title: Cell Culture System for Analysis of Genetic Heterogeneity Within Hepatocellular Carcinomas and Response to Pharmacologic Agents.
abstract: 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.
  
  
  
  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 fibroblast growth factor receptor (FGFR) 4 inhibitor.
  
  
  
  We identified a total of 3670 non-silent mutations (3192 missense, 94 splice-site variants, and 222 insertions or deletions) in the tumor samples. We observed considerable intratumor heterogeneity and branched evolution in all 10 tumors; the mean 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 FGFR 4; this observation was validated in 105 liver cancer cell lines (P = .0024).
  
  
  
  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 multiregional cancer samples, provides a platform to elucidate how intratumor heterogeneity affects sensitivity to different therapeutic agents.
authors.affiliation: Liver Cancer Institute, Zhongshan Hospital, and Key Laboratory of Carcinogenesis and Cancer Invasion (Ministry of Education), Fudan University, Shanghai, China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Gao
authors.affiliation: Liver Cancer Institute, Zhongshan Hospital, and Key Laboratory of Carcinogenesis and Cancer Invasion (Ministry of Education), Fudan University, Shanghai, China.
authors.forename: Zhi-Chao
authors.initials: ZC
authors.lastname: Wang
authors.affiliation: Liver Cancer Institute, Zhongshan Hospital, and Key Laboratory of Carcinogenesis and Cancer Invasion (Ministry of Education), Fudan University, Shanghai, China.
authors.forename: Meng
authors.initials: M
authors.lastname: Duan
authors.affiliation: Institute of Precision Medicine, 3D Medicines Inc., Shanghai, China.
authors.forename: Yi-Hui
authors.initials: YH
authors.lastname: Lin
authors.affiliation: Department of Psychiatry, Li Ka Shing Faculty of Medicine, the University of Hong Kong, Pokfulam, Hong Kong SAR, China.
authors.forename: Xue-Ya
authors.initials: XY
authors.lastname: Zhou
authors.affiliation: Cancer Theme, South Australian Health and Medical Research Institute and Department of Medicine, University of Adelaide, Adelaide, Australia.
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Worthley
authors.affiliation: Liver Cancer Institute, Zhongshan Hospital, and Key Laboratory of Carcinogenesis and Cancer Invasion (Ministry of Education), Fudan University, Shanghai, China.
authors.forename: Xiao-Ying
authors.initials: XY
authors.lastname: Wang
authors.affiliation: Institute of Precision Medicine, 3D Medicines Inc., Shanghai, China.
authors.forename: Gang
authors.initials: G
authors.lastname: Niu
authors.affiliation: School of Mathematical Sciences and Center for Statistical Science, Peking University, Beijing, China.
authors.forename: Yuchao
authors.initials: Y
authors.lastname: Xia
authors.affiliation: School of Mathematical Sciences and Center for Statistical Science, Peking University, Beijing, China.
authors.forename: Minghua
authors.initials: M
authors.lastname: Deng
authors.affiliation: Liver Cancer Institute, Zhongshan Hospital, and Key Laboratory of Carcinogenesis and Cancer Invasion (Ministry of Education), Fudan University, Shanghai, China.
authors.forename: Long-Zi
authors.initials: LZ
authors.lastname: Liu
authors.affiliation: Liver Cancer Institute, Zhongshan Hospital, and Key Laboratory of Carcinogenesis and Cancer Invasion (Ministry of Education), Fudan University, Shanghai, China.
authors.forename: Jie-Yi
authors.initials: JY
authors.lastname: Shi
authors.affiliation: Liver Cancer Institute, Zhongshan Hospital, and Key Laboratory of Carcinogenesis and Cancer Invasion (Ministry of Education), Fudan University, Shanghai, China.
authors.forename: Liu-Xiao
authors.initials: LX
authors.lastname: Yang
authors.affiliation: Liver Cancer Institute, Zhongshan Hospital, and Key Laboratory of Carcinogenesis and Cancer Invasion (Ministry of Education), Fudan University, Shanghai, China.
authors.forename: Shu
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Liver Cancer Institute, Zhongshan Hospital, and Key Laboratory of Carcinogenesis and Cancer Invasion (Ministry of Education), Fudan University, Shanghai, China.
authors.forename: Zhen-Bin
authors.initials: ZB
authors.lastname: Ding
authors.affiliation: Liver Cancer Institute, Zhongshan Hospital, and Key Laboratory of Carcinogenesis and Cancer Invasion (Ministry of Education), Fudan University, Shanghai, China; Cancer Center, Institute of Biomedical Sciences, Fudan University, Shanghai, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Department of Anatomy and Histology and Embryology, Shanghai Medical College, Fudan University, Shanghai, China.
authors.forename: Chun-Min
authors.initials: CM
authors.lastname: Liang
authors.affiliation: Cancer Research Institute, Xiangya School of Medicine, Central South University, Hunan, China.
authors.forename: Ya
authors.initials: Y
authors.lastname: Cao
authors.affiliation: Institute of Precision Medicine, 3D Medicines Inc., Shanghai, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Xiong
authors.affiliation: School of Mathematical Sciences and Center for Statistical Science, Peking University, Beijing, China.
authors.forename: Ruibin
authors.initials: R
authors.lastname: Xi
authors.affiliation: Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education), Shanghai Jiao Tong University, Shanghai, China. Electronic address: shiyongyong@gmail.com.
authors.forename: Yong-Yong
authors.initials: YY
authors.lastname: Shi
authors.affiliation: Liver Cancer Institute, Zhongshan Hospital, and Key Laboratory of Carcinogenesis and Cancer Invasion (Ministry of Education), Fudan University, Shanghai, China; Cancer Center, Institute of Biomedical Sciences, Fudan University, Shanghai, China. Electronic address: fan.jia@zs-hospital.sh.cn.
authors.forename: Jia
authors.initials: J
authors.lastname: Fan
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D001381:Azepines / Q000494:pharmacology; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology*; D045744:Cell Line, Tumor; D060965:Clonal Evolution; D056915:DNA Copy Number Variations; D004252:DNA Mutational Analysis; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D004354:Drug Screening Assays, Antitumor; D059472:Exome; D005346:Fibroblast Growth Factors / Q000235:genetics; D005784:Gene Amplification; D018740:Genetic Heterogeneity; D006801:Humans; D007191:Indazoles / Q000494:pharmacology; D008113:Liver Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology*; D020125:Mutation, Missense; D000071184:Pharmacogenomic Variants; D010802:Phylogeny; D061251:Primary Cell Culture; D012333:RNA, Messenger / Q000378:metabolism*; D051499:Receptor, Fibroblast Growth Factor, Type 4 / Q000037:antagonists & inhibitors; D017384:Sequence Deletion; D014230:Triazoles / Q000494:pharmacology
chemical_list: C561695:(+)-JQ1 compound; C570663:2-(4-(2-(5-(1-(3,5-dichloropyridin-4-yl)ethoxy)-1H-indazol-3yl)vinyl)-1H-pyrazol-1-yl)ethanol; D000970:Antineoplastic Agents; D001381:Azepines; C117476:FGF19 protein, human; D007191:Indazoles; D012333:RNA, Messenger; D014230:Triazoles; D005346:Fibroblast Growth Factors; C496371:FGFR4 protein, human; D051499:Receptor, Fibroblast Growth Factor, Type 4
keywords: Liver Cancer; Next-Generation Sequencing; Patient-Derived Cell Lines; Targeted Therapy