Sampling and sequencing of primary liver cancers developed from chronic hepatitis in HBV transgenic mice

We previously reported that repetitive injections of the agonist anti-CD137 mAb in HBV-transgenic mice consistently induced chronic hepatitis, fibrosis, cirrhosis, and, ultimately, adenoma and liver cancer, which closely mimics the pathogenic process of HCC developed from chronic hepatitis[29]. Nine months after five weeks of injection of the anti-CD137 mAb, multiple liver tumor nodules of various sizes were present in all treated mice. The cytological and histological characteristics of both hepatocellular adenoma (HCA) and carcinomas (HCC) were detected. Nodules with normal or larger hepatocytes with little cytoplasm and relatively hyperchromatic nuclei were arranged in one to two cell-thick distorted trabeculae were classified as HCA (Fig 1A), whereas nodules characterized by the uneven proliferation of hepatocytes, hemorrhage and necrosis, were classified as HCC (Fig 1B).

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Fig 1. Pathological analysis of tumor tissues and phylogenetic relationships of the three tumors in the M1 mouse.

HBV-transgenic mice were intraperitoneally injected with anti-CD137 Ab weekly up to five times. Live tumor nodules and adjacent non-tumor tissues were harvested at more than 11 months after the last injection. Representative tumor sections stained with H&E showing (A) hepatocellular adenomas (HCA), (B) hepatocellular carcinomas (HCC). Top: overview of tumor and pericancerous tissues. Bottom: zoomed-in view of the tumor in the top panel. White arrows indicate the uneven proliferation of hepatocytes. (C) Phylogeny of tumors from the M1 mouse based on validated SNVs. The ancestor lineage was defined as Ω. NS: nonsynonymous mutation, S: synonymous mutation. (D) Liver morphology and locations of 3 tumors in the liver of the M1 mouse.

https://doi.org/10.1371/journal.pone.0187551.g001

To study the genomic variations underlying primary HCC development, 25 liver tumor nodules larger than 3 mm in diameter and four adjacent non-tumor tissues were harvested by bulk sampling from four mice (M1, M2, M3 and M4) from 10 to 20 months after antibody injections (S1 Table and S1A Fig). The M1 mouse had only 3 macroscopic tumors, whereas the other mice had multiple tumor nodules. Except for six small nodules (T5-T9) from the M3 mouse, all other tissues were histologically analyzed using H&E staining. All analyzed nodules from the M1, M3 and M4 mice were HCC, whereas the six nodules from the M2 mouse included two adenomas (T2 and T5) and four hyperplasias (T1, T3, T 4 and T6) (S2 Fig). We performed WES for three tumor nodules (T) from each mouse, adjacent tissue samples from M2 and M3, and the peripheral blood DNA of M4 because a small tumor was found on M4N after the pathology analysis. M1T1, M1T2 and M1N were also submitted for WGS (S1 Table).

Genetic diversity among tumors and the independent origin of most tumors in the sequenced mice

For all WES sequenced samples, we obtained 56-fold mean coverage of whole-exome regions, with 84% of loci covered at > 10-fold, whereas the average depth was 23-fold for the whole genome-sequenced samples, with 93% coding regions (CDs) covered at > 10-fold (S2 Table). All candidate somatic SNVs within the CDs were further validated by Sequenom genotyping. In addition to the sequenced samples, 3, 6 and 4 additional tumor nodules were respectively harvested from the M2, M3 and M4 mice and used for validation (S1 Table). Overall, we identified 46 SNVs in the exomes of sequenced tumors, including 32 missense, 12 synonymous, 1 nonsense and 1 splicing mutation. The number of mutations ranged from 0 in nodules from M2T1 and M2T3, which were characterized as hyperplasic, to a maximum of 17 in M1T1. The other 9 tumors had 6 (M4T6), 5 (M1T2), 5 (M2T2), 5 (M4T1), 4 (M3T5), 3 (M1T1), 1 (M1T3), 1 (M3T2), and 1 (M4T4) mutations within CDs (Table 1). Of these mutations, the Hras Q61L mutation was the only one shared by three tumors from the M1 mouse. All other SNVs were only verified in the sequenced samples, such that no SNV was recurrent in other tumors. These data indicate the independent origin of the multiple tumors in the mice, except for the M1 mouse.

