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The increasing identification of driver oncogenic alterations and progress of targeted therapies addresses the need of comprehensive alternatives to standard molecular methods. The translation into clinical practice of next-generation sequencing (NGS) panels is actually challenged by the compliance of high quality standards for clinical accreditation. Herein, we present the analytical and clinical feasibility study of a hybridization capture-based NGS panel (Action OncoKitDx) for the analysis of somatic mutations, copy number variants (CNVs), fusions, pharmacogenetic SNPs and Microsatellite Instability (MSI) determination in formalin-fixed paraffin-embedded (FFPE) tumor samples. A total of 64 samples were submitted to extensive analytical validation for the identification of previously known variants. An additional set of 166 tumor and patient-matched normal samples were sequenced to assess the clinical utility of the assay across different tumor types. The panel demonstrated good specificity, sensitivity, reproducibility, and repeatability for the identification of all biomarkers analyzed and the 5% limit of detection set was validated. Among the clinical cohorts, the assay revealed pathogenic genomic alterations in 97% of patient cases, and in 82.7%, at least one clinically relevant variant was detected. The validation of accuracy and robustness of this assay supports the Action OncoKitDx’s utility in adult solid tumors.

Mechanical forces, hypoxia, and oxidative stress contribute to skin renewal, perfusion, and wound healing, but how are they regulating subcutaneous adipose-derived stem cells (ASCs) in the inflammatory microenvironment associated to skin repair and disorders is unknown. In this study, ASCs were isolated from lipoaspirate samples from plastic surgery patients, primary cultured and their differentiation and secretion of a panel of cytokines with pronounced effects on skin repair and angiogenesis were studied under mechanical stimulation by intermittent fluid flow, 1% hypoxia and oxidative stress by glutathione (GSH) depletion with buthionine sulfoximine (BSO) treatment. Mechanical action of fluid flow did not alter mesenchymal phenotype of CD90+ /CD29+ /CD44+ /CD34- /CD106- /CD45- ASCs; however, it remarkably induced ASC secretion of human umbilical vein endothelial cell (HUVEC) migration-stimulating factors. Multiplex Luminex assay further confirmed an increased secretion of VEGF, G-CSF, HGF, Leptin, IL-8, PDGF-BB, Angiopoietin-2, and Follistatin from mechanically-stimulated ASCs via cyclooxygenase-2. Consistent with this mechanism, GSH depletion and hypoxia also increased ASC secretion of VEGF, IL-8, leptin, Angiopoitein-2, and PDGF-BB. However, mechanical action of fluid flow abrogated VEGF and HUVEC migration-stimulating activity from GSH-depleted and hypoxic ASCs. Conversely, GSH depletion and hypoxia abrogated VEGF and HUVEC migration-stimulating activity from mechano-stimulated ASCs. Although mechanical action of fluid flow, hypoxia, and GSH-depletion had independent proangiogenic-stimulating activity on ASCs, mechanical stimulation had opposite effects on proangiogenic factor secretion from ASCs with and without oxidative stress. These data uncover the role of hypoxia and endogenous redox balance during the proangiogenic response of ASCs and other mesenchymal-derived cell types to mechanical action of interstitial fluid flow. J. Cell. Physiol. 232: 2158-2167, 2017. © 2016 Wiley Periodicals, Inc.

Decellularized natural bladder matrices (neobladders) represent an exciting means to regenerate the bladder following bladder cancer-associated cystectomy. In this study, we compare the evolution of decellularized matrices with recellularized matrices by seeding it with human adipose-derived mesenchymal stem cells (ADSC) after implantation following partial cystectomy in rats. We discovered significant anatomical differences since 10 days after neobladder implantation with the ADSC-containing matrices promoting a significant recovery of mature p63- and cytokeratin 7-positive urothelium. We also discovered significantly induced expression of the vimentin mesoderm marker in the submucosal layer in ADSC-seeded matrices. Interestingly, we found a higher expression of smooth muscle actin in transversal and longitudinal smooth muscle layers with ADSC-seeded matrices. Furthermore, ADSC also showed increased vascularization and nerve innervation of the neobladder as determined by the distribution of CD31 and S100β reactivity, respectively. We believe that ADSC and their paracrine-acting pro-regenerative secretome within decellularized matrices represent an efficient bladder substitution strategy; however, we require a fuller understanding of the mechanisms involved before clinical studies can begin.

