Cell Culture System for Analysis of Genetic Heterogeneity Within Hepatocellular Carcinomas and Response to Pharmacologic Agents

Abstract

Background & Aims No targeted therapies have been found to be effective against hepatocellular carcinoma (HCC), possibly due to the large degree of intratumor heterogeneity. We performed genetic analyses of different regions of HCCs to evaluate levels of intratumor heterogeneity and associate alterations with responses to different pharmacologic agents. Methods We obtained samples of HCCs (associated with hepatitis B virus infection) from 10 patients undergoing curative resection, before adjuvant therapy, at hospitals in China. We collected 4–9 spatially distinct samples from each tumor (55 regions total), performed histologic analyses, isolated cancer cells, and carried them low-passage culture. We performed whole-exome sequencing, copy-number analysis and high-throughput screening of the cultured primary cancer cells. We tested responses of an additional 105 liver cancer cell lines to a FGFR4 inhibitor. Results We identified a total of 3670 non-silent mutations (3192 missense, 94 splice-site variants, and 222 indels) in the tumor samples. We observed considerable intratumor heterogeneity and branched evolution in all 10 tumors; the average percentage of heterogeneous mutations in each tumor was 39.7% (range, 12.9%–68.5%). We found significant mutation shifts toward C>T and C>G substitutions in branches of phylogenetic trees among samples from each tumor (P < .0001). Of note, 14 of the 26 oncogenic alterations (53.8%) varied among subclones that mapped to different branches. Genetic alterations that can be targeted by existing pharmacologic agents (such as those in FGF19, DDR2, PDGFRA and TOP1) were identified in intratumor subregions from 4 HCCs and were associated with sensitivity to these agents. However, cells from the remaining subregions, which did not have these alterations, were not sensitive to these drugs. High-throughput screening identified pharmacologic agents to which these cells were sensitive, however. Overexpression of FGF19 correlated with sensitivity of cells to an inhibitor of FGFR4; this observation was validated in 105 liver cancer cell lines (P=.0024). Conclusions By analyzing genetic alterations in different tumor regions of 10 HCCs, we observed extensive intratumor heterogeneity. Our patient-derived cell line-based model, integrating genetic and pharmacologic data from multi-regional cancer samples, provides a platform to elucidate how intratumor heterogeneity affects sensitivity to different therapeutic agents.