The American Journal of Pathology, Vol. 180, No. 4, April 2012 Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. DOI: 10.1016/j.ajpath.2011.12.021 Gastrointestinal, Hepatobiliary, and Pancreatic Pathology Gene Deletions and Amplifications in Human Hepatocellular Carcinomas Correlation with Hepatocyte Growth Regulation Michael A. Nalesnik,* George Tseng,† and morbidity. Several chronic conditions are associated Ying Ding,†‡ Guo-Sheng Xiang,* with an increased incidence of HCC, including alcohol- Zhong-liang Zheng,* YanPing Yu,* ism, chronic infections with hepatitis B virus (HBV) or James W. Marsh,§ George K. Michalopoulos,* and hepatitis C virus (HCV), hemochromatosis, and metabolic Jian-Hua Luo* diseases. Common to all these conditions is that their chronic forms may cause liver cirrhosis and HCC can ‡ From the Departments of Pathology* and Surgery, and the often arise on this background. Because most of the Departments of Biostatistics,† and the Joint CMU-Pitt Ph.D. previously mentioned conditions do not involve genotoxic Program in Computational Biology,§ the Graduate School of agents and none of the associated viruses carry trans- Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania forming oncogenes, it is reasonable to assume that, for most HCCs, cirrhosis per se and associated irregularities induced in the hepatocyte microenvironment are some- Tissues from 98 human hepatocellular carcinomas how responsible for generating genomic or epigenetic (HCCs) obtained from hepatic resections were subjected alterations leading to carcinogenesis. Cirrhosis is asso- to somatic copy number variation (CNV) analysis. Most ciated with a higher rate of hepatocyte proliferation. HCC of these HCCs were discovered in livers resected for in rodents appears, in most conditions, associated with orthotopic transplantation, although in a few cases, the long-term hyperproliferation of hepatocytes.1 Several tumors themselves were the reason for the hepatecto- genomic abnormalities have been described in HCC, mies. Genomic analysis revealed deletions and amplifi- such as mutations in p53 and ␤-catenin, deletions in cations in several genes, and clustering analysis based areas of chromosome 1, and activation of specific onco- on CNV revealed five clusters. The LSP1 gene had the genes by HBV through insertional mutagenesis.2 Genes most cases with CNV (46 deletions and 5 amplifica- expressed by HBV or HCV may cause a long-term pro- tions). High frequencies of CNV were also seen in PT- motion effect, also facilitating the evolution of HCC.3 PRD (21/98), GNB1L (18/98), KIAA1217 (18/98), RP1- Studies4–6 of gene expression in HCC have demon- 1777G6.2 (17/98), ETS1 (11/98), RSU1 (10/98), strated subgroups of three clusters with differing gene TBC1D22A (10/98), BAHCC1 (9/98), MAML2 (9/98), expression profiles and prognosis. Despite several stud- RAB1B (9/98), and YIF1A (9/98). The existing literature ies related to gene expression profiling of hepatocyte- regarding hepatocytes or other cell types has connected derived tumors (adenomas or HCCs), there has been many of these genes to regulation of cytoskeletal archi- little analysis of genome-wide alterations underpinning tecture, signaling cascades related to growth regulation, and transcription factors directly interacting with nu- clear signaling complexes. Correlations with existing Supported by the Rangos Fund for Enhancement of Pathology Re- literature indicate that genomic lesions associated with search (RSG-08-137-01-CNE to Y.P.Y.) and grants from the NIH HCC at the level of resolution of CNV occur on many (5R01CA103958-05 and 5R01CA035373-27 to G.K.M. and R01CA098249 genes associated directly or indirectly with signaling to J.-H.L.). pathways operating in liver regeneration and hepato- Accepted for publication December 22, 2011. cyte growth regulation. (Am J Pathol 2012, 180:1495–1508; M.A.N. and G.T. contributed equally to this work. DOI: 10.1016/j.ajpath.2011.12.021) Supplemental material for this article can be found on http://ajp. amjpathol.org or at doi: 10.1016/j.ajpath.2011.12.021. Address reprint requests to George K. Michalopoulos, M.D., Ph.D., Hepatocellular carcinoma (HCC) is the most common S410 BST, Department of Pathology, University of Pittsburgh School of type of liver cancer and is associated with high mortality Medicine, Pittsburgh, PA 15241. E-mail: [email protected]. 