International pilot external quality assessment scheme for analysis and reporting of circulating tumour DNA

Abstract

BackgroundMolecular analysis of circulating tumour DNA (ctDNA) is becoming increasingly important in clinical treatment decisions. A pilot External Quality Assessment (EQA) scheme for ctDNA analysis was organized by four European EQA providers under the umbrella organization IQN Path, in order to investigate the feasibility of delivering an EQA to assess the detection of clinically relevant variants in plasma circulating cell-free DNA (cfDNA) and to analyze reporting formats.MethodsThirty-two experienced laboratories received 5 samples for EGFR mutation analysis and/or 5 samples for KRAS and NRAS mutation analysis. Samples were artificially manufactured to contain 3 mL of human plasma with 20 ng/mL of fragmented ctDNA and variants at allelic frequencies of 1 and 5%.ResultsThe scheme error rate was 20.1%. Higher error rates were observed for RAS testing when compared to EGFR analysis, for allelic frequencies of 1% compared to 5%, and for cases including 2 different variants. The reports over-interpreted wild-type results and frequently failed to comment on the amount of cfDNA extracted.ConclusionsThe pilot scheme demonstrated the feasibility of delivering a ctDNA EQA scheme and the need for such a scheme due to high error rates in detecting low frequency clinically relevant variants. Recommendations to improve reporting of cfDNA are provided.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-4694-x) contains supplementary material, which is available to authorized users.

From Somatic Variants Toward Precision Oncology: An Investigation of Reporting Practice for Next-Generation Sequencing-Based Circulating Tumor DNA Analysis

“…Similar results were also found in several recently published orthogonal comparison studies and the recent European pilot schemes, which also underscores the importance of analytically validated assays with sensitivity below 1% VAF . Thus, in the future, stricter experiments for the confirmation of performance parameters (especially LOD) need to be further developed in all laboratories by using materials with natural ctDNA characteristics and precise VAF rather than clinical samples or plasmids .…”

“…In comparison with the external quality assurance schemes that were organized by the IQN Path for ctDNA analysis , the overall performance of participants was poorer in our scheme. Several required laboratory performances (such as sample identification, detecting accuracy, and LOD reporting) and recommended laboratory performance (such as categorization of variants and/or medications and specific reporting preferences) for NGS‐based ctDNA analysis were found to be problematic.…”

“…However, because of the use of different NGS panels (commercial NGS panels or laboratory‐developed test panels), target enrichment strategies (targeted capture or multiplex PCR), sequencing platforms, improvement countermeasures, bioinformatics analysis pipelines, and databases (public databases or self‐built databases) by different laboratories, the detected variants and relevant information regarding medication in each report also differ . Although these laboratories usually self‐proclaim that they have accurately detected tumor‐specific alterations with high clinical sensitivity and specificity and have comprehensively reported the latest medication information, the authenticity and reliability of the clinical ctDNA analysis report is still doubtful . As accurate, comprehensive, and standardized test reports serve as the cornerstone for clinicians to identify the best treatment strategies for patients with cancer, the existing inconsistent reports have made this a challenging time for clinicians to exactly fathom out the reliable reports and effectively incorporate these reports into routine patient care .…”

Current status and future perspectives of liquid biopsy in non-small cell lung cancer

“…All participating laboratories had excellent performance with 100% accuracy for EGFR E19del, L858R, and T790M mutations ( Table 3). The 2017 European External Quality Assessments (EQA) scheme for ctDNA analysis that involved 32 laboratories from 16 countries showed an overall error rate of 42% for 1% of T790M/L858R and 7% for 5% of T790M/L858R [48]. One false-positive result was observed.…”

“…The rest stored plasma samples in a freezer. In the European EQA scheme, NGS (39%) is the most commonly used ctDNA test, followed by Cobas (26%) and ddPCR (23%) [48]. Fifty-five percent of the laboratories used QIAamp and 25% used Cobas for ctDNA extraction.…”