Abstract P4-01-10: Targeted exome sequencing of circulating tumor cells in breast cancer
Authors: Bains Pushpinder K. , Campo Daniel , Porras Tania B. , Lu Janice , Lang Julie E. 
Published: March 2020

Abstract
Background: Next-generation sequencing (NGS) has provided the means to comprehensively analyze somatic mutations in solid tumors. The major challenge is the accurate detection of somatic variants in low-quality DNA from formalin-fixed paraffin-embedded (FFPE) tissues and circulating tumor cells (CTCs) as white blood cells (WBCs) or other background cell populations confound variant identification. The aim of our study was to evaluate the feasibility of combining whole genome amplification (WGA) with hybridization capture-based, targeted exome sequencing in identifying clinically relevant variants in low-quality and limited input metastatic breast cancer (MBC) samples.
Methods: For clinical samples, we collected 7.5 mL of blood in Streck tubes and FFPE biopsies from 5 newly diagnosed MBC patients. The CTCs were isolated with the ANGLE Parsortix using a 10um cassette within 24h of blood draw. WBCs from the same patients were profiled to eliminate germline contaminants. We used spiked cell lines (CTC mimics) as a positive control. MDA-MB-231 cells (n=50) were spiked into 7.5ml healthy donor whole-blood. We used peripheral blood not processed on the Parsortix as a negative control (no spiked cells). We performed exome sequencing on a total of 20 samples (experimental samples: spike-in MDA-MB-231 samples (S1 and S2, n=2), positive control bulk MDA-MB-231 cells (P1 and P2, n=2) and negative control WBCs (WL1 and WL2, n=2); clinical samples: CTCs (n=5), MBC (n=4) and WBCs (n=5)). We used the Repli-G WGA method and the SeqCap EZ MedExome Probes that targets the human exome for ~4600 medically relevant genes, followed by sequencing using the Illumina HiSeq2500. Somatic mutations were called with GATK MuTect2 in clinical samples and Samtools using Partek Flow for experimental samples. The variant annotation was performed using SnpEff.
Results: The sequencing quality of all 6 spike-in experimental samples showed that &gt;75% of the reads were overlapping in genomic features. The total number of variants identified in P1 and P2 were 69,954 and 69,848, respectively. The number of variants identified in S1 and S2 was 51,359 and 50,798 respectively. We found a substantial overlap of 67% (47,201/ 69,954) common variants in P1 and S1. Likewise, an overlap of 64% common variants (45,392/69,848) was detected in P2 and S2. We analyzed the pattern of somatic variants in both coding and noncoding regions, resulting in the identification of 8786 variants in MBC (n=4) and 638 variants in CTCs (n=5). With further filtering steps, we detected 1959 coding variants in MBC and 118 variants in CTCs. SnpEff predicted that 74% of these variants in CTCs (88/118) and 14% in MBC (274/1959) had a high impact on protein function. A total of 25 common genes were also identified in CTCs and MBC in which variants were found in protein-coding regions. We matched a list of genes having coding variants in CTCs and MBC with the actionable gene panel of a commercial genomic testing platform (FoundationOne) and a precision oncology database (OncoKB) cancer gene list. The CTCs (3/5 samples) harbored alterations in a total of 11 FoundationOne genes and 9 OncoKB genes. We identified 54 actionable genes that showed overlap with FoundationOne and 104 genes with OncoKB in all 4 MBC samples. The copy number alterations (CNAs) analysis showed a larger number of amplifications in CTCs and deletions in MBC.
Conclusion: We established the feasibility of exome sequencing of medically relevant genes in detecting known and unknown variants in a breast cancer cell line spike in experiment. This workflow has further been validated on CTCs and MBC samples by sequencing both low-input material and low-quality FFPE samples. This approach has a high potential for the identification of the complete repertoire of mutations along with CNAs and for monitoring the presence of actionable alterations throughout the course of treatment.
Citation Format: Pushpinder K. Bains, Daniel Campo, Tania B. Porras, Janice Lu, Julie E. Lang. Targeted exome sequencing of circulating tumor cells in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-01-10.