1495 1496 Nalesnik et al AJP April 2012, Vol. 180, No. 4 the altered patterns of gene expression.7 We performed Tissue Processing, DNA Extraction, Amplicon this study to assess genetic alterations (deletions or am- Generation, and Labeling plifications) detectable by analysis of copy number vari- ation (CNV) and to examine the relationships, if any, to FFPE tissues were microdissected to achieve tumor pu- the known pathways controlling hepatocyte growth dur- rity Ͼ85%. The dissected tissues were incubated in xy- 8–14 ing liver regeneration and in hepatocyte cultures. We lene solution overnight to remove residual paraffin. DNA have used the Affymetrix (Santa Clara, CA) 6.0 single- was then extracted using the Qiagen tissue kit (Qiagen, nucleotide polymorphism (SNP) oligonucleotide arrays Valencia, CA). Amplicons were generated by performing for these studies. The method provides useful information linear PCR using random 12mers on 2-␮g DNA templates on the types of deletions and amplifications larger than using the following program: 94°C for 1 minute and then approximately 1000 bp and is much more detailed than 40 cycles of 94°C for 30 seconds, 45°C for 1 minute, and studies of large areas of loss of heterozygosity. In addi- 72°C for 2 minutes. The PCR products were then purified tion, this approach provides detailed information about and digested with DNase1 for fragmentation. The frag- the site of the genomic alterations within specific genes, mented DNA was then labeled with biotinylated cytosine. allowing correlations between the expected changes in protein structure and the change in function. Hybridization, Washing, and Scanning of the SNP 6.0 Chip Materials and Methods Fragmented DNA (250 ␮g) was hybridized with a pre- Case Material equilibrated Affymetrix chip at 50°C for 16 hours. After the Formalin-fixed, paraffin-embedded (FFPE) tissue speci- hybridization cocktails were removed, the chips were mens were obtained from the Surgical Pathology Ar- then washed in a fluidic station with low-stringency buffer chives of the University of Pittsburgh Medical Center, (six times SSPE, 0.01% Tween 20, and 0.005% antifoam) Pittsburgh, PA, in accordance with Institutional Review for 10 cycles (two mixes per cycle) and stringent buffer Board regulations (PRO-08050186). Anonymized sam- (100 mmol/L MES, 0.1 mol/L NaCl, and 0.01% Tween 20) ples spanned a 26-year period, from 1981 through 2006. for 4 cycles (15 mixes per cycle) and then stained with This time interval partially antedated establishment of the streptavidin-phycoerythrin. This was followed by incuba- Milan criteria15 and spanned a period during which HBV tion with biotinylated mouse anti-avidin antibody and infection was a more frequent indication for transplanta- restaining with streptavidin-phycoerythrin. The chips tion at our center. were scanned in a G7 scanner (Affymetrix Inc., Santa Clinical information was obtained via Honest Broker Clara, CA) to detect hybridization signals. from the EDIT Transplant Database and Surgical Pathol- ogy records. This included patient survival, tumor size, tumor recurrence after transplantation, underlying dis- ease, and ␣-fetoprotein levels. The 98 adult patients in- SYBR Green Real-Time Quantitation PCR cluded 22 females and 76 males. Samples were derived The LightCycler FastStart DNA Master SYBR-Green I kit from native livers at transplantation in all but six patients (Roche Applied Science, Indianapolis, IN) was used for who underwent surgical resection. Matched control tis- real-time PCR amplification. The reaction was performed sue was obtained from noncirrhotic tissue, usually from in a LightCycler machine (Roche Applied Science, Indi- hilar tissue, and did not include lymph nodes. Underlying anapolis, IN). A quantitation standard curve of normal diseases included HBV infection (n ϭ 22), HCV infection male DNA from 50,000 to 500,000 copies of genome was (n ϭ 21), ethanol use (n ϭ 13), hemochromatosis (n ϭ 3), generated using known amounts of template copies. and primary sclerosing cholangitis (n ϭ 2). Six patients Genomic DNA (100 ng) was used for all of the experi- had multiple underlying disorders (3 HBV and HCV, 2 HCV and ethanol use, and 1 HBV, HCV, and ethanol use), mental and control samples. HotStarTaq DNA polymer- and one patient each had sarcoid, granulomatous dis- ase (Qiagen, Valencia, CA) was activated with a 10- ease not further specified, type II glycogen storage dis- minute pre-incubation step at 95°C. Amplification for ease, autoimmune hepatitis, and nonalcoholic steato- LSP1 (leukocyte-specific protein 1; 5=-CAATGCTCCTC- hepatitis. In 26 patients, there was either no known TATCCAGCC-3= and 5=-ATGGAGGGGCACTGATTGGA- underlying disorder or insufficient data to establish an 3=) and PTPRD (protein tyrosine phosphatase, receptor underlying disease. Tumors were solitary in 30 patients type, D; 5=-CGTAGAGATGCCCAGTATCTGC-3= and 5=- and multiple in 66 patients. No determination was possi- CTCTGATGAGGCTGCCATTGTG-3=) was performed with ble in the remaining two patients. All tumors were HCCs, 45 cycles of the following program: