Abstract P1-08-16: Deep sequencing of breast tumor biopsies reveals an association between pathologic complete response and reduction of TP53 clonal abundance upon brief exposure to therapy
Authors: Williams N , Varadan V , Miskimen K , Vadodkar A , Poruban D , Edelheit S , Gilmore H , Maximuk S , Sinclair N , Lezon-Geyda K , Abu-Khalaf M , Sikov W , Harris L 
Published: August 2014

Abstract
Background: Next generation deep sequencing has revealed the existence of intra-tumor heterogeneity within subsets of breast tumors. The clinical implications of intra-tumor heterogeneity are not fully understood, however subclonal heterogeneity likely plays a role in treatment resistance. We quantify the clonal abundance of somatic mutations in breast tumor biopsies using deep targeted amplicon sequencing and assess their changes over the course of preoperative therapy (PT). We also evaluate the association of changes in clonal abundance upon brief exposure (BE) to therapy with clinical outcome.
Methods: DNA from 69 breast tumor samples obtained from BE preoperative clinical trials BrUOG 211A/211B were sequenced. Patients received a run-in dose of bevacizumab(B), nab-paclitaxel(N) or trastuzumab (T), followed by combination biologic/chemotherapy (HER2- with B/carboplatin/N; HER2+ with T/carboplatin/N). We sequenced biopsy pairs obtained pre/post 10 day exposure to run-in targeted therapy and germline and surgical tumor DNA for a subset of patients upon completion of PT. A TruSeqCustom Amplicon (Illumina) for targeted enrichment sequencing that included 1183 amplicons covering either hotspot regions or whole exonic regions from 35 commoly mutated genes in breast cancer (TCGA, Stephens, 2012; Shah); a total of 101,484 bp of the genome was represented. Sequencing was performed using IlluminaMiSeq platform and analyzed for variant calls using IlluminaBasespace. High confidence somatic mutations were identified in samples with matched germline data using VarScan2. In the absence of matched normal DNA, germline variants were eliminated using dbSNP and the 1000 Genomes Project. Minor allele frequencies (MAF) of somatic aberrations were estimated as the percentage of reads matching the variant.
Results: Approximately 5 mutations on average were found baseline and post-exposure, with a maximum mutational burden of 15 mutations in one basal breast cancer. Recurrent somatic aberrations were observed in TP53 (42%), PIK3CA (16%) and FAT4 (13%), whereas sporadic aberrations were also seen in COL1A1, PTEN, CDH1. More than 85% of samples harboring TP53 mutations exhibited MAF≥40%. Similar high clonal abundance (MAF &gt;50%) was observed for FAT4 mutations whereas PIK3CA mutations exhibited only subclonal frequencies (MAF≤30%). We evaluated changes in clonal architecture upon BE to therapy by scoring for a change in MAF of at least 10% from baseline to post-exposure sample. We scored a total of 16 cases for clonal abundance changes in TP53 mutations upon exposure to therapy. We found 6 cases that exhibited ≥10% reduction in MAF, of which 4 achieved pCR (p = 0.03) and the remaining 2 achieved RCB I. This association was independent of therapy arm and BE regimen.
Conclusions: We found that a reduction in TP53 clonal abundance upon BE to PT is associated with clinical outcome. We are currently integrating whole genome copy-number profiles with the deep sequencing data to more accurately assess clonal architecture and changes upon exposure to therapy. Clonal changes upon BE to therapy may provide early readouts of therapy benefit and provide biological insights into mechanisms of action.
Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-08-16.