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. 2022 Mar 17;12(3):729.
doi: 10.3390/diagnostics12030729.

Analytic and Clinical Validation of a Pan-Cancer NGS Liquid Biopsy Test for the Detection of Copy Number Amplifications, Fusions and Exon Skipping Variants

Audrey Audetat  1 , Chérie Tschida  1 , Sarah Kreston  1 , Adam Stephen  1 , Brittany D'Alessio  1 , Madeline Bondy  1 , Leisa Jackson  1 , Hestia Mellert  1 , Niki Givens  1 , Ubaradka G Sathyanarayana  1 , Gary A Pestano  1
Affiliations

Analytic and Clinical Validation of a Pan-Cancer NGS Liquid Biopsy Test for the Detection of Copy Number Amplifications, Fusions and Exon Skipping Variants

Audrey Audetat et al. Diagnostics (Basel). .

Abstract

Liquid biopsies are an integral part of the diagnosis of cancer. Here, we have extended previous validation studies of a new targeted NGS panel to include the detection of copy number amplifications (CNAs), fusions, and exon skipping variants. Detection of these gene classes included specimens from clinical and healthy donors and cell lines (fusions: ROS1, EML4-ALK, NTRK1; exon skipping: MET exon 14; CNAs: HER2, CDK6, EGFR, MYC, and MET). The limit of detection (LOD) for fusion/skipping was 42 copies (QC threshold was three copies) and was verified using three additional fusion/skipping variants. LOD for CNAs was 1.40-fold-change (QC threshold = 1.15-fold change) and was verified with three additional CNAs. In repeatability and intermediate precision (within lab) studies, all fusion/skipping variants were detected in all runs and all days of testing (n = 18/18; 100%); average CV for repeatability was 20.5% (range 8.7-34.8%), and for intermediate precision it was 20.8% (range 15.7-30.5%). For CNAs, 28/29 (96.6%) copy gains were detected. For CNAs, the average CV was 1.85% (range 0% to 5.49%) for repeatability and 6.59% (range 1.65% to 9.22%) for intermediate precision. The test panel meets the criteria for being highly sensitive and specific and extends its utility for the serial detection of clinically relevant variants in cancer.

Keywords: CNA (copy number amplifications); cell-free nucleic acid (cfNA); exon-skipping; fusions; liquid biopsy; next generation sequencing (NGS); non-small cell lung cancer (NSCLC).

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Control charts for CNA and fusion/skipping variant types. (a) CCDC6-RET fusion, (b) EML4-ALK fusion, (c) CD74-ROS1 fusion, (d) ERBB2/HER2 amplification, and (e) MYC amplification detected within the positive control over seven days of testing.
Figure 2
Figure 2
QC threshold evaluation and limit of detection verification. (a) Fusion/skipping variant types measured as molecular copies and (b) CNA variant types measured as fold-change were evaluated using the pre-set QC thresholds in the Oncomine Pan-Cancer assay bioinformatic pipeline and assessed detection at or near the pre-determined LOD.
Figure 3
Figure 3
Repeatability and intermediate precision study results for the fusion/skipping variant type. (a) Repeatability, (b) inter-day, (c) inter-operator, and (d) inter-instrument variability were evaluated using a high, medium, and low contrived specimen.
Figure 4
Figure 4
Repeatability and intermediate precision study results for the CNA variant type. (a) Repeatability, (b) inter-day, (c) inter-operator, and (d) inter-instrument variability were evaluated using two contrived specimens representing five unique amplification events. Overall, the study demonstrated acceptable consistency in the detection of both the CNA and fusion/exon-skipping variant types in biologically relevant specimens.
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