Abstract P1-01-10: Exome sequencing of circulating tumor cells in metastatic breast cancer
Authors: Ignatiadis M , Rothé F , Peeters D , Rouas G , Smeets D , Haan J , Lambrechts D , Campbell P , Piccart M , Voet T , Dirix L , Venet D , Sotiriou C 
Published: March 2017

Abstract
Aim: We interrogated whether Circulating Tumor Cells (CTCs) can complement metastatic biopsies for genomic analyses.
Patients and Methods: We compared single nucleotide variants (SNVs) and copy number aberrations (CNAs) identified using whole exome sequencing (WES) of DNA from frozen tumor tissue (primary/metastasis), amplified DNA from CTCs and normal DNA from 3 metastatic breast cancer (BC) patients (pts). All samples of the same patient were collected at the same timepoint. CTC isolation was performed using CellSearch and DEPArray systems followed by whole genome amplification (Ampli1 kit). WES was performed using the Illumina HiSeq2000 with 200X targeted coverage. Reads were aligned using bwa. SNVs had to be called by both Haplotype Caller (vs. reference genome) and Strelka (vs. paired normal). CNAs were determined by counting reads in 1MB windows and by comparing tumor/CTC samples with normal DNA. Pairwise concordance of CNAs profiles of different samples from the same patient was assessed using Spearman correlation (ρ). Significance of ρ differences between pts was obtained by Kruskal-Wallis test. Orthogonal validation for selected SNVs was performed.
Results: We studied 3 patients from the 3 major BC subtypes, patient (pt)1 with ER-/HER2+ BC (samples collected at diagnosis, initially metastatic disease), pt2 with triple-negative BC (samples collected 2 years from diagnosis) and pt3 with ER+/HER2- BC (samples collected 8 years from diagnosis).
We first compared tumor tissue and CTCs for SNVs. For pt1, of the 77 SNVs identified in the tumor, 51 were found on at least one of 12 CTCs samples. For pt2, of the 62 SNVs identified in the tumor, 19 were found on at least 1 of 11 CTCs samples. For pt3, of the 225 SNVs identified in the tumor, 48 were found on at least 1 of 3 CTCs samples. Interestingly, by increasing the number of CTCs analyzed, we increased the % of identified SNVs from synchronous tumor tissue. SNVs with high variant allele fraction (VAF) in tumor tissue were detected significantly more often in CTCs: 22% of the SNVs with VAFs &lt;20% were found at least once, compared to 53% and 74% of SNVs with VAFs &gt;20% and &gt;40%, respectively (p=10-12, Fisher exact test).
Then, we compared tumor tissue and CTCs for CNAs. As time from diagnosis of metastatic disease to samples collection increased, we observed significantly higher heterogeneity within CTCs from the same patient (median ρ between CTCs was 86% for pt1, 84% for pt2 and 28% for pt3, p&lt;0.01) and between CTCs and tumor tissue from the same patient (median ρ was 78% for pt1, 67% for pt2 and 21% for pt3, p&lt;10-4). Interestingly, in pt3 one CTC was more similar to the metastasis than the other 2 (ρ of 53%, 21% and 21%). When a phylogenetic tree was constructed for pt3 by combining SNVs and CNAs data, three clones were identified: one clone with an AKT1 (E17K) and a TP53 (R248W) mutation and a 8p deletion, a second clone with the above profile plus an 8q amplification and a third clone with an AKT1 and an ESR1 (Y537N) mutation and 1p deletion. The metastasis was similar with the first clone.
Conclusions: These data suggest that tumor tissue and single CTC exome sequencing analyses provide complementary information to map tumor heterogeneity. Further validation for potential clinical applications is needed.
Citation Format: Ignatiadis M, Rothé F, Peeters D, Rouas G, Smeets D, Haan J, Lambrechts D, Campbell P, Piccart M, Voet T, Dirix L, Venet D, Sotiriou C. Exome sequencing of circulating tumor cells in metastatic breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-01-10.