One Step Nucleic Acid Amplification (OSNA) Lysate Samples Are Suitable to Establish a Transcriptional Metastatic Signature in Patients with Early Stage Hormone Receptors-Positive Breast Cancer.

The One Step Nucleic Acid Amplification (OSNA) is being adopted worldwide for sentinel lymph nodes (SLNs) staging in breast cancer (BC). As major disadvantage, OSNA precludes prognostic information based on structural evaluation of SLNs. Our aim is to identify biomarkers related to tumor-microenvironment interplay exploring gene expression data from the OSNA remaining lysate. This study included 32 patients with early stage hormone receptors-positive BC. Remaining OSNA lysates were prepared for targeted RNA-sequencing analysis. Identification of differentially expressed genes (DEGs) was performed by DESeq2 in R and data analysis in STATA. The results show that, in metastatic SLNs, several genes were upregulated: KRT7, VTCN1, CD44, GATA3, ALOX15B, RORC, NECTIN2, LRG1, CD276, FOXM1 and IGF1R. Hierarchical clustering analysis revealed three different clusters. The identified DEGs codify proteins mainly involved in cancer aggressiveness and with impact in immune response. The overexpression of the immune suppressive genes VTCN1 and CD276 may explain that no direct evidence of activation of immune response in metastatic SLNs was found. We show that OSNA results may be improved incorporating microenvironment-related biomarkers that may be useful in the future for prognosis stratification and immunotherapy selection. As OSNA assay is being implemented for SLNs staging in other cancers, this approach could also have a wider utility.

Influence of clinical factors on the protective or deleterious impact of genetic variants in orthodontically induced external root resorption: an observational study.

BACKGROUND: Prediction of susceptibility to Orthodontically Induced External Apical Root Resorption (OIEARR) has been hampered by the complex architecture of this multifactorial phenotype. The aim of this study was to analyze the impact of the interaction of multiple variables in the susceptibility to OIEARR. METHODS: The study evaluated 195 patients requiring orthodontic treatment. Nine clinical and treatment variables, single nucleotide polymorphisms (SNPs) from five genes and variables interactions were analyzed as risk factors for OIEARR using a multiple linear regression model. RESULTS: The model explained 29% of OIEARR variability (ANOVA: p < 0.01). Duration of treatment was the most important predictor and gender was the second, closely followed by premolar extraction. For genes encoding osteoprotegerin (OPG), the receptor activator of nuclear factor κ B (RANK) and the IL1 receptor antagonist (IL1RN), the effect of analyzed variants changed from protective to deleterious depending on the duration of treatment and the age of the patient. CONCLUSIONS: This work shows that in OIEARR the impact of genetic susceptibility factors is dynamic changing according to clinical variables.

Clinical and Genetic Risk Factors for Drug-Induced Liver Injury Associated with Anti-Tuberculosis Treatment-A Study from Patients of Portuguese Health Centers.

Drug-induced liver injury (DILI) is an unpredictable and feared side effect of antituberculosis treatment (AT). The present study aimed to identify clinical and genetic variables associated with susceptibility to AT-associated hepatotoxicity in patients with pulmonary tuberculosis treated with a standard protocol. Of 233 patients enrolled, 90% prospectively, 103 developed liver injury: 37 with mild and 66 with severe phenotype (DILI). All patients with mild hepatitis had a RUCAM score ≥4 and all patients with DILI had a RUCAM score ≥ 6. Eight clinical variables and variants in six candidate genes were assessed. A logistic multivariate regression analysis identified four risk factors for AT-DILI: age ≥ 55 years (OR:3.67; 95% CI:1.82−7.41; p < 0.001), concomitant medication with other hepatotoxic drugs (OR:2.54; 95% CI:1.23−5.26; p = 0.012), NAT2 slow acetylator status (OR:2.46; 95% CI:1.25−4.84; p = 0.009), and carriers of p.Val444Ala variant for ABCB11 gene (OR:2.06; 95%CI:1.02−4.17; p = 0.044). The statistical model explains 24.9% of the susceptibility to AT-DILI, with an 8.9 times difference between patients in the highest and in the lowest quartiles of risk scores. This study sustains the complex architecture of AT-DILI. Prospective studies should evaluate the benefit of NAT2 and ABCB11 genotyping in AT personalization, particularly in patients over 55 years.

A new application to one-step nucleic acid amplification-discarded sample in breast cancer: Preliminary results.