Tumors are complex tissues that interact in many different ways. Tissue biopsies provide a great amount of information and remain the gold standard for tumor diagnosis. However, they cannot always be performed due to the invasive nature of the procedure and in such circumstances, a liquid biopsy could provide a solution. Liquid biopsy is defined as the search of biomarkers in peripheral blood. To date, there are three main research fields: (1) circulating tumor cells (CTCs); (2) circulating free tumor nucleic acids (cfNA) and (3) exosomes, small vesicles containing various types of signaling molecules capable of modulating a tumor-immune response. In recent years, exosomes have arisen as a powerful tool both to further our understanding of cancer biology and to improve clinical management. We review how the isolation and study of exosomes from liquid biopsies may affect clinical practice.

Since the proper activation of T cells requires the physical interaction with target cells through the formation of immunological synapses (IS), an alteration at this level could be a reason why tumors escape the immune response. As part of their life cycle, it is thought that T cells alternate between a static phase, the IS, and a dynamic phase, the immunological kinapse (IK), depending on high or low antigen sensing. Our investigation performed in tissue samples of human glioma shows that T cells are able to establish synapsing interactions not only with glioma tumorigenic cells, but also with stromal myeloid cells. Particularly, the IS displaying a T cell receptor-rich (TCR-rich) central supramolecular activation cluster (cSMAC) is preferentially established with stromal cells, as opposed to malignant cells. Conversely, T cells in the malignant areas showed distinct morphometric parameters compared with nonneoplastic tissue - the former characterized by an elongated shape, well-suited to kinaptic dynamics. Importantly, high-resolution 3-dimensional analyses demonstrated the existence of bona-fide IK preferentially arranged in malignant areas of the tumor. This imbalance of IS/IK states between these 2 microenvironments reveals the low antigenic sensing of T cells when patrolling tumorigenic cells and reflects the immunoevasive environment of the tumor.

Cryoplasty may reduce the incidence of post-angioplasty restenosis in peripheral atherosclerotic arteries. Our study is looking to investigate the mid-term effects (4 weeks) of an FDA-approved cryoplasty catheter (PolarCath®, Boston Scientific) compared to a conventional angioplasty catheter using a hypercholesterolemic rabbit model of arterial restenosis based on diet plus vessel injury. Thirty-six normolipidemic, 3-month old male New Zealand White rabbits were used. Balloon angioplasty was performed on left external iliac arteries on day 1. Animals were fed with a hypercholesterolemic diet for 60 days. On day 120, three groups of animals were established: conventional PTA (percutaneous transluminal angioplasty) was applied on the PTA group; the CRY group was treated with the PolarCath® cryoplasty system and no treatment was given to a control (CTR) group. A broad variety of atheromatous lesions were observed 30 days after treatment, presenting significant differences between groups. Most of the complicated lesions were found in the CRY group, while advanced and early lesions were more often appreciated in the CTR and PTA groups, respectively. The histomorphometric evaluation of the arteries showed significant differences between the CRY group and the other two groups, with the highest percentage of IEM (internal elastic membrane) injury, vascular stenosis and ratio intima/media being registered on animals treated with cryoplasty. Intravascular cryotherapy induces complicated lesions in arterial walls 30 days after treatment in a hypercholesterolemic rabbit model based on diet plus vessel injury. Cryoplasty leads to the production of severe fibrosis and mineralisation and stenosis compared to a conventional angioplasty.

