Liquid biopsy for disease monitoring in non-small cell lung cancer: The link between biology and the clinic

Introduction: Cell-free DNA (cfDNA) analysis offers a non-invasive method to identify sensitising and resistance mutations in advanced Non-Small Cell Lung Cancer (NSCLC) patients. Next-generation sequencing (NGS) of circulating free DNA (cfDNA) is a valuable tool for mutations detection and disease' s clonal monitoring. Material and methods: An amplicon-based targeted gene NGS panel was used to analyse 101 plasma samples of advanced non-small cell lung cancer (NSCLC) patients with known oncogenic mutations, mostly EGFR mutations, serially collected at different clinically relevant time points of the disease. Results: The variant allelic frequency (VAF) monitoring in consecutive plasma samples demonstrated different molecular response and progression patterns. The decrease in or the clearance of the mutant alleles was associated with response and the increase in or the emergence of novel alterations with progression. At the best response, the median VAF was 0% (0.0% to 3.62%), lower than that at baseline, with a median of 0.53% (0.0% to 9.9%) (p = 0.004). At progression, the VAF was significantly higher (median 4.67; range: 0.0–36.9%) than that observed at the best response (p = 0.001) and baseline (p = 0.006). These variations anticipated radiographic changes in most cases, with a median time of 0.86 months. Overall, the VAF evolution of different oncogenic mutations predicts clinical outcomes. Conclusion: The targeted NGS of circulating tumour DNA (ctDNA) has clinical utility to monitor treatment response in patients with advanced lung adenocarcinoma.This work was supported by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020; and by FCT—Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação in the framework of the projects “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274), and “Transferencia horizontal de resistencia à terapia: mudança de paradigma na monitorização de pacientes com cancro” (PTDC/DTPPIC/2500/2014); and by Norte Portugal Regional Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), in the framework of the project NORTE-01-0145-FEDER-000029

Targeted gene next-generation sequencing panel in patients with advanced lung adenocarcinoma: Paving the way for clinical implementation

Identification of targetable molecular changes is essential for selecting appropriate treatment in patients with advanced lung adenocarcinoma. Methods: In this study, a Sanger sequencing plus Fluorescence In Situ Hybridization (FISH) sequential approach was compared with a Next-Generation Sequencing (NGS)-based approach for the detection of actionable genomic mutations in an experimental cohort (EC) of 117 patients with advanced lung adenocarcinoma. Its applicability was assessed in small biopsies and cytology specimens previously tested for epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) mutational status, comparing the molecular changes identified and the impact on clinical outcomes. Subsequently, an NGS-based approach was applied and tested in an implementation cohort (IC) in clinical practice. Using Sanger and FISH, patients were classified as EGFR-mutated (n = 22, 18.8%), ALK-mutated (n = 9, 7.7%), and unclassifiable (UC) (n = 86, 73.5%). Retesting the EC with NGS led to the identification of at least one gene variant in 56 (47.9%) patients, totaling 68 variants among all samples. Still, in the EC, combining NGS plus FISH for ALK, patients were classified as 23 (19.7%) EGFR; 20 (17.1%) KRAS; five (4.3%) B-Raf proto-oncogene (BRAF); one (0.9%) Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2); one (0.9%) STK11; one (0.9%) TP53, and nine (7.7%) ALK mutated. Only 57 (48.7%) remained genomically UC, reducing the UC rate by 24.8%. Fourteen (12.0%) patients presented synchronous alterations. Concordance between NGS and Sanger for EGFR status was very high (¿ = 0.972; 99.1%). In the IC, a combined DNA and RNA NGS panel was used in 123 patients. Genomic variants were found in 79 (64.2%). In addition, eight (6.3%) EML4-ALK, four (3.1%), KIF5B-RET, four (3.1%) CD74-ROS1, one (0.8%) TPM3-NTRK translocations and three (2.4%) exon 14 skipping MET Proto-Oncogene (MET) mutations were detected, and 36% were treatable alterations. Conclusions: This study supports the use of NGS as the first-line test for genomic profiling of patients with advanced lung adenocarcinoma.We acknowledge the work of the members of our department, especially doctors from the thoracic oncology unit, oncology and pulmonology nurses and, patients and their relatives. N. Martins would like to thank the Portuguese Foundation for Science and Technology (FCT-Portugal) for the Strategic project ref. UID/BIM/04293/2013 and "NORTE2020—Northern Regional Operational Program" (NORTE-01-0145-FEDER-000012)

Factors associated with involuntary hospital admissions in technology-dependent children

