Single-Cell DNA Sequencing Reveals Chromosomal Diversity in HCC and a Novel Model of HCC Evolution

See “Single cell DNA sequencing reveals punctuated and gradual clonal evolution in hepatocellular carcinoma ” by Guo L, Yi X, Chen L, et al, on page 238. See “Single cell DNA sequencing reveals punctuated and gradual clonal evolution in hepatocellular carcinoma ” by Guo L, Yi X, Chen L, et al, on page 238. Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths in the world.1Sung H. Ferlay J. Siegel R.L. et al.Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.CA Cancer J Clin. 2021; 71: 209-249Crossref PubMed Scopus (5875) Google Scholar Effective treatment is challenging, and the best therapies can extend a patient’s life by only a matter of months.2Kudo M. Finn R.S. Qin S. et al.Lenvatinib versus sorafenib in first-line treatment of patients with unresectable hepatocellular carcinoma: a randomised phase 3 non-inferiority trial.Lancet. 2018; 391: 1163-1173Abstract Full Text Full Text PDF PubMed Scopus (1727) Google Scholar,3Llovet J.M. Ricci S. Mazzaferro V. et al.Sorafenib in advanced hepatocellular carcinoma.N Engl J Med. 2008; 359: 378-390Crossref PubMed Scopus (8111) Google Scholar In recent years, single-cell characterization of a limited number of cells suggested that chromosome copy number alterations (CNAs) occur in HCC and are a source of intratumor heterogeneity (ITH).4Duan M. Hao J. Cui S. et al.Diverse modes of clonal evolution in HBV-related hepatocellular carcinoma revealed by single-cell genome sequencing.Cell Res. 2018; 28: 359-373Crossref PubMed Scopus (51) Google Scholar,5Hou Y. Guo H. Cao C. et al.Single-cell triple omics sequencing reveals genetic, epigenetic, and transcriptomic heterogeneity in hepatocellular carcinomas.Cell Res. 2016; 26: 304-319Crossref PubMed Scopus (279) Google Scholar Unfortunately, our understanding of the scope and mechanisms of CNA-driven diversity in HCC is limited. For example, how common are CNAs among patients with HCC and within individual tumors? How do these heterogeneous HCC cells evolve during tumor formation? In the current issue of Gastroenterology, Guo and colleagues6Guo L. Yi X. Chen L. et al.Single cell DNA sequencing reveals punctuated and gradual clonal evolution in hepatocellular carcinoma.Gastroenterology. 2022; 162: 238-252Abstract Full Text Full Text PDF Scopus (1) Google Scholar tackle these questions by characterizing HCC heterogeneity at the single-cell level. They performed single-cell DNA sequencing (scDNA-seq) of HCC cells, and using a combination of modeling, validation, and patient tracking, they demonstrated extensive CNA-mediated tumor heterogeneity and identified a novel mechanism of HCC evolution. Characterization of CNA in whole tissues or tumors can underestimate the degree heterogeneity.7Duncan A.W. Hanlon Newell A.E. Bi W. et al.Aneuploidy as a mechanism for stress-induced liver adaptation.J Clin Invest. 2012; 122: 3307-3315Crossref PubMed Scopus (114) Google Scholar In cases of nonclonal aneuploidy, random gain or loss of a chromosome in one cell is “canceled out” by normal chromosome numbers in other cells, leading to the appearance of euploidy. Identification of CNA with single-cell resolution is important to detect cell-to-cell variation in heterogeneous tissues.8Navin N.E. The first five years of single-cell cancer genomics and beyond.Genome Res. 2015; 25: 1499-1507Crossref PubMed Scopus (211) Google Scholar Although throughput is relatively low (current technologies allow analysis of tens to hundreds of cells), scDNA-seq directly measures copy number and provides high-resolution mapping, which permits detection of chromosome regions of all sizes. Guo et al.6Guo L. Yi X. Chen L. et al.Single cell DNA sequencing reveals punctuated and gradual clonal evolution in hepatocellular carcinoma.Gastroenterology. 2022; 162: 238-252Abstract Full Text Full Text PDF Scopus (1) Google Scholar first performed scDNA-seq on 1275 HCC cells from 10 patients, which is more than 10 times as many HCC cells as previously reported. Although non-HCC cells adjacent to tumors were euploid, HCC cells were composed of a mixture of euploid, pseudoeuploid (limited CNA), and aneuploid (extensive CNA) cells. The degree and composition of CNA varied among patients (33%–94%) and within each tumor, indicating interpatient heterogeneity as well as ITH. For comparison, this high-degree heterogeneity in HCC contrasts with triple-negative breast cancer in which ITH is more restricted.9Gao R.L. Davis A. McDonald T.O. et al.Punctuated copy number evolution and clonal stasis in triple-negative breast cancer.Nat Genet. 2016; 48: 1119-1130Crossref PubMed Scopus (254) Google Scholar Second, the authors investigated evolution of HCC and compared 2 models that could account for CNA diversity: punctuated and gradual copy number tumor evolution. Punctuated copy number evolution refers to rapid accumulation of discrete chromosome alterations and is followed by expansion of individual clones. Punctuated evolution has been proposed to describe the emergence of tumors with low ITH, like triple-negative breast cancer.9Gao R.L. Davis A. McDonald T.O. et al.Punctuated copy number evolution and clonal stasis in triple-negative breast cancer.Nat Genet. 