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.

Tana, a Healthcare Chatbot to Help Patients During the COVID-19 Pandemic at a University Hospital in Argentina.

A Chatbot or Conversational Agent is a computer application that simulates the conversation with a human person (by text or voice), giving automated responses to people's needs. In the healthcare domain, chatbots can be beneficial to help patients, as a complement to care by health personnel, especially in times of high demand or constrained resources such as the COVID-19 Pandemic. In this paper we share the design and implementation of a healthcare chatbot called Tana at the Hospital Italiano de Buenos Aires. Considering best practices and being aware of possible unintended consequences, we must take advantage of information and communication technologies, such as chatbots, to analyze and promote useful conversations for the health of all people.

Next generation sequencing to decipher concurrent loss of PMS2 and MSH6 in colorectal cancer.

BACKGROUND: Immunohistochemistry (IHQ) is commonly used for the detection of mismatch repair proteins deficiency (MMRD). One very infrequent abnormal pattern of MMR protein expression is the loss of PMS2 and MSH6, with intact expression of MLH1 and MSH2. CASE PRESENTATION: We review the frequency of this MMRD IHC pattern among 108 colorectal (CRCs) and 35 endometrial cancers in our files with loss of expression of at least one protein, and present two CRCs showing loss of PMS2 and MSH6 protein expression (1.9% of CRCs). NGS analysis of these tumours identified PMS2 mutations (R134* germline mutation in one tumour and M1R and c.1239delA somatic mutation in the other) as the primary event and somatic MSH6 mutation (c.3261dupC) as the secondary event in both tumours. CONCLUSIONS: This study suggests that Next Generation Sequencing (NGS) tumour analysis should be considered in the algorithm of Lynch syndrome screening to detect MMR gen somatic mutation in inconclusive cases.

Molecular Heterogeneity of Endometrioid Ovarian Carcinoma: An Analysis of 166 Cases Using the Endometrial Cancer Subrogate Molecular Classification.

Endometrioid ovarian carcinoma (EOC) has clinical and biological differences compared with other histologic types of ovarian carcinomas, but it shares morphologic and molecular features with endometrioid endometrial carcinoma. To analyze the molecular heterogeneity of EOC according to the new molecular classification of endometrial cancer and to evaluate the prognostic significance of this molecular classification, we have analyzed 166 early-stage EOC by immunohistochemistry for mismatch repair proteins and p53 expression, and by Sanger sequencing for the exonuclease domain of polymerase epsilon (POLE EDM). In addition, we have carried out next-generation sequencing analysis of tumors with POLE EDM mutations to confirm the ultramutated profile. Eight tumors carried POLE EDM mutations and were classified as ultramutated (5%), 29 showed mismatch repair deficiency and were classified as hypermutated (18%), 16 tumors had a mutated pattern of p53 expression and were classified as p53 abnormal (11%), and 114 tumors did not have any of the previous alterations and were classified as no specific type (66%). Five tumors showed >1 classification criteria. The frequencies of ultramutated and hypermutated tumors were lower in EOC compared with the frequency reported in endometrial cancer. Subrogate molecular groups differed in both morphologic features (histologic grade, squamous and morular metaplasia, and necrosis) and immunohistochemical expression of several biomarkers (ARID1A, nuclear ß-catenin, estrogen receptors, Napsin A, and HINF1B). In addition, the number of CD8 tumor-infiltrating lymphocytes was higher in ultramutated and hypermutated tumors. The most commonly mutated genes in the ultramutated group were ARID1A (100%), PIK3R1, PTEN, BCOR, and TP53 (67% each), whereas no mutations were detected in KRAS. Although the prognosis did not differ among subgroups in the multivariate analysis, a trend toward a better prognosis in POLE-mutated and a worse prognosis in p53 abnormal tumors was observed. In addition, this classification could have important therapeutic implications for the use of immunotherapy in tumors classified as ultramutated and hypermutated.

Mismatch Repair Deficiency in Ovarian Carcinoma: Frequency, Causes, and Consequences.

Mismatch repair deficiency (MMRD) is involved in the initiation of both hereditary and sporadic tumors. MMRD has been extensively studied in colorectal cancer and endometrial cancer, but not so in other tumors, such as ovarian carcinoma. We have determined the expression of mismatch repair proteins in a large cohort of 502 early-stage epithelial ovarian carcinoma entailing all the 5 main subtypes: high-grade serous carcinoma, endometrioid ovarian carcinoma (EOC), clear cell carcinoma (CCC), mucinous carcinoma, and low-grade serous carcinoma. We studied the association of MMRD with clinicopathologic and immunohistochemical features, including tumor-infiltrating lymphocytes in EOC, the histologic type in which MMRD is most frequent. In addition, MLH1 promoter methylation status and massive parallel sequencing were used to evaluate the proportion of sporadic and Lynch syndrome-associated tumors, and the most frequently mutated genes in MMRD EOCs. MMRD occurred only in endometriosis-associated histologic types, and it was much more frequent in EOC (18%) than in CCC (2%). The most frequent immunohistochemical pattern was loss of MLH1/PMS2, and in this group, 80% of the cases were sporadic and secondary to MLH1 promoter hypermethylation. The presence of somatic mutations in mismatch repair genes was the other mechanism of MMRD in sporadic tumors. In this series, the minimum estimated frequency of Lynch syndrome was 35% and it was due to germline mutations in MLH1, MSH2, and MSH6. ARID1A, PTEN, KTM2B, and PIK3CA were the most common mutated genes in this series. Interestingly, possible actionable mutations in ERRB2 were found in 5 tumors, but no TP53 mutations were detected. MMRD was associated with younger age and increased tumor-infiltrating lymphocytes. Universal screening in EOC and mixed EOC/CCC is recommended for the high frequency of MMRD detected; however, for CCC, additional clinical and pathologic criteria should be evaluated to help select cases for analysis.

The Frequency and Prognostic Significance of the Histologic Type in Early-stage Ovarian Carcinoma: A Reclassification Study by the Spanish Group for Ovarian Cancer Research (GEICO).

The frequency and prognostic significance of the histologic type in early-stage ovarian cancer (OC) is not as well established as in advanced stages. In addition, histologic typing based only on morphologic features may be difficult, especially in high-grade tumors. In this study, we have analyzed a prospective cohort of 502 early-stage OCs to investigate their frequency, immunohistochemical characteristics, and survival of the 5 main histologic types. Histotype was assigned according to not only the morphologic features but also according to the expression pattern of WT1, p53, Napsin A, and progesterone receptors. In addition, an extended panel including p16, ß-catenin, HER2, Arid1A, HINF1B, CK7, CDX2, and CK20 was used to refine the diagnosis in difficult cases. In this series, the frequency of the 5 major histologic types was as follows: endometrioid carcinoma, 32.7%; clear cell carcinoma, 25.1%; high-grade serous carcinoma (HGSC), 24.7%; mucinous carcinoma, 10.2%; low-grade serous carcinoma, 4.6%; and others, 2.8%. The combination of morphology and immunohistochemistry allowed the reclassification of 23% of OCs. The lowest concordance was found between samples initially diagnosed as endometrioid, but finally classified as high-grade serous tumors (22% error rate). Endometrioid carcinoma was the most favorable histologic type, whereas HGSC and low-grade serous carcinoma had the worst prognosis. Clear cell carcinoma with abnormal p53 immunostaining pattern also had poor prognosis. Although histologic grade was not a prognostic factor among early-stage endometrioid OCs, distinction between grade 3 endometrioid OC and HGSC is recommended, taking into account differences in prognosis and molecular alterations that can guide different treatments.

Actin dynamics and the Bmp pathway drive apical extrusion of proepicardial cells.

The epicardium, the outer mesothelial layer enclosing the myocardium, plays key roles in heart development and regeneration. During embryogenesis, the epicardium arises from the proepicardium (PE), a cell cluster that appears in the dorsal pericardium (DP) close to the venous pole of the heart. Little is known about how the PE emerges from the pericardial mesothelium. Using a zebrafish model and a combination of genetic tools, pharmacological agents and quantitative in vivo imaging, we reveal that a coordinated collective movement of DP cells drives PE formation. We found that Bmp signaling and the actomyosin cytoskeleton promote constriction of the DP, which enables PE cells to extrude apically. We provide evidence that cell extrusion, which has been described in the elimination of unfit cells from epithelia and the emergence of hematopoietic stem cells, is also a mechanism for PE cells to exit an organized mesothelium and fulfil their developmental fate to form a new tissue layer, the epicardium.

Dedifferentiated endometrial carcinomas with neuroendocrine features: a clinicopathologic, immunohistochemical, and molecular genetic study.

