Potential conflict of interest: KW, SS, SD, TX, ZZ, YW, DP, WJY, PAR, MM and JX are or were employed by Pfizer Inc. KW, SS, SD, YW, and DP own stock in Pfizer.
Genomic landscape of copy number aberrations enables the identification of oncogenic drivers in hepatocellular carcinoma
Version of Record online: 1 JUL 2013
Copyright © 2013 by the American Association for the Study of Liver Diseases
Volume 58, Issue 2, pages 706–717, August 2013
How to Cite
Wang, K., Lim, H. Y., Shi, S., Lee, J., Deng, S., Xie, T., Zhu, Z., Wang, Y., Pocalyko, D., Yang, W. J., Rejto, P. A., Mao, M., Park, C.-K. and Xu, J. (2013), Genomic landscape of copy number aberrations enables the identification of oncogenic drivers in hepatocellular carcinoma. Hepatology, 58: 706–717. doi: 10.1002/hep.26402
Data availability: Both gene expression and SNP genotyping array data have been deposited to Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession numbers GSE36376 and GSE38326, respectively.
- Issue online: 29 JUL 2013
- Version of Record online: 1 JUL 2013
- Accepted manuscript online: 18 MAR 2013 04:05AM EST
- Manuscript Accepted: 13 MAR 2013
- Manuscript Received: 26 SEP 2012
Additional Supporting Information may be found in the online version of this article.
Figure S1. Association between somatic CNA, mRNA expression and clinical outcome. (A) Distribution of genome-wide cis-correlation between somatic CNAs and mRNA expression levels across the HCCs (red) and those obtained from the permutated dataset where sample labels were randomly scrambled (blue). (B) Cumulative Distribution of Cox regression p-values for associating somatic CNAs to clinical outcomes including both disease specific survival (DSS, blue) and disease-free survival (DFS, red), in comparison to same distributions calculated from a permutated dataset where sample labels were randomly scrambled (“DSS perm” in green and “DFS perm” in black). X-axis of the plot shows the –log10 of the Cox regression p-value cutoffs, and Y-axis is the number of genes with a p-value smaller than the corresponding cutoff on the X-axis.
Figure S2. Pair-wise DNA/DNA correlations reveal significant associations between unlinked loci. Pair-wise Pearson correlations computed from ∼20k gene copy number are ordered by genes' chromosomal positions through the genome on the X and Y axes with red indicating a positive correlation and blue indicating a negative correlation. The red diagonal represents the correlation of genes with themselves.
Figure S3. Distributions of GISTIC2 peak statistics. (A) Peak size distribution and comparison between amplification and deletion peaks. (B) Relationship between peak frequency and peak size for amplification peaks. (C) Relationship between peak frequency and peak size for deletion peaks. (D) Relationship between peak frequency and peak amplitude. Peak frequencies were calculated based on copy number cutoffs of 3 and 1.3 for amplification and deletion peaks, respectively. Peak amplitudes were taken as the average copy number of a peak among patients called positive for the peak. (E) Distribution of cis-correlations for genes not in any GISTIC2 peak, in deletion or amplification peaks. P-values shown were based on two-sample t-tests.
Figure S4. Frequent somatic copy number alterations in critical signaling pathways in HCC.
Figure S5. Immunostaining in HCCs for BCL9 and MTDH. HRP, original magnification x200) showing high levels of immunoreactivity for BCL9 in the nucleus (A) and MTDH in the cytoplasm (B).
Figure S6. Inferred copy numbers and expression levels of BCL9 and MTDH in a panel of 30 HCC cell line models. (A) BCL9; (B) MTDH. Cell lines colored in green were used as amplified models for each candidate driver in the functional validation, and those in pink were used as controls (i.e. copy number neutral with respect to the target).
