Sample pooling strategies for SARS-CoV-2 detection.

The worldwide COVID-19 pandemic outburst has caused a serious public health issue with increasing needs of accurate and rapid diagnostic and screening testing. This situation requires an optimized management of the chemical reagents, the consumables, and the human resources, in order to respond accurately and effectively, controlling the spread of the disease. Testing on pooled samples maximizes the number of tested samples, by minimizing the time and the lab supplies needed. The general conceptualization of the pooling method is based on mixing samples together in a batch. Individual testing is needed only if a specific pool exhibits a positive result. The development of alternative hybrid methods, based on "in house" protocols, utilizing commercially available consumables, in combination with a reliable pooling method would provide a solution, focusing on the better exploitation of the personnel and the lab supplies, allowing for rapid screening of a population in a reasonably short time.

Carotid Disease and Ageing: A Literature Review on the Pathogenesis of Vascular Senescence in Older Subjects.

Aging is a natural process that affects all systems of the human organism, leading to its inability to adapt to environmental changes. Advancing age has been correlated with various pathological conditions, especially cardiovascular and cerebrovascular diseases. Carotid artery (CA) is mainly affected by age-induced functional and morphological alterations causing atheromatous disease. The evolvement of biomedical sciences has allowed the elucidation of many aspects of this condition. Symptomatic carotid disease (CD) derives from critical luminar stenosis or eruption of an atheromatous plaque due to structural modifications of the vessels, such as carotid intima-media thickening. At a histologic level, the aforementioned changes are mediated by elastin fragmentation, collagen deposition, immune cell infiltration, and accumulation of cytokines and vasoconstrictors. Underlying mechanisms include chronic inflammation and oxidative stress, dysregulation of cellular homeostatic systems, and senescence. Thus, there is an imbalance in components of the vessel wall, which fails to counteract exterior stress stimuli. Consequently, arterial relaxation is impaired and atherosclerotic lesions progress. This is a review of current evidence regarding the relationship of aging with vascular senescence and CD. A deeper understanding of these mechanisms can contribute to the production of efficient prevention methods and targeted therapeutic strategies.

Clinical significance of AGE-RAGE axis in colorectal cancer: associations with glyoxalase-I, adiponectin receptor expression and prognosis.

BACKGROUND: Advanced glycation end products (AGEs) and their receptor RAGE emerge as important pathogenic contributors in colorectal carcinogenesis. However, their relationship to the detoxification enzyme Glyoxalase (GLO)-I and Adiponectin receptors (AdipoR1, AdipoR2) in colorectal carcinoma (CRC) is currently understudied. In the present study, we investigated the expression levels of the above molecules in CRC compared to adjacent non-tumoral tissue and their potential correlation with clinicopathological characteristics and patients' survival. METHODS: We analyzed the immunohistochemical expression of AGE, RAGE, GLO-1, AdipoR1 and AdipoR2 in 133 primary CRC cases, focusing on GLO-I. The tumour MSI status was further assessed in mucinous carcinomas. Western immunoblotting was employed for validation of immunohistochemical data in normal and tumoral tissues as well in three CRC cell lines. An independent set of 55 patients was also used to validate the results of univariate survival analysis regarding GLO-I. RESULTS: CRC tissue showed higher intensity of both AGE and RAGE expression compared with normal colonic mucosa which was negative for GLO-I in most cases (78 %). Western immunoblotting confirmed AGE, RAGE and GLO-I overexpression in tumoral tissue. GLO-I expression was directly related to RAGE and inversely related to AGE immunolabeling. There was a trend towards higher expression of all markers (except for RAGE) in the subgroup of mucinous carcinomas which, although of borderline significance, seemed to be more prominent for AdipoR1 and AGE. Additionally, AGE, AdipoR1 and Adipo R2 expression was related to tumor grade, whereas GLO-1 and AdipoR1 to T-category. In survival analysis, AdipoR2 and GLO-I overexpression predicted shortened survival in the entire cohort and in early stage cases, an effect which for GLO-I was reproduced in the validation cohort. Moreover, GLO-I emerged as an independent prognosticator of adverse significance in the patients' cohort. CONCLUSIONS: We herein provide novel evidence regarding the possible interactions between the components of AGE-RAGE axis, GLO-I and adiponectin receptors in CRC. AGE and AdipoR1 are possibly involved in colorectal carcinogenesis, whereas AdipoR2 and GLO-I emerged as novel independent prognostic biomarkers of adverse significance for patients with early disease stage. Further studies are warranted to extend our observations and investigate their potential therapeutic significance.

