from a similar multiplexing assay. We quantitatively assessed immune infiltrates in microscopic distances within the carcinoma, in a close distance of 50 microns, and in more distant areas. Results: Regarding prognostics, high CD3+ and CD8+ densities in intra-tumoral and close subregions pointed toward a favorable outcome. However, TCGA subtyping outperforms prognostication of CD3 and CD8 based parameters. Different CD3+ and CD8+ densities were significantly associated with the TCGA subgroups, but not consistently for histopathological parameter. In the testing cohort, intra-tumoral densities of less than 50 intra-tumoral CD8+ cells/mm2 were the most suitable parameter to assume a POLEwt, irrespective of an MMRdef, NSMP or p53abn background. An application to the validation cohort corroborates these findings with an overall sensitivity of 95.5%. Discussion: Molecular confirmation of POLEmut cases remains the gold standard. Even if CD3+ and CD8+ cell densities appeared less prognostic than TCGA, low intra-tumoral CD8+ values predict a POLE wild-type at substantial percentage rates, but not vice versa. This inverse correlation might be useful to increase pretest probabilities in consecutive POLE testing. Molecular subtyping is currently not conducted in one-third of cases deemed low-risk based on conventional clinical and histopathological parameters. However, this percentage could potentially be increased to two-thirds by excluding sequencing of predicted POLE wild-type cases, which could be determined through precise quantification of intra-tumoral CD8+ cells.

Weakly supervised deep learning to predict recurrence in low-grade endometrial cancer from multiplexed immunofluorescence images.

Predicting recurrence in low-grade, early-stage endometrial cancer (EC) is both challenging and clinically relevant. We present a weakly-supervised deep learning framework, NaroNet, that can learn, without manual expert annotation, the complex tumor-immune interrelations at three levels: local phenotypes, cellular neighborhoods, and tissue areas. It uses multiplexed immunofluorescence for the simultaneous visualization and quantification of CD68 + macrophages, CD8 + T cells, FOXP3 + regulatory T cells, PD-L1/PD-1 protein expression, and tumor cells. We used 489 tumor cores from 250 patients to train a multilevel deep-learning model to predict tumor recurrence. Using a tenfold cross-validation strategy, our model achieved an area under the curve of 0.90 with a 95% confidence interval of 0.83-0.95. Our model predictions resulted in concordance for 96,8% of cases (κ = 0.88). This method could accurately assess the risk of recurrence in EC, outperforming current prognostic factors, including molecular subtyping.

In-depth quantitative proteomics analysis revealed C1GALT1 depletion in ECC-1 cells mimics an aggressive endometrial cancer phenotype observed in cancer patients with low C1GALT1 expression.

BACKGROUND: Endometrial cancer (EC) is the most common cancer of the female reproductive organs. Despite the good overall prognosis of most low-grade ECs, FIGO I and FIGO II patients might experience tumor recurrence and worse prognosis. The study of alterations related to EC pathogenesis might help to get insights into underlying mechanisms involved in EC development and progression. METHODS: Core tumoral samples were used to investigate the role of C1GALT1 in EC by immunohistochemistry (IHC). ECC-1 cells were used as endometrioid EC model to investigate the effect of C1GALT1 depletion using C1GALT1 specific shRNAs. SILAC quantitative proteomics analyses and cell-based assays, PCR, qPCR, WB, dot-blot and IHC analyses were used to identify, quantify and validate dysregulation of proteins. RESULTS: Low C1GALT1 protein expression levels associate to a more aggressive phenotype of EC. Out of 5208 proteins identified and quantified by LC-MS/MS, 100 proteins showed dysregulation (log2fold-change ≥ 0.58 or ≤-0.58) in the cell protein extracts and 144 in the secretome of C1GALT1 depleted ECC-1 cells. Nine dysregulated proteins were validated. Bioinformatics analyses pointed out to an increase in pathways associated with an aggressive phenotype. This finding was corroborated by loss-of-function cell-based assays demonstrating higher proliferation, invasion, migration, colony formation and angiogenesis capacity in C1GALT1 depleted cells. These effects were associated to the overexpression of ANXA1, as demonstrated by ANXA1 transient silencing cell-based assays, and thus, correlating C1GALT and ANXA1 protein expression and biological effects. Finally, the negative protein expression correlation found by proteomics between C1GALT1 and LGALS3 was confirmed by IHC. CONCLUSION: C1GALT1 stably depleted ECC-1 cells mimic an EC aggressive phenotype observed in patients and might be useful for the identification and validation of EC markers of progression.