We analysed the expression of cyclins A2, B1, D1, and E1 by immunohistochemistry and numerical aberrations in CCND1 gene by fluorescence in situ hybridization technique in 67 primary oral squamous cell carcinomas (OSCC). Cyclin A2 expression was observed in 54 (83.1%) tumours, cyclin D1 in 58 (89.2%), cyclin B1 in 39 (60%), and cyclin E in 21 (32.8%). CCND1 region analysis revealed 26 (43.3%) tumours with the presence of numerical aberrations which were correlated with cyclin D1 high expression (Rho = 0.48; p < 0.001). Twenty-nine (45.3%) tumours were classified as high proliferative tumours assessed by Ki-67 protein expression and correlated with tumours with high expression of cyclin A2 (Rho = 0.30; p = 0.016) and cyclin B1 (Rho = 0.37; p = 0.003). In multivariate analysis for an overall five-year survival (OS), we found an adverse independent prognostic value for cyclin A2 high expression (p = 0.031) and for advanced tumour stage (p < 0.001). Our results confirm that several cyclins are commonly expressed in OSCC. CCND1 gene is abnormal in more than one-third of the cases and is frequently associated with cyclin D1 high expression. Moreover, cyclin A2 high expression is an independent indicator of worse OS suggesting that this protein may serve as a reliable biological marker to identify high-risk subgroups with poor prognosis.

Cartilage degeneration is associated with degenerative bone and joint processes in severe osteoarthritis (OA). Spontaneous cartilage regeneration is extremely limited. Often the treatment consists of a partial or complete joint implant. Adipose-derived stem cell (ASC) transplantation has been shown to restore degenerated cartilage; however, regenerative differences of ASC would depend on the source of adipose tissue. The infra- and suprapatellar fat pads surrounding the knee offer a potential autologous source of ASC for patients after complete joint substitution. When infrapatellar- and suprapatellar-derived stromal vascular fractions (SVF) were compared, a significantly higher CD105 (+) population was found in the suprapatellar fat. In addition, the suprapatellar SVF exhibited increased numbers of colony formation units and a higher population doubling in culture compared to the infrapatellar fraction. Both the suprapatellar- and infrapatellar-derived ASC were differentiated in vitro into mature adipocytes, osteocytes, and chondrocytes. However, the suprapatellar-derived ASC showed higher osteogenic and chondrogenic efficiency. Suprapatellar-derived ASC transplantation in a severe OA mouse model significantly diminished the OA-associated knee inflammation and cartilage degenerative grade, significantly increasing the production of glycosaminoglycan and inducing endogenous chondrogenesis in comparison with the control group. Overall, suprapatellar-derived ASC offer a potential autologous regenerative treatment for patients with multiple degenerative OA.

Mechanical forces, hypoxia, and oxidative stress contribute to skin renewal, perfusion, and wound healing, but how are they regulating subcutaneous adipose-derived stem cells (ASCs) in the inflammatory microenvironment associated to skin repair and disorders is unknown. In this study, ASCs were isolated from lipoaspirate samples from plastic surgery patients, primary cultured and their differentiation and secretion of a panel of cytokines with pronounced effects on skin repair and angiogenesis were studied under mechanical stimulation by intermittent fluid flow, 1% hypoxia and oxidative stress by glutathione (GSH) depletion with buthionine sulfoximine (BSO) treatment. Mechanical action of fluid flow did not alter mesenchymal phenotype of CD90

Background Folliculo-sebaceous cystic hamartoma (FSCH) is an uncommon skin condition presenting as a slow-growing papulo-nodular lesion, in or around the nose. Most cases are not clinically suspected and only histopathological examination allows the diagnosis. Pathological features include a dermal-located infundibulo-cystic structure with sebaceous glands radiating around, a stromal component encircling the epithelial structures, with clefts between the lesional epithelial and stromal parts, as well as between this and the adjacent dermis. Results We report eight patients with the diagnosis of FSCH (5 females and 3 males), with ages ranging from 35 to 77 years. Most cases (5 out of 8) were located in or around the nose and sizes were comprised between 0.6 and 1.2 cm. Lesions had grown for long periods of time, up to ten years in one case. Immunohistochemistry showed staining for p63 in the epithelial component of all lesions, while CD10 was only present in some sebocytes. CD34 and Factor XIIIa positive cells were present in the lesional stroma. Staining for androgen and alpha-estrogen receptors was also usually noticed. Conclusions FCSH is a hamartomatous skin lesion, clinically indistinct but with well-defined histopathological features. Immunohistochemistry shows a profile very close to normal sebaceous glands. © 2008 Blackwell Munksgaard.

