Sensitivity, specificity, and accuracy of a liquid biopsy approach utilizing molecular amplification pools

Jessica Garcia,Cristian Ionescu-Zanetti,Florence Geiguer,Sébastien Couraud,Nick Kamps-Hughes,Léa Payen,Brice Sarver

doi:10.1038/s41598-021-89592-8

Abstract

Circulating cell-free DNA (cfDNA) has the potential to be a specific biomarker for the therapeutic management of lung cancer patients. Here, a new sequencing error-reduction method based on molecular amplification pools (MAPs) was utilized to analyze cfDNA in lung cancer patients. We determined the accuracy of MAPs plasma sequencing with respect to droplet digital polymerase chain reaction assays (ddPCR), and tested whether actionable mutation discovery is improved by next-generation sequencing (NGS) in a clinical setting. This study reports data from 356 lung cancer patients receiving plasma testing as part of routine clinical management. Sequencing of cfDNA via MAPs had a sensitivity of 98.5% and specificity 98.9%. The ddPCR assay was used as the reference, since it is an established, accurate assay that can be performed contemporaneously on the same plasma sample. MAPs sequencing detected somatic variants in 261 of 356 samples (73%). Non-actionable clonal hematopoiesis-associated variants were identified via sequencing in 21% of samples. The accuracy of this cfDNA sequencing approach was similar to that of ddPCR assays in a clinical setting, down to an allele frequency of 0.1%. Due to broader coverage and high sensitivity for insertions and deletions, sequencing via MAPs afforded important detection of additional actionable mutations.

Highlights

Commercial introduction of liquid biopsy testing in o ncology[2,3,4], mutation detection performance limitations remain[5,6,7]
The discrepancy between data sets in challenging sample cohorts highlights the need for further orthogonal testing in the clinical setting and sensitivity/specificity improvements, especially in the 0.1%-1% allelic fraction (AF) range, where most of the false positives and false negatives have been found in orthogonal studies[6]
The use of a molecular amplification pools (MAPs)-based confidence score eliminates a majority of false positive calls in the 0.1–1% AF range

Summary

Introduction

Commercial introduction of liquid biopsy testing in o ncology[2,3,4], mutation detection performance limitations remain[5,6,7]. We present clinical validation for a recently-introduced sequencing error-reduction method based on molecular amplification pools (MAPs) that has shown improved analytical accuracy[14]. This methodology tracks variants present in large collections of molecules, as opposed to single molecule UMIs. Used in conjunction with the ERASE-Seq variant caller (Fluxion Biosciences, Alameda, CA, USA), MAPs provide a widely-applicable approach to ultrasensitive NGS test development. Sensitivity and specificity were measured with respect to ddPCR testing performed as part of standard clinical work-up This retrospective study reports data from 356 lung cancer patients receiving plasma testing as part of routine clinical management and utilizing the MAPs approach. The advantages in terms of additional actionable variant discovery by NGS as compared to both ddPCR and tissue sequencing were quantified

Methods

Results

Conclusion