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After undergoing remodeling, uterine spiral arteries turn into wide, flexible tubes, with low resistance. If remodeling does not occur, spontaneous abortions, intrauterine growth restriction, and pregnancy-related hypertensive disorders can ensue. Arterial transformation begins at a very early gestational stage; however, second quarter pregnancy histopathological samples have yet to pinpoint the exact moment when abnormal remodeling transpires. We examined 100 samples, taken from consecutive abortions at 12-23 gestational weeks. Following Pijnenborg and Smith guidelines, blinded pathologists analyzed clinical data on remodeling stages. Lab results showed that arterial remodeling is not synchronic in all vessels; a single sample can include various remodeling stages; neither is remodeling homogenous in a single vessel: change may be occurring in one part of the vessel, but not in another. To our knowledge, no one has published this finding. In the examined age group, Smith stage IV predominates; around week 14, substantial muscle and endothelium loss takes place. After week 17, endovascular or fibrin trophoblast does not usually occur. Although scant consensus exists on what defines preeclampsia etiology, it is clear that it involves abnormal remodeling in decidua vessels. Improved understanding requires further knowledge on both the physiological and pathological aspects of the remodeling process. We observed that muscle and endothelial tissues disappear from weeks 14-17, after which time reendothelization predominates. We list the expected proportion of spiral artery changes for each gestational age which, to date, has not been available.

Inhibidor de MNK1; Oncología Inhibidor de MNK1; Oncologia MNK1 inhibitor; Oncology Targeting the kinases MNK1 and MNK2 has emerged as a valuable strategy in oncology. However, most of the advanced inhibitors are acting in an adenosine triphosphate (ATP)-competitive mode, precluding the evaluation of different binding modes in preclinical settings. Using rational design, we identified and validated the 4,6-diaryl-pyrazolo[3,4-b]pyridin-3-amine scaffold as the core for MNK inhibitors. Signaling pathway analysis confirmed a direct effect of the hit compound EB1 on MNKs, and in line with the reported function of these kinases, EB1 only affects the growth of tumor but not normal cells. Molecular modeling revealed the binding of EB1 to the inactive conformation of MNK1 and the interaction with the specific DFD motif. This novel mode of action appears to be superior to the ATP-competitive inhibitors, which render the protein in a pseudo-active state. Overcoming this paradoxical activation of MNKs by EB1 represents therefore a promising starting point for the development of a novel generation of MNK inhibitors. This work was supported by the Instituto de Salud Carlos III (PI17/02247), (PI20/01687), and CIBERONC (CB16/12/00363). S.R.y.C. acknowledges support from the Generalitat de Catalunya (2017-9015-385045). E. Bou-Petit thanks the Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya (2017 FI_B2 00139) and the European Social Funds for her predoctoral fellowship.

Objective Ki67 is a prognostic and predictive marker in breast cancer (BC). However, manual scoring (MS) by visual assessment suffers from high inter-observer variability which limits its clinical use. Here, we developed a new digital image analysis (DIA) workflow, named KiQuant for automated scoring of Ki67 and investigated its equivalence with standard pathologist's assessment. Methods Sequential immunohistochemistry of Ki67 and cytokeratin, for precise tumor cell recognition, were performed in the same section of 5 tissue microarrays containing 329 tumor cores from different breast cancer subtypes. Slides were digitalized and subjected to DIA and MS for Ki67 assessment. The intraclass correlation coefficient (ICC) and Bland-Altman plot were used to evaluate inter-observer reproducibility. The Kaplan-Meier analysis was used to determine the prognostic potential. Results KiQuant showed an excellent correlation with MS (ICC:0.905,95%CI:0.878–0.926) with satisfactory inter-run (ICC:0.917,95%CI:0.884–0.942) and inter-antibody reproducibilities (ICC:0.886,95%CI:0.820–0.929). The distance between KiQuant and MS increased with the magnitude of Ki67 measurement and positively correlated with analyzed tumor area and breast cancer subtype. Agreement rates between KiQuant and MS within the clinically relevant 14% and 30% cut-off points ranged from 33% to 44% with modest interobserver reproducibility below the 20% cut-off (0.606, 95%CI:0.467–0.727). High Ki67 by KiQuant correlated with worse outcome in all BC and in the luminal subtype (P = 0.028 and P = 0.043, respectively). For MS, the association with survival was significant only in 1 out of 3 observers. Conclusions KiQuant represents an easy and accurate methodology for Ki67 measurement providing a step toward utilizing Ki67 in the clinical setting., Highlights • Automated Ki67 scoring workflow improved reproducibility. • Sequential immunohistochemistry in the same section for precise cell recognition. • Use of a tumor mask for automatic tumor region selection. • Outperform pathologist-based Ki67 scoring in prognostic prediction.

The experience of recent months in the study of the pathology associated with COVID-19 infection is showing histological alterations similar to other infections associated with previous pandemics, such as diffuse alveolar damage, signs of endotheliitis, inflammatory infiltrates and fibrin microthrombi. In the samples of patients with COVID-19, a greater number of cases with signs of endothelitis and a greater number of microthrombi stand out, both at the level of the lung parenchyma and in other locations, such as the colon, myocardium, liver … The study of the biopsies of patients who have overcome the acute phases of COVID is showing the presence of interstitial inflammatory infiltrates at the lung level and areas of fibrosis, and various factors that may predict the greater or lesser probability of the development of fibrosis are being studied pulmonary in such patients.

Objectives The human papilloma virus (HPV) test is recommended in the posttreatment follow-up of cervical intraepithelial neoplasia. The aim of the study was to assess whether the intraoperative HPV (IOP-HPV) test had a similar diagnostic accuracy that HPV test performed at 6 months to predict high-grade squamous intraepithelial lesion (HSIL) recurrence. Materials and methods In a prospective cohort study, 304 women diagnosed with HSIL by biopsy and/or endocervical curettage before treatment and/or confirmation in the histological specimen were included. Immediately after surgery, HPV testing was performed. This test was compared with the test at 6 months and other predictors of recurrence. Patients were followed for 24 months. An economic analysis was performed to compare the costs of IOP-HPV and HPV test at 6 months. Results Recurrence rate of HSIL was 6.2% (19 patients). The diagnostic accuracy of the IOP-HPV test to predict HSIL recurrence at 24 months was similar to the HPV test at 6 months, with comparative sensitivities of 100% versus 86.7%, specificities of 82.0% versus 77.9%, positive predictive values of 27.1% versus 18.1%, and negative predictive values of 100% versus 99.0%. Direct economic saving per high-grade intraepithelial lesion patient was 172.8 O. Conclusions The HPV test performed after loop electrosurgical resection procedure predicted recurrence of HSIL at 24 months with a similar diagnostic accuracy than the HPV test at 6 months. The use of the IOP-HPV test in the management of HSIL will allow early detection of the risk of recurrent disease and to save costs because of potential suppression of the need of HPV and follow-up controls at 6 months.

Objective: to assess new strategies in order to promptly diagnose small intestine diverticulitis. Methods: we thoroughly analyzed a case series of small intestine diverticulitis in order to describe three of the typical presentations of such disease (intestinal perforation, intestinal obstruction and obstruction developing perforation). A systematic review of the literature was then conducted in five different scientific databases using six different keywords. Imaging technique's sensitivity for small intestine diverticulitis was assessed, as well as the frequency of signs and symptoms of such pathology. A complete description of the signs and symptoms of acute abdomen-type pathologies was also investigated in order to provide differential traits to distinguish small intestine diverticulitis. Results: No pathognomonic signs or symptoms were detected for the pathology, although we found that all the other pathologies in the “acute abdomen” type of diseases have distinguishing traits that could be used to perform a differential diagnosis of small intestine diverticulitis. Moreover, contrast-enhanced computed tomography is the best imaging technique for the confirmation of such disease, when guided by an initial suspicion. Conclusion: Small intestine diverticulitis cannot be diagnosed by clinical exploration only, but patients that have acute abdomen characteristics, and are negative to signs and symptoms that are characteristic of the other diseases in its type should be suspected for such disease. The aforementioned suspicion may guide a better imaging technique selection and exploration through it.