2016; 48: 1119-1130Crossref PubMed Scopus (254) Google Scholar Gradual copy number evolution refers to the slow accumulation of chromosome alterations that leads to formation of diverse cellular subsets. Maximum-parsimony phylogenetic trees were calculated using CNA events in all the HCC cells. As predicted, the punctuated model failed to adequately model tumor evolution, but, surprisingly, the gradual model also failed. However, each model predicted aspects of HCC chromosome diversity, so the authors combined punctuated and gradual evolution into a dual-phase copy number evolution (DPCNE) model. Curve fitting revealed that the DPCNE model outperformed the other models from all patients with HCC with a high degree of confidence. The DPCNE model was subsequently validated with tumors from additional patients with HCC and in patients with colorectal cancer and triple-negative breast cancer (where it surpassed the established gradual model). Thus, modeling suggested that euploid cells underwent a burst of punctuated evolution followed by gradual changes to generate ITH seen in HCC and other cancers. Next, the DPCNE model revealed that the length of gradual phase evolution varied among patients. Some patients had a shortened gradual phase, which correlated with low CNA and ITH. In contrast, HCC from other patients (called the “G-group”) had an extended gradual phase that contributed to the highest levels of CNA and ITH. Intriguingly, stratification of patients based on the duration of gradual phase evolution revealed distinct outcomes. Tumors from G-group patients were characterized by genes associated with poor prognosis, such as chromosome-level amplification of ARID2 and/or loss of TSC1 and WNK2. Furthermore, disease-free survival was reduced in G-group patients (12 months) compared with patients with shortened gradual phase (28.5 months), indicating that a prolonged gradual phase contributes to higher ITH and rapid tumor recurrence. Finally, the authors devised a clever strategy to identify driver genes associated with G-group HCC. They found differentially expressed genes (1) located on regions of chromosome amplification/deletion, and (2) associated with rapid tumor recurrence. Among 47 candidate genes, the most promising hit was CAD, the gene encoding carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase and is involved in pyrimidine synthesis.10Rabinovich S. Adler L. Yizhak K. et al.Diversion of aspartate in ASS1-deficient tumours fosters de novo pyrimidine synthesis.Nature. 2015; 527: 379-383Crossref PubMed Scopus (163) Google Scholar CAD messenger RNA and protein expression were elevated in HCC tissues, and tumors with the highest CAD expression correlated with shortened disease-free survival. Although the role of CAD in HCC progression is unknown, identification of CAD as a potential biomarker or drug target illustrates a novel approach to finding HCC drivers based on CNA events. In summary, Guo et al.6Guo L. Yi X. Chen L. et al.Single cell DNA sequencing reveals punctuated and gradual clonal evolution in hepatocellular carcinoma.Gastroenterology. 2022; 162: 238-252Abstract Full Text Full Text PDF Scopus (1) Google Scholar demonstrate the power of scDNA-seq to characterize aberrations in chromosome number in HCC. CNA events are pervasive, and there is tremendous chromosomal heterogeneity among patients with HCC and among cells within a single tumor. The data indicate that HCC develops though a 2-step evolution model involving both punctuated and gradual accumulation of CNAs. Patients with a prolonged gradual phase have the highest ITH and poorest outcomes. Consideration of HCC from the perspective of CNA (or aneuploidy) is potentially paradigm shifting, but the data should be interpreted cautiously. The total number of patients and HCC cells analyzed is relatively small, and scDNA-seq on more cells from more patients (eg, with different stages of HCC, with different etiologies) is urgently needed. Development of high-throughput technologies for scDNA-seq will accelerate this work. Finally, the presence of heterogeneous aneuploid cells within each tumor may explain why kinase inhibitors like Sorafenib and Lenvatinib are only modestly effective.2Kudo M. Finn R.S. Qin S. et al.Lenvatinib versus sorafenib in first-line treatment of patients with unresectable hepatocellular carcinoma: a randomised phase 3 non-inferiority trial.Lancet. 2018; 391: 1163-1173Abstract Full Text Full Text PDF PubMed Scopus (1727) Google Scholar,3Llovet J.M. Ricci S. Mazzaferro V. et al.Sorafenib in advanced hepatocellular carcinoma.N Engl J Med. 2008; 359: 378-390Crossref PubMed Scopus (8111) Google Scholar When a dominant pathway is blocked in a majority of cells, even a small number of drug-resistant aneuploid cells with selective advantage can potentially drive tumor growth. Future work should focus on therapies that target all aneuploid HCC cells. The “evolutionary trap,” where a chromosomally diverse tumor population is first homogenized (or induced to near-clonality) and then the dominate CNA is targeted for elimination, is an exciting approach that should be considered.11Chen G. Mulla W.A. Kucharavy A. et al.Targeting the adaptability of heterogeneous aneuploids.Cell. 2015; 160: 771-784Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar Single-Cell DNA Sequencing Reveals Punctuated and Gradual Clonal Evolution in Hepatocellular CarcinomaGastroenterologyVol. 162Issue 1PreviewThis study revealed that copy number alteration accumulation in hepatocellular carcinoma follows a novel dual-phase copy number evolution model. Hepatocellular carcinoma that relies most heavily on the gradual phase may be the most severe. Full-Text PDF