Interrogation of Chromosome 13Q12-14 in Esophageal Squamous Cell Carcinoma

8 The Open Pathology Journal, 2012, 6, 8-16 Open Access Interrogation of Chromosome 13q12-14 in Esophageal Squamous Cell Carcinoma Heidi S. Erickson1,2, Jaime Rodriguez-Canales1, Paul S. Albert3, Kris Yala4, Sumana Mukherjee1, Nan Hu5, Alisa M. Goldstein5, Rodrigo F. Chuaqui1, Stephen A. Hewitt4, Philip R. Taylor5 and *,1 Michael R. Emmert-Buck 1Pathogenetics Unit, Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA 2Thoracic/Head & Neck Medical Oncology, Division of Cancer Medicine, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA 3Biometric Research Branch, Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20852, USA 4Tissue Array Research Program, Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA 5Genetic Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA Abstract: Previous studies of esophageal squamous cell carcinoma (ESCC) suggested chromosome region 13q12-14 harbors a familial ESCC gene. DNA sequencing of the BRCA2 gene, located on 13q12, showed evidence of both germline and tumor specific alterations but the frequency of changes was low and did not fit the classic Knudsen two-hit gene inactivation model. To further investigate chromosome 13q12-14 in ESCC, quantitative expression measurements were performed on BRCA2 and 11 neighboring genes in matched normal epithelium and tumor from 17 cases. Transcript analysis showed normal levels of five genes, tumor down-regulation of two genes (TNFRS19 and TPT1), and tumor up- regulation of five genes, including BRCA2. No evidence of BRCA2 loss-of-function was detected based on reduced mRNA in tumor cells. Between 13q12.3 (KATNAL1) and 13q12.3-q13 (CCNA1) five adjacent genes showed increased mRNA expression raising the possibility of a DNA amplicon; however, qPCR analysis showed normal DNA amounts in this region. CCNA1 transcript was significantly up-regulated in tumors and was thus further interrogated at the protein level by immunohistochemistry. CCNA1 staining was restricted to normal basal epithelium and was not expressed in more superficial, differentiated regions. In contrast, the CCNA1 protein was ubiquitously and highly expressed throughout tumor foci. Overall, these data from a relatively small number of cases (17) suggest that TNFRS19 and TPT1 deserve further investigation as candidate tumor suppressor genes in esophageal cancer in a larger patient series; BRCA2 mRNA is increased in the tumors, likely as a compensatory response to the marked DNA damage that is present in these lesions; and, CCNA1 was identified as a novel up-regulated gene in ESCC. Keywords: Esophagus, carcinoma, squamous cell, chromosome 13q, BRCA2, genomic region, gene expression, upregulated region, microdissected tissue. INTRODUCTION chromosome 13 [1-3]. Additionally, a statistically significant difference in allelic loss on chromosome 13 was observed Esophageal squamous cell carcinoma (ESCC) is the fifth between sporadic versus familial cases suggesting this leading cause of cancer death in the world, with a chromosome may harbor an inherited TSG [4, 5]. significantly higher incidence in certain geographic areas such as Shanxi Province in China. Epidemiological studies ESCC shows extensive chromosomal instability thus it suggest that both environment and genetics influence ESCC has not been possible to precisely map a minimal deletion development and there is evidence for Mendelian inheritance interval; however, both the tumor LOH data and the results of a tumor suppressor gene (TSG) [1, 2]. Genomic studies of comparing allelic loss in sporadic versus familial cases point ESCC have identified several chromosomal regions with to the genomic region spanning chromosome 13q12-14 as high levels of loss of heterozygosity (LOH), including the most likely location of a putative esophageal cancer gene [6-11]. Direct DNA sequencing of selected candidates to date has not identified a classically inactivated TSG on *Address correspondence to this author at the Pathogenetics Unit, Laboratory chromosome 13, although the data on BRCA2 are difficult to of Pathology, National Cancer Institute, National Institutes of Health, interpret as missense mutations have been identified in over Bethesda, Maryland 20892, USA; Tel: (301) 496-2912; Fax: (301) 594- 10% of tumors, and in the germline of 5% of patients with a 7582; E-mail: [email protected] family history of ESCC [12-14]. Moreover, similar results of 1874-3757/12 2012 Bentham Open Interrogation of Chromosome 13q12-14 in Esophageal Squamous Cell Carcinoma The Open Pathology Journal, 2012, Volume 6 9 BRCA2 germline mutations have been observed in high-risk Chromosome 13q Genes