Cáncer de mama; Metástasis; Pez cebra Càncer de mama; Metàstasi; Peix zebra Breast cancer; Metastasis; Zebrafish Background: Cancer metastasis is a deathly process, and a better understanding of the different steps is needed. The shedding of circulating tumor cells (CTCs) and CTC-cluster from the primary tumor, its survival in circulation, and homing are key events of the metastasis cascade. In vitro models of CTCs and in vivo models of metastasis represent an excellent opportunity to delve into the behavior of metastatic cells, to gain understanding on how secondary tumors appear. Methods: Using the zebrafish embryo, in combination with the mouse and in vitro assays, as an in vivo model of the spatiotemporal development of metastases, we study the metastatic competency of breast cancer CTCs and CTC-clusters and the molecular mechanisms. Results: CTC-clusters disseminated at a lower frequency than single CTCs in the zebrafish and showed a reduced capacity to invade. A temporal follow-up of the behavior of disseminated CTCs showed a higher survival and proliferation capacity of CTC-clusters, supported by their increased resistance to fluid shear stress. These data were corroborated in mouse studies. In addition, a differential gene signature was observed, with CTC-clusters upregulating cell cycle and stemness related genes. Conclusions: The zebrafish embryo is a valuable model system to understand the biology of breast cancer CTCs and CTC-clusters. This work was supported by Roche-Chus Joint Unit (IN853B 2018/03) funded by Axencia Galega de Innovación (GAIN), Consellería de Economía, Emprego e Industria. I.M.-P. is funded by the Training Program for Academic Staff fellowship (FPU16/01018), from the Ministry of Education and Vocational Training, Spanish Government. P.H. is funded by a Predoctoral fellowship (IN606A-2018/019) from Axencia Galega de Innovación (GAIN, Xunta de Galicia). N.C.-U. is funded by Axudas Predoutorais do IDIS (Instituto de Investigación Sanitaria de Santiago).

Introduction Despite all treatment advances, lung cancer is still the main cause of death worldwide. Treatment for resectable stage IIIA remains controversial including definitive chemoradiotherapy and induction treatment followed by surgery. After definitive chemoradiation up to 35% of patients will relapse locally. Experience with salvage resection after definitive chemoradiotherapy in lung cancer is limited. We present our experience in 27 patients who underwent surgical resection after definitive treatment. Patients and methods Between January 2007 and December 2016, 27 patients were evaluated in our department for surgical resection after receiving definitive chemoradiation treatment in different institutions. We conducted a retrospective study gathering the following data: age, gender, clinical and pathologic stage, histology, chemotherapy treatment regimen, radiotherapy dosage, surgical procedure and complications. Time between surgical resection and last follow-up was used to calculate Overall Survival (OS). Disease-Free Survival (DFS) was calculated from surgical resection to diagnosis of relapse. Results Most of the patients were men with a median age of 56.09 years. Median follow-up time was 46.94 months. All patients received platinum-based chemotherapy regimen and high-dose radiotherapy, except for one patient who received 45 Gy. Lobectomy and bilobectomy was performed in 7 patients each, and pneumonectomy in 13. Complications appeared in 5 patients. Bronchopleural fistula appeared in two patients, and only one death in the early postoperative period. The analysis showed an OS of 75.56 months, with 1-year, 3-year and 5-year survival of 74.1%, 57.8% and 53.3% respectively. Conclusion Salvage surgery in selected patients is technically feasible, with low morbidity and mortality rates and good long-term outcomes.

Background and objectives Although there is scientific evidence demonstrating causation of human papilloma virus (HPV) on squamous cell carcinoma of head and neck, its percentage of causality on the anatomic region remains in dispute. This study was developed with the objectives of evaluating the relationship between HPV and oral and oropharyngeal squamous cell carcinomas (OOSCC), and of studying the correlation between HPV detection tests (PCR and p16). Material and method Retrospective study of patients treated for OOSCC during 2011, with a follow-up of 6 years. The sample was divided into 2 groups according to HPV positivity, detected by 2 techniques: p16 by immunohistochemistry and PCR. Demographic and clinical variables were analysed using SPSS® 22.0, considering p Results We analysed 155 patients affected by OOSCC (mean age of 62.7, where 69% were males). Twenty six cases were p16+ (16.8%) and 19 cases PCR+ (12.3%), The HPV+ tumours were located predominantly in the oropharynx (42.1%, p = 0.017) and demonstrated the tendency to be more frequent in males, with higher incidence in younger patients, lower in smokers and drinkers, and higher when patients have a greater cervical lymph node involvement at the time of diagnosis. The PCR+ patients had higher survival (p = 0.024), as did the p16+ (p = 0.005). Conclusions The incidence of HPV in OOSCC is low (12.3%), but the clinical presentation and prognosis of the HPV+ patient differs from the classic smoker and/or drinker, which implies assessing the management of these patients independently. The p16 staining has a great diagnostic capacity to predict HPV (95.5%), although the detection of the HPV genome is still the gold standard technique.

Resumen Antecedentes y objetivos Aunque existe evidencia cientifica que demuestra la relacion de causalidad del virus papiloma humano (VPH) sobre el carcinoma escamoso de cabeza y cuello, su porcentaje de causalidad en las distintas regiones anatomicas permanece todavia en controversia. El presente estudio tiene como objetivos evaluar la relacion del VPH con el carcinoma escamoso de cavidad oral y orofaringe (CECOO) en nuestra poblacion de referencia, y estudiar la correlacion entre diferentes pruebas de deteccion del VPH basadas en metodos de PCR e inmunohistoquimica. Material y metodo Estudio retrospectivo en pacientes tratados de CECOO durante el ano 2011, con un seguimiento de 6 anos. La muestra se dividio en 2 grupos segun la positividad a VPH, detectado mediante 2 tecnicas: p16 por inmunohistoquimica y PCR. Se analizaron variables demograficas y clinicas mediante SPSS® 22.0, considerando una significacion estadistica con p Resultados Se analizaron 155 pacientes afectos de CECOO (edad media de 62,7 anos y un 69% varones). Veintiseis casos resultaron p16+ (16,8%) y 19 casos PCR+ (12,3%). Los tumores VPH+ se localizaron predominantemente en orofaringe (42,1%; p = 0,017) y mostraron una tendencia a ser mas frecuentes en el sexo masculino, mayor incidencia en pacientes mas jovenes, menos en fumadores y bebedores, y mayor afectacion ganglionar cervical en el momento diagnostico. Los pacientes PCR+ presentaron mayor supervivencia (p = 0,024), al igual que los p16+ (p = 0,005). Conclusiones La incidencia de VPH en CECOO en nuestro entorno actualmente es baja (12,3%), pero la presentacion clinica y el pronostico del paciente VPH+ difiere del clasico paciente fumador y/o bebedor, lo que implica valorar el manejo de estos pacientes de forma independiente. La tincion inmunohistoquimica para p16 tiene una gran capacidad diagnostica para predecir el VPH (95,5%), aunque la herramienta de referencia sigue siendo la deteccion de secuencias del genoma del VPH.

One of the most severe complications after successful cancer therapy is the development of therapy-related myeloid neoplasms (t-MN). Constitutional genetic variation is likely to impact on t-MN risk. We aimed to evaluate if polymorphisms in the p53 pathway can be useful for predicting t-MN susceptibility. First, an association study revealed that the Pro variant of the TP53 Arg72Pro polymorphism and the G allele of the MDM2 SNP309 were associated with t-MN risk. The Arg variant of TP53 is more efficient at inducing apoptosis, whereas the Pro variant is a more potent inductor of cell cycle arrest and DNA repair. As regards MDM2 SNP309, the G allele is associated with attenuation of the p53 apoptotic response. Second, to evaluate the biological effect of the TP53 polymorphism, we established Jurkat isogenic cell lines expressing p53Arg or p53Pro. Jurkat p53Arg cells presented higher DNA damage and higher apoptotic potential than p53Pro cells, after treatment with chemotherapy agents. Only p53Pro cells presented t(15;17) translocation and del(5q). We suggest that failure to repair DNA lesions in p53Arg cells would lead them to apoptosis, whereas some p53Pro cells, prone to cell cycle arrest and DNA repair, could undergo misrepair, generating chromosomal abnormalities typical of t-MN.

Gastric cancer constitutes the third leading cause of cancer mortality worldwide and tumors with diffuse histology have poor prognosis and low response rates to therapy. Missense mutations in the RHOA (Ras homolog family member A) GTPase have recently been identified in 24% of patients with diffuse gastric cancer. RHOA mutations are not randomly distributed along the coding sequence, but enriched in specific and recurrent hotspots. Substitution in codon 42 (Y42C) occurs in approximately 50% of the patients in which RHOA is mutated. RHOA orchestrates cellular processes such as proliferation, differentiation, migration and invasion, whose deregulation is essential for the onset, maintenance and progression of tumors. However, the functional role of wild type RHOA and RHOA mutants remains poorly characterized in gastric cancer. We used isogenic cell lines systems and animal models to study the impact of RHOA in the gastric tumorigenesis. RHOA loss strongly increased the proliferation, migration and invasion of diffuse gastric cancer cells in vitro, but also when growing as subcutaneous xenografts in immunodeficient mice, and in experimental mouse models of peritoneal and lung metastasis. Stomach tumor burden was also increased in transgenic mice conditionally expressing a dominant negative form of RHOA in the gastrointestinal tract (RHOA T19N). Moreover, cells overexpressing wild-type RHOA or G14V RHOA (a constitutive active form of the GTPase) exhibited reduced tumorigenic features both in vitro and in vivo. Conversely, diffuse gastric cancer cells engineered for overexpressing RHOA Y42C mutant, displayed enhanced growth and invasion in vitro. Importantly, stomach cancer tumors formed more efficiently in a transgenic mouse model in which we targeted the expression of Y42C RHOA mutant to the gastric epithelium. Collectively, these results demonstrate that RHOA has tumor-suppressive activity in diffuse gastric cancer, while the recurrent Y42C hotspot RHOA mutation found in these tumors is oncogenic. Citation Format: Agueda Martinez-Barriocanal, Higinio Dopeso, Lizbeth M. Jiménez-Flores, Juliana C. Santos, Estefania Anguita, Josipa Bilic, Rocio Nieto, Elia García-Vidal, Manuel Sánchez Martín, Stefania Landolfi, Kazuto Kobayashi, Javier Hernandez-Losa, Simo Schwartz, Santiago Ramón y Cajal, Diego Arango. Role of RHOA in diffuse gastric cancer tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 847.

Background: Non-small cell lung cancer (NSCLC) is usually diagnosed at stages IIIB-IV, with a median overall survival that does not exceed two years. In contrast, patients diagnosed at early and locally advanced stages (I-IIIA) can undergo surgery and have a significantly better prognosis. Imaging technologies often detect lung nodules of unknown significance that pose a diagnostic challenge. In a proof-of-concept study, based on a 76-patient cohort, we developed a preliminary mRNA expression signature in plasma that discriminated healthy individuals from early-stage NSCLC patients with AUC=0.98. Here, we aimed to expand the training cohort, to refine the diagnostic signature and to prospectively validate the final signature in the clinical setting. Methods: Two hundred and thirty individuals with pulmonary nodules suspicious of lung cancer have been enrolled in the training cohort. All of them underwent bronchoscopy, fine needle aspiration, percutaneous or surgical biopsy to confirm the diagnosis. Circulating-free RNA (cfRNA) has been isolated from plasma using an automatic extraction method (Qiasymphony, Qiagen). Purified cfRNA has been quantified using Qubit®, retrotranscribed and pre-amplified with 14 cycles using the Low RNA Input Amplification kit (NanoString Technologies). Gene expression analysis has been performed on the nCounter platform using the PanCancer IO360࣪ panel (NanoString Technologies), which can detect 770 transcripts related to tumor biology, micro-environment and the immune system. Results: One hundred twenty-six patients have been analyzed so far; plasma samples have been successfully analyzed by nCounter in all cases. Ongoing analysis reveal differential patterns of gene expression in early-stage NSCLC patients versus non-cancer individuals. Using a bioinformatics recursive feature elimination algorithm, we have selected a diagnostic signature with an area under the ROC curve of 0.89. The signature scores derived from the algorithm are significantly different between the non-cancer and NSCLC cases. Final results of the training and validation cohort will be presented at the meeting Conclusions: Plasma RNA expression signatures can be a useful tool to guide clinical decision in patients with pulmonary nodules suspicious of malignancy, orienting towards surgery or observation. Citation Format: Ana María Giménez Capitán, Pablo Rubisntein, Andrés Aguilar-Hernández, María González-cao, Irene Moya, Santiago Viteri, Carlos Cabrera, Santiago Ramón y Cajal, Karina Loor, Mario Culebras, Irene Sansano, Federico Rubisntein, Joselyn Valarezo, Clara Mayo-de las-Casas, Carlos Pedraz, Joseph Beechem, Sarah Warren, Rafael Rosell, Miguel Ángel Molina-Vila. Prospective validation of a mRNA signature in plasma for the diagnosis of early stage lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1424.

SUMMARY To inform on the correlates of protection against SARS-CoV-2, we studied T cell functions, migration patterns and apoptosis associated with antigen responses in three groups of patients during acute infection. T cell functional profiles against SARS-CoV-2 depended on the targeted viral protein and clinical outcome. Hospitalization and disease severity were associated with predominant IFNg and IL-4 responses, as well as increased responses against S peptides and apoptosis, while non-hospitalized patients were characterized by IL-10 secretion, to which a particular subset expressing high levels of CCR7 contributed abundantly. Importantly, lung-resident memory T cells were strongly detected in convalescent patients, in which contemporary blood did not reflect tissue resident profiles. Our results suggest that a balanced anti-inflammatory antiviral response promoted by non-spike proteins may be key to favor infection resolution without major complications. Still, these immune responses can migrate and establish in the lung as resident memory T cells, affecting future protection.

Alzheimer; CD2AP; Enfermedad de Pick Alzheimer's disease; CD2AP; Pick's disease Alzheimer; CD2AP; Malaltia de Pick Genome-wide association studies have described several genes as genetic susceptibility loci for Alzheimer's disease (AD). Among them, CD2AP encodes CD2-associated protein, a scaffold protein implicated in dynamic actin remodeling and membrane trafficking during endocytosis and cytokinesis. Although a clear link between CD2AP defects and glomerular pathology has been described, little is known about the function of CD2AP in the brain. The aim of this study was to analyze the distribution of CD2AP in the AD brain and its potential associations with tau aggregation and β-amyloid (Aβ) deposition. First, we performed immunohistochemical analysis of CD2AP expression in brain tissue from AD patients and controls (N = 60). Our results showed granular CD2AP immunoreactivity in the human brain endothelium in all samples. In AD cases, no CD2AP was found to be associated with Aβ deposits in vessels or parenchymal plaques. CD2AP neuronal inclusions similar to neurofibrillary tangles (NFT) and neuropil thread-like deposits were found only in AD samples. Moreover, immunofluorescence analysis revealed that CD2AP colocalized with pTau. Regarding CD2AP neuronal distribution, a hierarchical progression from the entorhinal to the temporal and occipital cortex was detected. We found that CD2AP immunodetection in neurons was strongly and positively associated with Braak neurofibrillary stage, independent of age and other pathological hallmarks. To further investigate the association between pTau and CD2AP, we included samples from cases of primary tauopathies (corticobasal degeneration [CBD], progressive supranuclear palsy [PSP], and Pick's disease [PiD]) in our study. Among these cases, CD2AP positivity was only found in PiD samples as neurofibrillary tangle-like and Pick body-like deposits, whereas no neuronal CD2AP deposits were detected in PSP or CBD samples, which suggested an association of CD2AP neuronal expression with 3R-Tau-diseases. In conclusion, our findings open a new road to investigate the complex cellular mechanism underlying the tangle conformation and tau pathology in the brain. This work was funded by Instituto de Salud Carlos III (ISCIII) (PI17/00275, PI20/00465), cofinanced by the European Regional Development Fund (FEDER). The Neurovascular Research Laboratory is part of the INVICTUS+ network, ISCIII, Spain (RD16/0019/0021). M.H.-G. is supported by the Miguel Servet Programme, ISCIII, Spain (CPII17/00010)

