Simultaneous identification of clinically relevant single nucleotide variants, copy number alterations and gene fusions in solid tumors by targeted next-generation sequencing

Context.— An increasing number of molecular laboratories are implementing next-generation sequencing platforms to identify clinically actionable and relevant genomic alterations for precision oncology. Objective.— To describe the validation studies as per New York State–Department of Health (NYS-DOH) guidelines for the Oncomine Comprehensive Panel v2, which was originally tailored to the National Cancer Institute Molecular Analysis for Therapy Choice (NCI-MATCH) trial. Design.— Accuracy, precision, and reproducibility were investigated by using 130 DNA and 18 RNA samples from cytology cell blocks; formalin-fixed, paraffin-embedded tissues; and frozen samples. Analytic sensitivity and specificity were tested by using ATCC and HapMap cell lines. Results.— High accuracy and precision/reproducibility were observed for single nucleotide variants and insertion/deletions. We also share our experience in the detection of gene fusions and copy number alterations from an amplicon-based sequencing platform. After sequencing analysis, variant annotation and report generation were performed by using the institutional knowledgebase. Conclusions.— This study serves as an example for validating a comprehensive targeted next-generation sequencing assay with both DNASeq and RNASeq components for NYS-DOH.

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Validation of OncoPanel: A Targeted Next-Generation Sequencing Assay for the Detection of Somatic Variants in Cancer

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2016-0527-oa ◽

2017 ◽

Vol 141 (6) ◽

pp. 751-758 ◽

Cited By ~ 130

Author(s):

Elizabeth P. Garcia ◽

Alissa Minkovsky ◽

Yonghui Jia ◽

Matthew D. Ducar ◽

Priyanka Shivdasani ◽

...

Keyword(s):

Next Generation Sequencing ◽

Copy Number ◽

Cancer Biology ◽

Next Generation ◽

Analytical Validation ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Insertions And Deletions ◽

Targeted Next Generation Sequencing ◽

Generation Sequencing

Context.— The analysis of somatic mutations across multiple genes in cancer specimens may be used to aid clinical decision making. The analytical validation of targeted next-generation sequencing panels is important to assess accuracy and limitations. Objective.— To report the development and validation of OncoPanel, a custom targeted next-generation sequencing assay for cancer. Design.— OncoPanel was designed for the detection of single-nucleotide variants, insertions and deletions, copy number alterations, and structural variants across 282 genes with evidence as drivers of cancer biology. We implemented a validation strategy using formalin-fixed, paraffin-embedded, fresh or frozen samples compared with results obtained by clinically validated orthogonal technologies. Results.— OncoPanel achieved 98% sensitivity and 100% specificity for the detection of single-nucleotide variants, and 84% sensitivity and 100% specificity for the detection of insertions and deletions compared with single-gene assays and mass spectrometry–based genotyping. Copy number detection achieved 86% sensitivity and 98% specificity compared with array comparative genomic hybridization. The sensitivity of structural variant detection was 74% compared with karyotype, fluorescence in situ hybridization, and polymerase chain reaction. Sensitivity was affected by inconsistency in the detection of FLT3 and NPM1 alterations and IGH rearrangements due to design limitations. Limit of detection studies demonstrated 98.4% concordance across triplicate runs for variants with allele fraction greater than 0.1 and at least 50× coverage. Conclusions.— The analytical validation of OncoPanel demonstrates the ability of targeted next-generation sequencing to detect multiple types of genetic alterations across a panel of genes implicated in cancer biology.

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Abstract LB-227: Guideline adherent clinical validation of a comprehensive DNA/RNA panel (523 genes-TruSight Oncology 500) for determination of single nucleotide variants (SNV’s), small insertions or deletions (Indels), copy number variations (CNV’s), splice variations (SV’s), gene fusions (GF’s), tumor mutation burden (TMB) and micro-satellite instability (MSI) on anext-generation sequencing (NGS)platform in a CLIA setting

10.1158/1538-7445.sabcs18-lb-227 ◽

2019 ◽

Author(s):

Ravindra Kolhe ◽

Pankaj Ahluwalia ◽

Saleh Heneidi ◽

Sudha Ananth ◽

Vamsi Kota ◽

...

Keyword(s):

Copy Number ◽

Copy Number Variations ◽

Clinical Validation ◽

Gene Fusions ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Tumor Mutation Burden ◽

Mutation Burden ◽

Generation Sequencing

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Analytical and Clinical Validation of an Amplicon-based Next Generation Sequencing Assay for Ultrasensitive Detection of Circulating Tumor DNA

10.1101/2021.08.03.21261575 ◽

2021 ◽

Author(s):

Jonathan Poh ◽

Kao Chin Ngeow ◽

Michelle Pek ◽

Kian-Hin Tan ◽

Jing Shan Lim ◽

...

Keyword(s):

Next Generation Sequencing ◽

Copy Number ◽

Point Mutations ◽

Circulating Tumor Dna ◽

Clinical Validation ◽

Gene Fusions ◽

Copy Number Alterations ◽

Next Generation ◽

Tumor Dna ◽

Generation Sequencing

Next-generation sequencing of circulating tumor DNA presents a promising approach to cancer diagnostics, complementing conventional tissue-based diagnostic testing by enabling minimally invasive serial testing and broad genomic coverage through a simple blood draw to maximize therapeutic benefit to patients. LiquidHALLMARK® is an amplicon-based next-generation sequencing assay developed for the genomic profiling of plasma-derived cell-free DNA. The comprehensive 80-gene panel profiles point mutations, insertions/deletions, copy number alterations, and gene fusions, and further detects oncogenic viruses (EBV and HBV) and microsatellite instability. Here, the analytical and clinical validation of the assay is reported. Analytical validation using reference genetic materials demonstrated a sensitivity of 99.38% for point mutations and 95.83% for insertions/deletions at 0.1% variant allele frequency (VAF), and a sensitivity of 91.67% for gene fusions at 0.5% VAF, with high specificity even at 0.1% VAF (99.11% per-base). The limit of detection for copy number alterations, EBV, HBV, and microsatellite instability were also empirically determined. Orthogonal comparison of EGFR variant calls made by LiquidHALLMARK and a reference allele-specific PCR method for 355 lung cancer specimens revealed an overall concordance of 93.80%, while external validation with cobas® EGFR Mutation Test v2 for 50 lung cancer specimens demonstrated an overall concordance of 84.00%, with a 100% concordance rate for EGFR variants above 0.4% VAF. Clinical application of LiquidHALLMARK in 1,592 consecutive patients demonstrated a high detection rate (74.8% alteration-positive in cancer samples) and broad actionability (50.0% of cancer samples harboring alterations with biological evidence for actionability). Among ctDNA-positive lung cancers, 72.5% harbored at least one biomarker with a guideline-approved drug indication. These results establish the high sensitivity, specificity, accuracy, and precision of the LiquidHALLMARK assay and supports its clinical application for blood-based genomic testing.

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