Abstract 3968: Comparison of NGS and digital PCR for the detection of point mutations in breast tumor and plasma samples
Authors: Rothé Françoise , Rouas Ghizlane , Coureau Michelle , Maetens Marion , Brown David , Sotiriou Christos , Ignatiadis Michail 
Published: July 2016

Abstract
Background
Over the past years, tumor derived alterations in plasma has emerged as a promising cancer biomarker. The clinical applications envisioned with liquid biopsies range from monitoring of treatment response to disease progression and acquired resistance. However, the analysis of circulating tumor DNA (ctDNA) is challenging owing to the small fraction of tumor specific DNA relative to background levels of non-tumor derived DNA and therefore requires highly specific and sensitive techniques.
Experimental procedures
The Ion AmpliSeqTM Cancer Hotspot Panel v2, covering 2,800 COSMIC mutations from 50 cancer genes was used to analyze 31 primary tumor and metastatic lesions along with 19 matched plasma samples from 13 breast cancer (BC) patients receiving neoadjuvant treatment and 6 advanced stage metastatic BC patients. Digital PCR (dPCR) experiments were set up using mutation-specific assays (Biorad) for the detection of 13 hotspot mutations in PIK3CA, TP53, PTEN, AKT1, CDKN2A and STK11 genes previously identified in the solid tumor samples using next generation sequencing (NGS).
Results
The specificity and sensitivity of the mutation-specific dPCR assays were tested in dilution curves using synthetic oligonucleotides and tumor samples bearing the mutation of interest. Mutations could be robustly detected in plasma samples using dPCR with a sensitivity as low as 0.1% and a sensitivity of 0.5% using NGS. All mutations previously identified using NGS were validated using dPCR in tumor (n = 27) as well as in plasma samples (n = 5). Moreover, dPCR identified 4 additional mutations in 4 plasma samples in the neoadjuvant setting and 4 in the metastatic setting, 2 in tumor and 2 in plasma samples. The mutant allelic frequencies (MAF) of these newly identified mutations were very low (range 0.2%-1.8%) close to the detection limit of the technology. Through the use of dPCR, the detection rate of plasma ctDNA increased from 8% using NGS to 38% in the neoadjuvant setting and from 67% using NGS to 100% in the metastatic setting thereby highlighting the greater sensitivity of the dPCR technology. The MAF assessed by NGS and dPCR were highly correlated both in the neoadjuvant and metastatic (Spearman ρ = 0.84 and ρ = 0.97) settings, a higher concordance between NGS and dPCR being observed when the MAF are high.
Conclusions
The use of dPCR allows the detection of low-frequency tumor-specific mutations in plasma with a greater sensitivity compared to NGS providing the high accuracy and sensitivity essential for liquid biopsies.
Citation Format: Françoise Rothé, Ghizlane Rouas, Michelle Coureau, Marion Maetens, David Brown, Christos Sotiriou, Michail Ignatiadis. Comparison of NGS and digital PCR for the detection of point mutations in breast tumor and plasma samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3968.