Abstract P4-02-01: High-depth sequencing of circulating tumor DNA to interrogate the genetics of residual micro-metastatic disease prior to relapse in early breast cancer
Authors: Ng Charlotte KY , Weigelt Britta , Garcia-Murillas Isaac , Schiavon Gaia , Hrebien Sarah , Cutts Rosalind J , Osin Peter , Nerurkar Ashutosh , Kozarewa Iwanka , Armisen Garrido Javier , Dowsett Mitch , Smith Ian E , Reis-Filho Jorge S , Turner Nicholas C 
Published: May 2015

Abstract
Background: The identification of early stage breast cancers that are at high risk of relapse after apparently curative treatment of the primary tumor would allow tailored adjuvant therapy approaches to prevent relapse. We sought to define whether high-depth targeted sequencing of circulating cell-free plasma DNA could be used to interrogate the genetics of residual micro-metastatic disease (RMD) persisting after neoadjuvant treatment in patients with early stage breast cancer.
Methods: In a cohort of 31 patients with early breast cancer receiving neoadjuvant chemotherapy, with no evidence of metastasis at presentation, we collected tumor tissue at baseline with the metastatic relapse if it occurred, and serial plasma samples at baseline, post-surgery, and every 6 months in follow-up. Serial plasma samples were subject to digital PCR mutation tracking to examine for circulating tumor DNA (ctDNA). Four patients had ctDNA detectable prior to clinical relapse. Primary tumor-derived DNA (from 4 cases), serial plasma DNA samples (from 4 cases), and metastases derived DNA (from 2 cases) were subjected to massively parallel sequencing (Illumina HiSeq2000) targeting all exons of 273 genes frequently mutated in breast cancers and/ or a custom AmpliSeq cancer panel (IonTorrent).
Results: Targeted sequencing revealed the presence of 1 to 20 somatic mutations in the early breast cancers at baseline, including 3 PIK3CA H1047L/R and 2 TP53 pathogenic mutations, all of which were detected in the subsequent plasma DNA samples taken prior to relapse. However, in one case an ESR1 E380Q mutation found in the baseline primary breast cancer was undetectable by targeted sequencing in all 3 subsequent plasma samples and in the metastasis; this mutation could not be identified in the metastasis by digital PCR. In two cases plasma DNA sequencing revealed no additional mutations to those identified in the primary tumor, whereas in the other two cases, plasma DNA targeted capture sequencing revealed divergence in the genetics of RMD. In these cases, 1 and 5 somatic mutations were found in the plasma DNA but were not detected in the respective baseline tumors. In particular, an activating FGFR1 K656E mutation, which was not detected by targeted capture sequencing in the primary breast cancer at 355x sequencing depth, was found at a mutant allele fraction (MAF) of 2.6% in the plasma 12 months post-surgery, and was subsequently detected in the distant metastasis at a MAF of 43.4% by targeted sequencing and digital PCR.
Conclusions: Our results provide evidence of clonal shifts in response to neoadjuvant systemic therapy of early breast cancers. In addition, we demonstrate that high-depth targeted capture massively parallel sequencing analysis of plasma ctDNA may help predict the genotype of recurrence prior to the onset of clinically overt metastasis.
Citation Format: Charlotte KY Ng, Britta Weigelt, Isaac Garcia-Murillas, Gaia Schiavon, Sarah Hrebien, Rosalind J Cutts, Peter Osin, Ashutosh Nerurkar, Iwanka Kozarewa, Javier Armisen Garrido, Mitch Dowsett, Ian E Smith, Jorge S Reis-Filho, Nicholas C Turner. High-depth sequencing of circulating tumor DNA to interrogate the genetics of residual micro-metastatic disease prior to relapse in early breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-02-01.