Dataset Information

Development, testing and validation of a targeted NGS-panel for the detection of actionable mutations in lung cancer (NSCLC) using anchored multiplex PCR technology in a multicentric setting.

ABSTRACT: Lung cancer is a paradigm for a genetically driven tumor. A variety of drugs were developed targeting specific biomarkers requiring testing for tumor genetic alterations in relevant biomarkers. Different next-generation sequencing technologies are available for library generation: 1) anchored multiplex-, 2) amplicon based- and 3) hybrid capture-based-PCR. Anchored multiplex PCR-based sequencing was investigated for routine molecular testing within the national Network Genomic Medicine Lung Cancer (nNGM). Four centers applied the anchored multiplex ArcherDX-Variantplex nNGMv2 panel to re-analyze samples pre-tested during routine diagnostics. Data analyses were performed by each center and compiled centrally according to study design. Pre-defined standards were utilized, and panel sensitivity was determined by dilution experiments. nNGMv2 panel sequencing was successful in 98.9% of the samples (N = 90). With default filter settings, all but two potential MET exon 14 skipping variants were identified at similar allele frequencies. Both MET variants were found with an adapted calling filter. Three additional variants (KEAP1, STK11, TP53) were called that were not identified in pre-testing analyses. Only total DNA amount but not a qPCR-based DNA quality score correlated with average coverage. Analysis was successful with a DNA input as low as 6.25 ng. Anchored multiplex PCR-based sequencing (nNGMv2) and a sophisticated user-friendly Archer-Analysis pipeline is a robust and specific technology to detect tumor genetic mutations for precision medicine of lung cancer patients.

SUBMITTER: Kumbrink J

PROVIDER: S-EPMC11006983 | biostudies-literature | 2024

REPOSITORIES: biostudies-literature

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Development, testing and validation of a targeted NGS-panel for the detection of actionable mutations in lung cancer (NSCLC) using anchored multiplex PCR technology in a multicentric setting.

Kumbrink Jörg J Demes Melanie-Christin MC Jeroch Jan J Bräuninger Andreas A Hartung Kristin K Gerstenmaier Uwe U Marienfeld Ralf R Hillmer Axel A Bohn Nadine N Lehning Christina C Ferch Ferdinand F Wild Peter P Gattenlöhner Stefan S Möller Peter P Klauschen Frederick F Jung Andreas A

Pathology oncology research : POR 20240328

Lung cancer is a paradigm for a genetically driven tumor. A variety of drugs were developed targeting specific biomarkers requiring testing for tumor genetic alterations in relevant biomarkers. Different next-generation sequencing technologies are available for library generation: 1) anchored multiplex-, 2) amplicon based- and 3) hybrid capture-based-PCR. Anchored multiplex PCR-based sequencing was investigated for routine molecular testing within the national Network Genomic Medicine Lung Cance ...[more]

PMID: 38605929

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Project description:BackgroundWith the introduction of Olaparib treatment for BRCA-deficient recurrent ovarian cancer, testing for somatic and/or germline mutations in BRCA1/2 genes in tumor tissues became essential for treatment decisions. In most cases only formalin-fixed paraffin-embedded (FFPE) samples, containing fragmented and chemically modified DNA of minor quality, are available. Thus, multiplex PCR-based sequencing is most commonly applied in routine molecular testing, which is predominantly focused on the identification of known hot spot mutations in oncogenes.MethodsWe compared the overall performance of an adjusted targeted capture-based enrichment protocol and a multiplex PCR-based approach for calling of pathogenic SNVs and InDels using DNA extracted from 13 FFPE tissue samples. We further applied both strategies to seven blood samples and five matched FFPE tumor tissues of patients with known germline exon-spanning deletions and gene-wide duplications in BRCA1/2 to evaluate CNV detection based solely on panel NGS data. Finally, we analyzed DNA from FFPE tissues of 11 index patients from families suspected of having hereditary breast and ovarian cancer, of whom no blood samples were available for testing, in order to identify underlying pathogenic germline BRCA1/2 mutations.ResultsThe multiplex PCR-based protocol produced inhomogeneous coverage among targets of each sample and between samples as well as sporadic amplicon drop out, leading to insufficiently or non-covered nucleotides, which subsequently hindered variant detection. This protocol further led to detection of PCR-artifacts that could easily have been misinterpreted as pathogenic mutations. No such limitations were observed by application of an adjusted targeted capture-based protocol, which allowed for CNV calling with 86% sensitivity and 100% specificity. All pathogenic CNVs were confirmed in the five matched FFPE tumor samples from patients carrying known pathogenic germline mutations and we additionally identified somatic loss of the second allele in BRCA1/2. Furthermore we detected pathogenic BRCA1/2 variants in four the eleven FFPE samples from patients of whom no blood was available for analysis.ConclusionsWe demonstrate that an adjusted targeted capture-based enrichment protocol is superior to commonly applied multiplex PCR-based protocols for reliable BRCA1/2 variant detection, including CNV-detection, using FFPE tumor samples.

Project description:BackgroundCirculating tumor DNA (ctDNA) has emerged as a promising biomarker for noninvasive cancer diagnostics, particularly in the context of metastatic non-small-cell lung cancer (NSCLC). Detecting targetable variants through ctDNA analysis offers the potential to guide treatment decisions, especially in cases where tissue samples are insufficient or unavailable.MethodIn this study, we developed and validated a next-generation sequencing panel targeting 101 cancer-related genes (101-test) to detect somatic variants in ctDNA from a large cohort of Chinese patients with metastatic NSCLC. The performance of the 101-test was assessed by evaluating its limit of detection (LOD), accuracy, and precision in identifying molecular variants. Additionally, the concordance between ctDNA and tissue samples for detecting targetable variants was analyzed in 904 patients.ResultsThe 101-test demonstrated a LOD of 0.38% for single-nucleotide variants (SNVs), 0.33% for insertions and deletions (InDels), and 0.33% for fusions. Sensitivity was 98.3% for SNVs, 100% for InDels, and 100% for fusions when compared to digital droplet PCR (ddPCR)/breakpoint PCR reference methods. The by-variant sensitivity for somatic variants was 97.5%, with a specificity of 99.9% between tumor-only and tumor-normal analyses. In a real-world cohort, the concordance between ctDNA and tissue samples for identifying targetable variants was 72.2% (457/633). Notably, the EGFR S768I variant, recently recommended by clinical guidelines, achieved an 80% concordance rate. Furthermore, 4.3% of patients (27/633) with targetable variants were identified exclusively through ctDNA testing.ConclusionThe ctDNA-based 101-test is a highly sensitive and specific tool for detecting targetable variants in metastatic NSCLC, particularly in cases with insufficient tissue samples. The findings support the use of ctDNA testing as a reliable and complementary method to traditional tissue-based molecular analysis, enhancing the precision of treatment strategies for NSCLC patients.

Dataset Information

Development, testing and validation of a targeted NGS-panel for the detection of actionable mutations in lung cancer (NSCLC) using anchored multiplex PCR technology in a multicentric setting.

Publications

Development, testing and validation of a targeted NGS-panel for the detection of actionable mutations in lung cancer (NSCLC) using anchored multiplex PCR technology in a multicentric setting.

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