Lin Guo1, Xianfu Yi2, Lu Chen3, Ti Zhang4, Hua Guo5, Ziye Chen5, Jinghui Cheng6, Qi Cao6, Hengkang Liu6, Chunyu Hou6, Lisha Qi7, Zhiyan Zhu8, Yucun Liu9, Ruirui Kong6, Chong Zhang10, Xiaohua Zhou11, Zemin Zhang10, Tianqiang Song4, Ruidong Xue12, Ning Zhang13. 1. Laboratory of Tumor Cell Biology, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China; Department of Genetics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China. 2. School of Biomedical Engineering and Technology, Department of Bioinformatics, The Province and Ministry Co-Sponsored Collaborative Innovation Center for Medical Epigenetics, Tianjin Medical University, Tianjin, China. 3. Laboratory of Tumor Cell Biology, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China; Department of Hepatobiliary Cancer, Liver Cancer Research Center, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin's Clinical Research Center for Cancer, Tianjin, China. 4. Department of Hepatobiliary Cancer, Liver Cancer Research Center, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin's Clinical Research Center for Cancer, Tianjin, China. 5. Laboratory of Tumor Cell Biology, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China. 6. Translational Cancer Research Center, Peking University First Hospital, Beijing, China. 7. Department of Pathology, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China. 8. Tianjin Research Center of Basic Medical Science, Tianjin Medical University, Tianjin, China. 9. Division of General Surgery, Peking University First Hospital, Beijing, China. 10. Beijing International Center for Mathematical Research, Peking University, Beijing, China. 11. BIOPIC, Beijing Advanced Innovation Center for Genomics, and School of Life Sciences, Peking University, Beijing, China. 12. Translational Cancer Research Center, Peking University First Hospital, Beijing, China. Electronic address: rxue@hsc.pku.edu.cn. 13. Laboratory of Tumor Cell Biology, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China; Translational Cancer Research Center, Peking University First Hospital, Beijing, China. Electronic address: zhangning@bjmu.edu.cn.
Abstract
BACKGROUND & AIMS: Copy number alterations (CNAs), elicited by genome instability, are a major source of intratumor heterogeneity. How CNAs evolve in hepatocellular carcinoma (HCC) remains unknown. METHODS: We performed single-cell DNA sequencing (scDNA-seq) on 1275 cells isolated from 10 patients with HCC, ploidy-resolved scDNA-seq on 356 cells from 1 additional patient, and single-cell RNA sequencing on 27,344 cells from 3 additional patients. Three statistical fitting models were compared to investigate the CNA accumulation pattern. RESULTS: Cells in the tumor were categorized into the following 3 subpopulations: euploid, pseudoeuploid, and aneuploid. Our scDNA-seq analysis revealed that CNA accumulation followed a dual-phase copy number evolution model, that is, a punctuated phase followed by a gradual phase. Patients who exhibited prolonged gradual phase showed higher intratumor heterogeneity and worse disease-free survival. Integrating bulk RNA sequencing of 17 patients with HCC, published datasets of 1196 liver tumors, and immunohistochemical staining of 202 HCC tumors, we found that high expression of CAD, a gene involved in pyrimidine synthesis, was correlated with rapid tumorigenesis and reduced survival. The dual-phase copy number evolution model was validated by our single-cell RNA sequencing data and published scDNA-seq datasets of other cancer types. Furthermore, ploidy-resolved scDNA-seq revealed the common clonal origin of diploid- and polyploid-aneuploid cells, suggesting that polyploid tumor cells were generated by whole genome doubling of diploid tumor cells. CONCLUSIONS: Our work revealed a novel dual-phase copy number evolution model, showed HCC with longer gradual phase was more severe, identified CAD as a promising biomarker for early recurrence of HCC, and supported the diploid origin of polyploid HCC.
BACKGROUND & AIMS: Copy number alterations (CNAs), elicited by genome instability, are a major source of intratumor heterogeneity. How CNAs evolve in hepatocellular carcinoma (HCC) remains unknown. METHODS: We performed single-cell DNA sequencing (scDNA-seq) on 1275 cells isolated from 10 patients with HCC, ploidy-resolved scDNA-seq on 356 cells from 1 additional patient, and single-cell RNA sequencing on 27,344 cells from 3 additional patients. Three statistical fitting models were compared to investigate the CNA accumulation pattern. RESULTS: Cells in the tumor were categorized into the following 3 subpopulations: euploid, pseudoeuploid, and aneuploid. Our scDNA-seq analysis revealed that CNA accumulation followed a dual-phase copy number evolution model, that is, a punctuated phase followed by a gradual phase. Patients who exhibited prolonged gradual phase showed higher intratumor heterogeneity and worse disease-free survival. Integrating bulk RNA sequencing of 17 patients with HCC, published datasets of 1196 liver tumors, and immunohistochemical staining of 202 HCC tumors, we found that high expression of CAD, a gene involved in pyrimidine synthesis, was correlated with rapid tumorigenesis and reduced survival. The dual-phase copy number evolution model was validated by our single-cell RNA sequencing data and published scDNA-seq datasets of other cancer types. Furthermore, ploidy-resolved scDNA-seq revealed the common clonal origin of diploid- and polyploid-aneuploid cells, suggesting that polyploid tumor cells were generated by whole genome doubling of diploid tumor cells. CONCLUSIONS: Our work revealed a novel dual-phase copy number evolution model, showed HCC with longer gradual phase was more severe, identified CAD as a promising biomarker for early recurrence of HCC, and supported the diploid origin of polyploid HCC.