Zika virus (ZikV) is a flavivirus that infects neural tissues, causing congenital microcephaly. ZikV has evolved multiple mechanisms to restrict proliferation and enhance cell death, although the underlying cellular events involved remain unclear. Here we show that the ZikV-NS5 protein interacts with host proteins at the base of the primary cilia in neural progenitor cells, causing an atypical non-genetic ciliopathy and premature neuron delamination. Furthermore, in human microcephalic fetal brain tissue, ZikV-NS5 persists at the base of the motile cilia in ependymal cells, which also exhibit a severe ciliopathy. Although the enzymatic activity of ZikV-NS5 appears to be dispensable, the amino acids Y25, K28, and K29 that are involved in NS5 oligomerization are essential for localization and interaction with components of the cilium base, promoting ciliopathy and premature neurogenesis. These findings lay the foundation for therapies that target ZikV-NS5 multimerization and prevent the developmental malformations associated with congenital Zika syndrome., The work in E.M.’s laboratory was supported by grants BFU2016-77498-P, BFU2016-77498-P, and La Maratò de TV3 foundation 201833-10. M.S. holds a Ramón Y Cajal fellowship (RYC2018-025379-I). J.B.-A is a recipient of a BES-2017-080050 PhD scholarship. The work in N.V.’s laboratory was supported by grants BIO2017-83906-P, Maria de Maeztu Unit of Excellence MDM-2014-0435 (MCIU), and La Maratò de TV3 foundation 201833-10. The work in N.A.’s laboratory was supported by NIH/NINDS R00NS089859 and IDDRC-NPDA (CHOP/Penn).

[Background] Cancer is a rapidly evolving, multifactorial disease that accumulates numerous genetic and epigenetic alterations. This results in molecular and phenotypic heterogeneity within the tumor, the complexity of which is further amplified through specific interactions between cancer cells. We aimed to dissect the molecular mechanisms underlying the cooperation between different clones., [Methods] We produced clonal cell lines derived from the MDA-MB-231 breast cancer cell line, using the UbC-StarTrack system, which allowed tracking of multiple clones by color: GFP C3, mKO E10 and Sapphire D7. Characterization of these clones was performed by growth rate, cell metabolic activity, wound healing, invasion assays and genetic and epigenetic arrays. Tumorigenicity was tested by orthotopic and intravenous injections. Clonal cooperation was evaluated by medium complementation, co-culture and co-injection assays., [Results] Characterization of these clones in vitro revealed clear genetic and epigenetic differences that affected growth rate, cell metabolic activity, morphology and cytokine expression among cell lines. In vivo, all clonal cell lines were able to form tumors; however, injection of an equal mix of the different clones led to tumors with very few mKO E10 cells. Additionally, the mKO E10 clonal cell line showed a significant inability to form lung metastases. These results confirm that even in stable cell lines heterogeneity is present. In vitro, the complementation of growth medium with medium or exosomes from parental or clonal cell lines increased the growth rate of the other clones. Complementation assays, co-growth and co-injection of mKO E10 and GFP C3 clonal cell lines increased the efficiency of invasion and migration., [Conclusions] These findings support a model where interplay between clones confers aggressiveness, and which may allow identification of the factors involved in cellular communication that could play a role in clonal cooperation and thus represent new targets for preventing tumor progression., SRYC acknowledges support from Fondo de Investigaciones Sanitarias (FIS; PI17/02247 and PI14/01320), Centro de Investigación Biomédica en Red de Cáncer (CIBERONC; CB16/12/00363) and Generalitat de Catalunya (AGAUR; 2017 SGR 1799 and 2014 SGR 1131). AMP is funded by a FP7 Marie Sklodowska-Curie COFUND program under Grant Agreement n° 267128 (INCOMED program). ADL is funded by a “Juan Rodés” contract (JR17/00016) from ISCIII.

Simple Summary The gap junction protein connexin 43 (Cx43) facilitates direct intercellular communication and displays a complex dichotomous role in cancer. Here, we used breast cancer cell lines and CRISPR/Cas9 technology to dissect some of the poorly understood multimodal functions of Cx43 in this setting. Our data show that Cx43 directly modulates the formation of pro-tumorigenic tunneling nanotubes (TNTs), which are cellular protrusions that connect non-adjacent cells to facilitate cellular communication, including the direct cell–cell transfer of organelles. We identify several important cancer signaling pathways that affect TNT formation and show that this effect is strongly influenced by the presence or absence of Cx43. Finally, we demonstrate that Cx43 can affect TNT formation by modulating the cellular secretome. This work provides important insight into the pro-tumorigenic role of Cx43 and its interconnections with TNTs. Abstract Connexin 43 (Cx43) forms gap junctions that mediate the direct intercellular diffusion of ions and small molecules between adjacent cells. Cx43 displays both pro- and anti-tumorigenic properties, but the mechanisms underlying these characteristics are not fully understood. Tunneling nanotubes (TNTs) are long and thin membrane projections that connect cells, facilitating the exchange of not only small molecules, but also larger proteins, organelles, bacteria, and viruses. Typically, TNTs exhibit increased formation under conditions of cellular stress and are more prominent in cancer cells, where they are generally thought to be pro-metastatic and to provide growth and survival advantages. Cx43 has been described in TNTs, where it is thought to regulate small molecule diffusion through gap junctions. Here, we developed a high-fidelity CRISPR/Cas9 system to knockout (KO) Cx43. We found that the loss of Cx43 expression was associated with significantly reduced TNT length and number in breast cancer cell lines. Notably, secreted factors present in conditioned medium stimulated TNTs more potently when derived from Cx43-expressing cells than from KO cells. Moreover, TNT formation was significantly induced by the inhibition of several key cancer signaling pathways that both regulate Cx43 and are regulated by Cx43, including RhoA kinase (ROCK), protein kinase A (PKA), focal adhesion kinase (FAK), and p38. Intriguingly, the drug-induced stimulation of TNTs was more potent in Cx43 KO cells than in wild-type (WT) cells. In conclusion, this work describes a novel non-canonical role for Cx43 in regulating TNTs, identifies key cancer signaling pathways that regulate TNTs in this setting, and provides mechanistic insight into a pro-tumorigenic role of Cx43 in cancer.

