Nonoptical massive parallel DNA sequencing of BRCA1 and BRCA2 genes in a diagnostic setting.

The introduction of the benchtop massive parallel sequencers made it possible for the majority of clinical diagnostic laboratories to gain access to this fast evolving technology. In this study, using the Ion Torrent Personal Genome Machine, we present a strategy for the molecular diagnosis of hereditary breast and ovarian cancer and respective analytical validation. The methodology relies on a multiplex PCR amplification of the BRCA1 and BRCA2 genes combined with a variant prioritization pipeline, designed to minimize the number of false-positive calls without the introduction of false-negative results. A training set of samples was used to optimize the entire process, and a second set was used to validate and independently evaluate the performance of the workflow. Performing the study in a blind manner relative to the variants in the samples and using conventional Sanger sequencing as standard, the workflow resulted in a strategy with a maximum analytical sensitivity ≥98.6% with a confidence of 95% and a specificity of 96.9%. Importantly, no true variant was missed. This study presents a comprehensive massive parallel sequencing-Sanger sequencing based strategy, which results in a high analytical sensitivity assay that provides a time- and cost-effective strategy for the identification of mutations in the BRCA1 and BRCA2 genes.

Citizens' Perception on Air Quality in Portugal-How Concern Motivates Awareness.

This study aimed to understand the knowledge of Portuguese citizens about air quality and the extent to which the concerns about specific environmental problems can motivate their acquaintance of information. Moreover, this study also allowed to understand which information about air quality needs further dissemination to provide the citizens with all the available tools and the correct knowledge. For this, a national online survey about air quality perception was conducted, where 1131 answers were obtained and two different populations were compared: the general population and a sub-population from an urban-industrial area of Lisbon metropolitan area that had experienced frequent air pollution events in the past. Air pollution was considered the environmental topic of higher concern among this sub-population (61.4%), while in the general population it ranked thirdly (27.4%). Generally, the sub-population showed higher knowledge about air quality than the general population, with 61% being able to identify at least one air pollutant. The perception of the local air quality was also very different between populations, with 61% of the sub-population considering it poor or very poor, while only 14% of the general population had the same perception, which highlights the different levels of concern between populations. A weak knowledge about air pollutants (50% of the general population could not identify any air pollutant) and an erroneous perception of the contribution of the different pollution sources to air quality levels were found. More than 50% of the respondents of both populations were considered to not have enough information regarding the air quality in their area of residence, with the national air quality database being unknown to almost everyone. Overall, strong efforts should be made to increase the awareness about the importance of air quality, which may promote a higher acceptance of the implementation of future actions to improve air quality.

Spatial Distribution of Air Pollution, Hotspots and Sources in an Urban-Industrial Area in the Lisbon Metropolitan Area, Portugal-A Biomonitoring Approach.

This study aimed to understand the influence of industries (including steelworks, lime factories, and industry of metal waste management and treatment) on the air quality of the urban-industrial area of Seixal (Portugal), where the local population has often expressed concerns regarding the air quality. The adopted strategy was based on biomonitoring of air pollution using transplanted lichens distributed over a grid to cover the study area. Moreover, the study was conducted during the first period of national lockdown due to COVID-19, whereas local industries kept their normal working schedule. Using a set of different statistical analysis approaches (such as enrichment and contamination factors, Spearman correlations, and evaluation of spatial patterns) to the chemical content of the exposed transplanted lichens, it was possible to assess hotspots of air pollution and to identify five sources affecting the local air quality: (i) a soil source of natural origin (based on Al, Si, and Ti), (ii) a soil source of natural and anthropogenic origins (based on Fe and Mg), (iii) a source from the local industrial activity, namely steelworks (based on Co, Cr, Mn, Pb, and Zn); (iv) a source from the road traffic (based on Cr, Cu, and Zn), and (v) a source of biomass burning (based on Br and K). The impact of the industries located in the study area on the local air quality was identified (namely, the steelworks), confirming the concerns of the local population. This valuable information is essential to improve future planning and optimize the assessment of particulate matter levels by reference methods, which will allow a quantitative analysis of the issue, based on national and European legislation, and to define the quantitative contribution of pollution sources and to design target mitigation measures to improve local air quality.

Clinical Application of Next-Generation Sequencing of Plasma Cell-Free DNA for Genotyping Untreated Advanced Non-Small Cell Lung Cancer.

