Gastroenterology

Gastroenterology

Volume 150, Issue 4, April 2016, Pages 998-1008
Gastroenterology

Original Research
Full Report: Basic and Translational—Liver
Variable Intra-Tumor Genomic Heterogeneity of Multiple Lesions in Patients With Hepatocellular Carcinoma

https://doi.org/10.1053/j.gastro.2015.12.033Get rights and content

Background & Aims

Many patients with hepatocellular carcinoma (HCC) have multiple lesions (primary tumors, intrahepatic metastases, multiple occurrences, satellite nodules, and tumor thrombi); these have been associated with a poor prognosis and tumor recurrence after surgery. We investigated the clonal relationship among these lesions on the basis of genetic features.

Methods

We collected 43 lesions and 10 matched control samples (blood or nontumorous liver) from 10 patients with hepatitis B virus–associated HCC treated at Tianjin Cancer Hospital (China) from January 2013 through May 2014. We performed exome and low-depth, whole-genome sequencing on these samples. Genomic aberrations, including somatic mutations and copy number variations, were identified using germline DNA as control. We compared the genetic features of different lesions from each patient and constructed phylogenetic trees to depict their evolutionary histories.

Results

In each patient, mutations shared by all the lesions were called ubiquitous mutations. The percentage of ubiquitous mutations varied from 8% to 97% among patients, indicating variation in the extent of intratumor heterogeneity. Branched evolution was evident, with somatic mutations, hepatitis B virus integrations, and copy number variations identified on both the trunks and branches of the phylogenetic trees. Intrahepatic metastases and tumor thrombi contained some, but not all, of the mutations detected in their matched primary lesions. By contrast, satellite nodules shared approximately 90% of mutations detected in primary lesions. In a patient with multicentric tumors, 6 lesions were assigned to 2 distinct groups, based on significant differences in genetic features. In another patient with combined hepatocellular and intrahepatic cholangiocarcinoma, the physically separate HCC and cholangiocarcinoma lesions shared 102 mutations.

Conclusions

The extent of intratumor heterogeneity varies considerably among patients with HCC. Therefore, sequence analysis of a single lesion cannot completely characterize the genomic features of HCC in some patients. Genomic comparisons of multiple lesions associated with HCCs will provide important information on the genetic changes associated with tumor progression.

Section snippets

Patient Samples

MLs and matched blood or adjacent noncancerous liver tissues were collected from HCC patients treated with surgical resection at Tianjin Cancer Hospital from January 2013 to May 2014. Informed consent was obtained from patients enrolled in this study. This research was approved by the institutional review boards of Tianjin Cancer Hospital and Peking University. For further information, see the Supplementary Materials and Methods section.

DNA Isolation, Library Preparation, and Next-Generation Sequencing

DNA (50 ng to 1 μg) was extracted from each sample and

Genomic Landscape of 10 HBV-Associated HCC Patients

A total of 53 samples were collected, comprising 43 lesions and 10 matched samples of noncancerous liver tissues or blood (Supplementary Table 1). On average, we obtained 99x exome coverage by uniquely mapping 100-bp reads from paired-end sequencing (Supplementary Table 2). A total of 1474 nonsynonymous and 496 synonymous somatic mutations were identified (Supplementary Figure 2). Validation of a set of nonsynonymous mutations randomly selected from each patient via Sanger sequencing yielded an

Discussion

Genomic sequencing of MLs in HCC patients allowed us to assess intratumor heterogeneity and recapitulate the clonal relationship of these lesions. In this study, we reported the variable extent of ITH, in mutations, HBV integrations, and CNVs that existed among different patients. Given that driver events can be located on either trunks or branches of phylogenetic trees, genomic profiling of only one lesion cannot capture the full genomic landscape, including all driver events, of certain HCC

Acknowledgments

The authors thank all patients who participated in this study. Liver images were obtained from http://pie.med.utoronto.ca/VLiver/ with permission.

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    Conflicts of interest The authors disclose no conflicts.

    Funding Supported by the National High Technology Research and Development Program of China (863 program, 2015AA020403 to F.B.), the National Basic Research Program of China (973 program, 2011CB933100 to N.Z.), the National Science Fund for Distinguished Young Scholars (81125019 to N.Z.), and the Recruitment Program of Global Youth Experts (F.B.).

    Author names in bold designate shared co-first authors.

    ∗

    Authors share co-first authorship.

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