Metastasis, the spread of malignant cells from a primary tumour to distant sites, causes 90% of cancer-related deaths. The integrin ITGB3 has been previously described to play an essential role in breast cancer metastasis, but the precise mechanisms remain undefined. We have now uncovered essential and thus far unknown roles of ITGB3 in vesicle uptake. The functional requirement for ITGB3 derives from its interactions with heparan sulfate proteoglycans (HSPGs) and the process of integrin endocytosis, allowing the capture of extracellular vesicles and their endocytosis-mediated internalization. Key for the function of ITGB3 is the interaction and activation of focal adhesion kinase (FAK), which is required for endocytosis of these vesicles. Thus, ITGB3 has a central role in intracellular communication via extracellular vesicles, proposed to be critical for cancer metastasis., The integrin ITGB3 has been described to play an essential role in breast cancer metastasis, but the precise mechanisms remain undefined. Here the authors describe thus far unknown roles of ITGB3 in the uptake of extracellular vesicles, required for colony growth of breast cancer cells.

We present 783 surgical resections of typical and atypical carcinoid tumors of the lung identified in the pathology files of 20 different pathology departments. All cases were critically reviewed for clinical and pathological features and further correlated with clinical outcome. Long-term follow-up was obtained in all the patients and statistically analyzed to determine significance of the different parameters evaluated. Of the histopathological features analyzed, the presence of mitotic activity of 4 mitoses or more per 2mm2, necrosis, lymphatic invasion and lymph node metastasis were identified as statistically significant. Tumors measuring 3 cm or more were also identified as statistically significant and correlate with clinical outcome. Based on our analysis, we consider that the separation of low and intermediate grade neuroendocrine neoplasms of the lung needs to be readjusted in terms of mitotic count as the risk of over grading these neoplasms exceeds 10% under the current criteria. We also consider that tumor size is an important feature to be considered in the assessment of these neoplasms and together with the histological grade of the tumor offers important features that can be correlated with clinical outcome.

Thirteen cases of primary pulmonary adenofibromas are presented. The patients are 8 women and 5 men between the ages of 41 and 73 years (average: 57 y). The patients presented with nonspecific symptomatology or their tumor was identified during routine chest films. A wedge resection was performed in all cases with lymph node sampling. Grossly, the tumors varied in size from 1 to 2.5 cm in greatest dimension. The entire tumor was histologically evaluated in all cases. All the tumors shared similar histologic features namely leaf-like/phyllodes-like growth patterns with varying areas of sclerosis, focal inflammation, and entrapped epithelium. A wide panel of immunohistochemical studies was performed including epithelial, neural, muscle, and vascular markers, all of which showed negative staining. The tumors were positive only for vimentin in the stroma and keratin in the entrapped epithelium. Further evaluation in 6 cases using in situ hybridization for the solitary fibrous tumor was performed and was negative. Clinical follow-up in all the patients showed no evidence of recurrence or metastatic disease, during a period of 12 to 36 months. The current cases highlight the unusual occurrence of pulmonary adenofibromas and the importance of separating these tumors from other tumors that may have the potential to recur or metastasize. The use of proper immunohistochemical stains/molecular analysis aids in the proper classification of these tumors.

Approximately 30% of strokes are cryptogenic despite an exhaustive in-hospital work-up Analysis of clot composition following endovascular treatment could provide insight into stroke etiology. Objective: To evaluate different population of lymphocytes in the clot analyses obtained in the hyper acute phase of stroke, as an early marker of etiology of ischemic stroke. Material and Methods: Consecutively, intracranial clots were obtained in the hyper acute phase of stroke in endovascular treatment with solitaire device and analyzed by flow cytometry quantifying the relative proportion ( RP ) of main leukocyte, monocyte, granulocyte and total lymphocyte populations and lymphocyte sub populations of B line (CD19), T line (CD3, CD4, CD8), Cytotoxic and Natural Killer. The results were expressed as percentages (%). All patients included were studied with complete diagnostic work up that included at least complete arteriography , aortic Angio CT , complete cardiac holter monitoring until one month form hospital discharge and transthoracic echocardiography , The etiology of stroke was categorized: cardiomebolic ( major structural heart disease and Atrial Fibrillation) atherosclerotic ( AT) (severe symptomatic arterial stenosis or complicated aortic atheromatosis ulceration confirmed by trans esophageal echocardiography ) ,infrequent causes or unknown etiology . Results: 80 patients were included. 22, 5% AT (n = 18) strokes were associated with higher RP lymphocytes PR (16.55 % vs. 5. 37 % p < 0,001), CD4 RP (5.74% vs. 2.03 % p = 0.001) and RP CD 19 (2.26 % vs. 0.37 % p< 0,001) in clot cytometry Analysis than rest of of strokes etiologies (n= 72). Multivariate logistic regression analysis showed that RP CD 19 (OR 3.51; 95% CI, 1, 81-6.81) was an independent predictor of atherosclerotic stroke .The ROC curve showed AUC (0.83) (95% CI, 0.72-0.95) for RP CD 19 for diagnosis of atherosclerotic strokes with flow cytometry of the clot ,obtained in emergent endovascular treatment. Conclusion: High expression of lymphocytes B in intracranial thrombi is a marker of atherosclerotic etiology.

Introduction Axillary staging (pN) is a strong predictor of outcome in early stage breast cancer yet following the publication of the Z0011 trial there has been an increasing tendency to spare lymph node dissection. Automated molecular detection of cytokeratin 19mRNA by one-step nucleic acid amplification (OSNA) has been demonstrated to be an accurate method to assess sentinel lymph node (SLN) metastasis. In this study we compare histological and molecular methods following complete axillary lymph node dissection (cALND), determine whether molecular axillary staging affects survival, and evaluate the predictive and prognostic value of total tumor load in ALND (AD-TTL) and in all positive nodes (G-TTL). Material and methods Axillary lymph nodes were collected from 102 patients with primary breast cancer with histological confirmation of axillary involvement (cN+) or positive SLN. The central 1-mm portion of each non-SLN was processed for hematoxylin-eosin staining and the remaining tissue was analyzed by OSNA. Results Non-SLNs were diagnosed as positive in 72 out of 102 patients (70.6 %) on OSNA compared with only 53 (52 %) on histology (p < 0.01). Thirteen patients would have changed staging if the diagnoses provided had been by molecular methods (p < 0.01), but without a change in prognosis. AD-TTL and G-TTL were predictive of recurrence and mortality. Conclusions Compared to molecular detection, histological examination significantly underestimates the frequency of axillary node metastases. However, the increase in pN did not show a clinical effect on survival in those patients., This study was supported by a Grant from Sysmex España S.L. The sponsor had no role in the study design, analysis, or interpretation of the data.

Pyrazol-3-amine is a scaffold present in a large number of compounds with a wide range of biological activities and, in many cases, the heterocycle is C4–C5 fused to a second ring. Among the different reactions used for the decoration of the pyrazole ring, Ullmann and acylation have been widely applied. However, there is some confusion in the literature regarding the regioselectivity of such reactions (substitution at N1 or N2 of the pyrazole ring) and no predictive rule has been so far established. As a part of our work on 3-amino-pyrazolo[3,4-b]pyridones 13, we have studied the regioselectivity of such reactions in different C4–C5 fused pyrazol-3-amines. As a rule of thumb, the Ullmann and acylation reactions take place, predominantly, at the NH and non-protonated nitrogen atom of the pyrazole ring respectively, of the most stable initial tautomer (1H- or 2H-pyrazole), which can be easily predicted by using DFT calculations.