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Table 1. Validated somatic SNVs in tumors from mice M1, M2, M3 and M4.

https://doi.org/10.1371/journal.pone.0187551.t001

The three tumors of the M1 mouse shared the Hras mutation, suggesting that they have a common origin. Their phylogenetic relationship was constructed based on the validated point mutations (Fig 1C). In addition to the Hras mutation, T1 and T2 had 16 and 4 of their own SNVs, respectively, whereas no extra SNV was identified in T3. To verify the SNV distribution within tumors, we validated the SNVs of T1 and T2 in 12 and 19 micro-dissected samples of the T1 and T2 tumors, respectively. Most of the SNVs were validated by Sequenom genotyping, except a few that failed (S4 Table). For SNV frequencies that were validated in micro-dissected samples, there was no significant difference among micro-dissected samples and the bulk sample in T1 (Kruskal-Wallis test, P value, 0.99) and T2 (Kruskal-Wallis test, P value, 0.81). The specific SNVs for each tumor were nearly validated in all micro-sections of the tumor, suggesting that these specific mutations accumulated at a very early stage of clone expansion.

For the whole-genome level variations of tumors from M1, we identified 1163 and 943 SNVs for M1T1 and M1T2, respectively. Only 56 SNVs, representing 6% of the total SNVs in M1T1 and M1T2, were shared by both tumors (S3 Table). For the liver specimen, M1T1 and M1T2 were located on the middle liver lobe, whereas T3 was found on a margin of the left liver lobe (Fig 1D). The common origin of the three tumors from M1 suggests that their ancestor cell population was able to migrate within the liver at a very early stage prior to tumor cell population expansion. To define the chromosomal aberrations in the tumorigenesis of primary HCC, we assessed somatic CNAs of M1T1 and M1T2 based on the read depth of the whole genome sequencing data compared to that of the normal control M1N. A large fraction of the genome had undergone alterations in M1T1, including deletions of Chr4, Chr8, Chr9, Chr13, Chr14, and Chr17 and a 45 M region within ChrX and gains of Chr7, a 50 M region of Chr10, Chr15, and Chr16 and a region of approximately 50 M of Chr18 (S3 Fig). However, no obvious CNA was found in M1T2. SVs of M1T1 and M1T2 were predicted based on the whole genome sequencing data. After validation of the deletions by PCR and Sanger sequencing, we found one 57 kb deletion, including the second exon of Gpr19 and exons 1–3 of Cdkn1b on Chr6; two deletions of 257 kb, including exons 28–37 of Gtf3c1 and exons 1–28 of D430042o09rik, and of 25 kb containing exons 1–11 of Gsk3a and exons 1–4 of Erf on Chr7; and one deletion approximately 57 kb, comprising exons 2–5 of Egfr on Chr11 in M1T1 (S5 Table). No SVs were found in M1T2. The more frequent occurrence of CNAs and SVs in M1T1 is consistent with it having the most SNVs, suggesting that M1T1 is more genomically unstable than M1T2.

Allele fractions of SNVs, tumor cell fraction and cell ploidy in liver tumors with multiple mutations

In tumors, somatic mutations of similar frequencies may reside in the same population of cells, which may have descended from the same founder; therefore, the clustering of mutation frequency may represent different subclones within a single tumor[17]. Although we only detected 46 SNVs, we found that the allele fraction of different SNVs varied from low (approximately 0.1) to high (> 0.3) in the same tumors that contained more than 3 mutations in the CDs, such as M1T1, M1T2, M2T2, M3T1, M3T5, M4T1 and M4T6 (Table 1). We used violin plots to illustrate the allelic fraction densities of somatic mutations in each tumor. The violin plots of SNV distributions indicate the existence of subclones in those tumors with multiple SNVs (Fig 2A).

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Fig 2. Allelic fraction density and ploidy of tumor cells.

Violin plots illustrating the allelic fraction density of validated somatic mutations in each tumor (A) and of the estimated SNVs at the whole genome level in M1 (B). (C) Estimated ploidy of M1T1 based on sequencing data by Sequenza. (D) DNA ploidy of M5T1, as determined by flow cytometry.

https://doi.org/10.1371/journal.pone.0187551.g002

For the whole-genome level variations of M1T1 and M1T2, the 56 SNVs shared by both tumors had a higher frequency than their specific SNVs. Using allele fractions of 0.3 as the cut-off point, we found that 82% of shared mutations in T1 were above the cut-off, with a mean value of 0.42, and 59% of common mutations in T2 were above the cut-off, with a mean value of 0.35 (S4 Table and Fig 2B). The relatively higher allele fraction, 0.35 to 0.42, of their shared mutations suggests that the tumor cell fraction of those two samples is quite high, approximately 70% to 82%, which is consistent with tumor cell purities of 70%-90% in clinical HCC samples[9]. The allele fraction of specific SNVs below the 0.3 cut-off was 60% in M1T1 and 75% in M1T2. Moreover, the average allele fraction was 0.23 for M1T1 and 0.25 for M1T2 (S4 Table and Fig 2B). The allele fraction was relatively higher for shared SNVs and lower for specific SNVs in M1, suggesting that the specific mutations were gained after splitting from their common ancestor and indicate the existence of subclones in M1T1 and M1T2 (Fig 2B).

However, when we used the sequence data without distinguishing between common mutations and specific mutations in M1T1 and M1T2, the tumor cell purity was estimated to be approximately 50% because most SNVs have a medium allele fraction of 0.2–0.25, which is consistent with results estimated using only tumor-specific mutations. In addition, except for M4T1 and M4T4, which were polyploid, the karyotypes of all other tumors were nearly diploid, as estimated based on the sequenced dataset (Fig 2C and S4A Fig), although abundant CNAs were found in M1T1. The estimated diploid karyotypes were confirmed by flow cytometry analysis with a diploid lymphoma cell line as a control (Fig 2D and S4B Fig).

Functional annotation of the mutated genes

Most individual liver tumors only have 1–6 mutated genes that have been identified in liver tumor models, and the question is whether these mutated genes are drivers for tumorigenesis. To evaluate their tumorigenic capacity, we used Ingenuity Pathway Analysis (IPA) to investigate molecular/cellular functions, diseases and disorders based on all 46 mutated genes. A total of 26 mutated genes were mapped to 17 terms that describe different molecular and cellular functions, such as cellular function and maintenance, cellular development, cell morphology, cellular assembly and organization, cellular growth and proliferation, cell signaling, cellular movement, cell cycle, cell death and survival (Fig 3). We also found 37 genes related to different types of cancer in the IPA database, and the functional annotation showed that they are associated with cancer-specific biological processes, such as tumorigenesis, transformation, development, and invasion (S6 Table). In summary, most mutated genes found in the mouse HCC samples may play important roles in tumor progression or suppression, although the mechanism by which these mutations contributes to the tumorigenesis of HCC requires further studies.

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Fig 3. Cellular and molecular functional analysis of the mutated genes.

Ingenuity Pathway Analysis (IPA) was used to investigate the molecular/cellular functions of the 46 mutated genes, 26 of which were mapped to 17 terms that describe different molecular and cellular functions.

https://doi.org/10.1371/journal.pone.0187551.g003

Mutation frequency and expression of mutated genes in clinical HCC samples

The number of mutated genes found in mouse HCC samples was lower than in clinical HCC samples. To explore the mutation frequency in human HCCs, 1128 patients with liver tumors from six research projects of the ICGC were enrolled (S7 Table). Of the 46 mutated genes in the mouse liver tumor, all 41 human homolog genes were mutated in at least one patient (Fig 4A). LRP1B was the most frequently mutated gene (359 of 926 patients), followed by ROBO2, FGF13, MAST4 and SPHKAP, which were mutated in more than 20% of patients (Fig 4A and S8 Table). LRP1B is a tumor suppressor and may regulate cell motility via the RhoA/Cdc42 pathway and actin cytoskeleton reorganization[30], but the exact role of LRP1B in HCC development has not been reported. ROBO2 is also a candidate tumor suppressor gene[31]; thus far, there is no clear evidence that FGF13, MAST4 and SPHKAP are associated with cancer development.

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Fig 4. Mutation frequency, gene expression and clinical outcome analysis based on the ICGC database.

(A) Mutation percentage of the mutated genes found in the mouse model and patients with liver tumors. (B) Correlation of the gene expression level of ANXA11, PDE2A, PPP3CC, RYR1, SLC9A5, and NAA15 and overall survival for patients with liver cancer. P values are based on Spearman’s rank test (two-sided). The solid lines represent a linear trend. (C) Kaplan–Meier survival curves based on TSSK3, PDE2A, MAD2L1BP, PLOD2 and FGF13 expression. The median gene expression value was used as a cut-off point for each gene to divide patients into high and low gene expression groups. The hazard ratio with 95% confidence interval and P value of the log-rank test are given for each gene.

https://doi.org/10.1371/journal.pone.0187551.g004

For the gene expression and clinical outcome analysis, we used Spearman’s rank test (P < 0.05) to analyze the correlation between global gene expression and patient survival. The results showed that the expression of ANXA11, PDE2A, PPP3CC, RYR1 and SLC9A5 was positively correlated with the patient survival, whereas expression of NAA15 was negatively correlated with patient survival (Fig 4B). To further elucidate the relationships between the differential expression of these genes and patient survival, two patient groups with high and low expression of each gene were compared using a Kaplan-Meier survival plot. Based on log-rank P values < 0.05, patients with high expression of TSSK3 or PDE2A had a longer survival time than those with low expression of both genes; in contrast, patients with high expression of MAD2L1BP, PLOD2 or FGF13 tended to have a shorter survival time than those with low expression of these genes (Fig 4C). Of these genes, PLOD2 expression has been significantly correlated with tumor size and macroscopic intrahepatic metastasis and has also been identified as a significant, independent factor of poor prognosis[32].

Primary HCC mouse model and collection of the HCC tissues

HBV-transgenic mice C57BL/6J-TgN (Alb1 HBV) 44Bri [43] were purchased from the Jackson Laboratory (Bar Harbor, ME) and maintained under specific pathogen-free conditions in the animal facility at the Institute of Biophysics, Chinese Academy of Sciences. HBV-transgenic mice, 2 months old, were intraperitoneally injected with 100 mg of anti-CD137 Ab (clone 2A) weekly up to five times[29]. Four male mice (M1, M2, M3 and M4) were euthanized at aged 13 months or older, and all nodules on the liver larger than 3 mm in diameter were subjected to bulk sampling (S1A Fig). In addition to one sample obtained from each tumor, 15 and 22 micro-sections (each section is approximately 20,000 cells) were obtained from two tumors (M1T1 and M1T2) in the M1 mouse (S1B Fig) by performing micro-dissections[11]. All mouse and tumor sample information is listed in S1 Table. All studies involving animals were approved by the Institutional Laboratory Animal Care and Use Committee at the Institute of Biophysics, Chinese Academy of Sciences.

Pathology analysis

Pathological analysis was performed in all tissues used for sequencing to confirm the occurrence of tumors. After paraffin embedding, tissue sections (5 μm) were stained with hematoxylin and eosin (H&E).

Library preparation, whole-exome capture, WGS and WES

Genomic DNA from bulk samples and micro-sections was extracted using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) and TIANampMicro DNA Kit (Tiangen, Beijing, China), respectively. Libraries for the samples from the M2, M3 and M4 mice were constructed using the traditional method with 3 μg DNA as each input, which was sheared to generate fragments between 200 and 300 bp. DNA fragments were end-repaired, ligated with adapters, and amplified following the standard protocol of Paired-End DNA Sample Prep Kit (Illumina). To prepare the libraries from the M1 mouse samples, we used the modified EZ-Tn5 transposase-based method to fragment double-stranded DNA, with 20 ng genomic DNA as each input[11]. After fragmentation, we amplified the libraries used for exome capture and the WGS of M1 with 8 and 10 cycles, respectively. The amplified libraries were purified using the QIAquick Gel Extraction Kit (Qiagen).

Four DNA libraries from each mouse were barcoded with different indexes and equally pooled together. According to the manufacturer’s instructions, 800 ng of pooled DNA libraries were captured using the SureSelectXT Mouse All Exon Kit (Agilent), except custom blockers were used for the M1 libraries[11]. The captured libraries were amplified by PCR for 10 cycles and purified using the QIAquick Gel Extraction Kit (Qiagen). The insert size and the concentration of purified libraries for sequencing were examined using an Agilent Bioanalyzer and qRT-PCR. Paired-end (2×100 bp) multiplex sequencing of samples on the Illumina HiSeq2000 platform was performed.