ESCC populations in both Iran and India [15, 16]. Twelve genes spanning 27.5 million bp between 13q11- One strategy to assess the status of candidate TSGs in a 13q14 were selected for analysis (Fig. 1, Table 1). The genes genomic interval of interest is to evaluate transcript levels. were chosen such that they spanned the genomic region of Alterations in mRNA in tumors can occur due to several interest on chromosome 13 and because reports in the different mechanisms, including homozygous DNA deletion, literature suggested a potential role in cell growth [3, 8, 21, aberrant epigenetic regulation, decreased dosage due to 22]. haploinsufficiency, or decreased stability of mutated mRNAs [17]. In the present study we performed quantitative RT- PCR (qRT-PCR) analysis on 12 genes on 13q12-14, 13p13 including BRCA2, to assess if any of the candidates exhibited 13p12 LATS2 20446577 – 20533654 bp loss-of-function based on reduced expression of mRNA. The EFHA1 20964839 – 21076309 bp 13p11.2 gene set was selected to include genomic locations across the TNFRSF19 23042723 – 23148232 bp region of interest on chromosome 13, and genes with RNF6 25604253 – 25694508 bp potential growth related functions. 13q12.12 13q12.2 KATNAL1 29674767 – 29779584 bp MATERIALS AND METHODS HSP1 30608762 – 30634117 bp 13q13.1 Clinical Tissue Specimens 13q13.3 BRCA2 31787617 – 31871809 bp 13q14.12 RFC3 33290234 – 33438695 bp Twenty-four cases comprising 52 frozen tissue blocks of 13q14.2 CCNA1 35904633 – 35915008 bp newly diagnosed and untreated human esophageal squamous cell carcinoma (ESCC) were obtained from patients who 13q21.1 ELF1 40404164 – 40454418 bp underwent esophageal resection at Shanxi Cancer Hospital TPT1 44809304 – 44813297 bp 13q21.31 and Institute, Taiyuan in Shanxi Province of China. The RB1 47775912 – 47954023 bp specimens were collected under an IRB-approved protocol 13q21.33 and transferred to the National Cancer Institute (NCI). The 13q22.2 tissues were evaluated by a pathologist (JRC) and blocks from seventeen cases were selected for study based upon 13q31.1 both histologic criteria (presence of ESCC epithelium and 13q31.3 matched normal squamous epithelium) and microdissection criteria (> 10,000 normal epithelial or tumor cells). 13q32.2 13q33.1 Tissue Microdissection 13q33.3 Matched normal epithelium and tumor were dissected from histological sections using laser capture microdissection (LCM) as described previously [18]. Frozen tissue samples were hematoxylin and eosin (H&E) stained Fig. (1). Schematic of chromosome 13 showing the location of the prior to LCM and approximately 10,000 cells were procured twelve genes that were analyzed by qRT-PCR in patient-matched from each block (equivalent to ~3,000 LCM shots per tissue normal and tumor samples. type), immediately placed into lysis buffer, and stored at - Quantitative Reverse Transcription PCR (qRT-PCR) 80oC until RNA extraction. A replicate microdissection of all Gene Expression samples was obtained for a second qRT-PCR analysis of BRCA2 to assess reproducibility. Additional frozen tissue RT was conducted on all RNA samples using TaqMan sections were available in five of seventeen cases (case RT Reagents (Applied Biosystems, Inc. (ABI), Foster City, numbers 9, 10, 11, 12, and 16) and were manually CA, USA; Cat # N808-0234), with random hexamers as the microdissected for subsequent DNA extraction. RT primers. cDNA was used immediately for all PCR reactions, with remaining cDNA stored at -80o C. RNA Isolation, Quantitation, and Qualitation Fourteen commercially available optimized TaqMan RNA extraction and isolation from samples were primer/probe sets (Assays-on-Demand, ABI) were used in conducted as previously described [19, 20]. Following RNA singleplex qPCR reactions. Beta-actin (ACTB) has been isolation, 3 μl aliquots per sample were taken for RNA shown to be the most stable endogenous control gene quantity and quality measurement and used immediately. All between esophageal tumor and normal tissue [23]: therefore, remaining RNA samples were stored at -80o C. Quantitation ACTB was used as the endogenous control housekeeping and qualitation of individual sample total RNA was gene for normalization. The assays for LATS2, EFHA1, conducted using NanoDrop (NanoDrop Technologies, TNFRSF19, RNF6, KATNAL1, HSP1, BRCA2, RFC3, Wilmington, DE, USA) and Bioanalyzer (Agilent CCNA1, ELF1, TPT1, and RB1 are cDNA specific. To assess Technologies, Inc., Santa Clara, CA, USA) equipment, reproducibility, two BRCA2 primer/probes sets, each specific respectively. Sample quality was assessed by RNA integrity to a different exon of the gene (exon

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