Figure S7. Association to clinical outcomes and AJCC tumor stages for the putative CNA drivers BCL9 and MTDH. Panels (A-C) show data for BCL9; panels (D-F) show data for MTDH. Patients were separated into two groups based on the amplification status of BCL9 and MTDH. Differences in disease-specific and disease-free survival were assessed by Kaplan-Meier curves and the associated log rank test. For association with AJCC tumor stage, a linear trend test was performed (p-values shown in parenthesis).
|hep26402-sup-0002-supptab1.doc||439K||Table S1. Major demographic and clinicopathological parameters of the HCC cohort. For AJCC T Stage, categories "3a" and "3b" were merged as "3". For BCLC stage, categories "0", "A1", "A2", "A3" and "A4" were merged into 1; categories "B", "C" and "D" were coded as 2, 3 and 4, respectively.|
|hep26402-sup-0003-supptab2.doc||37K||Table S2. Cell lines used in this study and their sources. SIBS – Shanghai Institutes of Biological Sciences (Shanghai, China); HSRRB – Japan Health Science Research Resources (Japan); ATCC – American Type Culture Collection (Virginia, USA); CrownBio – Crown Bioscience Inc. (Beijing, China)|
|hep26402-sup-0004-supptab3a.doc||335K||Table S3. All CNA peaks predicted by GISTIC2 analysis. "Peak frequency" was calculated using copy number cutoffs of 2.3 and 1.7 for copy number gains and losses, respectively. "High peak frequency" was calculated using cutoffs 3.0 and 1.3 for amplifications and deletions, respectively. Full sets of genes under each CNA peak can be found in "Wang_HCC_CNA_landscape_Table_S3_full.docx".|
|hep26402-sup-0005-supptab3b.doc||335K||Table S3. All CNA peaks predicted by GISTIC2 analysis. "Peak frequency" was calculated using copy number cutoffs of 2.3 and 1.7 for copy number gains and losses, respectively. "High peak frequency" was calculated using cutoffs 3.0 and 1.3 for amplifications and deletions, respectively. Column "Peak affected genes" lists the full set of genes covered under each CNA peak, whereas the column "CGC genes in peak" only shows those that belong to Cancer Gene Census.|
|hep26402-sup-0006-supptab4.doc||346K||Table S4. Association of average copy number of the focal amplification and deletion peaks identified by GISTIC2 to clinical and outcome variables. Values shown in the table are signed nominal p-values. For age, gender, etiology, tumor size, serum AFP, cirrhosis, intrahepatic metastasis and vascular invasion, two-sample t-tests were used: a positive p-value indicates higher average copy number of the peak in the group stated in the header. AJCC T stage, BCLC stage, Child-Pugh class and Edmondson grade were treated as ordinal variable and linear trend tests were performed: a positive p-value indicate a positive trend and vice versa. Cox regression analysis was used to evaluate the association to disease specific overall survival and disease free survival: a positive p-value indicates that increased copy number is associated with poor outcome. Order of the peaks in this table is the same as that of Table S2. The two genes validated in this study, BCL9 and MTDH, are in amplification peak #2 and #51, respectively. Significant associations at a nominal p-value <0.05 level are shaded in red for positive associations and blue for negative associations.|
|hep26402-sup-0007-supptab5a.doc||243K||Table S5. All pathways enriched among cis-acting genes in CNA peaks. Full sets of genes that map to each pathway can be found in "Wang_HCC_CNA_landscape_Table_S5_full.docx".|
|hep26402-sup-0008-supptab5b.doc||243K||Table S5. All pathways enriched among cis-acting genes in CNA peaks. The column "Genes" lists full sets of genes that map to each pathway.|
|hep26402-sup-0009-supptab6.doc||57K||Table S6. Candidate driver genes selected based on focal CNA, expression changes and model availability. "Amplification frequency" is calculated based on inferred copy numbers of individual genes (which may differ from the GISTIC2 peak frequency).|
|hep26402-sup-0010-supptab7.doc||249K||Table S7. Somatic copy number, gene expression and IHC staining results for BCL9. For definition of stain positivity please refer to Supplementary Methods.|
|hep26402-sup-0011-supptab8.doc||249K||Table S8. Somatic copy number, gene expression and IHC staining results for MTDH. For definition of stain positivity please refer to Supplementary Methods.|
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