Dataset Information

Analytical validation of a next generation sequencing liquid biopsy assay for high sensitivity broad molecular profiling.

ABSTRACT: Circulating tumor DNA (ctDNA) analysis is being incorporated into cancer care; notably in profiling patients to guide treatment decisions. Responses to targeted therapies have been observed in patients with actionable mutations detected in plasma DNA at variant allele fractions (VAFs) below 0.5%. Highly sensitive methods are therefore required for optimal clinical use. To enable objective assessment of assay performance, detailed analytical validation is required. We developed the InVisionFirst™ assay, an assay based on enhanced tagged amplicon sequencing (eTAm-Seq™) technology to profile 36 genes commonly mutated in non-small cell lung cancer (NSCLC) and other cancer types for actionable genomic alterations in cell-free DNA. The assay has been developed to detect point mutations, indels, amplifications and gene fusions that commonly occur in NSCLC. For analytical validation, two 10mL blood tubes were collected from NSCLC patients and healthy volunteer donors. In addition, contrived samples were used to represent a wide spectrum of genetic aberrations and VAFs. Samples were analyzed by multiple operators, at different times and using different reagent Lots. Results were compared with digital PCR (dPCR). The InVisionFirst assay demonstrated an excellent limit of detection, with 99.48% sensitivity for SNVs present at VAF range 0.25%-0.33%, 92.46% sensitivity for indels at 0.25% VAF and a high rate of detection at lower frequencies while retaining high specificity (99.9997% per base). The assay also detected ALK and ROS1 gene fusions, and DNA amplifications in ERBB2, FGFR1, MET and EGFR with high sensitivity and specificity. Comparison between the InVisionFirst assay and dPCR in a series of cancer patients showed high concordance. This analytical validation demonstrated that the InVisionFirst assay is highly sensitive, specific and robust, and meets analytical requirements for clinical applications.

SUBMITTER: Plagnol V

PROVIDER: S-EPMC5854321 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Analytical validation of a next generation sequencing liquid biopsy assay for high sensitivity broad molecular profiling.

Plagnol Vincent V Woodhouse Samuel S Howarth Karen K Lensing Stefanie S Smith Matt M Epstein Michael M Madi Mikidache M Smalley Sarah S Leroy Catherine C Hinton Jonathan J de Kievit Frank F Musgrave-Brown Esther E Herd Colin C Baker-Neblett Katherine K Brennan Will W Dimitrov Peter P Campbell Nathan N Morris Clive C Rosenfeld Nitzan N Clark James J Gale Davina D Platt Jamie J Calaway John J Jones Greg G Forshew Tim T

PloS one 20180315 3

Circulating tumor DNA (ctDNA) analysis is being incorporated into cancer care; notably in profiling patients to guide treatment decisions. Responses to targeted therapies have been observed in patients with actionable mutations detected in plasma DNA at variant allele fractions (VAFs) below 0.5%. Highly sensitive methods are therefore required for optimal clinical use. To enable objective assessment of assay performance, detailed analytical validation is required. We developed the InVisionFirst™ ...[more]