Heterogeneidad intratumoral; Inteligencia artificial; Terapias antitumorales Heterogeneïtat intratumoral; Intel·ligència artificial; Teràpies antitumorals Intratumor heterogeneity; Artificial intelligence; Antitumor therapeutics In this review, we highlight the role of intratumoral heterogeneity, focusing on the clinical and biological ramifications this phenomenon poses. Intratumoral heterogeneity arises through complex genetic, epigenetic, and protein modifications that drive phenotypic selection in response to environmental pressures. Functionally, heterogeneity provides tumors with significant adaptability. This ranges from mutual beneficial cooperation between cells, which nurture features such as growth and metastasis, to the narrow escape and survival of clonal cell populations that have adapted to thrive under specific conditions such as hypoxia or chemotherapy. These dynamic intercellular interplays are guided by a Darwinian selection landscape between clonal tumor cell populations and the tumor microenvironment. Understanding the involved drivers and functional consequences of such tumor heterogeneity is challenging but also promises to provide novel insight needed to confront the problem of therapeutic resistance in tumors. SRYC received support from Fondo de Investigaciones Sanitarias (PI14/01320 and PI17/02247), Redes Temáticas de Investigación Cooperativa en Salud (RD12/0036/0057), CIBERONC (CB16/12/00363), and Generalitat de Catalunya (AGAUR, 2017 SGR 1799 and 2014 SGR 1131). TA received support from Instituto de Salud Carlos III (grants PI16/00772 and CPII16/00042), co-financed by the European Regional Development Fund (ERDF).

Resumen Los hibernomas cardiacos son muy infrecuentes, y generalmente son infra-diagnosticados pre-mortem debido a su similitud clinico-radiologica con otras lesiones intracardiacas ocupantes de espacio. Ademas, los hibernomas cardiacos pueden ser asintomaticos y pueden escapar a la deteccion en los estudios radiologicos. Presentamos el caso de una mujer de 81 anos quien fallecio como consecuencia de embolismo tumoral pulmonar. Reportamos este caso con el fin de familiarizar a residentes y patologos con las neoplasias intracardiacas. Se discute su dificultad diagnostica, ademas de la embolia tumoral pulmonar como complicacion de las neoplasias intracardiacas.

Current anticancer paradigms largely target driver mutations considered integral for cancer cell survival and tumor progression. Although initially successful, many of these strategies are unable to overcome the tremendous heterogeneity that characterizes advanced tumors, resulting in the emergence of resistant disease. Cancer is a rapidly evolving, multifactorial disease that accumulates numerous genetic and epigenetic alterations. This results in wide phenotypic and molecular heterogeneity within the tumor, the complexity of which is further amplified through specific interactions between cancer cells and the tumor microenvironment. In this context, cancer may be perceived as an "ecomolecular" disease that involves cooperation between several neoplastic clones and their interactions with immune cells, stromal fibroblasts, and other cell types present in the microenvironment. This collaboration is mediated by a variety of secreted factors. Cancer is therefore analogous to complex ecosystems such as microbial consortia. In the present article, we comment on the current paradigms and perspectives guiding the development of cancer diagnostics and therapeutics and the potential application of systems biology to untangle the complexity of neoplasia. In our opinion, conceptualization of neoplasia as an ecomolecular disease is warranted. Advances in knowledge pertinent to the complexity and dynamics of interactions within the cancer ecosystem are likely to improve understanding of tumor etiology, pathogenesis, and progression. This knowledge is anticipated to facilitate the design of new and more effective therapeutic approaches that target the tumor ecosystem in its entirety.

Endometrial cancer (EC) is the fourth most common cancer in women in developed countries. The identification of sensitive and specific biomarkers to improve early detection of EC is crucial for an appropriate management of this disease, in which 30% of patients are diagnosed only at advanced stages, which is associated with high levels of morbidity and mortality. Despite major efforts and investments made to identify EC biomarkers, no protein has yet reached the stage of clinical application. Areas covered: This review gathers the numerous candidate biomarkers for EC diagnosis proposed in proteomic studies published from 1978 to 2017. Additionally, we summarize limitations associated with the proteomic technologies and study designs employed in those articles. Finally, we address new perspectives in EC biomarker research, including the comprehensive knowledge of previously suggested candidate biomarkers in conjunction with novel mass spectrometry-based proteomic technologies with enhanced sensitivity and specificity not yet applied to EC studies and a directed clinical perspective in the study design. Expert commentary: These ingredients could be the recipe to accelerate the application of protein biomarkers in the clinic.

Introduction Lymphadenectomy is recommended during surgery for papillary thyroid carcinoma when there is evidence of cervical lymph node metastasis (therapeutic) or in high-risk patients (prophylactic) such as those with T3 and T4 tumors of the TNM classification. Selective sentinel lymph node biopsy may improve preoperative diagnosis of nodal metastases. Objective To analyze the results of selective sentinel lymph node biopsy in a group of patients with papillary thyroid carcinoma and no evidence of nodal involvement before surgery. Patients and method A retrospective, single-center study in patients with papillary thyroid carcinoma and no clinical evidence of lymph node involvement who underwent surgery between 2011 and 2013. The sentinel node was identified by scintigraphy. When the sentinel node was positive, the affected compartment was removed, and when sentinel node was negative, central lymph node dissection was performed. Results Forty-three patients, 34 females, with a mean age of 52.3 (±17) years, were enrolled. Forty-six (27%) of the 170 SNs resected from 24 (55.8%) patients were positive for metastasis. In addition, 94 (15.6%) out of the 612 lymph nodes removed in the lymphadenectomies were positive for metastases. Twelve of the 30 (40%) low risk patients (cT1N0 and cT2N0) changed their stage to pN1, whereas 12 of 13 (92%) high risk patients (cT3N0 and cT4N0) changed to pN1 stage. Conclusions Selective sentinel lymph node biopsy changes the stage of more than 50% of patients from cN0 to pN1. This confirms the need for lymph node resection in T3 and T4 tumors, but reveals the presence of lymph node metastases in 40% of T1–T2 tumors.

Resumen Introduccion La linfadenectomia en la cirugia del carcinoma papilar de tiroides se aconseja cuando hay evidencia de metastasis ganglionar cervical (terapeutica) o en pacientes de alto riesgo (profilactica), como en los tumores T3 y T4 de la clasificacion TNM. La tecnica de la biopsia selectiva del ganglio centinela puede mejorar el diagnostico prequirurgico de las metastasis ganglionares. Objetivo Analizar el resultado de la biopsia selectiva del ganglio centinela en un grupo de pacientes con carcinoma papilar de tiroides T sin evidencia de afectacion ganglionar antes de la cirugia. Pacientes y metodo Estudio retrospectivo, unicentrico en el que se incluyeron los pacientes intervenidos entre los anos 2011-2013 que fueran clinicamente N0. La identificacion del ganglio centinela se realizo mediante tecnica isotopica. En todos los casos, se practico linfadenectomia del compartimento afecto si el ganglio centinela era positivo, y del compartimento central en caso de ganglio centinela negativo. Resultados Se incluyeron 43 pacientes, 34 mujeres, con una edad media de 52,3 (±17) anos. De los 170 ganglios centinela resecados, 46 (27%) fueron positivos para metastasis, que correspondian a 24 (55,8%) pacientes. En las linfadenectomias se resecaron 612 ganglios. De ellos, 96 (15,6%) fueron positivos para metastasis. Doce de los treinta (40%) pacientes cT1N0 y cT2N0 pasaron a pN1 tras la biopsia selectiva del ganglio centinela, mientras que 12 de los 13 (92%) pacientes cT3N0 y cT4N0, acabaron siendo pN1. Conclusiones La biopsia selectiva del ganglio centinela recalifica mas del 50% de pacientes de cN0 a pN1. Se confirma la necesidad de vaciamiento ganglionar en los tumores T3 y T4, pero pone al descubierto la presencia de metastasis linfaticas en el 40% de los T1-T2.

Resumen La salpingitis xantogranulomatosa es un proceso inflamatorio poco frecuente, caracterizado por un infiltrado de la mucosa por tejido inflamatorio de granulacion conteniendo histiocitos de citoplasma espumoso. Este tipo de inflamacion es excepcional a nivel del tracto genital femenino y clasicamente se ha asociado a infecciones bacterianas cronicas, inmunosupresion u otras causas de inflamacion cronica. Pero tambien se ha visto en relacion con el carcinoma endometrial, la endometriosis, cuerpos extranos y enfermedad inflamatoria pelvica cronica. El hecho de que acompane o no a endometriosis genital ha llevado a la diferenciacion de 2 tipos de entidades: la salpingitis xantogranulomatosa, sin endometriosis acompanante, y la salpingiosis pseudoxantomatosa. Presentamos 2 casos con su histologia e inmunohistoquimica, y se ha revisado la literatura sobre este tipo de inflamaciones cronicas.