The one-step nucleic acid amplification (OSNA) assay is a molecular method used for detecting breast cancer (BC) metastasis in sentinel lymph nodes (SLNs). However, this method has a major disadvantage, since it prevents tissue structure analysis, while only one molecular marker can be evaluated, namely cytokeratin 19 mRNA. The aim of the present study was to evaluate whether an OSNA-discarded sample could be suitable for the gene expression analysis of the SLN microenvironment. The remaining intermediate phase of the centrifuged SLN homogenate obtained from the OSNA assay of samples from two patients with BC was used for mRNA extraction. Subsequently, the expression of five genes, namely forkhead box, cluster of differentiation 4 and three control genes, was determined by reverse transcription-quantitative PCR analysis. The results demonstrated that high-quality RNA was extracted. Therefore, this RNA may be used for gene expression analyses to predict novel molecular biomarkers associated with immuno-inflammatory microenvironment.

Nonsyndromic hereditary gingival fibromatosis: Characterization of a family and review of genetic etiology.

Our aim is to describe a family with a nonsyndromic form of hereditary gingival fibromatosis (HGF) and discuss genetic characteristics of this rare disease by reviewing reported cases. A mother and three descendants were diagnosed with HGF. There was marked variable expressivity: from severe generalized gingival overgrowth in a 16-year-old boy (the proband) to minimal manifestations in the mother. The proband was submitted to gingivectomy and gingivoplasty. In younger siblings, the disease remained stable for 5 years, suggesting that clinical surveillance is a good option. The diagnosis was supported by histopathological examination. Analysis of this family and literature-reported cases supports that HGF most frequently shows an autosomal dominant inheritance with high penetrance and variable expressivity. Neomutations and gonadal mosaicism do not seem to be a rare event. Although five loci have been mapped by linkage analysis, only two genes, SOS1 and REST, were identified in four families.

Effect of miR-34b overexpression on the radiosensitivity of non-small cell lung cancer cell lines.

BACKGROUND: The miR-34 family, under-expressed in-non small cell lung cancers (NSCLCs), are effectors of p53 activation upon irradiation of cells. We evaluated whether the miR-34b overexpression modulates the NSCLCs response to radiation. MATERIALS AND METHODS: NSCLC cell lines A549 with V-KI-RAS2 Kirsten Rat Sarcoma viral oncogene (KRAS) codon 12 mutation and with wild type p53, and H1299, not expressing p53, were irradiated after transfection with pre-miR-34b. Cell survival was assessed by clonogenic survival assays. The apoptosis and the cell cycle were evaluated by flow cytometry. RESULTS: In the A549 cell line, overexpression of miR-34b significantly reduced cell survival at lower than 4 Gy radiation doses. There was a significant reduction in B-cell CLL/lymphoma 2 (BCL2) expression but no significant differences were observed in the apoptotic cell population or the cycle profile. No significant effect was recorded in the H1299 irradiated cells. CONCLUSION: In the p53 wild type, KRAS mutated NSCLC cells, the overexpression of miR-34b increases radiosensitivity at low doses of radiation.

Impairment of breast cancer cell invasion by COX-2-specific inhibitor NS398: roles of CXCR4 and of uPA system.

Inhibition of cyclooxygenase-2 (COX-2) is known to impair cancer cell metastatic behaviour, but the mechanisms involved largely remain elusive. We aimed to analyse whether the antimetastatic effect of COX-2 inhibition in breast cancer cells could be explained by variations in the expression levels of chemokine receptor CXCR4, vascular endothelium growth factor (VEGF) and UPA/UPAR components of the urokinase plasminogen activator system (uPAR). Breast cancer cell line MDA-MB-231 was exposed to COX-2-specific inhibitor NS398. Experimental data were assessed using Matrigel invasion tests, qRT-PCR, ELISA, flow cytometry and MTT test. Exposure to NS398 had no major effect on cell viability, apoptosis or VEGF production. Cell invasion was significantly decreased with reductions ranging from of 3.6% with 10 µM NS398 to 81.04% with 100 µM NS398. CXCR4 membrane expression was significantly reduced by 18% (P < 0.05) when cells were treated with 100 µM of NS398 for 72 h. UPA mRNA levels were significantly reduced to 78 and 63% after treatment with 10 µM NS398 for 48 and 72 h, respectively (P < 0.05). UPAR mRNA levels also decreased with mild NS398 concentrations, reaching the lowest level of 56% with 50 µM of NS398 for 48 h (P < 0.05). With NS398 higher concentrations, UPAR and UPA expression levels increased. According to our results, impairment of expression of CXCR4, UPA and UPAR differentially contribute to the antimetastatic effect of COX-2 inhibitors depending on drug concentration.