Published OnlineFirst February 8, 2016; DOI: 10.1158/0008-5472.CAN-15-0338 Cancer Integrated Systems and Technologies Research Genomic Landscape of Somatic Alterations in Esophageal Squamous Cell Carcinoma and Gastric Cancer Nan Hu1, Mitsutaka Kadota2, Huaitian Liu2, Christian C. Abnet1, Hua Su1, Hailong Wu2, Neal D. Freedman1, Howard H. Yang2, Chaoyu Wang1, Chunhua Yan2, Lemin Wang1, Sheryl Gere2, Amy Hutchinson1,3, Guohong Song2, Yuan Wang4, Ti Ding4, You-Lin Qiao5, Jill Koshiol1, Sanford M. Dawsey1, Carol Giffen6, Alisa M. Goldstein1, Philip R. Taylor1, and Maxwell P. Lee2 Abstract Gastric cancer and esophageal cancer are the second and that oxidation of guanine may be a potential mechanism sixth leading causes of cancer-related death worldwide. Multi- underlying cancer mutagenesis. Furthermore, we identified ple genomic alterations underlying gastric cancer and esoph- genes with mutations in gastric cancer and ESCC, including ageal squamous cell carcinoma (ESCC) have been identified, well-known cancer genes, TP53, JAK3, BRCA2, FGF2, FBXW7, but the full spectrum of genomic structural variations and MSH3, PTCH, NF1, ERBB2, and CHEK2, and potentially novel mutations have yet to be uncovered. Here, we report the results cancer-associated genes, KISS1R, AMH, MNX1, WNK2,and of whole-genome sequencing of 30 samples comprising tumor PRKRIR. Finally, we identified recurrent chromosome altera- and blood from 15 patients, four of whom presented with tions in at least 30% of tumors in genes, including MACROD2, ESCC, seven with gastric cardia adenocarcinoma (GCA), and FHIT,andPARK2 that were often intragenic deletions. These four with gastric noncardia adenocarcinoma. Analyses revealed structural alterations were validated using the The Cancer that an A>CmutationwascommoninGCA,andinadditionto Genome Atlas dataset. Our studies provide new insights into the preferential nucleotide sequence of A located 5 prime to the understanding the genomic landscape, genome instability, mutation as noted in previous studies, we found enrichment of and mutation profile underlying gastric cancer and ESCC T in the 5 prime base. The A>C mutations in GCA suggested development. Cancer Res; 76(7); 1714–23. Ó2016 AACR. Introduction region have been attributed to these diseases (3). However, the cause of the high rates and geographical overlap of these two Gastric cancer and esophageal cancer cause an estimated anatomically adjacent but histologically distinct tumors has not 783,000 and 407,000 deaths, respectively, each year, and repre- been determined. Gastric cancers in this area occur primarily sent the second and sixth leading causes of cancer-related death in the uppermost portion of the stomach and are referred to as worldwide (1). In China, gastric cardia adenocarcinoma (GCA) GCA, whereas those in the remainder of the stomach are referred and esophageal squamous cell carcinoma (ESCC) occur together to as gastric noncardia adenocarcinoma (GNCA). In addition to in the Taihang Mountains of north central China, including being anatomically adjacent, GCA and ESCC share many of the Shanxi and Henan Provinces, at some of the highest rates reported same etiologic risk factors, and before the widespread use of for any cancer (2), and historically over 20% of all deaths in this endoscopy and biopsy, they were diagnosed as a single disease referred to as "esophageal cancer" (4). The reason for the high 1Division of Cancer Epidemiology and Genetics, National Cancer Insti- rates of GCA and ESCC in this geographic area and their relation to tute, NIH, Bethesda, Maryland. 2Center for Cancer Research, National each other remains unclear, but there are almost certainly com- 3 Cancer Institute, NIH, Bethesda, Maryland. Cancer Genomics mon etiologically important environmental exposures, and a Research Laboratory, Leidos, Gaithersburg, Maryland. 4Shanxi Cancer Hospital, Taiyuan, Shanxi, PR China. 5Cancer Institute, Chinese Acad- recent genome-wide association study of germline DNA found emy of Medical Sciences, Beijing, PR China. 