[Undergraduate Pathology: Results of a Survey on Undergraduate Teaching of Pathology in Spanish Medical Schools]. / Docencia de pregrado en Anatomía Patológica: Resultados de la encuesta sobre la docencia de la asignatura de Anatomía Patológica en los estudios de pregrado de las facultades de medicina españolas.

In order to ascertain the present state of undergraduate pathology teaching in Spain, the Spanish Society of Pathology sent a survey to the coordinators of the subject in every Medical School. The survey consisted of a form created using Google Forms tool and covered various aspects of teaching, such as the different syllabi, methodology and resources. 62% of the 55 Medical Schools contacted participated in the study (76% public and 25% private). In about half of cases, Pathology was taught as a single subject, while in the rest it was divided into General and Special Pathology. Only 18% integrated other clinical subjects into Pathology teaching and only 55% coordinated the timing of the course in order to coordinate with the content of other clinical subjects. We present the results of the survey together with all the accompanying comments and reflections, which highlight the heterogeneity of the Pathology syllabus in Spanish Medical Schools. We consider that if undergraduate Pathology is taught in an attractive, stimulating and clinically relevant manner, more students would be motivated to choose Pathology as their future speciality. Our main recommendations would be to emphasize the clinical application of Pathology and offer opportunities to gain practical, hands-on experience in Pathology departments.

Docencia de pregrado en Anatomía Patológica: Resultados de la encuesta sobre la docencia de la asignatura de Anatomía Patológica en los estudios de pregrado de las facultades de medicina españolas / Undergraduate Pathology: Results of a Survey on Undergraduate Teaching of Pathology in Spanish Medical Schools

Desde la Sociedad Española de Anatomía Patológica se ha realizado una encuesta entre las facultades de medicina españolas con el fin de obtener una imagen global del estado de la docencia de la asignatura de Anatomía Patológica en pregrado valorando los diferentes planes de estudio, la metodología empleada y la dotación de recursos. La encuesta se envió a los coordinadores de la asignatura en las diferentes facultades de medicina españolas a través de un formulario creado con la herramienta Formularios de Google.El 62% de las 55 unidades docentes encuestadas participaron en el estudio (76% universidades públicas y 24% privadas). Prácticamente en la mitad de los casos, la Anatomía Patológica se imparte como asignatura única, y en la otra mitad como una asignatura general y otra especial. Solo un 18% de las facultades contempla la asignatura especial integrada con otras asignaturas clínicas, y en general, solo en un 55% de los centros con Anatomía Patológica Especial, el contenido está coordinado con el de otras asignaturas clínicas.Los resultados de este estudio ponen en evidencia la heterogeneidad de la formación ofrecida en la asignatura de Anatomía Patológica para los estudiantes de Medicina en las diversas universidades españolas. Solamente planteando una asignatura en pregrado que sea atractiva y estimulante podremos motivar suficientemente a los estudiantes para elegir Anatomía Patológica como especialidad. En este artículo presentamos los resultados de la encuesta con todos los comentarios recogidos y las reflexiones planteadas a partir de los mismos. Las principales medidas propuestas están relacionadas con insistir en el carácter asistencial de la especialidad y en ofrecer rotaciones prácticas en los servicios de Anatomía Patológica.(AU)

In order to ascertain the present state of undergraduate pathology teaching in Spain, the Spanish Society of Pathology sent a survey to the coordinators of the subject in every Medical School. The survey consisted of a form created using Google Forms tool and covered various aspects of teaching, such as the different syllabi, methodology and resources.62% of the 55 Medical Schools contacted participated in the study (76% public and 25% private). In about half of cases, Pathology was taught as a single subject, while in the rest it was divided into General and Special Pathology. Only 18% integrated other clinical subjects into Pathology teaching and only 55% coordinated the timing of the course in order to coordinate with the content of other clinical subjects.We present the results of the survey together with all the accompanying comments and reflections, which highlight the heterogeneity of the Pathology syllabus in Spanish Medical Schools. We consider that if undergraduate Pathology is taught in an attractive, stimulating and clinically relevant manner, more students would be motivated to choose Pathology as their future speciality. Our main recommendations would be to emphasize the clinical application of Pathology and offer opportunities to gain practical, hands-on experience in Pathology departments.(AU)

Villoglandular Pattern in HPV-associated Endocervical Adenocarcinoma is Associated With Excellent Prognosis: A Reappraisal of 31 Cases Using IECC and Silva Pattern Classification.

