Human Cancer Biology Chromosomal Instability and Copy Number Alterations in Barrett's Esophagus and Esophageal Adenocarcinoma Thomas G. Paulson,1,2 Carlo C. Maley,4,5 Xiaohong Li,2 Hongzhe Li,6 Carissa A. Sanchez,1,2 Dennis L. Chao,3 Robert D. Odze,7 Thomas L. Vaughan,2,8 Patricia L. Blount,1,2,10 and Brian J. Reid1,2,9,10 Abstract Purpose: Chromosomal instability, as assessed by many techniques, including DNA content aneuploidy, loss of heterozygosity, and comparative genomic hybridization, has consistently been reported to be common in cancer and rare in normal tissues. Recently, a panel of chromosome instability biomarkers, including loss of heterozygos- ity and DNA content, has been reported to identify patients at high and low risk of pro- gression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA), but required multiple platforms for implementation. Although chromosomal instability in- volving amplifications and deletions of chromosome regions have been observed in nearly all cancers, copy number alterations (CNA) in premalignant tissues have not been well characterized or evaluated in cohort studies as biomarkers of cancer risk. Experimental Design: We examined CNAs in 98 patients having either BE or EA using Bacterial Artificial Chromosome (BAC) array comparative genomic hybridization to char- acterize CNAs at different stages of progression ranging from early BE to advanced EA. Results: CNAs were rare in early stages (less than high-grade dysplasia) but were pro- gressively more frequent and larger in later stages (high-grade dysplasia and EA), in- cluding high-level amplifications. The number of CNAs correlated highly with DNA content aneuploidy. Patients whose biopsies contained CNAs involving >70 Mbp were at increased risk of progression to DNA content abnormalities or EA (hazards ratio, 4.9; 95% confidence interval, 1.6-14.8; P = 0.0047), and the risk increased as more of the genome was affected. Conclusions: Genome-wide analysis of CNAs provides a common platform for the eval- uation of chromosome instability for cancer risk assessment as well as for the identifi- cation of common regions of alteration that can be further studied for biomarker discovery. Authors' Affiliations: 1Divisions of Human Biology, 2Public Health Sciences, Barrett's esophagus (BE) is a premalignant condition in which and 3The Vaccine and Infectious Disease Institute, Fred Hutchinson Cancer the squamous epithelium that normally lines the esophagus 4 Research Center, Seattle, Washington; Molecular and Cellular Oncogenesis is replaced with an intestinal metaplasia as a result of chronic Program, Systems Biology Division, The Wistar Institute, Philadelphia, Pennsylvania; 5Genomics and Computational Biology Graduate Program gastroesophageal reflux disease. Patients with BE have at least and Cellular and Molecular Biology Graduate Program, University of a 15-fold increased risk for the development of esophageal Pennsylvania, Philadelphia, Pennsylvania; 6Department of Biostatistics and adenocarcinoma (EA; ref. 1), a cancer that has increased in in- Epidemiology, University of Pennsylvania School of Medicine, Philadelphia, cidence by >600% over the past 30 years (2). Treatment options 7 Pennsylvania; Department of Pathology, Brigham and Women's Hospital, for EA are limited, and the majority of patients who develop EA Boston, Massachusetts; and 8Departments of Epidemiology, 9Genome Sciences, and 10Medicine, University of Washington, Seattle, Washington present initially with advanced disease, with 5-year survival Received 9/29/08; revised 2/20/09; accepted 2/20/09; published OnlineFirst rates of 13.7% (3). Patients with BE are typically placed in sur- 5/5/09. veillance programs for the early detection of cancer, but the rate Grant support: NIH K07CA089147, PO1CA91955, and the Ryan Hill Founda- of progression from BE to EA is estimated to be only 0.7% per tion (T.G. Paulson). year (4), and the vast majority of patients with BE will neither The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in develop nor die from EA (5). Thus, there is a strong clinical accordance with 18 U.S.C. Section 1734 solely to indicate this fact. need for biomarkers that can discriminate between those who Note: Supplementary data for this article are available at Clinical Cancer are unlikely to progress to cancer, those who should be reas- Research Online (http://clincancerres.aacrjournals.org/). sured and removed from frequent surveillance because of their Requests for reprints: Brian J. Reid, Fred Hutchinson Cancer Research low risk, and those at higher risk, who need frequent surveil- Center, 1100 Fairview Avenue North, C1-157, Seattle, WA. Phone: 206- 667-4615; Fax: 206-667-6132; E-mail: [email protected]. lance or intervention to prevent cancer. F 2009 American Association for Cancer Research. Chromosomal instability involving DNA copy number alter- doi:10.1158/1078-0432.CCR-08-2494 ations (CNA) are frequently observed in many types of cancer, www.aacrjournals.org 3305 Clin Cancer Res 2009;15(10) May 15, 2009 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2009 American Association for Cancer Research. Human Cancer Biology of tumor suppressor genes and DNA content biomarkers, in- Translational Relevance cluding CDKN2A (LOH, methylation, mutation), TP53 (LOH, mutation), tetraploidy, and aneuploidy (26). Only the chromo- Barrett's esophagus (BE) is the only known precur- some instability biomarkers, 9p LOH, 17p LOH, tetraploidy, sor to esophageal adenocarcinoma (EA), but the vast and aneuploidy provided independent cancer risk assessment majority of patients with BE will die of unrelated in multivariate analyses. However, this panel required a combi- causes. The identification of biomarkers that discrim- nation of platforms, including short tandem repeat polymorph- inate between patients at low versus high risk of pro- isms for LOH and DNA content flow cytometry, which would gressing to cancer is necessary to improve patient be difficult to implement clinically. outcomes. Here, we report an array comparative Here, we report for the first time the evaluation of genome- genomichybridization analysis of copy number al- wide chromosome instability analysis of copy number altera- terations in a cohort of 98 patients with either prema- tions using Bacterial Artificial Chromosome (BAC) array CGH lignant BE or EA. In addition to determining the in 174 samples from a cohort of 98 patients with diagnoses frequency and locations of deletions and amplifica- ranging from BE negative for dysplasia to advanced EA, a pop- tions occurring before the development of cancer, ulation representative of the range of BE stages of neoplastic genome-wide analysis of copy number alterations progression and a sample size that provides statistical power can identify DNA content aneuploid populations, as to quantify early and relatively rare CNA events. Bacterial Artifi- well as patients at risk for progression to DNA con- cial Chromosome array CGH allows genome-wide analysis of tent abnormalities or EA. Array comparative ge- copy number alterations and much more precise location of nomichybridization provides a single platform for gains and deletions than traditional CGH (27). DNA content the validation of chromosomal instability as a bio- flow cytometric data and patient characteristics were also avail- marker for cancer risk assessment that can be further able for each of the samples allowing us to validate array CGH evaluated in larger studies. as a measure of aneuploidy, a previously validated biomarker of progression from BE to EA (28). We further investigated array CGH as a common platform to assess chromosomal instability including those of the pancreas, lung, colon, breast, and pros- in a prospective biomarker validation study. This study extends tate, among others (6). CNAs have been used as biomarkers previous discovery research from many sources into a transla- for cancer prognosis in multiple studies (7, 8), but there are tional research cohort study (25), demonstrating that ge- few longitudinal studies of CNAs as predictors of progression nome-wide assessment of copy number identifies BE patients to cancer. Most studies analyzing CNAs that occur during neo- with an increased risk for progression. plastic progression in vivo primarily examine cancer samples. CNAs in patients with EA have been examined primarily by tra- Materials and Methods ditional comparative genomic hybridization (CGH; refs. 9–20). Traditional CGH studies of EA have typically reported wide- Study subjects and tissue acquisition. The Seattle Barrett's Esophagus spread alterations throughout the genome, but with low resolu- Study was approved by the Human Subjects Division of the University tion with respect to the specific chromosomal regions being of Washington in 1983 and renewed annually thereafter with reciproc- ity from the Fred Hutchinson Cancer Research Center Institutional affected. Recently, Nancarrow et al. reported a study of EA using Review Board from 1993 to 2001. Since 2001, the study has been ap- single nucleotide polymorphism arrays, confirming widespread proved by the Fred Hutchinson Cancer Research Center Institutional and extensive chromosomal alterations in advanced EAs (21). Review Board with reciprocity from the University of Washington The utility of CNAs as biomarkers of risk assessment for Human Subjects Division. The 72 noncancer participants in this