A Rapid, Shallow Whole Genome Sequencing Workflow Applicable to Limiting Amounts of Cell-Free DNA

Background: Somatic copy number alterations (sCNAs) acquired during the evolution of breast cancer provide valuable prognostic and therapeutic information. Here we present a workflow for screening sCNAs using picogram amounts of cell-free DNA (cfDNA) and single circulating tumor cells (CTCs).

Methods: We repurposed the Ion ReproSeq PGS™ preimplantation genetic testing kit to perform shallow whole genome sequencing on 178 cfDNA samples (300 pg) and individual CTCs from 10 MBC patients with metastatic breast cancer (MBC) recovered by CellSearch®/DEPArray™. Results were analyzed using a tailored ichorCNA workflow.

Results: sCNAs were detected in cfDNA of 41/105 (39%) patients with MBC and 3/23 (13%) primary breast cancers on follow-up (PBC FU), all of whom subsequently relapsed. In 8 of 10 MBCs, individual CTCs had a higher copy number count than matched cfDNA. The median tumor fraction detected by ichorCNA was 0.34 (range 0.17-0.58) for MBC and 0.36 (range 0.31-0.37) for PBC FU. Patients with detectable tumor fraction (≥ 0.1) and TFx and OncomineTM variants had significantly lower overall survival rates (P values P = 0.002 and P < 0.0001 for the log-rank test, respectively).

Conclusions: The ReproSeq PGS assay is rapid, at approximately $120 per sample, providing both a sCNA profile and estimation of the tumor DNA fraction from limiting cfDNA template (300pg) and individual CTCs. The approach could be used to examine the copy number landscape over time to guide treatment decisions, support future trial designs, and be applied to low volume blood spot samples enabling remote monitoring.