Villoglandular adenocarcinoma of the cervix is a rare histologic entity that typically develops in young women, characterized by an association with oral contraceptives and excellent prognosis, though this point is controversial. These tumors have not been studied in the context of the International Endocervical Adenocarcinoma Criteria and Classification (IECC) or Silva Pattern Classification. We analyzed 31 cases that met strict diagnostic criteria, including being completely excised with negative margins. These were categorized according to IECC and Silva Pattern Classification and the association with various pathologic parameters analyzed. Most patients were young with a mean age of 41.1 (range 25-79). There were 14 (45.2%) pattern A, 11 (35.5%) pattern B, and 6 (19.3%) pattern C cases. Only 1 of 22 patients (4.5%) presented with lymph node metastasis at the time of diagnosis (pattern C, stage IB1) and 3 (9.7%) had lymphovascular invasion (2 pattern C, 1 pattern B). Overall survival was 100%, while recurrence-free survival was 96.2% for the entire cohort with only 1 case (3.2%) recurring 25 mo after surgery (IB2, pattern B). Kaplan Meier analysis (log rank test) revealed no significant correlation for recurrence-free survival at 5 and 10 yr associated with depth of invasion, tumor size, Silva pattern, FIGO stage, lymphovascular invasion, or lymph node metastasis. Cox univariate analysis demonstrated no independent prognostic factors predicting recurrence-free survival. These results indicate that completely excised villoglandular adenocarcinoma generally has an excellent prognosis and when Silva Pattern Classification is applied, those tumors that potentially have a higher chance for adverse outcomes can be identified.

The association between the tumor immune microenvironments and clinical outcome in low-grade, early-stage endometrial cancer patients.

Endometrial tumors show substantial heterogeneity in their immune microenvironment. This heterogeneity could be used to improve the accuracy of current outcome prediction tools. We assessed the immune microenvironment of 235 patients diagnosed with low-grade, early-stage endometrial cancer. Multiplex quantitative immunofluorescence was carried out to measure CD8, CD68, FOXP3, PD-1, and PD-L1 markers, as well as cytokeratin (CK), on tissue microarrays. Clustering results revealed five robust immune response patterns, each associated with specific immune populations, cell phenotypes, and cell spatial clustering. Most samples (69%) belonged to the immune-desert subtype, characterized by low immune cell densities. Tumor-infiltrating lymphocyte (TIL)-rich samples (4%) displayed high CD8+ T-cell infiltration, as well as a high percentage of CD8/PD-1+ cells. Immune-exclusion samples (19%) displayed the lowest CD8+ infiltration combined with high PD-L1 expression levels in CK+ tumor cells. In addition, they demonstrated high tumor cell spatial clustering as well as increased spatial proximity of CD8+ /PD-1+ and CK/PD-L1+ cells. FOXP3 and macrophage-rich phenotypes (3% and 4% of total samples) displayed relatively high levels of FOXP3+ regulatory T-cells and CD68+ macrophages, respectively. These phenotypes correlated with clinical outcomes, with immune-exclusion tumors showing an association with tumor relapse. When compared with prediction models built using routine pathological variables, models optimized with immune variables showed increased outcome prediction capacity (AUC = 0.89 versus 0.78) and stratification potential. The improved prediction capacity was independent of mismatch repair protein status and adjuvant radiotherapy treatment. Further, immunofluorescence results could be partially recapitulated using single-marker immunohistochemistry (IHC) performed on whole tissue sections. TIL-rich tumors demonstrated increased CD8+ T-cells by IHC, while immune-exclusion tumors displayed a lack of CD8+ T-cells and frequent expression of PD-L1 in tumor cells. Our results demonstrate the capability of the immune microenvironment to improve standard prediction tools in low-grade, early-stage endometrial carcinomas. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The Clonal Relationship Between the Ductal and Lobular Components of Mixed Ductal-Lobular Carcinomas Suggested a Ductal Origin in Most Tumors.