PMID: 29543828

Similar Datasets

Project description:To assess the feasibility and utility of circulating tumor DNA (ctDNA) by amplicon-based next-generation sequencing (NGS) analysis in the daily clinical setting in a cohort of patients with advanced non-small-cell lung cancer (NSCLC), as an alternative approach to tissue molecular profiling. In this single-center prospective study, treatment-naïve and previously treated patients with advanced NSCLC were enrolled. Clinical validation of ctDNA using amplicon-based NGS analysis (with a 36-gene panel) was performed against standard-of-care tissue molecular analysis in treatment-naïve patients. The feasibility, utility, and prognostic value of ctDNA as a dynamic marker of treatment efficacy was evaluated. Results of tissue molecular profile were blinded during ctDNA analysis. Of 214 patients with advanced NSCLC who were recruited, 156 were treatment-naïve patients and 58 were pretreated patients with unknown tissue molecular profile. ctDNA screening was successfully performed for 91% (n = 194) of all patients, and mutations were detected in 77% of these patients. Tissue molecular analysis was available for 111 patients (52%), and tissue somatic mutations were found for 78% (n = 87) of patients. For clinically relevant variants, concordance agreement between ctDNA and tumor tissue analysis was 95% among 94 treatment-naïve patients who had concurrent liquid and tumor biopsy molecular profiles. Sensitivity and specificity were 81% and 97%, respectively. Of the 103 patients with no tissue available, ctDNA detected potential actionable mutations in 17% of patients; of these, 10% received personalized treatment. ctDNA kinetics correlated with response rate and progression-free survival in 31 patients treated with first-line platinum-based chemotherapy. These real-world data from a prospective study endorse ctDNA molecular profile by amplicon-based NGS as an accurate and reliable tool to detect and monitor clinically relevant molecular alterations in patients with advanced NSCLC.

Project description: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 (cfDNA). The comprehensive 80-gene panel profiles point mutations, insertions/deletions, copy number alterations, and gene fusions, and further detects oncogenic viruses (Epstein-Barr virus (EBV) and hepatitis B virus (HBV)) and microsatellite instability (MSI). 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. In non-cancer samples, a high specificity (≥99.9999% per-base) was observed. The limit of detection for copy number alterations, EBV, HBV, and MSI were also empirically determined. Orthogonal comparison of epidermal growth factor receptor (EGFR) variant calls made by LiquidHALLMARK and a reference allele-specific polymerase chain reaction (AS-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% circulating tumor DNA (ctDNA)-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.

Project description:BackgroundComprehensive genomic profiling has become standard clinical practice in the management of advanced lung cancer. In addition to tissue and plasma, other body fluids are also being actively explored as alternative sources of tumor DNA. This study investigated the utility of induced sputum obtained from patients with non-small-cell lung cancer (NSCLC) for somatic variation profiling.MethodsOur study included 41 treatment-naïve patients diagnosed with locally advanced to advanced NSCLC between October 2018 and June 2019. Capture-based targeted sequencing was performed on matched tumor, plasma, and induced sputum samples of 41 patients using a 168-gene panel. We analyzed the somatic variations detected from each sample type and the concordance of variations detected between matched samples. The concordance rate was defined as the proportion of the total number of variations detected from one sample type relative to the reference sample type.ResultsComparative analysis on the somatic variation detection using matched tumor samples as a reference revealed detection rates of 76.9% for plasma, 72.4% for sputum-supernatant, and 65.7% for sputum-sediment samples. Plasma, sputum-supernatant, and sputum-sediment achieved positive predictive values of 73.3%, 80.4%, and 55.6% and sensitivities of 50.0%, 36.9%, 31.3%, respectively, relative to tumor samples for 168 genes. Sputum-supernatants had significantly higher concordance rates relative to matched tumor samples (69.2% vs. 37.8%; P=0.031) and maximum allelic fraction (P<0.001) than their matched sputum-sediments. Sputum-supernatants had comparable detection rates (71.4% vs. 67.9%; P=1.00) but with significantly higher maximum allelic fraction than their matched plasma samples (P=0.003). Furthermore, sputum-supernatant from smokers had a significantly higher maximum allelic fraction than sputum-supernatant from non-smokers (P=0.021).ConclusionsOur study demonstrated that supernatant fraction from induced sputum is a better sampling source than its sediment and performs comparably to plasma samples. Induced sputum from NSCLC patients could serve as an alternative media for next-generation sequencing (NGS)-based somatic variation profiling.

Dataset Information

Analytical validation of a next generation sequencing liquid biopsy assay for high sensitivity broad molecular profiling.

Publications

Analytical validation of a next generation sequencing liquid biopsy assay for high sensitivity broad molecular profiling.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets