scholarly journals Implementation of Next Generation Sequencing-Based Liquid Biopsy for Clinical Molecular Diagnostics in Non-Small Cell Lung Cancer (NSCLC) Patients

Diagnostics ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1468
Author(s):  
Elisabetta Zulato ◽  
Valeria Tosello ◽  
Giorgia Nardo ◽  
Laura Bonanno ◽  
Paola Del Bianco ◽  
...  
Keyword(s):  
Liquid Biopsy ◽  
Single Nucleotide Variants ◽  
Clinical Genetic ◽  
Single Nucleotide ◽  
Circulating Free Dna ◽  
Somatic Alterations ◽  
High Consistency ◽  
Nsclc Patients ◽  
Actionable Variants

Genetic screening of somatic mutations in circulating free DNA (cfDNA) opens up new opportunities for personalized medicine. In this study, we aim to illustrate the implementation of NGS-based liquid biopsy in clinical practice for the detection of somatic alterations in selected genes. Our work is particularly relevant for the diagnosis and treatment of NSCLC. Beginning in 2020, we implemented the use of Roche’s Avenio ctDNA expanded panel in our diagnostic routine. In this study, we retrospectively review NGS-based clinical genetic tests performed in our laboratory, focusing on key analytical parameters. Avenio ctDNA kits demonstrated 100% sensitivity in detecting single nucleotide variants (SNVs) at >0.5% variant allele frequency (VAF), and high consistency in reproducibility. Since 2020, we performed cfDNA genotyping test in 86 NSCLC patients, and we successfully sequenced 96.5% (83/86) of samples. We observed consistency in sequencing performance based upon sequencing depth and on-target rate. At least one gene variant was identified in 52 samples (63%), and one or more actionable variants were detected in 21 out of 83 (25%) of analysed patients. We demonstrated the feasibility of implementing an NGS-based liquid biopsy assay for routine genetic characterization of metastatic NSCLC patients.

scholarly journals Peptide-Affinity Precipitation of Extracellular Vesicles and Cell-Free DNA Improves Sequencing Performance for the Detection of Pathogenic Mutations in Lung Cancer Patient Plasma

2020 ◽  
Vol 21 (23) ◽  
pp. 9083
Author(s):  
Catherine Taylor ◽  
Simi Chacko ◽  
Michelle Davey ◽  
Jacynthe Lacroix ◽  
Alexander MacPherson ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Extracellular Vesicles ◽  
Liquid Biopsy ◽  
Nsclc Patient ◽  
Egfr Mutations ◽  
Single Nucleotide Variants ◽  
Cell Free Dna ◽  
Free Dna ◽  
Nsclc Patients ◽  
Peptide Affinity

Liquid biopsy is a minimally-invasive diagnostic method that may improve access to molecular profiling for non-small cell lung cancer (NSCLC) patients. Although cell-free DNA (cf-DNA) isolation from plasma is the standard liquid biopsy method for detecting DNA mutations in cancer patients, the sensitivity can be highly variable. Vn96 is a peptide with an affinity for both extracellular vesicles (EVs) and circulating cf-DNA. In this study, we evaluated whether peptide-affinity (PA) precipitation of EVs and cf-DNA from NSCLC patient plasma improves the sensitivity of single nucleotide variants (SNVs) detection and compared observed SNVs with those reported in the matched tissue biopsy. NSCLC patient plasma was subjected to either PA precipitation or cell-free methods and total nucleic acid (TNA) was extracted; SNVs were then detected by next-generation sequencing (NGS). PA led to increased recovery of DNA as well as an improvement in NGS sequencing parameters when compared to cf-TNA. Reduced concordance with tissue was observed in PA-TNA (62%) compared to cf-TNA (81%), mainly due to identification of SNVs in PA-TNA that were not observed in tissue. EGFR mutations were detected in PA-TNA with 83% sensitivity and 100% specificity. In conclusion, PA-TNA may improve the detection limits of low-abundance alleles using NGS.

Identification and characterization of human xylosyltransferase II promoter single nucleotide variants

2015 ◽  
Vol 458 (4) ◽  
pp. 901-907
Author(s):  
Isabel Faust ◽  
Kai Oliver Böker ◽  
Christina Eirich ◽  
Dagmar Akkermann ◽  
Joachim Kuhn ◽  
...  
Keyword(s):  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Identification And Characterization

scholarly journals Ultra-sensitive mutation detection and genome-wide DNA copy number reconstruction by error corrected circulating tumour DNA sequencing

2017 ◽  
Author(s):  
Sonia Mansukhani ◽  
Louise J. Barber ◽  
Sing Yu Moorcraft ◽  
Michael Davidson ◽  
Andrew Woolston ◽  
...  
Keyword(s):  
Error Correction ◽  
Copy Number ◽  
Parallel Evolution ◽  
Tumour Tissue ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Target Region ◽  
Circulating Free Dna ◽  
Genome Wide ◽  
Variant Frequency

AbstractMinimally invasive circulating free DNA (cfDNA) analysis can portray cancer genome landscapes but highly sensitive and specific genetic approaches are necessary to accurately detect mutations with often low variant frequencies. We developed a targeted cfDNA sequencing technology using novel off-the-shelf molecular barcodes for error correction, in combination with custom solution hybrid capture enrichment. Modelling based on cfDNA yields from 58 patients shows that our assay, which requires 25ng of cfDNA input, should be applicable to >95% of patients with metastatic colorectal cancer. Sequencing of a 163.3 kb target region including 32 genes detected 100% of single nucleotide variants with 0.15% variant frequency in cfDNA spike-in experiments. Molecular barcode error correction reduced false positive mutation calls by 98.6%. In a series of 28 patients with metastatic colorectal cancers, 80 out of 91 (88%) mutations previously detected by tumour tissue sequencing were called in the cfDNA. Call rates were similar for single nucleotide variants and small insertions/deletions. Mutations only called in cfDNA but not detectable in matched tumour tissue included, among others, a subclonal resistance driver mutation to anti-EGFR antibodies in theKRASgene, multiple activatingPIK3CAmutations in each of two patients (indicative of parallel evolution), andTP53mutations originating from clonal haematopoiesis. Furthermore, we demonstrate that cfDNA off-target read analysis allows the reconstruction of genome wide copy number aberration profiles from 71% of these 28 cases. This error-corrected ultra-deep cfDNA sequencing assay with a target region that can be readily customized enables broad insights into cancer genomes and evolution.

scholarly journals Genomic and transcriptomic somatic alterations of hepatocellular carcinoma in non-cirrhotic livers

2021 ◽  
Author(s):  
Zachary L Skidmore ◽  
Jason Kunisaki ◽  
Yiing Lin ◽  
Kelsy C Cotto ◽  
Erica K Barnell ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Copy Number Variants ◽  
Genomic Analysis ◽  
Structural Variants ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Tert Promoter Mutations ◽  
Somatic Alterations ◽  
Differential Gene ◽  
Promoter Mutations

Background: Liver cancer is the second leading cause of cancer-related deaths worldwide. Hepatocellular carcinoma (HCC) risk factors include chronic hepatitis, cirrhosis, and alcohol abuse, whereby tumorigenesis is induced through inflammation and subsequent fibrotic response. However, a subset of HCC arises in non-cirrhotic livers. We characterized the genomic and transcriptomic landscape of non-cirrhotic HCC to identify features underlying the disease's development and progression. Methods: Whole genome and transcriptome sequencing was performed on 30 surgically resectable tumors comprised of primarily of non-cirrhotic HCC and adjacent normal tissue. Using somatic variants, capture reagents were created and employed on an additional 87 cases of mixed cirrhotic/non-cirrhotic HCC. Cases were analyzed to identify viral integrations, single nucleotide variants (SNVs), insertions and deletions (INDELS), copy number variants, loss of heterozygosity, gene fusions, structural variants, and differential gene expression. Results: We detected 3,750 SNVs/INDELS and extensive CNVs and expression changes. Recurrent TERT promoter mutations occurred in >52% of non-cirrhotic discovery samples. Frequently mutated genes included TP53, CTNNB1, and APOB. Cytochrome P450 mediated metabolism was significantly downregulated. Structural variants were observed at MACROD2, WDPCP, and NCKAP5 in >20% of samples. Furthermore, NR1H4 fusions involving gene partners EWSR1, GNPTAB, and FNIP1 were detected and validated in 2 non-cirrhotic samples. Conclusion: Genomic analysis can elucidate mechanisms that may contribute to non-cirrhotic HCC tumorigenesis. The comparable mutational landscape between cirrhotic and non-cirrhotic HCC supports previous work suggesting a convergence at the genomic level during disease progression. It is therefore possible genomic-based treatments can be applied to both HCC subtypes with progressed disease.