The relationship between the ductal and lobular components of invasive ductolobular carcinomas (IDLC) has not been fully elucidated. In this study, the molecular alterations of both components were analyzed in a series of 20 IDLC that were selected, not only by morphologic criteria, but also by the loss of E-cadherin expression in the lobular component. We found that 80% of tumors shared alterations of driver genes in both components, being PIK3CA the most common alteration. In addition, 45% of IDLC carried CDH1 mutations in their lobular component that were absent in the ductal component. Fluorescent in situ hybridization analysis of the CDH1 gene excluded homozygous CDH1 loss as a frequent cause of E-cadherin loss in tumors without CDH1 mutations. In addition, no pathogenic mutations of catenin genes were detected in this series of tumors. In 25% of tumors, actionable mutations in PIK3CA , AKT1 , and ERBB2 were found in only 1 component. Altogether, our results confirm that most IDLC derive from invasive carcinoma of no special type, in which a population of cells lose E-cadherin and acquire a lobular phenotype. The frequency of CDH1 mutations in IDLC appears to be lower than in conventional invasive lobular carcinomas, suggesting the implication of alternative mechanisms of E-cadherin loss. Moreover, molecular heterogeneity between ductal and lobular areas suggests the need for molecular characterization of both components to guide targeted therapies.

Neutrophil Extracellular Traps, Local IL-8 Expression, and Cytotoxic T-Lymphocyte Response in the Lungs of Patients With Fatal COVID-19.

BACKGROUND: Excessive inflammation is pathogenic in the pneumonitis associated with severe COVID-19. Neutrophils are among the most abundantly present leukocytes in the inflammatory infiltrates and may form neutrophil extracellular traps (NETs) under the local influence of cytokines. NETs constitute a defense mechanism against bacteria, but have also been shown to mediate tissue damage in a number of diseases. RESEARCH QUESTION: Could NETs and their tissue-damaging properties inherent to neutrophil-associated functions play a role in the respiratory failure seen in patients with severe COVID-19, and how does this relate to the SARS-CoV-2 viral loads, IL-8 (CXCL8) chemokine expression, and cytotoxic T-lymphocyte infiltrates? STUDY DESIGN AND METHODS: Sixteen lung biopsy samples obtained immediately after death were analyzed methodically as exploratory and validation cohorts. NETs were analyzed quantitatively by multiplexed immunofluorescence and were correlated with local levels of IL-8 messenger RNA (mRNA) and the density of CD8+ T-cell infiltration. SARS-CoV-2 presence in tissue was quantified by reverse-transcriptase polymerase chain reaction and immunohistochemistry analysis. RESULTS: NETs were found in the lung interstitium and surrounding the bronchiolar epithelium with interindividual and spatial heterogeneity. NET density did not correlate with SARS-CoV-2 tissue viral load. NETs were associated with local IL-8 mRNA levels. NETs were also detected in pulmonary thrombi and in only one of eight liver tissues. NET focal presence correlated negatively with CD8+ T-cell infiltration in the lungs. INTERPRETATION: Abundant neutrophils undergoing NETosis are found in the lungs of patients with fatal COVID-19, but no correlation was found with viral loads. The strong association between NETs and IL-8 points to this chemokine as a potentially causative factor. The function of cytotoxic T-lymphocytes in the immune responses against SARS-CoV-2 may be interfered with by the presence of NETs.

The Clinical Impact of Neoadjuvant Endocrine Treatment on Luminal-like Breast Cancers and Its Prognostic Significance: Results from a Single-Institution Prospective Cohort Study.

Purpose: Neoadjuvant endocrine treatment (NET) has become a useful tool for the downstaging of luminal-like breast cancers in postmenopausal patients. It enables us to increase breast- conserving surgery (BCS) rates, provides an opportunity for us to assess in vivo NET effectiveness, and allows us to study any biological changes that may act as valid biomarkers. The purpose of this study was to evaluate the safety and effectiveness of NET, and to assess the role of Ki67 proliferation rate changes as an indicator of endocrine responsiveness. Methods: From 2016 to 2020, a single-institution cohort of patients, treated with NET and further surgery, was evaluated. In patients with Ki67 ≥ 10%, a second core biopsy was performed after four weeks. Information regarding histopathological and clinical changes was gathered. Results: A total of 115 estrogen receptor-positive (ER+)/HER2-negative patients were included. The median treatment duration was 5.0 months (IQR: 2.0−6.0). The median maximum size in the surgical sample was 40% smaller than the pretreatment size measured by ultrasound (p < 0.0001). The median pretreatment Ki67 expression was 20.0% (IQR: 12.0−30.0), and was reduced to 5.0% (IQR: 1.8−10.0) after four weeks, and to 2.0% (IQR: 1.0−8.0) in the surgical sample (p < 0.0001). BCS was performed on 98 patients (85.2%). No pathological complete responses were recorded. A larger Ki67 fold change after four weeks was significantly related to a PEPI score of zero (p < 0.002). No differences were observed between luminal A- and B-like tumors, with regard to fold change and PEPI score. Conclusions: In our cohort, NET was proven to be effective for tumor size and Ki67 downstaging. This resulted in a higher rate of conservative surgery, aided in therapeutic decision making, provided prognostic information, and constituted a safe and well-tolerated approach.