Undifferentiated endometrial carcinoma is an aggressive type of uterine cancer, which is occasionally associated with a low-grade endometrioid carcinoma component. This combination is referred to as "dedifferentiated endometrioid endometrial carcinoma." Neuroendocrine expression may occur in undifferentiated endometrial carcinoma, but its significance in dedifferentiated endometrial carcinomas is unknown. To gain insight into the pathogenesis of these tumors we have analyzed the immunophenotype (ARID1A, MLH1, PMS2, MSH2, MSH6, p53, ß-catenin, SMARCB1, synaptophysin, chromogranin A, and CD56) and mutational status (PTEN, KRAS, PIK3CA, TP53 and POLE) of 4 dedifferentiated endometrial carcinomas with strong and diffuse neuroendocrine expression. All tumors demonstrated neuroendocrine expression in ≥70% of the cells in the undifferentiated carcinoma areas. Loss of expression of at least 1 DNA mismatch repair protein was observed in 2 cases, and p53 immunoreaction was aberrant (mutated/inactivated) in one case. All carcinomas were negative for ß-catenin and maintained nuclear SMARCB1 (INI1) and ARID1A expression. Three tumors shared identical endometrioid molecular profile (PTEN and/or PIK3CA mutations) in both components. One tumor had POLE exonuclease domain mutation in the undifferentiated component. In one case, TP53 mutation was found exclusively in the undifferentiated component. Two patients died with peritoneal carcinomatosis and abdominal metastases, respectively; one patient died of a renal failure without evidence of disease, and the last patient is alive and free of disease at 3.3 years. Dedifferentiated endometrial carcinomas with neuroendocrine features are clinically and molecularly heterogeneous tumors. Probably, these carcinomas might acquire undifferentiated phenotype through mutations in TP53 and POLE.

Differential Lectin Binding Patterns Identify Distinct Heart Regions in Giant Danio ( Devario aequipinnatus) and Zebrafish ( Danio rerio) Hearts.

Lectins are carbohydrate-binding proteins commonly used as biochemical and histochemical tools to study glycoconjugate (glycoproteins, glycolipids) expression patterns in cells, tissues, including mammalian hearts. However, lectins have received little attention in zebrafish ( Danio rerio) and giant danio ( Devario aequipinnatus) heart studies. Here, we sought to determine the binding patterns of six commonly used lectins-wheat germ agglutinin (WGA), Ulex europaeus agglutinin, Bandeiraea simplicifolia lectin (BS lectin), concanavalin A (Con A), Ricinus communis agglutinin I (RCA I), and Lycopersicon esculentum agglutinin (tomato lectin)-in these hearts. Con A showed broad staining in the myocardium. WGA stained cardiac myocyte borders, with binding markedly stronger in the compact heart and bulbus. BS lectin, which stained giant danio coronaries, was used to measure vascular reconstruction during regeneration. However, BS lectin reacted poorly in zebrafish. RCA I stained the compact heart of both fish. Tomato lectin stained the giant danio, and while low reactivity was seen in the zebrafish ventricle, staining was observed in their transitional cardiac myocytes. In addition, we observed unique staining patterns in the developing zebrafish heart. Lectins' ability to reveal differential glycoconjugate expression in giant danio and zebrafish hearts suggests they can serve as simple but important tools in studies of developing, adult, and regenerating fish hearts.

Molecular genetic heterogeneity in undifferentiated endometrial carcinomas.

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 tumors, 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 tumors 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) tumors 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) tumors 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 tumors. These results indicate that undifferentiated and dedifferentiated endometrial carcinomas are molecularly heterogeneous tumors, which may have prognostic value.

Analysis of the dynamic co-expression network of heart regeneration in the zebrafish.

The zebrafish has the capacity to regenerate its heart after severe injury. While the function of a few genes during this process has been studied, we are far from fully understanding how genes interact to coordinate heart regeneration. To enable systematic insights into this phenomenon, we generated and integrated a dynamic co-expression network of heart regeneration in the zebrafish and linked systems-level properties to the underlying molecular events. Across multiple post-injury time points, the network displays topological attributes of biological relevance. We show that regeneration steps are mediated by modules of transcriptionally coordinated genes, and by genes acting as network hubs. We also established direct associations between hubs and validated drivers of heart regeneration with murine and human orthologs. The resulting models and interactive analysis tools are available at http://infused.vital-it.ch. Using a worked example, we demonstrate the usefulness of this unique open resource for hypothesis generation and in silico screening for genes involved in heart regeneration.

