Discovery of New Targets to Control Metastasis in Pancreatic Cancer by Single-cell Transcriptomics Analysis of Circulating Tumor Cells.

Metastasis development is the leading cause of cancer-related mortality in pancreatic ductal adenocarcinoma (PDAC) and yet, few preclinical systems to recapitulate its full spreading process are available. Thus, modeling of tumor progression to metastasis is urgently needed. In this work, we describe the generation of highly metastatic PDAC patient-derived xenograft (PDX) mouse models and subsequent single-cell RNA-sequencing (RNA-seq) of circulating tumor cells (CTC), isolated by human HLA sorting, to identify altered signaling and metabolic pathways, as well as potential therapeutic targets. The mouse models developed liver and lung metastasis with a high reproducibility rate. Isolated CTCs were highly tumorigenic, had metastatic potential, and single-cell RNA-seq showed that their expression profiles clustered separately from those of their matched primary and metastatic tumors and were characterized by low expression of cell-cycle and extracellular matrix-associated genes. CTC transcriptomics identified survivin (BIRC5), a key regulator of mitosis and apoptosis, as one of the highest upregulated genes during metastatic spread. Pharmacologic inhibition of survivin with YM155 or survivin knockdown promoted cell death in organoid models as well as anoikis, suggesting that survivin facilitates cancer cell survival in circulation. Treatment of metastatic PDX models with YM155 alone and in combination with chemotherapy hindered the metastatic development resulting in improved survival. Metastatic PDX mouse model development allowed the identification of survivin as a promising therapeutic target to prevent the metastatic dissemination in PDAC.

Sample Management: Stability of Plasma and Serum on Different Storage Conditions.

BACKGROUND AND OBJECTIVE: The analytes stability on serum and plasma are critical for clinical laboratory, especially if there is a delay in their processing or if they need to be stored for future research. The objective of this research was to determine the stability of K3EDTA-plasma and serum on different storage conditions. MATERIALS AND METHODS: A total of thirty healthy adults were studied. The serum/plasma samples were centrifuged at 2000g for 10 minutes. Immediately after centrifugation, the serum/plasma analytes were assayed in primary tubes using a Cobas c501 analyzer (T0); the residual serum/plasma was stored at either 2-8°C or -20°C for 15 (T15) and 30 days (T30).Mean concentrations changes in respect of initial concentrations (T0) and the reference change values were calculated. For assessing statistical difference between samples, the Wilcoxon ranked-pairs test was applied. RESULTS: We evidenced instability for total bilirubin, uric acid, creatinine and glucose at T15 and T30 and stored at -20°C (p<0.05). However, potential clinical impact significance were observed only for total bilirrubin T30 at -20°C, and creatinine T30 at 2-8°C. CONCLUSIONS: Our results had shown that storage samples at -20°C is a better way to preserve glucose, creatinine, and uric acid. Therefore, laboratories should freeze their samples as soon as possible to guarantee proper stability when there is need to repeat analysis, verify a result, or add a laboratory testing.

¿Los odontólogos de nuestro medio siguen correctamente las pautas de profilaxis de endocarditis infecciosa recomendadas? / Prophylaxis of infective endocarditis in dentistry: analysis of the Situation after almost a decade of clinical practice guidelines

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Exome Sequencing of Plasma DNA Portrays the Mutation Landscape of Colorectal Cancer and Discovers Mutated VEGFR2 Receptors as Modulators of Antiangiogenic Therapies.

