Correlation Between Circulating Tumor Cell DNA Genomic Alterations and Mesenchymal CTCs or CTC-Associated White Blood Cell Clusters in Hepatocellular Carcinoma

Abstract

Purpose: Liquid biopsy is attracting attention as a method of real-time monitoring of patients with tumors. It can be used to understand the temporal and spatial heterogeneity of tumors and has good clinical application prospects. We explored a new type of circulating tumor cell (CTC) enrichment technology combined with next-generation sequencing (NGS) to analyze the correlation between genomic alterations in circulating tumor cells of hepatocellular carcinoma and the counts of mesenchymal CTCs and CTC-associated white blood cell (CTC-WBC) clusters.

Methods: We collected peripheral blood samples from 29 patients with hepatocellular carcinoma from January 2016 to December 2019. We then used the CanPatrol™ system to capture and analyze mesenchymal CTCs and CTC-WBC clusters for all the patients. A customized Illumina panel was used for DNA sequencing and the Mann-Whitney U test was used to test the correlation between mesenchymal CTCs, CTC-WBC cluster counts, and specific genomic changes.

Results: At least one somatic hotspot mutation was detected in each of the 29 sequenced patients. A total of 42 somatic hot spot mutations were detected in tumor tissue DNA, and 39 mutations were detected in CTC-DNA, all of which included common changes in PTEN, MET, EGFR, RET, and FGFR3. The number of mesenchymal CTCs was positively correlated with the somatic genomic alterations in the PTEN and MET genes (PTEN, P = 0.021; MET, P = 0.008, Mann-Whitney U test) and negatively correlated with the somatic genomic alterations in the EGFR gene (P = 0.006, Mann-Whitney U test). The number of CTC-WBC clusters was positively correlated with the somatic genomic alterations in RET genes (P = 0.01, Mann-Whitney U test) and negatively correlated with the somatic genomic alterations in FGFR3 (P = 0.039, Mann-Whitney U test).

Conclusions: We report a novel method of a CTC enrichment platform combined with NGS technology to analyze genetic variation, which further demonstrates the potential clinical application of this method for spatiotemporal heterogeneity monitoring of hepatocellular carcinoma. We found that the number of peripheral blood mesenchymal CTCs and CTC-WBC clusters in patients with hepatocellular carcinoma was related to a specific genome profile.

Keywords: CTC-associated WBC (CTC-WBC) cluster; hepatocellular carcinoma; liquid biopsy; mesenchymal CTC; next-generation sequencing (NGS).

Figure 1

Representative images of various subtypes of CTCs and CTC-WBC clusters. Epithelial cells are positive for EpCAM and CK8/18/19 (red fluorescence), mesenchymal cells are positive for vimentin/twist (green fluorescence), and mixed cells show red fluorescence and green fluorescence. Nuclei were labeled with 40,6-diamidino- 2-phenylindole (DAPI) (blue fluorescence). White blood cells were labeled with CD45 (white fluorescence).

Figure 2

The landscape of genome alteration using tissue and circulating tumor cells (CTCs) for targeted next-generation sequencing. (A) The heat map on the left shows the genomic change landscape of the tissue sample, and the right is the genomic change landscape of the CTC sample. The four colors indicate different mutation types, indels (blue), missense mutations (red), synonymous mutations (green), and meaningless mutations (black). (B) Venn diagram of genomic alterations using tissue and CTCs for targeted next-generation sequencing.

Figure 3

Genomic alterations associated with mesenchymal CTCs. The number of mesenchymal CTCs was positively correlated with the somatic genomic alterations in PTEN and MET genes (PTEN, P = 0.021; MET, P  = 0.008, Mann–Whitney U test), and negatively correlated with the somatic genomic alterations in EGFR (P = 0.006, Mann–Whitney U test).

Figure 4

Genomic alterations associated with mesenchymal CTC-WBC clusters. The number of CTC-WBC clusters was positively correlated with the somatic genomic alterations in RET genes (P = 0.01, Mann–Whitney U test) and negatively correlated with the somatic genomic alterations in FGFR3 (P = 0.039, Mann–Whitney U test).