A core microRNA signature associated with inducers of the epithelial-to-mesenchymal transition.

Although it is becoming clear that certain miRNAs fulfil a fundamental role in the regulation of the epithelial-to-mesenchymal transition (EMT), a comprehensive study of the miRNAs associated with this process has yet to be performed. Here, we profiled the signature of miRNA expression in an in vitro model of EMT, ectopically expressing in MDCK cells one of seven EMT transcription factors (SNAI1, SNAI2, ZEB1, ZEB2, TWIST1, TWIST2 or E47) or the EMT inducer LOXL2. In this way, we identified a core subset of deregulated miRNAs that were further validated in vivo, studying endometrial carcinosarcoma (ECS), a tumour entity that represents an extreme example of phenotypic plasticity. Moreover, epigenetic silencing through DNA methylation of miRNA genes of the miR-200 family and miR-205 that are down-regulated during EMT was evident in both the in vitro (MDCK transfectants) and in vivo (ECS) models of EMT. The strong correlation between expression and DNA methylation suggests a major role for this epigenetic mark in the regulation of the miR-141-200c locus.

TNF receptors regulate vascular homeostasis in zebrafish through a caspase-8, caspase-2 and P53 apoptotic program that bypasses caspase-3.

Although it is known that tumor necrosis factor receptor (TNFR) signaling plays a crucial role in vascular integrity and homeostasis, the contribution of each receptor to these processes and the signaling pathway involved are still largely unknown. Here, we show that targeted gene knockdown of TNFRSF1B in zebrafish embryos results in the induction of a caspase-8, caspase-2 and P53-dependent apoptotic program in endothelial cells that bypasses caspase-3. Furthermore, the simultaneous depletion of TNFRSF1A or the activation of NF-κB rescue endothelial cell apoptosis, indicating that a signaling balance between both TNFRs is required for endothelial cell integrity. In endothelial cells, TNFRSF1A signals apoptosis through caspase-8, whereas TNFRSF1B signals survival via NF-κB. Similarly, TNFα promotes the apoptosis of human endothelial cells through TNFRSF1A and triggers caspase-2 and P53 activation. We have identified an evolutionarily conserved apoptotic pathway involved in vascular homeostasis that provides new therapeutic targets for the control of inflammation- and tumor-driven angiogenesis.

Comprehensive genomic analysis of a BRCA2 deficient human pancreatic cancer.

Capan-1 is a well-characterised BRCA2-deficient human cell line isolated from a liver metastasis of a pancreatic adenocarcinoma. Here we report a genome-wide assessment of structural variations and high-depth exome characterization of single nucleotide variants and small insertion/deletions in Capan-1. To identify potential somatic and tumour-associated variations in the absence of a matched-normal cell line, we devised a novel method based on the analysis of HapMap samples. We demonstrate that Capan-1 has one of the most rearranged genomes sequenced to date. Furthermore, small insertions and deletions are detected more frequently in the context of short sequence repeats than in other genomes. We also identify a number of novel mutations that may represent genetic changes that have contributed to tumour progression. These data provide insight into the genomic effects of loss of BRCA2 function.

Building the vertebrate heart - an evolutionary approach to cardiac development.

The vertebrate heart is unique among the blood pumps described in metazoans. In contrast to the myoepithelial tubes found in most animal phyla, the vertebrate heart is made up of multilayered myocardial cells surrounded by connective tissue derived from epicardium and endocardium, and endowed with complex valvular, coronary vessel and conduction systems. Despite these profound differences, a common genetic program seems to underlie the specification and differentiation of all the cardiac tissues. In this article, we will review the similarities in the transcriptional networks and signalling mechanisms regulating cardiac development in different animals, as well as the origin of the main differences existing between vertebrate and invertebrate hearts. We will pay special attention to the hypotheses concerning the evolutionary origin of the endothelium and the epicardium from ancestral blood cells and pronephric progenitors, respectively. We can summarize the transition between the invertebrate and the vertebrate heart as the result of the thickening of the primarily myoepithelial cardiac tube which was concomitant with: 1) an inner lining by an endothelium with the ability to transform into mesenchyme; 2) an outer lining derived from an ancestral pronephric glomerular primordium with vasculogenic potential; 3) a neural crest cell population which reaches the heart from the pharyngeal region; 4) the incorporation of new myocardium at both ends from a second heart field and 5) the formation of specialized chambers. The complex interactions between all these elements originated an exceptionally powerful blood pump which allowed vertebrates to reach their characteristically large size and activity.