6Information Management that the same SNPs in the PLCE1 gene had the strongest associa- Services, Inc., Silver Spring, Maryland. tions with risk for both GCA and ESCC (5). This led to our Note: Supplementary data for this article are available at Cancer Research concurrent examination of these two cancers plus GNCA in the Online (http://cancerres.aacrjournals.org/). current study. Corresponding Authors: Philip R. Taylor, Genetic Epidemiology Branch, Division Recent advances in next-generation sequencing technology of Cancer Epidemiology and Genetics, National Cancer Institute, 9609 Medical have revolutionized how we study cancer genomes. The iden- Center Drive, Rm 6E444 MSC 90892, Rockville, MD 90892-9769. Phone: 240- tification of IDH1/2 mutations, initially in glioma (6, 7) and 276-7235; Fax: 240-276-7832; E-mail: [email protected]; and Maxwell P. Lee, more recently in many other cancers such as AML (8), has [email protected] transformed our understanding of cancer by relating mutations doi: 10.1158/0008-5472.CAN-15-0338 to metabolic control and epigenetic regulation (9). IDH1/2 Ó2016 American Association for Cancer Research. encodes isocitrate dehydrogenases (IDH), which convert 1714 Cancer Res; 76(7) April 1, 2016 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst February 8, 2016; DOI: 10.1158/0008-5472.CAN-15-0338 Somatic Alterations in Esophageal and Gastric Cancer isocitrate to 2-oxoglutarate. But mutant IDHs produce 2-hydro- Materials and Methods xyglutarate, which inhibits the methyl cytosine hydroxylase Study population TET2 as well as H3K36 demethylases, thus changes the global This study was approved by the Institutional Review Boards of epigenetic landscape. Whole genome sequencing (WGS) is the Shanxi Cancer Hospital, the Cancer Institute and Hospital of particularly useful for elucidating complex genomic changes, the Chinese Academy of Medical Sciences (CICAMS, Shanxi, PR including translocations, inversions, tandem duplications, and China), and the U.S. National Cancer Institute (NCI, Bethesda, large deletions. The importance of these structural changes has MD). Fifteen cases were analyzed with WGS. These cases came from been well documented in the case of BCR-ABL in leukemia, a larger study sample and were selected on the basis of high-quality, TMPRESS2-ERG in prostate cancer (10), and EML4-ALK in lung sufficiently large amount of DNA (requiring at least 10 mg) avail- cancer (11). able for WGS, and patients being deceased. Three cases with ESCC, For gastric adenocarcinoma and ESCC, several publications seven cases with GCA, and four cases with GNCA diagnosed have reported genomic scale analyses of cancers using exome between 1998 and 2001 in the Shanxi Cancer Hospital in Taiyuan, or WGS technology. Wang and colleagues performed exome Shanxi Province, PR China, were recruited to participate in this sequencing of 22 gastric cancer samples and identified fre- study. One ESCC case from Yaocun Commune Hospital in Linxian, quent mutations in ARID1A (12). Mutations in ARID1A were Henan Province was also recruited. None of the cases had therapy particularly high in gastric cancers with microsatellite insta- before their surgical resection. After obtaining informed consent, bility (MSI; 83%) or with Epstein-Barr virus (EBV) infection cases were interviewed to obtain information on demographics, (73%). Exome sequencing of 15 gastric adenocarcinomas and cancer risk factors (e.g., detailed family history of cancer), and their matched normal DNAs by Zang and colleagues also clinical information. Only deceased cases were selected for study. identified frequent mutations of ARID1A (13). In addition, Clinical data are described in Supplementary Table S1. they found 5% of gastric cancer contained FAT4 mutations. A study by Agrawal and colleagues reported exomic sequencing Biological specimen collection and processing of 11 esophageal adenocarcinomas (EAC) and 12 ESCCs Venous blood (10 mL) was taken from each case before surgery NOTCH1 and found frequent mutations in ESCC (14). and germline DNA from whole blood was extracted and purified Nagarajan and colleagues performed WGS analysis for two using the standard phenol/chloroform method. Tumors obtained gastric cancer samples and found three mutational signatures during surgery were snap-frozen in liquid nitrogen and stored at (15). Dulak and colleagues did exome sequencing for 149 À130C until used. The specimens were chosen for this study EAC tumor-normal pairs and WGS for 15 EACs and matched based on two criteria: (i) histologic diagnosis of ESCC or gastric > normals. They found a high prevalence of A C transversions at cancer confirmed by pathologists at the Shanxi Cancer Hospital or AAdinucleotides(16).ArecentstudybyWangandcolleagues CICAMS, and the NCI; (ii) availability of high purity tumor tissue – analyzed 100 tumor normal pairs of gastric cancer with (at least >75%). WGS and identified MUC6, CTNNA2, GLI3, RNF43,and RHOA fi as signi cantly mutated driver genes. They found that Tissue DNA isolation RHOA fi mutations are speci c for diffuse-type tumors (17). DNA from frozen tumors was extracted using AllPrep DNA/ Recently, the International Cancer Genome Consortium re- RNA/Protein Mini Kit (Qiagen, Inc.). DNA was dissolved in 100 search team published a study involving WGS of 17 ESCC mL Buffer BE. Concentrations of DNA were measured with the cases and exome sequencing of 71 ESCC cases, which iden- NanoDrop