Prognostic markers of inflammation in endometrioid and clear cell ovarian cancer.

OBJECTIVES: Cancer-related systemic inflammation has been associated with prognosis in multiple cancer types. Conversely, local inflammation, which is characterized by dense intratumoral immune infiltrates, is a favorable predictor of survival outcome. However, these survival associations are not well established in ovarian cancer, particularly in the less frequent endometrioid and clear cell endometriosis associated histotypes. METHODS: This retrospective study included 119 patients (63 endometrioid and 56 clear cell ovarian carcinomas). We performed a comprehensive survival association analysis of both systemic (neutrophil-to-lymphocyte ratio or presence of endometriosis) and local inflammation markers (CD3+ and CD8+ tumor infiltrating lymphocytes) using multivariate Cox proportional hazards models that account for confounding factors. RESULTS: Medium to high levels of intraepithelial CD8+ tumor infiltrating lymphocytes are associated with longer survival in endometrioid ovarian cancer (p=0.04). In addition, we found that intraepithelial CD8+ tumor infiltrating lymphocytes are prognostic in clear cell ovarian cancer (p=0.02), and that intraepithelial CD3+ tumor infiltrating lymphocytes are also associated with improved outcome (p=0.02). Furthermore, intratumoral CD3+ and CD8+ tumor infiltrating lymphocytes showed improved prognosis in the endometrioid subtype (p<0.1). No prognostic value was observed for systemic immune markers. CONCLUSIONS: In this study, patients with endometrioid and clear cell ovarian cancer with moderate to high CD8+ and CD3+ intraepithelial tumor infiltrating lymphocytes had longer overall survival. Higher expression of intratumoral CD3+ and CD8+ tumor infiltrating lymphocytes also showed an improved outcome in endometrioid ovarian cancer. In contrast, systemic inflammation, evaluated by neutrophil-to-lymphocyte ratio or presence of endometriosis, did not have a prognostic impact in these histologic subtypes.

Antitumoral Effect of Plocabulin in High Grade Serous Ovarian Carcinoma Cell Line Models.

Ovarian cancer (OC) is a life-threatening tumor and the deadliest among gynecological cancers in developed countries. First line treatment with a carboplatin/paclitaxel regime is initially effective in the majority of patients, but most advanced OC will recur and develop drug resistance. Therefore, the identification of alternative therapies is needed. In this study, we employed a panel of high-grade serous ovarian cancer (HGSOC) cell lines, in monolayer and three-dimensional cell cultures. We evaluated the effects of a novel tubulin-binding agent, plocabulin, on proliferation, cell cycle, migration and invasion. We have also tested combinations of plocabulin with several drugs currently used in OC in clinical practice. Our results show a potent antitumor activity of plocabulin, inhibiting proliferation, disrupting microtubule network, and decreasing their migration and invasion capabilities. We did not observe any synergistic combination of plocabulin with cisplatin, doxorubicin, gemcitabine or trabectedin. In conclusion, plocabulin has a potent antitumoral effect in HGSOC cell lines that warrants further clinical investigation.

Aryl-hydrocarbon receptor-interacting protein regulates tumorigenic and metastatic properties of colorectal cancer cells driving liver metastasis.