Detection of somatic SNVs

Paired-end reads in FastQ format were aligned to the mouse reference sequence (mm9) using the Burrows-Wheeler Aligner (BWA)[44]. The Genome Analysis Toolkit (GATK) was used to re-calibrate the read quality[45], and Picard was used to mark the reads from PCR duplicates. WGS and WES data statistics are given in S2 Table.

With the normal control, somatic SNVs for each tumor were detected using Samtools[46] and VarScan[47]. WGS data from M1N served as a normal control to call SNVs from M1 WES data. In addition to Varscan’s built-in filters, the following filtering criteria were applied to identify candidate somatic mutations of WES: (i) a minimum of 10× coverage required in both tumor and normal samples, (ii) variant present on both strands with total reads ≥ 3 in the tumor, (iii) a variant allele frequency (VAF) in tumor DNA ≥ 10%, (iv) reads with more than two variants were removed, and (vii) variants listed in dbSNP132 were removed. For the WGS data for M1, we used the following criteria to filter SNVs: (i) a minimum of 10× coverage required in both tumor and normal sample, (ii) variant present on both strands with total read ≥ 4 in the tumor, and (iii) a VAF in the tumor ≥ 14%. In addition, we manually checked all candidate SNVs at CDs, which were submitted for Sequenom genotyping validation. All validated SNVs are shown in Table 1, and the SNVs of M1T1 and M1T2 at the whole genome level are presented in S3 Table.

SNVs validation by Sequenom genotyping

Genomic positions for all validated SNVs were retrieved using mm9 as a reference. The detailed procedures of primer design, multiplexed PCR and allele-specific extension, and VAF calculation of Sequenom genotyping were performed according to Ling et al.[11]. After validation, we used the R package ggplot to draw the violin plots to illustrate the allelic fraction densities of somatic mutations in each tumor, i.e., the width of the shaded area represents the proportion of data located there. For the SNVs validated in micro-dissected samples, we used the Kruskal-Wallis test[48] to compare their frequencies among all micro-dissected samples and the bulk sample in T1 and T2.

Detection of CNAs and SVs and estimation of tumor cell purity and ploidy

Sequenza was used to detect the somatic CNAs and to estimate tumor cell purity and ploidy[49]. First, we used Samtools to convert the Bam file of DNA sequencing data into the Pileup format. Second, the paired tumor and normal Pileup files were processed by sequenza-utils, which extracts sequencing depth, determines homozygous and heterozygous positions in the normal specimen, and calculates the variant alleles and allelic frequency from the tumor specimen. The sequenza-utils output was further processed using the Sequenza R package 2.1.1 to provide the segmented copy number data, cellularity, and ploidy estimates for each sample. We used Crest[50] to detect the SVs in M1T1 and M1T2 based on the WGS data. Deletions were further validated by PCR and Sanger sequencing.

DNA ploidy analysis by flow cytometry

Two tumors and a non-tumor tissue sample from the M5 mouse were mechanically dissociated in phosphate-buffered saline followed by filtration through a piece of fine nylon mesh (75 μm pore size) and centrifugation to remove debris and cell clumps. The single cell suspensions were fixed in cold 70% ethanol followed by staining using propidium iodide (Sigma) (50 g/ml in PBS) as a DNA-specific fluorochrome. Flow cytometric analysis was performed with BD FACSCalibur.

Functional analysis of the mutated genes

IPA was used to analyze the 46 mutated genes for their molecular/cellular functions and relationship with diseases and disorders. To explore the clinical significance of the mutated genes, we used ICGC data to investigate the mutation rates of these genes in human HCC. A total of 1128 patients with HCC from six projects (Liver Hepatocellular carcinoma—TCGA, Liver Cancer—FR, Liver Cancer—RIKEN, Liver Cancer—NCC, Benign Liver Tumour, and Liver Cancer—Hepatocellular macronodules) were included.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 6.0 (GraphPad Software, Inc). Spearman’s rank test (two-sided) was used to analyze the correlation of the gene expression level and overall survival for patients with liver cancer. In addition, we used the median gene expression value as the bifurcating point for each gene to divide patients into high and low gene expression groups. The two patient groups were compared using a Kaplan-Meier survival plot for each gene, and the hazard ratio with 95% confidence intervals and log-rank P value were calculated.