Comprehensive Molecular Analysis of NSCLC; Clinicopathological Associations.

BACKGROUND: Selection of NSCLC patients for targeted therapy is currently based upon the presence of sensitizing mutations in EGFR and EML4/ALK translocations. The heterogeneity of molecular alterations in lung cancer has led to the ongoing discovery of potential biomarkers and targets in order to improve survival. AIM: This study aimed to detect alterations in EGFR, KRAS, BRAF, PIK3CA, MET-gene copy number and ALK rearrangements in a large cohort of 956 NSCLC patients of Hellenic origin using highly sensitive techniques and correlations with clinicopathological characteristics. RESULTS: Mutations were detected in EGFR 10.6% (101 out of 956 samples), KRAS 26.5% (191 out of 720 samples), BRAF 2.5% (12 out of 471 samples), PIK3CA 3.8% (7 out of 184 samples), MET gene amplification was detected in 18% (31 out of 170) and ALK rearrangements in 3.7% (4 out of 107 samples). EGFR mutations were detected in exon 19 (61.4% of mutant cases), exon 21 p.Leu858Arg (19.8%), exon 20 (15.8%), exon 18 (2.9%) and were correlated with gender histology, smoking status and TTF1 staining. p.Thr790Met mutant cases (3.9%) displayed concurrent mutations in exons 19 or 21. Negative TTF-1 staining showed strong negative predictive value for the presence of EGFR mutations. KRAS mutations were associated with histology, the most common mutation being p.Gly12Cys (38%). DISCUSSION: In conclusion, only 89 patients were eligible for EGFR -TKIs and ALK inhibitors therapy, whereas 257 patients showed other alterations, highlighting the necessity for a detailed molecular profiling potentially leading to more efficient individualized therapies for NSCLC patients.

Polycystin-1 and polycystin-2 are involved in the acquisition of aggressive phenotypes in colorectal cancer.

The polycystins PC1 and PC2 are emerging as major players in mechanotransduction, a process that influences all steps of the invasion/metastasis cascade. We hypothesized that PC1 and PC2 facilitate cancer aggressiveness. Immunoblotting, RT-PCR, semi-quantitative and quantitative real-time PCR and FACS analyses were employed to investigate the effect of polycystin overexpression in colorectal cancer (CRC) cells. The impact of PC1 inhibition on cancer-cell proliferation was evaluated through an MTT assay. In vitro data were analyzed by Student's t-test. HT29 human xenografts were treated with anti-PC1 (extracellular domain) inhibitory antibody and analyzed via immunohistochemistry to determine the in vivo role of PC1 in CRC. Clinical significance was assessed by examining PC1 and PC2 protein expression in CRC patients (immunohistochemistry). In vivo and clinical data were analyzed by non-parametric tests, Kaplan-Meier curves, log-rank test and Cox model. All statistical tests were two-sided. PC1 overexpression promotes epithelial-to-mesenchymal transition (EMT) in HCT116 cells, while PC2 overexpression results in upregulation of the mTOR pathway in SW480 cells. PC1 inhibition causes reduced cell proliferation in CRC cells inducing tumor necrosis and suppressing EMT in HT29 tumor xenografts. In clinical study, PC1 and PC2 overexpression associates with adverse pathological parameters, including invasiveness and mucinous carcinomas. Moreover, PC1 overexpression appears as an independent prognostic factor of reduced recurrence-free survival (HR = 1.016, p = 0.03) and lowers overall survival probability, while aberrant PC2 expression predicts poor overall survival (p = 0.0468). These results support, for the first time, a direct link between mechanosensing polycystins (PC1 and PC2) and CRC progression.

DNA repair systems in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) represents the most common soft tissue sarcoma in children and adolescent population. There are two major histological subtypes, embryonal (ERMS) and alveolar (ARMS), differing in cytogenetic and morphological features. RMS pathogenesis remains controversial and several cellular mechanisms and pathways have been implicated. Application of intense chemo- and radio-therapy improves survival rates for RMS patients, but significant efficacy has not been proved as DNA damage induced-resistance frequently occurs. The present review is aimed at summarizing the current evidence on DNA repair systems, implications in RMS development, focusing on gene expression alterations and point mutations of genes encoding for DNA repair enzymes. Understanding of DNA repair systems involvement in RMS pathogenesis could diversify RMS patients and provide novel individualized therapeutic targets.

