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.

中文翻译：

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单细胞 DNA 测序揭示肝细胞癌中点状和渐进的克隆进化

背景与目标

由基因组不稳定性引起的拷贝数改变（CNA）是肿瘤内异质性的主要来源。CNA如何在肝细胞癌（HCC）中进化仍然未知。

方法

我们对分离自 10 名 HCC 患者的 1275 个细胞进行了单细胞 DNA 测序 (scDNA-seq)，对另外 1 名患者的 356 个细胞进行了倍性解析 scDNA-seq，并对另外 3 名患者的 27,344 个细胞进行了单细胞 RNA 测序。比较了三种统计拟合模型以研究 CNA 积累模式。

结果

肿瘤细胞分为以下3个亚群：整倍体、假整倍体和非整倍体。我们的 scDNA-seq 分析表明，CNA 积累遵循双阶段拷贝数进化模型，即一个标点阶段，然后是一个渐进阶段。渐进期延长的患者显示出更高的肿瘤内异质性和更差的无病生存期。整合 17 例 HCC 患者的大量 RNA 测序、已发表的 1196 例肝肿瘤数据集和 202 例 HCC 肿瘤的免疫组化染色，我们发现CAD的高表达，一种参与嘧啶合成的基因，与快速的肿瘤发生和降低的存活率相关。我们的单细胞 RNA 测序数据和已发表的其他癌症类型的 scDNA-seq 数据集验证了双相拷贝数进化模型。此外，倍性解析的 scDNA-seq 揭示了二倍体和多倍体非整倍体细胞的共同克隆起源，表明多倍体肿瘤细胞是由二倍体肿瘤细胞的全基因组加倍产生的。

结论

我们的工作揭示了一种新的双相拷贝数进化模型，显示渐进期较长的 HCC 更严重，将CAD确定为 HCC 早期复发的有希望的生物标志物，并支持多倍体 HCC 的二倍体起源。