Volume 150, Issue 4, Pages 998-1008 (April 2016) Variable Intra-Tumor Genomic Heterogeneity of Multiple Lesions in Patients With Hepatocellular Carcinoma  Ruidong Xue, Ruoyan Li, Hua Guo, Lin Guo, Zhe Su, Xiaohui Ni, Lisha Qi, Ti Zhang, Qiang Li, Zemin Zhang, Xiaoliang Sunney Xie, Fan Bai, Ning Zhang  Gastroenterology  Volume 150, Issue 4, Pages 998-1008 (April 2016) DOI: 10.1053/j.gastro.2015.12.033 Copyright © 2016 AGA Institute Terms and Conditions

Figure 1 Research strategy for genomic comparison of MLs in HCC patients. Schematic illustration of MLs in the liver. The positions of the portal vein (purple) and bile duct (green) are shown. First, surgeons collected the MLs via hepatectomy. DNA was isolated from these lesions and subjected to library preparation and NGS. Finally, genomic information was extracted from the sequence data. BDTT, bile duct tumor thrombus; IM, intrahepatic metastasis; MCT, multicentric tumor; N, adjacent noncancerous tissue or blood; P, primary tumor; PVTT, portal vein tumor thrombus; SN, satellite nodule. Gastroenterology 2016 150, 998-1008DOI: (10.1053/j.gastro.2015.12.033) Copyright © 2016 AGA Institute Terms and Conditions

Figure 2 Spatial distribution of somatic nonsynonymous mutations in MLs of 10 HCC patients. Heatmaps show the regional distribution of all nonsynonymous mutations. Gene names are listed on the right of each heatmap. Mutation (purple) or wild type (light purple) of each gene is shown. Column next to heatmap show three categories of mutations: mutation present in all regions (blue), in more than one but not all (green), or in one region (brown). Potential driver mutations are indicated on these regions. The percentage of ubiquitous mutations of each individual is indicated in parentheses after the patient identification. Lesion names in the form of patient identification + lesion type are indicated at the bottom of the figure. For example, P1P is the primary lesion of patient 1, whereas P2IM1 is the first IM of patient 2. B, bile duct tumor thrombus; N, noncancerous tissue or blood; P, primary tumor; SN, satellite nodule; T, portal vein tumor thrombus. Gastroenterology 2016 150, 998-1008DOI: (10.1053/j.gastro.2015.12.033) Copyright © 2016 AGA Institute Terms and Conditions

Figure 3 Whole-genome CNVs of 10 HCC patients. (A) Heatmap of copy number states across the genome for all tumor samples from 10 HCC patients. The colors in the heatmap represent log2(CN/ploidy) values. A gain (red) was defined as a copy number at least one greater than ploidy, whereas a loss (blue) was defined as a copy number at least one less than ploidy. (B) Distribution of Genomic Identification of Significant Targets in Cancer regions in HCC as reported by The Cancer Genome Atlas. Red, amplification; blue, deletion; grey, normal. Recurrent CNV regions are listed above the heatmap, and lesion names are listed on the left. (C) Number of recurrent CNVs and percentage of ubiquitous CNVs in each patient. Gastroenterology 2016 150, 998-1008DOI: (10.1053/j.gastro.2015.12.033) Copyright © 2016 AGA Institute Terms and Conditions

Figure 4 Phylogenetic trees of 10 HCC patients. Phylogenetic trees were constructed using a maximum parsimony algorithm based on all nonsynonymous mutations identified in each patient. The length of each line is proportional to the number of nonsynonymous mutations. Arrows indicate the acquisition of potential driver events during tumor evolution. Recurrent CNVs detected in each case are mapped to trunks or branches (red and blue represent amplification and deletion, respectively). Each CNV region corresponds to a The Cancer Genome Atlas–highlighted region in Figure 3B (1p*, 1p36.31-1p36.31; 1p**, 1p36.11; 4q*, 4q21.21-4q22.2; 4q**, 4q35.1). The red boxes indicate genes with HBV integration. Possible convergent evolution events are underlined in green. Patient IDs and lesion names are labeled in each tree. Gastroenterology 2016 150, 998-1008DOI: (10.1053/j.gastro.2015.12.033) Copyright © 2016 AGA Institute Terms and Conditions

Figure 5 Genomic analyses of MCTs in P8. (A) Mutation heatmap of P8. Mutation (purple) or wild type (light purple) of each gene is shown. (B) Phylogenetic tree of MLs. The length of each line is proportional to the number of nonsynonymous mutations. Arrows indicate the acquisition of potential driver events during tumor evolution. The red boxes indicate genes with HBV integration. Possible convergent evolutionary events are underlined in green. (C) Comparison of the mutation spectra of PG and MG based on all point mutations. The numbers of mutations in each group and the P value for the comparison are shown above the bars. (D) Linear plots of CNVs. A circular binary segmentation algorithm was used to segment the CNV data (green lines). (E) Breakpoint analysis of chromosome 8q. Blue arrows indicate the breakpoints of the 8q amplification events. Gastroenterology 2016 150, 998-1008DOI: (10.1053/j.gastro.2015.12.033) Copyright © 2016 AGA Institute Terms and Conditions

Figure 6 Pathologic and genomic analyses of MLs in P5. (A) Spatial locations of lesions in P5 are indicated. Lesion names and staining markers are indicated in the pathologic graphs. Scale bar: 100 μm. CK19, cytokeratin 19; GPC3, glypican-3. (B) Venn diagram of nonsynonymous mutations across the lesions. (C) Phylogenetic tree of MLs. The length of each line is proportional to the number of nonsynonymous mutations. Lesion names are labeled in the tree. Arrows indicate the acquisition of potential driver events during tumor evolution. Gastroenterology 2016 150, 998-1008DOI: (10.1053/j.gastro.2015.12.033) Copyright © 2016 AGA Institute Terms and Conditions