Abstract OBJECTIVE To identify the factors associated with involuntary hospital admissions of technology-dependent children, in the municipality of Ribeirão Preto, São Paulo State, Brazil. METHOD A cross-sectional study, with a quantitative approach. After an active search, 124 children who qualified under the inclusion criteria, that is to say, children from birth to age 12, were identified. Data was collected in home visits to mothers or the people responsible for the children, through the application of a questionnaire. Analysis of the data followed the assumptions of the Generalized Linear Models technique. RESULTS 102 technology-dependent children aged between 6 months and 12 years participated in the study, of whom 57% were male. The average number of involuntary hospital admissions in the previous year among the children studied was 0.71 (±1.29). In the final model the following variables were significantly associated with the outcome: age (OR=0.991; CI95%=0.985-0.997), and the number of devices (OR=0.387; CI95%=0.219-0.684), which were characterized as factors of protection and quantity of medications (OR=1.532; CI95%=1.297-1.810), representing a risk factor for involuntary hospital admissions in technology-dependent children. CONCLUSION The results constitute input data for consideration of the process of care for technology-dependent children by supplying an explanatory model for involuntary hospital admissions for this client group

The effect of hexose ratios on metabolite production in Saccharomyces cerevisiae strains obtained from the spontaneous fermentation of mezcal

Mezcal from Tamaulipas (Me´xico) is produced by spontaneous alcoholic fermentation using Agave spp. musts, which are rich in fructose. In this study eight Saccharomyces cerevisiae isolates obtained at the final stage of fermentation from a traditional mezcal winery were analysed in three semisynthetic media. Medium M1 had a sugar content of 100 g l-1 and a glucose/fructose (G/F) of 9:1. Medium M2 had a sugar content of 100 g l-1 and a G/F of 1:9. Medium M3 had a sugar content of 200 g l-1 and a G/F of 1:1. In the three types of media tested, the highest ethanol yield was obtained from the glucophilic strain LCBG-3Y5, while strain LCBG-3Y8 was highly resistant to ethanol and the most fructophilic of the mezcal strains. Strain LCBG-3Y5 produced more glycerol (4.4 g l-1) and acetic acid (1 g l-1) in M2 than in M1 (1.7 and 0.5 g l-1, respectively), and the ethanol yields were higher for all strains in M1 except for LCBG-3Y5, -3Y8 and the Fermichamp strain. In medium M3, only the Fermichamp strain was able to fully consume the 100 g of fructose l-1 but left a residual 32 g of glucose l-1. Regarding the hexose transporters, a high number of amino acid polymorphisms were found in the Hxt1p sequences. Strain LCBG-3Y8 exhibited eight unique amino acid changes, followed by the Fermichamp strain with three changes. In Hxt3p, we observed nine amino acid polymorphisms unique for the Fermichamp strain and five unique changes for the mezcal strains

Irresponsiveness of two retinoblastoma cases to conservative therapy correlates with up- regulation of hERG1 channels and of the VEGF-A pathway

<p>Abstract</p> <p>Background</p> <p>Treatment strategies for Retinoblastoma (RB), the most common primary intraocular tumor in children, have evolved over the past few decades and chemoreduction is currently the most popular treatment strategy. Despite success, systemic chemotherapeutic treatment has relevant toxicity, especially in the pediatric population. Antiangiogenic therapy has thus been proposed as a valuable alternative for pediatric malignancies, in particolar RB. Indeed, it has been shown that vessel density correlates with both local invasive growth and presence of metastases in RB, suggesting that angiogenesis could play a pivotal role for both local and systemic invasive growth in RB. We present here two cases of sporadic, bilateral RB that did not benefit from the conservative treatment and we provide evidence that the VEGF-A pathway is significantly up-regulated in both RB cases along with an over expression of hERG1 K⁺channels.</p> <p>Case presentation</p> <p>Two patients showed a sporadic, bilateral RB, classified at Stage II of the Reese-Elsworth Classification. Neither of them got benefits from conservative treatment, and the two eyes were enucleated. In samples from both RB cases we studied the VEGF-A pathway: VEGF-A showed high levels in the vitreous, the <it>vegf-a, flt-1, kdr</it>, and <it>hif1-α </it>transcripts were over-expressed. Moreover, both the transcripts and proteins of the hERG1 K⁺channels turned out to be up-regulated in the two RB cases compared to the non cancerous retinal tissue.</p> <p>Conclusions</p> <p>We provide evidence that the VEGF-A pathway is up-regulated in two particular aggressive cases of bilateral RB, which did not experience any benefit from conservative treatment, showing the overexpression of the <it>vegf-a</it>, <it>flt-1</it>, <it>kdr </it>and <it>hif1-α </it>transcripts and the high secretion of VEGF-A. Moreover we also show for the first time that the <it>herg1 </it>gene transcripts and protein are over expressed in RB, as occurs in several aggressive tumors. These results further stress the relevance of the VEGF-A pathway in RB and the correlation with hERG1, making aggressive and recurrent RB cases good candidates for antiangiogenesis therapies based on the targeting of VEGF-A.</p