Histological seasonal changes in ovaries of Spotted tinamous (Nothura maculosa Tinamidae, Temminck, 1815) related to gonadotrope population / Cambios histológicos estacionales en ovarios de perdiz común (Nothura maculosa Tinamidae, Temminck, 1815)) y su relación con la población de gonadotropas

Nothura maculosa is a South American Tinamidae with a marked seasonal reproductive pattern. This work describes ovarian seasonal changes in this species related to gonadotrope (GTHs) population. Ovary and pituitary samples were collected monthly from adult birds during four annual periods. They were fixed in Bouin's solution and processed for light microscopy. The data of post-fixation gonadal weight were analysed using STATISTIX 4.0. Histological sections of the ovaries were stained with H/E, PAS and Goldner-Masson trichrome. Single and double immunostaining were applied on pituitary sections with anti-chicken-FSH and anti-chicken-LH antibodies. The samples were analysed in quarterly periods of the year, Pl: March-May (resting stage); P2: June-August (developing stage); P3: September-November (reproductive stage); P4: December-February (involutive stage). Ovary weight (ow) significantly varied among periods (p<0.001). During Pl, only primordial and previtellogenic follicles were observed, ow 0.09±0.01 g (n=25); during P2, developing follicles with signs of vitellogenesis were detected, ow 0.13+0.01 g (n=14); during P3, maximum follicular development was found, ow 0.9 +/- 0.15 g (n=39); P4 exhibited great variability in follicular stages, ow 0.18+0.18 g (n=19). Involutive atresia was observed in all the periods, while bursting atresia and post-ovulatory follicles were only characterized at P3 and P4. The GTHs containing few LH and FSH immunoreactive (ir) granules were predominant during P1-P2. The GTHs with LH ir granules were abundant in intermediate zone and caudal lobe in P3 and P4 while few cells contained both types of granules. The number of FSH cells was increased during P3 and P4. The histological ovarían changes were narrowly correlated with the variations in the gonadotrope population.

Nothura maculosa es un tinámido sudamericano que presenta marcada estacionalidad reproductiva. Este trabajo describe los cambios estacionales del ovario de esta especie, en relación con la población de gonadotropas (GTHs). Muestras de ovarios y pituitarias de ejemplares adultos fueron colectadas mensualmente durante cuatro años; se fijaron en solución de Bouin y procesadas para M.O. Los datos del peso gonadal posfijación fueron analizados usando STATISTIX 4.0. Los cortes de ovarios fueron coloreados con H/E, P.A.S. y Tricrómico de Goldner-Masson. En cortes de adenohipófisis se aplicó inmunocitoquímica simple y doble (sistema ABC, Vector Lab.), empleando anticuerpos anti-pollo FSH y anti-pollo LH. Las muestras se analizaron en períodos trimestrales de cada año (P): Pl: Marzo-Abril-Mayo (etapa de reposo), P2: Junio-Julio-Agosto (etapa de desarrollo), P3: Septiembre-Octubre-Noviembre (etapa reproductiva), P4: Diciembre-Enero-Febrero (etapa involutiva). El peso de los ovarios (PO) varió significativamente entre los periodos (p< 0.001). Durante Pl, sólo se observaron folículos primordiales y pre-vitelogénicos, PO 0.09 +/- 0.01 g (n=25); durante P2, se detectaron folículos en desarrollo con signos de vitelogénesis, PO 0.13+0.01 g (n=14); durante P3, se encontró máximo desarrollo folicular, PO 0.90+0.15 g (n=39); P4 exhibió gran variabilidad folicular, PO 0.18+0.18 g (n=19). La atresia involutiva se observó en todos los períodos, mientras que la atresia explosiva y los folículos postovulatorios caracterizaron a P3 y P4. Las GTHs conteniendo escasos granulos LH y FSH inmunoreactivos (ir) predominaron durante Pl y P2. Las GTHs con granulos LH¡> eran abundantes en la zona intermedia y en el lóbulo caudal en P3 y P4 mientras que escasas células contenían ambos tipos de granulos. El número de células FSH¡> se incrementó durante P3 y P4. Los cambios histológicos del ovario se correlacionaron estrechamente con las variaciones en la población de gonadotropas.