Direct Plasmonic Detection of Circulating RAS Mutated DNA in Colorectal Cancer Patients

2020 ◽  
Author(s):  
Roberta D’Agata ◽  
Noemi Bellassai ◽  
Matteo Allegretti ◽  
Andrea Rozzi ◽  
Saša Korom ◽  
...  
Keyword(s):  
Liquid Biopsy ◽  
Direct Detection ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Sandwich Hybridization ◽  
Healthy Donors ◽  
Order Of Magnitude ◽  
Sandwich Hybridization Assay ◽  
Colorectal Cancer Patients ◽  
Promising Avenue

By exploiting a liquid biopsy approach, we developed an ultrasensitive nanoparticle-enhanced plasmonic method for detecting RAS single nucleotide variants (SNVs) in the plasma of CRC patients. The PCR-free method we developed is based on an imaging platform and allows the direct detection of ~1 attomolar RAS sequences in plasma with a sandwich hybridization assay using peptide nucleic acids probes. The assay involves a simple pre-analytical procedure that does not require the extraction of tumor DNA from plasma and detects it in volumes as low as 40 uL of plasma, which is at least an order of magnitude smaller than that required by state of the art liquid biopsy technologies. The most prevalent RAS SNVs are detected in DNA from tumor tissue with 100% sensitivity and 83.33% specificity. Spike-in experiments in human plasma further encouraged assay application on clinical specimens. Assay performances were then proven in plasma from CRC patients and healthy donors, demonstrating its promising avenue for cancer monitoring.<br>

scholarly journals Characterization of single nucleotide variants of OPN3 gene in melanocytic nevi and melanoma

2021 ◽  
pp. 100006
Author(s):  
Wei Zhang ◽  
Jianglong Feng ◽  
Wen Zeng ◽  
Zhixu Zhou ◽  
Yu Wang ◽  
...  
Keyword(s):  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Melanocytic Nevi

Clinical performance of a comprehensive novel liquid biopsy test for identifying non-small cell lung cancer (NSCLC) patients for treatment with osimertinib.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 9553-9553
Author(s):  
Jhanelle Elaine Gray ◽  
Ji-Youn Han ◽  
Aino Telaranta-Keerie ◽  
Xiangning Huang ◽  
Alexander Kohlmann ◽  
...  
Keyword(s):  
Targeted Therapy ◽  
Liquid Biopsy ◽  
Clinical Performance ◽  
Cox Model ◽  
Progression Free Survival ◽  
Circulating Tumor Dna ◽  
Single Nucleotide Variants ◽  
Biomarker Testing ◽  
Nsclc Patients ◽  
Egfr Tki