Purpose: Despite the wide use of antiangiogenic drugs in the clinical setting, predictive biomarkers of response to these drugs are still unknown.Experimental Design: We applied whole-exome sequencing of matched germline and basal plasma cell-free DNA samples (WES-cfDNA) on a RAS/BRAF/PIK3CA wild-type metastatic colorectal cancer patient with primary resistance to standard treatment regimens, including inhibitors to the VEGF:VEGFR2 pathway. We performed extensive functional experiments, including ectopic expression of VEGFR2 mutants in different cell lines, kinase and drug sensitivity assays, and cell- and patient-derived xenografts.Results: WES-cfDNA yielded a 77% concordance rate with tumor exome sequencing and enabled the identification of the KDR/VEGFR2 L840F clonal, somatic mutation as the cause of therapy refractoriness in our patient. In addition, we found that 1% to 3% of samples from cancer sequencing projects harbor KDR somatic mutations located in protein residues frequently mutated in other cancer-relevant kinases, such as EGFR, ABL1, and ALK. Our in vitro and in vivo functional assays confirmed that L840F causes strong resistance to antiangiogenic drugs, whereas the KDR hot-spot mutant R1032Q confers sensitivity to strong VEGFR2 inhibitors. Moreover, we showed that the D717V, G800D, G800R, L840F, G843D, S925F, R1022Q, R1032Q, and S1100F VEGFR2 mutants promote tumor growth in mice.Conclusions: Our study supports WES-cfDNA as a powerful platform for portraying the somatic mutation landscape of cancer and discovery of new resistance mechanisms to cancer therapies. Importantly, we discovered that VEGFR2 is somatically mutated across tumor types and that VEGFR2 mutants can be oncogenic and control sensitivity/resistance to antiangiogenic drugs. Clin Cancer Res; 24(15); 3550-9. ©2018 AACR.

Laser-induced fluorescence in the diagnosis of pulp exposure and the influence of residual dentin thickness: An in vivo study.

PURPOSE: To clinically (a) determine whether laser-induced fluorescence (LIF) was able to assess pulp tissue health or disease in situations of pulp exposure; (b) evaluate the influence of different pulp tissue conditions upon LIF through dentin thicknesses of ≤1 mm; and (c) explore possible differences between the diagnostic performance of quantitative (q) and qualitative (ql) LIF. METHODS: 98 healthy subjects were scheduled for the treatment of caries. Three groups were established according to pulp tissue condition: Group A (n=30 teeth) (deep caries with healthy pulp tissue); Group B (n=30 teeth) (pulp necrosis); and Group C (n=30 teeth) (irreversible symptomatic acute pulpitis). The carious lesions were eliminated, and q and ql LIF measurements were made at two levels: measurement in dentin at < 1 mm from the pulp (A-D); and direct pulp exposure measurement (A-LP). In healthy pulp tissue at level A-LP, eight teeth with accidental pulp exposure were used. The Kruskal-Wallis test was used to evaluate the statistical significance of the differences in LIF readings among the three groups. The diagnostic performance of q and ql LIF in application to pulp tissue health or disease was assessed by calculating the sensitivity and specificity of the two tests at level A-LP. RESULTS: A significant correlation was observed between acute pulpitis and an increase in the q LIF values at level A-D (P= 0.004), but with no correlation to healthy pulp. Quantitative and qualitative LIF may be useful in diagnosing pulp tissue health or disease in situations of pulp exposure (A-LP).

Epstein-Barr virus transformation of human lymphoblastoid cells from patients with fragile X syndrome induces variable changes on CGG repeats size and promoter methylation.

BACKGROUND: Our understanding of fragile X syndrome can be improved by reversing the expression of the silenced fragile X mental retardation 1 (FMR1) gene in immortalized cells from these patients. Epstein-Barr virus (EBV) infection has been extensively used to transform B cells into a permanent lymphoblastoid cell line. METHODS: We immortalized B lymphocytes from three different fragile X patients and one normal male. We analyzed the CGG triplet repeats and methylation status of the FMR1 and interferon (IFN)-gamma promoter. We also assayed FMR1 mRNA levels by real-time PCR and FMR1 protein (FMRP) by Western blot. RESULTS: We observed that EBV transformation may induce the instability of CGG repeats and DNA demethylation that can lead to the modification of mRNA expression. CONCLUSIONS: EBV transformation may induce variable changes in the genome that can lead to the misinterpretations of experimental data obtained from these cells. Thus, periodic testing of DNA from immortalized cells should be routinely undertaken to detect undesired effects.