Differential expression of WT1 gene product in non-Hodgkin lymphomas.

The tumor suppressor gene wt1 (Wilms tumor 1) encodes a zinc finger transcription factor reported to be expressed in many tumors, including mesotheliomas, carcinomas, and acute leukemias. However, WT1 expression in non-Hodgkin lymphomas (NHLs) has not been studied. The authors assessed for WT1 expression in six lymphoma/leukemia cell lines using Western blot methods after subcellular fractionation. We also assessed for WT1 expression in 167 NHLs using immunohistochemical methods. The B-cell NHLs analyzed were 18 diffuse large B-cell lymphomas, 13 marginal zone B-cell lymphomas, 9 small lymphocytic lymphomas, (DLBCLs), 8 follicular lymphomas, 6 mantle cell lymphomas, 5 Burkitt lymphomas, 3 lymphoplasmacytic lymphomas, and 2 B-cell lymphoblastic lymphomas. The T-cell NHLs analyzed were 43 anaplastic large cell lymphomas (ALCLs), 26 peripheral T-cell lymphomas unspecified, 13 angioimmunoblastic T-cell lymphomas, 6 cutaneous ALCLs, 6 cases of mycosis fungoides, 5 extranodal NK/T-cell lymphomas of nasal type, and 4 T-cell lymphoblastic lymphomas. WT1 levels were higher in cytoplasmic extracts than in nuclear extracts of the Karpas 299 and SU-DHL-1 lymphoma cell lines but were higher in nuclear extracts than in the cytoplasmic extracts of the Jurkat, HH, U-937, and K562 leukemia cell lines. In NHLs, WT1 was positive in 4 of 5 (80%) Burkitt lymphomas, 9 of 12 (75%) ALK-positive ALCLs, 3 of 6 (50%) lymphoblastic lymphomas (2 of 4 T-cell, 1 of 2 B-cell), 14 of 31 (45%) ALK-negative ALCLs, 6 of 18 (33%) DLBCLs, and 1 of 6 (17%) cutaneous ALCLs. WT1 was negative in all other NHLs tested. WT1 immunoreactivity was primarily cytoplasmic in all positive NHLs except T-cell lymphoblastic lymphoma. In conclusion, WT1 protein is frequently detected in the cytoplasm of a subset of high-grade NHLs.

Differential expression and clinical significance of tyrosine-phosphorylated STAT3 in ALK+ and ALK- anaplastic large cell lymphoma.

PURPOSE: Recent data suggest that nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) activates signal transducers and activators of transcription 3 (STAT3) directly, and ALK expression correlates with STAT3 activation in non-Hodgkin's lymphomas. In this study, we evaluated comprehensively STAT3 activation status in anaplastic large cell lymphoma (ALCL) cell lines and pretreatment ALCL tumors. EXPERIMENTAL DESIGN: The study included five ALK(+)ALCL cell lines and 80 systemic ALCL tumors (31 ALK(+), 49 ALK(-)) that were formalin fixed and paraffin embedded. All 80 patients with systemic ALCL were treated with doxorubicin-based chemotherapy. The STAT3 activation status in cell lines was determined using Western blots and an antibody that reacts specifically with the phosphorylated tyrosine 705 of STAT3, pSTAT3(tyr705). In ALCL tumors, STAT3 was considered active when > or =20% of neoplastic cells show unequivocal nuclear immunostaining for pSTAT3(tyr705). RESULTS: All five ALK(+)ALCL cell lines showed strong pSTAT3(tyr705) expression on Western blots. In systemic ALCL, STAT3 activation was detected in 49 of 80 (61%) ALCL tumors: 26 of 31 (84%) ALK(+) tumors and 23 of 49 (47%) ALK(-) tumors. ALK expression correlated significantly with STAT3 activation (P < 0.0001). Clinical follow-up data were available for 72 patients. In the ALK(-) group, the lack of STAT3 activation correlated with a favorable 5-year overall survival (P = 0.0076) but not failure-free survival. In the ALK(+) group, patients with inactive STAT3 showed a trend toward longer overall survival (P = 0.09) and failure-free survival (P = 0.19). Importantly, all five ALK(+) ALCL patients with inactive STAT3 survived without treatment failure after a median follow-up of 83 months. CONCLUSIONS: STAT3 activation correlates with but is not strictly dependent on ALK expression in ALCL. Lack of STAT3 activation appears to correlate with a favorable clinical outcome in ALCL.