Tracking plasma DNA mutation dynamics in estrogen receptor positive metastatic breast cancer with dPCR-SEQ

Sunil Kumar,Gaorav P Gupta,Q Brent Chen,Xianming M Tan,Amy L Garrett,Carey K Anders,Daniel Lindsay,Lisa A Carey

doi:10.1038/s41523-018-0093-3

Abstract

Serial monitoring of plasma DNA mutations in estrogen receptor positive metastatic breast cancer (ER + MBC) holds promise as an early predictor of therapeutic response. Here, we developed dPCR-SEQ, a customized assay that utilizes digital PCR-based target enrichment followed by next-generation sequencing to analyze plasma DNA mutations in ESR1, PIK3CA, and TP53. We validated dPCR-SEQ in a prospective cohort of 58 patients with ER + MBC and demonstrate excellent concordance with hotspot ESR1 mutation abundance measured by conventional digital PCR. The dPCR-SEQ assay revealed ESR1, PIK3CA, and TP53 plasma ctDNA mutations in 55%, 32%, and 32% of the study patients, respectively. We also observed dynamic changes in ESR1, PIK3CA, and TP53 ctDNA mutant allele fraction (MAF) that were frequently discordant between the different genes. Thus, monitoring plasma DNA mutation dynamics using a dPCR-SEQ assay is feasible, accurate, and may be investigated as a biomarker of therapeutic response in ER + MBC.

Highlights

The detection and monitoring of circulating tumor DNA mutations in ER + MBC patients has emerged as a promising predictive biomarker of therapeutic sensitivity.[1,2,3,4,5,6] Digital PCR is an established and cost-effective technology to serially monitor plasma DNA mutation kinetics with exceptional accuracy, sensitivity, and specificity.[7]
We report the development of Digital PCR (dPCR)-SEQ, which utilizes digital PCR technology for target enrichment followed by nextgeneration sequencing (Fig. 1a, Supplementary Methods)
We designed a custom primer set for use in the Raindance Thunderbolts OpenSource platform to perform multiplexed amplification of ESR1 and TP53 coding regions and hotspot mutation regions in PIK3CA, PIK3R1, and POLE (Supplementary Table 1)

Summary

Introduction

The detection and monitoring of circulating tumor DNA (ctDNA) mutations in ER + MBC patients has emerged as a promising predictive biomarker of therapeutic sensitivity.[1,2,3,4,5,6] Digital PCR (dPCR) is an established and cost-effective technology to serially monitor plasma DNA mutation kinetics with exceptional accuracy, sensitivity, and specificity.[7]. The high cost and complexity of bioinformatics analysis of advanced NGS-based assays[9] are barriers for implementing serial NGS-based assessment of patients over time

Objectives

Methods

Results