Electrophysical properties of nanoporous cerium dioxide–water system

The impedance of nanoporous cerium dioxide with adsorbed water is investigated in the frequency range 103–104 Hz at temperatures near the water–ice phase transition. Here we show that the manifestation of impedance peculiarities at phase transition is caused by the dielectric constant of the matrix

HLA-DRB1 association with Henoch-Schonlein purpura

Objective: Henoch-Schönlein purpura (HSP) is the most common vasculitis in children but it is not exceptional in adults. Increased familial occurrence supports a genetic predisposition for HSP. In this context, an association with the human leukocyte antigen-HLA-DRB1*01 phenotype has been suggested in Caucasian individuals with HSP. However, data on the potential association of HSP with HLA-DRB1*01 were based on small case series. To further investigate this issue, we performed HLA-DRB1 genotyping of the largest series of HSP patients ever assessed for genetic studies in Caucasians. Methods: 342 Spanish patients diagnosed with HSP fulfilling the American College of Rheumatology and the Michel et al classification criteria, and 303 sex and ethnically matched controls were assessed. HLA-DRB1 alleles were determined using a PCR-Sequence-Specific-Oligonucleotide Probe (PCR-SSOP) method. Results: A statistically significant increase of HLA-DRB1*01 in HSP patients when compared with controls was found (43% vs 7%, respectively; p<0.001; odds ratio-OR=2.03 [1.43-2.87]). It was due to the increased frequency of HLA-DRB1*0103 phenotype in HSP (14% vs 2%; p<0.001; OR=8.27 [3.46-23.9]). These results remained statistically significant after adjusting for Bonferroni correction. In contrast, a statistically significant decreased frequency of the HLA-DRB1*0301 phenotype was observed in patients compared to controls (5.6% vs 18.1%, respectively; p<0.001, OR=0.26 [0.14-0.47]), even after adjustment for Bonferroni correction. No HLA-DRB1 association with specific features of the disease was found. Conclusion: Our study confirms an association of HSP with HLA-DRB1*01 in Caucasians. Also, a protective effect against the development of HSP appears to exist in Caucasians carrying the HLA-DRB1*03 phenotype

Observation of associated near-side and away-side long-range correlations in √sNN=5.02 TeV proton-lead collisions with the ATLAS detector

Two-particle correlations in relative azimuthal angle (Δϕ) and pseudorapidity (Δη) are measured in √sNN=5.02  TeV p+Pb collisions using the ATLAS detector at the LHC. The measurements are performed using approximately 1  μb-1 of data as a function of transverse momentum (pT) and the transverse energy (ΣETPb) summed over 3.1<η<4.9 in the direction of the Pb beam. The correlation function, constructed from charged particles, exhibits a long-range (2<|Δη|<5) “near-side” (Δϕ∼0) correlation that grows rapidly with increasing ΣETPb. A long-range “away-side” (Δϕ∼π) correlation, obtained by subtracting the expected contributions from recoiling dijets and other sources estimated using events with small ΣETPb, is found to match the near-side correlation in magnitude, shape (in Δη and Δϕ) and ΣETPb dependence. The resultant Δϕ correlation is approximately symmetric about π/2, and is consistent with a dominant cos⁡2Δϕ modulation for all ΣETPb ranges and particle pT

Effective detection of human adenovirus in hawaiian waters using enhanced pcr methods

<p>Abstract</p> <p>Background</p> <p>The current criteria for recreational water quality evaluation are primarily based on measurements of fecal indicator bacteria growth. However, these criteria often fail to predict the presence of waterborne human pathogenic viruses. To explore the possibility of direct use of human enteric viruses as improved human fecal contamination indicators, human adenovirus (HAdV) was tested as a model in this study.</p> <p>Findings</p> <p>In order to establish a highly sensitive protocol for effective detection of HAdV in aquatic environments, sixteen published PCR primer sets were re-optimized and comparatively evaluated. Primer sets nehex3deg/nehex4deg, ADV-F/ADV-R, and nested PCR primer sets hex1deg/hex2deg and nehex3deg/nehex4deg were identified to be the most sensitive ones, with up to 1,000 fold higher detection sensitivity compared to other published assays. These three PCR protocols were successfully employed to detect HAdV in both treated and untreated urban wastewaters, and also in 6 of 16 recreational water samples collected around the island of Oahu, Hawaii.</p> <p>Conclusions</p> <p>Findings from this study support the possible use of enteric viruses for aquatic environmental monitoring, specifically for the essential routine monitoring of Hawaiian beach waters using the optimized PCR protocol to detect HAdV at certain water sites to ensure a safe use of recreational waters.</p