Background Axillary staging (pN) is considered one of the most important prognostic factors in breast cancer patients. However, the Z0011 study data drastically reduced the number of surgical axillary dissections in a selected group of patients, limiting the prognostic information relating to axillary involvement to the sentinel lymph node (SLN). It is known that there is a relationship between SLN total tumour load (TTL) and axillary involvement. The objective of this study is to analyse the relationship between the TTL and outcomes in patients with early stage breast cancer. Patients and methods clinicopathological and follow-up data were collected from 950 patients with breast cancer between 2009 and 2010 on whom SLN analysis was conducted by molecular methods (One Step Nucleic Acid Amplification, Sysmex, Kobe, Japan). Results TTL (defined as the total number of CK19 mRNA copies in all positive SLN) correlates with disease free survival (HR, 1.08; p = 0.000004), with local recurrence disease free survival (HR = 1.07; p = 0.0014) and overall survival (HR: 1.08, p = 0.0032), clearly defining a low-risk group (TTL 4 CK19 mRNA copies/μL) versus a high-risk group (>2.5 × 10 4 CK 19 mRNA copies/μL). Conclusions SLN TTL permits the differentiation between two patient groups in terms of DFS and OS, independently of axillary staging (pN), age and tumour characteristics (size, grade, lymphovascular invasion). This new data confirms the clinical value of low axillary involvement and could partially replace the information that staging of the entire axilla provides in patients on whom no axillary lymph node dissection is performed.

Zika virus (ZikV) is a flavivirus that infects neural tissues, causing congenital microcephaly. ZikV has evolved multiple mechanisms to restrict proliferation and enhance cell death, although the underlying cellular events involved remain unclear. Here we show that the ZikV-NS5 protein interacts with host proteins at the base of the primary cilia in neural progenitor cells, causing an atypical non-genetic ciliopathy and premature neuron delamination. Furthermore, in human microcephalic fetal brain tissue, ZikV-NS5 persists at the base of the motile cilia in ependymal cells, which also exhibit a severe ciliopathy. While the enzymatic activity of ZikV-NS5 appears to be dispensable, the Y25, K28 and K29 residues in the protein, that are involved in NS5-oligomerization, are essential for the localization and interaction with components of the cilium base, promoting ciliopathy and premature neurogenesis. These findings lay the foundation to develop therapies that target ZikV-NS5-multimerization, preventing the developmental malformations associated with congenital Zika syndrome

Lipoblastoma-like tumor of the vulva (LBLTV) was first described as a benign mesenchymal neoplasia; it was not recognized as a separate diagnosis in the 2013 WHO classification of soft-tissue tumors. To date, only 19 cases have been reported. LBLTV differential diagnosis includes other tumors of the vulvoperineal region and tumors with adipocytic differentiation, most of which are benign and thus a misdiagnosis has few clinical consequences. However, LBLTV may also mimic some aggressive lipomatous neoplasms. We describe a case of LBLTV in a 28 year-old woman and review the literature.

The present study was supported by Fondo de Investigaciones Sanitarias (grant nos. PI17/02247 and PI14/01320), Centro de Investigación Biomédica en Red de Cáncer (grant no. CB16/12/00363), Generalitat de Catalunya (grant nos. 2017 SGR 1799 and 2014 SGR 1131), and the FP7 Marie Sklodowska‑Curie COFUND program (grant no. 267128; INCOMED program). Adenocarcinomas exhibit great heterogeneity, with many genetic and epigenetic alterations. The Kelch domain-containing protein 7B (KLHDC7B) has recently been identified as epigenetically modified and upregulated in breast cancer. The potential reversibility of epigenetic states offers exciting possibilities for novel cancer diagnostics and drugs. However, to properly evaluate specific inhibitors, the role of KLHDC7B in the development and progression of breast cancer should be established. With that objective in mind, the present study investigated a series of human breast tumours and correlated their clinicopathology, according to the Elston-Ellis modification of the Scarff-Bloom-Richardson (SBR) grading system, with KLHDC7B mRNA expression, analysed using quantitative PCR (qPCR). The results revealed that KLHDC7B was significantly upregulated in grade 3 tumours, and that KLHDC7B expression varied according to the tumour grade and the individual, being downregulated in well-differentiated and moderately-differentiated tumours (grade 1-2) and upregulated in poorly-differentiated tumours (grade 3). Immunohistochemical staining revealed that ductal tumours and tumours with a higher percentage of Ki67 positive cells showed the highest levels of KLHDC7B. Receptor expression, HER, p53 status, presence of metastasis, and vascular invasion showed no association with KLHDC7B expression. Previous studies have proposed KLHDC7B as an epigenetic marker of breast cancer. We propose that KLHDC7B should be used as a marker for poorly-differentiated tumours only; use of KLHDC7B without considering tumour grade could lead to an inaccurate diagnosis. Finally, we suggest the appropriate breast cancer cell lines to use to determine the functions of KLHDC7B. KLHDC7B expression was tested in the non-tumour cell line MCF-10A and in the breast cancer cell lines MCF-7, MDA-MB-231 and MDA-MB-468, using qPCR and western blotting. The results revealed that all tested cancer cell lines overexpressed KLHDC7B mRNA, but MDA-MB-468 exhibited a much lower level of protein expression relative to mRNA. Although the breast cancer cell lines used may be appropriate for studying KLHDC7B epigenetic status, MDA-MB-468 should be excluded from functional experiments.

The work of Santiago Ramón y Cajal is difficult to understand without knowing the personalities of Justo, his father, and his brother Pedro. His father practically forced the two brothers to study medicine. Thanks to that, Santiago was able to combine his artistic talent with histology and wonderfully describe cerebral architecture. Pedro was a faithful brother and above all a friend of Santiago, and they worked together for years. Pedro was able to demonstrate the theories of his brother in nonmammalian amniotes, concluding that the basic elements of the nervous system are common to these animals and he provided images that served Santiago to formulate the theory of dynamic polarization. Pedro, who decided to remain in the shadow of his brother, was a very complete doctor, pathologist and gynecologist, who made interesting contributions in all these fields and above all was a great humanist who left an important personal and scientific legacy. Anat Rec, 2019. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists. Anat Rec, 303:1189–1202, 2020. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Mercedes Olaya-C,1 Marta Garrido,2 Javier Hernandez-Losa,2–4 Marta Sesé,2–4 Paola Ayala-Ramirez,5 Rosa Somoza,2–4 Magda Jimena Vargas,6 Santiago Ramón y Cajal2–4 1Department of Pathology, Institute of Human Genetics, The Medical School, Pontificia Universidad Javeriana - Hospital Universitario San Ignacio, Bogota, Colombia; 2Pathology Department, Vall d’Hebron Hospital, Barcelona, Spain; 3Translational Molecular Pathology, Vall d’Hebron Research Institute (VHIR), Universitat Autònoma de Barcelona, Barcelona, Spain; 4Spanish Biomedical Research Network Center in Oncology (CIBERONC), Barcelona, Spain; 5Institute of Human Genetics, The Medical School, Pontificia Universidad Javeriana, Bogota, Colombia; 6Department of Pathology, The Medical School, Pontificia Universidad Javeriana - Hospital Universitario San Ignacio, Bogota, Colombia Introduction: The VEGF family has been identified as abnormal in preeclampsia (PE). Hypertensive disorders of pregnancy (HDP) are major contributors to maternal and neonatal morbidity and mortality worldwide; likewise, umbilical cord anatomical abnormalities (UCAA) are linked to poor neonatal outcomes. Based on the relationship described between PE and UCAA and the role of the VEGF family in PE, this study explored VEGF expression in placental and UC tissued from patients with PE and with UCAA.Methods: We performed an observational, analytical study on placentas, comparing protein and mRNA expression in four groups: patients with PE, patients with UC abnormalities, patients with both, and patients with none of them. Using immunohistochemistry, we studied VEGF A, VEGF R1 (FLT1), MMP1, and PLGF. With quantitative reverse transcription polymerase chain reaction we described mRNA expression of PLGF, VEGF and sFLT1, and sFLT1/PLGF ratio.Results: Forty newborns were included. Sixty-seven percent of mothers and 45% of newborns developed no complications. Immunohistochemistry was performed on UC and placental disc paraffin-embedded tissue; in the latter, the mRNA of the VEGF family was also measured. Statistically significant differences were observed among different expressions in both HDP and UCAA groups. Interestingly, the UCAA group exhibited lower levels of sFLT1 and VEGF-A in comparison with other groups, with significant P-value for sFLT1 (P=0000.1).Conclusion: The origin of UCAA abnormalities and their relation with HDP are still unknown. VEGF family alterations could be involved in both. This study provides the first approach related to molecules linked to UCAA. Keywords: preeclampsia, umbilical cord, VEGF, sFLT1, PLGF, stillbirth