BACKGROUND: Analysis of circulating tumor DNA (ctDNA) has remarkable potential as a non-invasive lung cancer molecular diagnostic method. This prospective study addressed the clinical value of a targeted-gene amplicon-based plasma next-generation sequencing (NGS) assay to detect actionable mutations in ctDNA in patients with newly diagnosed advanced lung adenocarcinoma. METHODS: ctDNA test performance and concordance with tissue NGS were determined, and the correlation between ctDNA findings, clinical features, and clinical outcomes was evaluated in 115 patients with paired plasma and tissue samples. RESULTS: Targeted-gene NGS-based ctDNA and NGS-based tissue analysis detected 54 and 63 genomic alterations, respectively; 11 patients presented co-mutations, totalizing 66 hotspot mutations detected, 51 on both tissue and plasma, 12 exclusively on tissue, and 3 exclusively on plasma. NGS-based ctDNA revealed a diagnostic performance with 81.0% sensitivity, 95.3% specificity, 94.4% PPV, 83.6% NPV, test accuracy of 88.2%, and Cohen's Kappa 0.764. PFS and OS assessed by both assays did not significantly differ. Detection of ctDNA alterations was statistically associated with metastatic disease (p = 0.013), extra-thoracic metastasis (p = 0.004) and the number of organs involved (p = 0.010). CONCLUSIONS: This study highlights the potential use of ctDNA for mutation detection in newly diagnosed NSCLC patients due to its high accuracy and correlation with clinical outcomes.

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.

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 (κ = 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.

Comprehensive massive parallel DNA sequencing strategy for the genetic diagnosis of the neuro-cardio-facio-cutaneous syndromes.

Variants in 11 genes of the RAS/MAPK signaling pathway have been causally linked to the neuro-cardio-facio-cutaneous syndromes group (NCFCS). Recently, A2ML1 and RIT1 were also associated with these syndromes. Because of the genetic and clinical heterogeneity of NCFCS, it is challenging to define strategies for their molecular diagnosis. The aim of this study was to develop and validate a massive parallel sequencing (MPS)-based strategy for the molecular diagnosis of NCFCS. A multiplex PCR-based strategy for the enrichment of the 13 genes and a variant prioritization pipeline was established. Two sets of genomic DNA samples were studied using the Ion PGM System: (1) training set (n =15) to optimize the strategy and (2) validation set (n = 20) to validate and evaluate the power of the new methodology. Sanger sequencing was performed to confirm all variants and low covered regions. All variants identified by Sanger sequencing were detected with our MPS approach. The methodology resulted in an experimental approach with a specificity of 99.0% and a maximum analytical sensitivity of ≥ 98.2% with a confidence of 99%. Importantly, two patients (out of 20) harbored described disease-causing variants in genes that are not routinely tested (RIT1 and SHOC2). The addition of less frequently altered genes increased in ≈ 10% the diagnostic yield of the strategy currently used. The presented workflow provides a comprehensive genetic screening strategy for patients with NCFCS in a fast and cost-efficient manner. This approach demonstrates the potential of a combined MPS-Sanger sequencing-based strategy as an effective diagnostic tool for heterogeneous diseases.

Heterozygous germline mutations in A2ML1 are associated with a disorder clinically related to Noonan syndrome.

Noonan syndrome (NS) is a developmental disorder characterized by short stature, facial dysmorphisms and congenital heart defects. To date, all mutations known to cause NS are dominant, activating mutations in signal transducers of the RAS/mitogen-activated protein kinase (MAPK) pathway. In 25% of cases, however, the genetic cause of NS remains elusive, suggesting that factors other than those involved in the canonical RAS/MAPK pathway may also have a role. Here, we used family-based whole exome sequencing of a case-parent trio and identified a de novo mutation, p.(Arg802His), in A2ML1, which encodes the secreted protease inhibitor α-2-macroglobulin (A2M)-like-1. Subsequent resequencing of A2ML1 in 155 cases with a clinical diagnosis of NS led to the identification of additional mutations in two families, p.(Arg802Leu) and p.(Arg592Leu). Functional characterization of these human A2ML1 mutations in zebrafish showed NS-like developmental defects, including a broad head, blunted face and cardiac malformations. Using the crystal structure of A2M, which is highly homologous to A2ML1, we identified the intramolecular interaction partner of p.Arg802. Mutation of this residue, p.Glu906, induced similar developmental defects in zebrafish, strengthening our conclusion that mutations in A2ML1 cause a disorder clinically related to NS. This is the first report of the involvement of an extracellular factor in a disorder clinically related to RASopathies, providing potential new leads for better understanding of the molecular basis of this family of developmental diseases.