Gene deletions and amplifications in human hepatocellular carcinomas: correlation with hepatocyte growth regulation

Abstract

Tissues from 98 human hepatocellular carcinomas (HCCs) obtained from hepatic resections were subjected to somatic copy number variation (CNV) analysis. Most of these HCCs were discovered in livers resected for orthotopic transplantation, although in a few cases, the tumors themselves were the reason for the hepatectomies. Genomic analysis revealed deletions and amplifications in several genes, and clustering analysis based on CNV revealed five clusters. The LSP1 gene had the most cases with CNV (46 deletions and 5 amplifications). High frequencies of CNV were also seen in PTPRD (21/98), GNB1L (18/98), KIAA1217 (18/98), RP1-1777G6.2 (17/98), ETS1 (11/98), RSU1 (10/98), TBC1D22A (10/98), BAHCC1 (9/98), MAML2 (9/98), RAB1B (9/98), and YIF1A (9/98). The existing literature regarding hepatocytes or other cell types has connected many of these genes to regulation of cytoskeletal architecture, signaling cascades related to growth regulation, and transcription factors directly interacting with nuclear signaling complexes. Correlations with existing literature indicate that genomic lesions associated with HCC at the level of resolution of CNV occur on many genes associated directly or indirectly with signaling pathways operating in liver regeneration and hepatocyte growth regulation.

Figure 1

Comparison of DNA fragmentation between fresh-frozen and FFPE tissues. Copy number distribution of frozen sample and its matched FFPE sample.

Figure 2

Localization of deletions and amplifications for each chromosome (chr) by moving-window analysis. For genomic visualization of CNV regions cumulated across sample population, we used moving windows of 100 kb and counted the number of cases that contained any CNV region overlapping with the window. The genome-wide CNV map is plotted with chromosomal locations on the x axis [arranged from the beginning of the chromosome (p region) to the end of the chromosome (q region)] and number of cases with CNV on the y axis. The number of cases (of a total of 98) with amplifications and deletions on the specific site is illustrated by red (upward) and blue (downward), respectively. The eight genomic locations (A–H) that contain no known genes and that have a significant number of deletions are noted.

Figure 3

Ideogram of chromosome deletions or amplifications. Each bar next to the specific chromosome ideogram demonstrates that at least one locus was detected to have a genome copy number alteration. The frequency of the events is not demonstrated but is shown in Figure 2. Red bars indicate amplifications; blue bars, deletions. The chromosome number is indicated at the bottom of each ideogram. A: Cases from male patients. B: Cases from female patients. The data for male patients were calculated while excluding the female patients, to avoid erroneous detection of a deficient amount of Y-chromosome genomic material because the Y chromosome is absent in females.

Figure 4

Clustering of the HCC cases based on CNV (amplifications and deletions). Cases were clustered according to 1324 CNV conditions in the exons or introns of 473 known genes from Partek preprocessing, ignoring areas that do not contain expressed genes. Genes that have CNV changes in <10% (or 9) samples were filtered out. The filtering reduces the data from 93 samples and 473 genes to 78 samples and 15 genes. For details, see Materials and Methods.

Figure 5

Box plots and a contingency table demonstrate tumor attributes correlated with cluster assignment. A: Tumor size. B: Log-transformed survival (not statistically significant). C: Fatty cell inclusion.

Figure 6

Genomic mapping of the deletions (blue) and amplifications (red) in LSP1 and PTPRD. Exons are represented by vertical bar; introns, horizontal line. The spans of deletions of LSP1 (A) or PTPRD (B) are indicated by a blue line; amplifications in LSP1 (A) are shown in red. The minimal number of markers used for detection and P values are indicated. All LSP1 CNVs are located in the basic C-terminal portion of the resulting protein. Most PTPRD deletions occur in a site between exons 24 and 25.