Malignant transformation in tumors is a complex process requiring accumulation of numerous oncogenic abnormalities. Brain tumors show considerable phenotypic and genetic heterogeneity. In a series comprising diffuse infiltrating astrocytomas (DIA) and reactive gliosis, we investigated the main factors associated with signaling pathways. We assessed expression levels and their association with tumor progression and survival. We studied 19 grade II astrocytomas, 25 anaplastic astrocytomas (grade III), 60 glioblastomas (grade IV), and 15 cases of reactive gliosis. Epidermal growth factor receptor (EGFR), pMAPK, 4E‐BP1, p4E‐BP1, pS6, eIF4E, and peIF4E expression levels were evaluated using immunohistochemistry. Expression levels were semiquantitatively evaluated using a histoscore. Immunohistochemistry and PCR were used for IDH1 mutations. Statistical analysis was based on the following tests: chi‐square, Student's t, Pearson correlation, Spearman's rho, and Mann–Whitney; ROC and Kaplan–Meier curves were constructed. A significant increase was observed between grades for expression of total and phosphorylated 4E‐BP1 and for eIF4E, Ki67, EGFR, and cyclin D1. Although expression of EGFR, eIF4E, and Ki67 correlated with survival, only peIF4E was an independent predictor of survival in the multivariate analysis. Combining the evaluation of different proteins enables us to generate helpful diagnostic nomograms. In conclusion, cell signaling pathways are activated in DIAs; peIF4E is an independent prognostic factor and a promising therapeutic target. Joint analysis of the expression of 4E‐BP1 and peIF4E could be helpful in the diagnosis of glioblastoma multiforme in small biopsy samples.

Purpose: Most hormone receptor (HR)+/HER2− breast cancer patients respond unfavorably to neoadjuvant chemotherapy (NAC); however, genomic tests may identify those patients who are likely to benefit. Using the Prosigna assay, we first evaluated the technical performance of core needle biopsy (CNB) tissues. We then determined whether Prosigna risk of relapse (ROR) score and intrinsic subtype predicted response to NAC in HR+/HER2− patients using CNB samples. Experimental Design: Using the NanoString's nCounter Dx analysis system and a development tissue sample set, we established tissue requirements and assay output variance. We then evaluated the concordance in subtype and correlation in ROR between CNBs and corresponding surgical resection specimens (SRS) in a second independent sample set. Finally, we analyzed 180 independent CNB samples from HR+/HER2− patients who were treated with NAC and correlated ROR and intrinsic subtype with pathologic response. Results: Intra- and interbiopsy variabilities were 2.2 and 6.8 ROR units, respectively. Subtype concordance within multiple CNBs was high for the 4- and 3-subtype classifications (k = 0.885 and 0.889, respectively). Correlation in Prosigna ROR score observed between paired CNBs and SRS was high (r ≥ 0.90), and subtype concordance was also high for the 4- and 3-subtype classifications (kappa = 0.81 and 0.91, respectively). Prosigna results obtained from the HR+/HER2− patient samples showed that both ROR (P = 0.047) and intrinsic subtype (OR LumA vs. non-LumA = 0.341, P = 0.037) were significant predictors of response to NAC. Conclusions: Prosigna ROR and intrinsic subtype are readily obtained from CNB samples in normal practice and reliably predict response to NAC in HR+/HER2− patients. Clin Cancer Res; 22(3); 560–6. ©2015 AACR.

Introduction: Previous studies have shown that levels of circulating tumor DNA (ctDNA) during therapy and post-surgery can stratify patients who exhibit effective responses versus those showing minimal residual disease. In this study, we performed longitudinal tracking of plasma TP53 and PIK3CA mutations prespecified from NGS analysis of tumor tissue specimens of HER2-positive and triple-negative (TN) breast cancer (BC) patients in order to demonstrate that ctDNA clearance during neoadjuvant treatment (NAT) correlates with effective response to treatment. Initially, we examined the degree to which clearance of TP53 and PIK3CA mutations in ctDNA during NAT correlated with pathologic complete response (pCR) in the resected specimen. Our findings suggest that effective ctDNA clearance may predict pCR. Moreover, these findings prompt further investigative trials that explore more fully the value of ctDNA monitoring to identify those patients who may avoid surgery following NAT. Patients and Methods: In initial analyses, tumor tissue samples from 18 TN or HER2 BC patients whoreceived NAT and showed significant response were analyzed for TP53 and PI3KCA mutations (Qiasymphony DNA midi and DNA Tissue Kits, respectively, QIAGEN). Plasma samples were obtained from all of these patients at diagnosis, after the 1st treatment cycle, during the middle of treatment, and prior to surgery. All TP53 and PIK3CA ctDNA analyses were carried out using the plasma SafeSEQ technology (Sysmex Inostics) and were only carried out on patient samples in which a specific TP53 or PIK3CA mutation had been initially identified in that patient’s tissue. All patients underwent a surgical excision after NAT. Results: Of the 18 patients, only 8 were found to have either a TP53 or a PIK3CA mutation (6 TP53 and 2 PI3KCA mutations). PI3KCA mutations were only detected in HER2-positive patients, whereas TP53 mutations were detected predominantly in TN patients (88%). After the 1st cycle of NAT, no mutations were detected in 6 (75%) patients while the 2 remaining (25%) demonstrated mutation clearance at the middle of treatment. Interestingly, one patient showed a TP53 baseline mutation (TP53 p.R273L) that was no longer detected after the 1st cycle of NAT, but a new mutation was detected at the time of surgery (TP53 p.R267W). Seven out of these 8 patients showing mutation clearance in ctDNA (88%) reached a pCR. Conclusions: ctDNA detected in plasma of patients with TN or HER2-positive BC showed a sharp decrease from the first cycle of NAT, reaching undetectable levels until the time of surgery and correlating with pCR. However, new mutations not previously present appeared, which may reflect the adaptation of the tumor to treatment. We are currently extending our analyses to reanalyze with an alternative NGS platform those tissue specimens for which no mutations were detected. Also, additional patient plasma and tissue specimens are undergoing analysis to evaluate the relationship between molecular MRD as determined by ctDNA and pCR. Citation Format: Nikaoly Ciriaco, Esther Zamora, Santiago Escrivá, Rosa Somoza, Javier Hernández-Losa, Santiago Ramón y Cajal, Martín Espinosa-Bravo, Vicente Peg. Clearance of ctDNA in triple-negative and Her2-positive breast cancer patients during neoadjuvant treatment is correlated with pathologic complete response [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B63.