Genomic alterations of cell-free DNA in early breast cancer patients with recurrence

Abstract Cell-free DNA (cfDNA), as a non-invasive strategy, provides substantial benefit to overcome tumor heterogeneity. Surveillance of recurrence after standard treatment in early breast cancer (BC) using cfDNA, enables to detect minimal residual disease (MRD), also to identify genomic alterations driving recurrences. We aimed to assess the role of cfDNA in detecting MRD by investigating genomic alterations of 1)primary, recurred tumor and 2)cfDNA at time of recurrence using deep targeted sequencing. Fifty-four early BC patients were enrolled prospectively between 2014 and 2017 at time of recurrence. Median disease free interval was 28.5 months (rage 6.2-49.8). 62.7% (32/51) were hormone receptor (HR) positive (28 HRpos/HER2neg, 4 HRpos/HER2pos), 11.8% (6/51) were HRneg/HER2pos and 25.5% (13/51) were triple negative BCs. 59.3% (32/54) patients developed loco-regional recurrence (15 local recurrence only, 13 regional only, 4 with both) and distant metastasis was observed among 40.7% (22/54) patients. Cell-free DNA was extracted from 5cc blood at time of recurrence. Deep targeted sequencing was performed using customized NGS panel –encompassing 426 cancer-related target coding region, 242 fusion and amplification-related region- of cfDNA and FFPE(formalin fixed paraffin embedded) tumor samples archived from surgical resection or biopsy. Deep targeted sequencing data was successfully performed in 72.1% (31/43) plasma samples and sequencing yield was significantly lower when stored for more than 2yrs (46.2% vs 83.3%). Mutations of cfDNA and tumor (primary, recurred) were analyzed. Mean sequencing depth of cfDNA and FFPE were x425.7 and x777.6 respectively. Median number of pathogenic mutations found in primary tumor, cfDNA and recurred tumor were 27(range 12-99), 25(range 8-85) and 9(range 0-23). Among mutations found in primary tumor, 27.4% were shared mutations (range 8.1%-72.7%) with recurred tumor and 26.1% were shared mutations (range 4.7%-69.2%) observed in cfDNA sample. Among mutations found in recurred tumor, 40.9% were observed in cfDNA (range 17.7-87.5%). In primary tumor, median number of mutations with allelic fraction (MAF)>10% were 12 (range 4-21) and at least one mutation was found in cfDNA at time of recurrence. Among mutations with MAF>10%, 59.4% and 69.1% were found in cfDNA and recurred tumor. Known oncogenic mutations of PIK3CA, TP53, GATA3, AKT1, ESR1, RELN, ERBB2, ERBB3, BRCA1 mutation were found. PIK3CA gene (p.H1047R) was found in two cases both in primary tumor and cfDNA at recurrence (MAF 11.4% vs 5.3% and 12.3% vs 15.4%) suggesting de novo driver mutation. One patient developed regional recurrence during adjuvant aromatase inhibitor with ESR1 V392I mutation in both cfDNA and recurred tumor (MAF 48.1 and 54.5%), while another patient's recurred tumor during aromatase inhibitor harbored ESR1 D538G mutation exclusively in recurred tumor with MAF <1%. Both patients had no ESR1 hotpot mutation in primary tumor. Our data showed sequencing yield of 83.3% in plasma samples within 2yr. Pathogenic mutations in primary tumor, especially when MAF>10%, half of them was observed in cfDNA at time of recurrence. ESR1 mutation should be included in cfDNA surveillance for patients undergoing endocrine therapy even absent in primary tumor. Citation Format: Kim J, Jo WK, Kim KY, Kim BJ, Lee SB, Lee HJ, Yu JH, Kim HJ, Chung IY, Ko BS, Kim S-B, Jung KH, Ahn JH, Chang S, Lee JW, Son BH, Ahn SH. Genomic alterations of cell-free DNA in early breast cancer patients with recurrence [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-01-11.