Resumen Introduccion El Sarcoma histiocitico (SH) es una neoplasia muy poco frecuente, con mucha controversia respecto a los criterios diagnosticos de esta entidad. Desde que las tecnicas inmunohistoquimicas y citogeneticas presentan una mayor disponibilidad universal, muchos casos que inicialmente se diagnosticaron como sarcoma histiocitico, han sido reclasificados como otras enfermedades. Presentacion del caso Describimos el caso de un sarcoma histiocitico que se presento como una masa abdominal. En la autopsia tambien se observo afectacion tumoral de pulmones, higado, bazo y multiples adenopatias. El examen histologico mostro proliferacion difusa de celulas grandes con areas de necrosis. Las celulas malignas eran de aspecto histiocitario y pleomorficas. Inmunohistoquimicamente, las celulas tumorales fueron positivas para tinciones contra marcadores histiociticos y negativas para marcadores mieloides, dendriticos, CD30, ALK1, y otros marcadores linfoides. En el estudio ultraestructural las celulas mostraron extensiones citoplasmicas interdigitantes, pero no granulos de Birbeck. Conclusiones El sarcoma histiocitico plantea diagnostico diferencial con otras neoplasias. El diagnostico en este caso, se basa en la morfologia, tecnicas inmunohistoquicas y ultraestructurales.

Resumen La Miocarditis de Celulas Gigantes (MCG) es una miocardiopatia poco frecuente, de posible causa inmunologica, con evolucion fatal por fallo cardiaco congestivo, cuya principal terapia es el tratamiento inmunosupresor o el trasplante. El diagnostico definitivo es histopatologico. El caso que se describe corresponde a una mujer de 59 anos, que mostro un cuadro clinico de infarto agudo de miocardio, con imagenes de coronariografia normales, realizandose el diagnostico «post-mortem» en la autopsia.

Background: Hibernoma or lipoma of brown fat is a rare benign tumor, representing 1.6% of the neoplasms of this tissue. Because of its histological characteristics can be wrongly classified as liposarcoma, therefore a correct differential diagnosis is necessary to provide appropriate treatment.Case presentation: The patient on which this case study is based is a 44-year-old male with a painless soft mass in his axilla located by his 4th and 5th ribs. The resected specimen did not have the classic macroscopic features of lipoma or fibrolipoma. Microscopically, the report described a proliferation of unilocular adipocytes with eccentric nucleus and, in less frequency, multilocular adipocytes with central nucleus. He had no recurrence after excision.Conclusions: Despite radiology studies and other technologies such as magnetic resonance imaging, computerized axial tomography (CAT), etc., the clinical diagnosis of hibernoma could be difficult. Lipoma-like hibernoma only have a few multilocular cells and can be wrongly classified as liposarcoma. Well-differentiated liposarcoma resembles it on low-power examination. Due to this it is especially important to perform a differential diagnosis with lipoma, fibroma, and even with liposarcoma. In this study we describe the histological features, the molecular markers and cytogenetic aspects that contribute to differentiate hibernoma from others tumors.

Ependymoma tumors likely derive from the ependymal cells lining the CNS ventricular system. In grade II ependymomas, tumor cells resemble typical ependymocytes, while anaplastic ependymomas are poorly differentiated. We studied three grade II and one anaplastic ependymoma, focusing on the ciliary structures. To unambiguously characterize the ultrastructure and number of cilia, we performed electron microscopy serial section analysis of individual cells. Differentiated ependymomas contained large basal bodies and up to three cilia, and lacked centrioles. Anaplastic ependymoma cells showed instead two perpendicularly oriented centrioles and lacked cilia or basal bodies. These findings could contribute to understand the mechanisms of ependymoma aggressiveness.