BACKGROUND: Liver metastasis is the primary cause of colorectal cancer (CRC)-associated death. Aryl-hydrocarbon receptor-interacting protein (AIP), a putative positive intermediary in aryl-hydrocarbon receptor-mediated signalling, is overexpressed in highly metastatic human KM12SM CRC cells and other highly metastatic CRC cells. METHODS: Meta-analysis and immunohistochemistry were used to assess the relevance of AIP. Cellular functions and signalling mechanisms mediated by AIP were assessed by gain-of-function experiments and in vitro and in vivo experiments. RESULTS: A significant association of high AIP expression with poor CRC patients' survival was observed. Gain-of-function and quantitative proteomics experiments demonstrated that AIP increased tumorigenic and metastatic properties of isogenic KM12C (poorly metastatic) and KM12SM (highly metastatic to the liver) CRC cells. AIP overexpression dysregulated epithelial-to-mesenchymal (EMT) markers and induced several transcription factors and Cadherin-17 activation. The former induced the signalling activation of AKT, SRC and JNK kinases to increase adhesion, migration and invasion of CRC cells. In vivo, AIP expressing KM12 cells induced tumour growth and liver metastasis. Furthermore, KM12C (poorly metastatic) cells ectopically expressing AIP became metastatic to the liver. CONCLUSIONS: Our data reveal new roles for AIP in regulating proteins associated with cancer and metastasis to induce tumorigenic and metastatic properties in colon cancer cells driving liver metastasis.

Prognosis Stratification Tools in Early-Stage Endometrial Cancer: Could We Improve Their Accuracy?

There are three prognostic stratification tools used for endometrial cancer: ESMO-ESGO-ESTRO 2016, ProMisE, and ESGO-ESTRO-ESP 2020. However, these methods are not sufficiently accurate to address prognosis. The aim of this study was to investigate whether the integration of molecular classification and other biomarkers could be used to improve the prognosis stratification in early-stage endometrial cancer. Relapse-free and overall survival of each classifier were analyzed, and the c-index was employed to assess accuracy. Other biomarkers were explored to improve the precision of risk classifiers. We analyzed 293 patients. A comparison between the three classifiers showed an improved accuracy in ESGO-ESTRO-ESP 2020 when RFS was evaluated (c-index = 0.78), although we did not find broad differences between intermediate prognostic groups. Prognosis of these patients was better stratified with the incorporation of CTNNB1 status to the 2020 classifier (c-index 0.81), with statistically significant and clinically relevant differences in 5-year RFS: 93.9% for low risk, 79.1% for intermediate merged group/CTNNB1 wild type, and 42.7% for high risk (including patients with CTNNB1 mutation). The incorporation of molecular classification in risk stratification resulted in better discriminatory capability, which could be improved even further with the addition of CTNNB1 mutational evaluation.

Clear Cell Carcinoma (CCC) of the Cervix Is a Human Papillomavirus (HPV)-independent Tumor Associated With Poor Outcome: A Comprehensive Analysis of 58 Cases.

Cervical clear cell carcinoma (CCC) is a rare human papillomavirus-independent adenocarcinoma. While recent studies have focused on gastric-type endocervical adenocarcinoma (GTA), little is known about CCC. A total of 58 (CCCs) were collected from 14 international institutions and retrospectively analyzed using univariable and multivariable methods and compared with 36 gastric-type adenocarcinomas and 173 human papillomavirus-associated (HPVA) endocervical adenocarcinoma (ECA) regarding overall survival (OS) and recurrence-free survival (RFS). Most cases were FIGO stage I (72.4%), with Silva C pattern of invasion (77.6%), and the majority were treated with radical surgery (84.5%) and adjuvant therapy (55.2%). Lymphovascular invasion was present in 31%, while lymph node metastasis was seen in 24.1%; 10.3% were associated with abdominopelvic metastases at the time of diagnosis; 32.8% had recurrences, and 19% died of disease. We did not find statistically significant differences in OS and RFS between CCC and GTA at 5 and 10 years (P=0.313 and 0.508, respectively), but there were significant differences in both OS and RFS between CCC and HPVA ECA (P=0.003 and 0.032, respectively). Also, OS and RFS in stage I clear cell and GTA were similar (P=0.632 and 0.692, respectively). Multivariate analysis showed that OS is influenced by the presence of recurrence (P=0.009), while RFS is influenced by the FIGO stage (P=0.025). Cervical CCC has poorer outcomes than HPVA ECA and similar outcomes to human papillomavirus-independent GTA. Oncologic treatment significantly influences RFS in univariate analysis but is not an independent prognostic factor in multivariate analysis suggesting that alternative therapies should be investigated.

SARS-CoV-2 infection associated with monoclonal gammopathy. A case report based on the study of minimally invasive ultrasound-guided autopsy.

