Development of a highly sensitive liquid biopsy platform to detect clinically-relevant cancer mutations at low allele fractions in cell-free DNA.

Gale D1, Lawson ARJ1, Howarth K1, Madi M1, Durham B1, Smalley S2, Calaway J2, Blais S2, Jones G2, Clark J1, Dimitrov P1, Pugh M1, Woodhouse S1, Epstein M1, Fernandez-Gonzalez A3, Whale AS3, Huggett JF3,4, Foy CA3, Jones GM3, Raveh-Amit H5, Schmitt K5, Devonshire A3, Green E1, Forshew T1, Plagnol V1, Rosenfeld N1.

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Abstract

INTRODUCTION:

Detection and monitoring of circulating tumor DNA (ctDNA) is rapidly becoming a diagnostic, prognostic and predictive tool in cancer patient care. A growing number of gene targets have been identified as diagnostic or actionable, requiring the development of reliable technology that provides analysis of multiple genes in parallel. We have developed the InVision™ liquid biopsy platform which utilizes enhanced TAm-Seq™ (eTAm-Seq™) technology, an amplicon-based next generation sequencing method for the identification of clinically-relevant somatic alterations at low frequency in ctDNA across a panel of 35 cancer-related genes.

MATERIALS AND METHODS:

We present analytical validation of the eTAm-Seq technology across two laboratories to determine the reproducibility of mutation identification. We assess the quantitative performance of eTAm-Seq technology for analysis of single nucleotide variants in clinically-relevant genes as compared to digital PCR (dPCR), using both established DNA standards and novel full-process control material.

RESULTS:

The assay detected mutant alleles down to 0.02% AF, with high per-base specificity of 99.9997%. Across two laboratories, analysis of samples with optimal amount of DNA detected 94% mutations at 0.25%-0.33% allele fraction (AF), with 90% of mutations detected for samples with lower amounts of input DNA.

CONCLUSIONS:

These studies demonstrate that eTAm-Seq technology is a robust and reproducible technology for the identification and quantification of somatic mutations in circulating tumor DNA, and support its use in clinical applications for precision medicine.