El adenocarcinoma de laringe es un tipo de tumor muy infrecuente, que supone solo un 0,35-0,5 % de las neoplasias laringeas malignas. Se caracteriza por su elevada agresividad y por su tendencia a producir metastasis en los ganglios linfaticos regionales y a distancia. Presentamos el caso de un varon de 64 anos de edad con un adenocarcinoma de laringe con metastasis ganglionares en el momento del diagnostico. Fue tratado con una combinacion de cirugia y radioterapia postoperatoria. Siete meses despues presento recidiva regional.

Laryngeal adenocarcinomas are extremely unusual (only 0.35 %-0.5 % of all laryngeal malignancies). It usually behaves as a highly malignant tumour, with tendency for local lymph node and distant organ metastases. We report a case of a 64-year-old man with an adenocarcinoma of the larynx with cervical lymph node involvement at the moment of the diagnosis. The patient was treated with surgery (total laryngectomy and cervical lymph node dissection) and post-operative radiotherapy. Seven months later, the patient had a local recurrence.

BACKGROUND: Breast cancer is a heterogenous disease that impacts racial/ethnic groups differently. Differences in genetic composition, lifestyles, reproductive factors, or environmental exposures may contribute to the differential presentation of breast cancer among Hispanic women. MATERIALS AND METHODS: A population-based study was conducted in the city of Santiago de Compostela, Spain. A total of 645 women diagnosed with operable invasive breast cancer between 1992 and 2005 participated in the study. Data on demographics, breast cancer risk factors, and clinico-pathological characteristics of the tumors were collected. Hormone receptor negative tumors were compared with hormone receptor postive tumors on their clinico-pathological characteristics as well as risk factor profiles. RESULTS: Among the 645 breast cancer patients, 78% were estrogen receptor-positive (ER+) or progesterone receptor-positive (PR+), and 22% were ER-&PR-. Women with a family history of breast cancer were more likely to have ER-&PR- tumors than women without a family history (Odds ratio, 1.43; 95% confidence interval, 0.91-2.26). This association was limited to cancers diagnosed before age 50 (Odds ratio, 2.79; 95% confidence interval, 1.34-5.81). CONCLUSIONS: An increased proportion of ER-&PR- breast cancer was observed among younger Spanish women with a family history of the disease.

A 35-year-old woman had a painful, umbilical mass. The nodule was removed and histopathological examination yielded the diagnosis of umbilical endometriosis attached to an umbilical hernia. The presentation of endometriosis as a solitary node in the skin, without involvement of any other location as well as the association with an umbilical hernia are uncommon features of this entity.

Laryngeal adenocarcinomas are extremely unusual (only 0.35 %-0.5 % of all laryngeal malignancies). It usually behaves as a highly malignant tumour, with tendency for local lymph node and distant organ metastases. We report a case of a 64-year-old man with an adenocarcinoma of the larynx with cervical lymph node involvement at the moment of the diagnosis. The patient was treated with surgery (total laryngectomy and cervical lymph node dissection) and post-operative radiotherapy. Seven months later, the patient had a local recurrence.

Pseudomyxoma peritonei describes the accumulation of mucinous material in the abdominal cavity. The main diagnostic problem appears when the primary site of origin could be appendix or ovary. In this paper describe clinicopathological features and biological markers that support appendiceal origin.