Coronavirus disease-2019 (COVID-19) is a global public health emergency with numerous clinical facets, including acute kidney injury and acute cerebrovascular disease. Further knowledge of its various pathogenic mechanisms is essential, including coagulation disorders. Monoclonal gammopathy is characterized by the overproduction of a monoclonal immunoglobulin caused by clonal proliferation. Using a postmortem study of ultrasound-guided percutaneous core biopsies, the aim of this report is to present our observations on the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection pathology associated with monoclonal gammopathy. The clinical presentation was acute renal failure. Pathological findings revealed kappa light chain cast nephropathy. SARS-CoV-2 immunohistochemistry was positive in some renal tubular cells. Another notable finding was the presence of a high density of alveolar megakaryocytes, which probably explained the final outcome (acute cerebrovascular disease). Immunohistochemical study for SARS-CoV-2 does not verify the pathogenic effect of the virus and thus its contribution to the acute kidney injury.

SARS-CoV-2 infection associated with monoclonal gammopathy. A case report based on the study of minimally invasive ultrasound-guided autopsy

Coronavirus disease-2019 (COVID-19) is a global public health emergency with numerous clinical facets, including acute kidney injury and acute cerebrovascular disease. Further knowledge of its various pathogenic mechanisms is essential, including coagulation disorders. Monoclonal gammopathy is characterized by the overproduction of a monoclonal immunoglobulin caused by clonal proliferation. Using a postmortem study of ultrasound-guided percutaneous core biopsies, the aim of this report is to present our observations on the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection pathology associated with monoclonal gammopathy. The clinical presentation was acute renal failure. Pathological findings revealed kappa light chain cast nephropathy. SARS-CoV-2 immunohistochemistry was positive in some renal tubular cells. Another notable finding was the presence of a high density of alveolar megakaryocytes, which probably explained the final outcome (acute cerebrovascular disease). Immunohistochemical study for SARS-CoV-2 does not verify the pathogenic effect of the virus and thus its contribution to the acute kidney injury.(AU)

La enfermedad por coronavirus de 2019 (COVID-19) es una emergencia sanitaria pública global con numerosas facetas clínicas que incluyen enfermedad renal aguda y enfermedad cerebrovascular aguda. Es necesario un conocimiento adicional de su mecanismo patogénico. Los trastornos de coagulación están claramente incluidos en dichos mecanismos. La gammapatía monoclonal se caracteriza por la sobreproducción de inmunoglobulina monoclonal causada por proliferación clonal. Utilizando un estudio postmortem de biopsias percutáneas ecoguiadas, el objetivo de este informe es presentar nuestras observaciones sobre la patología del síndrome respiratorio agudo severo por infección de coronavirus 2 (SARS-CoV-2) con gammapatía monoclonal. La presentación clínica fue insuficiencia renal aguda. Los hallazgos patológicos revelaron nefropatía por cilindros de cadenas ligeras kappa. La inmunohistoquímica de SARS-CoV-2 fue positiva en ciertas células tubulares renales. La presencia de megacariocitos alveolares (alta densidad) fue un hallazgo notable, que explica probablemente el resultado final del paciente (enfermedad cerebrovascular aguda). El estudio inmunohistoquímico frente a SARS-CoV-2 no verifica el efecto patogénico del virus y, por tanto, su contribución a la nefropatía aguda.(AU)

Molecular characterization of triple negative breast cancer formaldehyde-fixed paraffin-embedded samples by data-independent acquisition proteomics.

Triple negative breast cancer accounts for 15%-20% of all breast carcinomas and is clinically characterized by an aggressive phenotype and poor prognosis. Triple negative tumors do not benefit from targeted therapies, so further characterization is needed to define subgroups with potential therapeutic value. In this work, the proteomes of 125 formalin-fixed paraffin-embedded samples from patients diagnosed with non-metastatic triple negative breast cancer were analyzed using data-independent acquisition + in a LTQ-Orbitrap Fusion Lumos mass spectrometer coupled to an EASY-nLC 1000. 1206 proteins were identified in at least 66% of the samples. Hierarchical clustering, probabilistic graphical models and Significance Analysis of Microarrays were combined to characterize proteomics-based molecular groups. Two molecular groups were defined with differences in biological processes such as glycolysis, translation and immune response. These two molecular groups showed also several differentially expressed proteins. This clinically homogenous dataset may serve to design new therapeutic strategies in the future.