Abstract

Background:

Single-cell technologies address the critical issue of tumor heterogeneity in hepatocellular carcinoma (HCC), but face challenges such as complex cell isolation, need for fresh samples, and limited genomic coverage from low DNA content. To overcome these obstacles, we developed a robust workflow for single-nucleus DNA sequencing from frozen HCC tissue, utilizing a custom amplicon panel to elucidate genetic events in HCC clonal evolution.

Methods:

Under IRB approval, we collected eight paired frozen HCC tissue samples from the tumor core and edge of four treatment-naive patients. Enzymatic nuclei extraction generated single-nuclei droplet suspensions, followed by Tapestri platform barcoding, amplicon library creation, and Illumina sequencing (Fig 1A). We curated a consensus panel of HCC-specific genes from five public databases and designed 146 amplicons to target 204 mutations in 44 frequently mutated genes. Our analysis was augmented with bulk DNA sequencing data from six external studies comprising 1122 samples.

Results:

We isolated and genotyped 13,718 nuclei through single-nuclei targeted DNA sequencing, identifying 23 total single-nucleotide variants (SNVs), insertions, and deletions. TP53, PIK3CA, and TRMT9B were the most frequently mutated and 12 mutations (52%) occurred on chromosome 8. Using the variants identified in each patient, we compared the subclone architecture between the tumor core and edge. The dominant clone in the edge showed a 10.8-fold enrichment (SEM=3.5) versus the core (Fig 1B).

We also examined copy number variations (CNVs) relative to the least mutated clone in each sample and found 10 total CNVs in chromosomes 1, 4, 8, 13, 16, and 17. In five samples, there was a loss in chromosome 17 that corresponded to decreased ploidy of TP53. Chromosome 8 was frequently affected by copy number alterations, where ZFHX4, XKR9, and ZFAT were amplified while ERI1, PDGFRL, and XPO7 were deleted. These genes are clustered on either chromosome arm, suggesting that partial chromosome deletion contributes to tumor progression (Fig 1C).

Our workflow achieved comprehensive coverage of HCC-specific genetic events, as the alterations we identified were present in 50% of patients in the external HCC cohort (n=1122) (Fig 1D). Moreover, patients with alterations in genes found in the tumor edge had significantly poorer survival rates (median 70.1 vs 83.6 months, p=0.04).

Conclusion:

In summary, we optimized a novel workflow that enabled in-depth study of intratumoral clonal evolution in HCC through targeted sequencing of 13,718 single nuclei isolated from frozen tissue. We revealed a higher frequency of genetic alterations in the tumor core than edge, indicating the selection of invasive subclones during tumor progression. Our workflow overcomes the obstacles of traditional single-cell studies while achieving high-resolution insights that reflect true genetic alterations driving clonal evolution in HCC.