Tracking focal amplifications using plasma-Seq to identify driver genes in CRC

The analysis of circulating tumor DNA (ctDNA) is a promising tool in the management of colorectal cancer (CRC). Recently, we reported that shallow whole-genome sequencing of plasma DNA (plasma-Seq) identifies focal somatic copy number alterations (SCNA), which in addition to mutations are a driving force in many cancers.

Here we applied plasma-Seq to 150 metastatic CRC patients to track SCNA in response to therapy. We identified focal SCNA harboring known cancer driver genes, such as KRAS on chr12p12.1 or FGFR1 on chr8p11.23-p11.22. Moreover, we observed a recurrent focal amplification on 13q12.2 in 13 patients (8.7%). In order to identify the driver gene in this amplicon, we first we first checked for an association of gene expression and copy number in the TCGA or the CCLE CRC cell line datasets. Surprisingly, the well-known driver gene for hematological malignancies FLT3 did not show a correlation. Moreover, stable expression of FLT3 in Oxco2 cells did not lead to a significant change in proliferation suggesting that FLT3 may not function as a driver gene in CRC. Five of the six other candidate genes demonstrated a positive correlation. After siRNA silencing of these five genes in CRC cell lines with a 13q12.2 overrepresentation, only POLR1D knockdown resulted in significant reduction in cell viability. RNA-Seq revealed an upregulation of VEGFA, an important regulator of angiogenesis, and EREG a member of the epidermal growth factor family. Longitudinal plasma samples from patients receiving anti-VEGF treatment demonstrated dynamic changes of the chr13 amplicon indicating that increased expression of POLR1D in combination with upregulation of VEGFA and EREG may contribute to anti-VEGF treatment resistance.

Our data suggest that POLR1D acts as a potential driver gene in the 13q12.2 amplification and may affect cancer progression through VEGF and EGF pathways. These data highlight the utility of genome-wide ctDNA monitoring to identify novel driver genes in late-stage CRC.