9553 Background: Genotyping is required to identify cancer patients (pts) eligible for targeted therapy; however, many do not receive biomarker testing, in part due to limitations associated with tissue-only genotyping practices and the growing list of biomarkers recommended to be tested. Liquid biopsy overcomes many of these limitations but is not yet fully adopted. We report here the clinical performance of a comprehensive liquid biopsy test based on next generation sequencing (NGS) of circulating tumor DNA (ctDNA) for the identification of NSCLC patients with EGFR exon 19 deletions (ex19del) or L858R mutations ( EGFRm) or EGFR T790M, eligible for treatment with osimertinib. Methods: 441 (79%) of 556 pts randomized in FLAURA (NCT02296125; first-line osimertinib vs comparator EGFR TKI in EGFRm NSCLC) and 300 (72%) of 419 pts from AURA3 (NCT012151981; osimertinib vs chemotherapy in NSCLC pts with T790M at progression on EGFR TKI) were retrospectively tested with Guardant360 (G360), a 74-gene ctDNA NGS assay assessing single nucleotide variants, insertion-deletions, amplifications, and fusions in genes relevant to targeted therapy selection as well as microsatellite instability. Progression-free survival (PFS) of pts with EGFRm or T790M detected by G360 was compared to pts detected by the cobas EGFR Mutation Test (cobas) using tissue or plasma with an unadjusted cox model. Results: Treatment with osimertinib was associated with a significant PFS benefit relative to control therapy in NSCLC pts with EGFRm (FLAURA) and T790M (AURA3) detected using G360 (Table). Observed clinical benefit for pts with EGFRm or T790M detected by G360 was similar to that for pts with EGFRm or T790M identified by cobas using tissue or plasma specimens. Conclusions: This analysis demonstrates that G360 accurately identifies pts for osimertinib therapy while simultaneously providing comprehensive genotyping for other therapeutic molecular targets. The application of NGS liquid biopsy has the potential to increase rates of pts genotyped and access to precision medicine. [Table: see text]

scholarly journals Identification and Characterization of Splicing Defects by Single-Molecule Real-Time Sequencing Technology (PacBio)

2020 ◽  
Vol 7 (4) ◽  
pp. 477-481
Author(s):  
Marco Savarese ◽  
Talha Qureshi ◽  
Annalaura Torella ◽  
Pia Laine ◽  
Teresa Giugliano ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Genetic Diseases ◽  
Disease Genes ◽  
Test Results ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Dna Test ◽  
Splicing Defects

Although DNA-sequencing is the most effective procedure to achieve a molecular diagnosis in genetic diseases, complementary RNA analyses are often required. Reverse-Transcription polymerase chain reaction (RT-PCR) is still a valuable option when the clinical phenotype and/or available DNA-test results address the diagnosis toward a gene of interest or when the splicing effect of a single variant needs to be assessed. We use Single-Molecule Real-Time sequencing to detect and characterize splicing defects and single nucleotide variants in well-known disease genes (DMD, NF1, TTN). After proper optimization, the procedure could be used in the diagnostic setting, simplifying the workflow of cDNA analysis.

scholarly journals Genomic characterization of a novel SARS-CoV-2 lineage from Rio de Janeiro, Brazil

2020 ◽  
Author(s):  
Carolina M Voloch ◽  
Ronaldo da Silva F ◽  
Luiz G P de Almeida ◽  
Cynthia C Cardoso ◽  
Otavio J. Brustolini ◽  
...  
Keyword(s):  
Rio De Janeiro ◽  
Neutralizing Antibodies ◽  
Health Management ◽  
The State ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Viral Genomes ◽  
Genomic Characterization ◽  
Second Wave

AbstractIn this study, we report the sequencing of 180 new viral genomes obtained from different municipalities of the state of Rio de Janeiro from April to December 2020. We identified a novel lineage of SARS-CoV-2, originated from B.1.1.28, distinguished by five single-nucleotide variants (SNVs): C100U, C28253U, G28628U, G28975U, and C29754U. The SNV G23012A (E484K), in the receptor-binding domain of Spike protein, was widely spread across the samples. This mutation was previously associated with escape from neutralizing antibodies against SARS-CoV-2. This novel lineage emerged in late July being first detected by us in late October and still mainly restricted to the capital of the state. However, as observed for other strains it can be rapidly spread in the state. The significant increase in the frequency of this lineage raises concerns about public health management and continuous need for genomic surveillance during the second wave of infections.Article Summary LineWe identified a novel circulating lineage of SARS-CoV-2 in the state of Rio de Janeiro Brazil originated from B.1.1.28 lineage.

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