This study aims to evaluate the effects of ischemia on the myocardial fibers and the expression of the transcriptional factor for angiogenesis hypoxia-inducible factor-1 alpha (HIF-1alpha) in human heart specimens. We have prospectively analyzed the HIF-1alpha expression in human ischemic hearts with the ABC-inmunohistochemistry technique and amplification by biotinylated tyramide. The relationship between the expression of HIF-1alpha and the temporal evolution of ischemia has also been evaluated. As pathomorphological diagnosis of early myocardial ischemia has many problems in human autopsy material with less than 4 to 6 h after clinical onset, we suggest that HIF-1alpha is helpful in the early acute myocardial infarction diagnosis, so it stains necrotic areas within the first 2 h. The amplification procedure provides a higher intensity of the final staining without losing specificity. It is concluded that in normal cardiac fibers, basal expression of HIF-1alpha is not appreciable, but it steadily increases after ischemia. With regard to the practical applicability in forensic field, our observations suggest that positive immunohistochemical expression of HIF-1alpha on heart samples may be used as a reliable indicator of myocardial damage in cases without cardiac lesion evidence, using conventional microscopy. This method is especially useful and may provide definitive proof of myocardial ischemia in unexpected deaths without previous symptoms, or in forensic cases with a short period of clinical manifestations. In addition, it may have been involved in possible future cardiovascular therapies.

This study of innervation of the bone marrow in new-born rats demonstrates that major signs of differentiation occur in the nerves at the end of the second week after birth. Myelinated nerve fibers begin to acquire their myelin sheath at this time. The Schwann cells show abundant ergastoplasmic reticulum. Some of these cells separate individual axons and wrap them up with the double membranes that form the myelin sheath. From then on, the nerves of the marrow contain both myelinated and nonmyelinated fibers. Fibroblasts also differentiate during this time, producing collagen fibers around the nerves. Some fibroblasts are interconnected by desmosome-like structures. Fibroblasts and collagen fibers form the connective tissue sheaths of the nerve (perineurium and endoneurium). Upon completion of the myelin sheath by the Schwann cells and the connective tissue sheath by the fibroblasts, nerves of the marrow acquire the morphological characteristics of the peripheral nerves of the adult animal. The fine structure of the axons in contact with the muscle fibers of the arterial wall correspond to Type 2-a of Watari. These nerve fibers are considered to be of sympathetic type. The time of maturation of nerves in the bone marrow coincides with the beginning of responsiveness to stimulatory and inhibitory conditions demonstrated in this organ by other authors.

Nerves containing numerous Schwann cells can be found in bone marrow with routine histological methods. Thin nerve bundles and single nerve fibers containing Schwann cells can be identified histologically with help of methods which demonstrate nerve fibers and myelin. Smears of marrow stained with the May-Grünwald-Giemsa method are not adequate for demonstration of nerve fibers, but their satellite Schwann cells appear well stained. Histological, cytological and ultrastructural characteristics of these cells are described in detail. Blood-forming cells and nerve fibers with their Schwann cells lie in close proximity throughout the marrow. This circumstance suggests that interaction may take place between both elements.

Whole body X-irradiation of rats with 400 rad produced accumulation of glycogen in the nerves of the bone marrow. The glycogen was demonstrated as dense granules 200 to 400 A in diameter. These granules were present in myelinated, nonmyelinated axons and Schwann cells of the marrow nerves from the first to the thirtieth day after irradiation. About one third of the axons were affected. In normal animals only 5.9% of the nonmyelinated axons showed glycogen, while this substance was not demonstrable in myelinated fibers.

The morphological changes of erythroblasts in congenital dyserythropoietic anemia type I have been analyzed by light and electron microscopy. The results support our earlier assumption, that this hereditary disorder of erythropoiesis is a disease entity and is not identical with any of the previously described hereditary hematological diseases. Characteristic morphological aberrations consist of widening of nuclear membrane pores, condensation, vacuolization and disintegration of the nuclear chromatin, structural changes of the nucleolus, appearance of myelin figures, and final autolysis of the cells. All changes are restricted to erythroblastic cells beyond the stage of the proerythroblast. The biochemical basis of this abnormality is not known. It leads to severe ineffective erythropoiesis with anemia, increased total hemoglobin turnover, and secondary hemochromatosis.