ICANCER RESEARCH 58. 2456-2460. June I, 1998] Identification of Two Common Regions of Allelic Loss in Chromosome Arm 12q in Human Pancreatic Cancer1 Mitsuhiro Kimura,2 Toni Furukawa,2 Tadayoshi Abe, Toshimasa Yatsuoka, Emile M. Youssef, Tadaaki Yokoyama, Hong Ouyang, Yasuyuki Ohnishi, Makoto Sunamura, Masao Kobari, Seiki Matsuno, and Akira Horii3 Departments of Molecular Pathology ¡M.KL. T. F.. T. A., T. Ya.. E. M. Y.. T. Yo., H. O., A. H.¡ and Surgery I ¡M.KL, T. A., T. Ya.. T. Yo.. M. S.. M. Ko., S. M.], Tohoku University School of Medicine, Sendai, 980-8575: Venture Business Laboratory, Tohoku University, Sendai, 980-8576 /£.M. Y.j: and Central Institute for Experimental Animals. Kawasaki. 213-0027 ¡Y.O.¡,Japan ABSTRACT Using the method of microsatellite analysis, we studied 40 tissues with pancreatic (liutai adenocarcinoma and identified two commonly deleted regions on the long arm of chromosome 12. One (region A) was found between D12S81 and D12S1719 at 12q21 at a frequency of 67.5%, and the other (region B) was located between DÃŒ2S360and D12S78 at 12q22- q23.1 at a frequency of 60%; the latter was reported previously (M. Kimura, et al. Genes Chromosomes Cancer, 17: 88-93, 1996). The results of microsatellite analyses were verified by fluorescence in situ hybridiza tion. We further analyzed 19 pancreatic cancer cell lines by fluorescence in situ hybridization and found that 10 of them showed allelic loss at D12S81 and 6 showed allelic loss at D12S360. Yeast artificial chromosome contigs were constructed to cover the deleted regions. Region B was completely covered by a 650-kb yeast artificial chromosome clone. The frequently deleted regions in chromosome 12q in pancreatic cancer that were identified here may provide new avenues for isolating novel tumor suppressor genes. INTRODUCTION Pancreatic cancer is one of the leading causes of cancer death in Japan, as well as in Western countries. The poor prognosis is due to difficulty in diagnosis and inefficiency in surgical and/or multidisci- plinary treatment of the cancer ( 1). An understanding of the molecular mechanisms of pancreatic carcinogenesis would provide important molecular clues for the development of methods for early detection and efficient therapy of this very malignant disease. Recent cytoge- netic studies using karyotype analyses, as well as microsatellite anal yses, CGH,4 and FISH, suggest that many genetic alterations occur in several chromosomal regions other than those including MTS1, p53, and DPC4 (2-11). These results suggest possible involvement of unknown genes in pancreatic carcinogenesis. We previously per formed an allelotype analysis to search for localization of putative tumor suppressor genes and identified a 1-cM region of common allelic loss in 12q22-q23.1 (12). However, our CGH analysis sug gested the existence of a region of common allelic loss in 12q other than the one that we had identified by microsatellite analysis (13). Hence, we examined the entire region on the long arm of chromosome 12 with microsatellite analysis and FISH combined to construct a deletion map of the entire chromosome 12q arm in pancreatic ductal Received 11/26/97; accepted 4/2/98. 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 accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported in part by the Ministry of Education. Science, Sports and Culture of Japan, the Vehicle Racing Commemorative Foundation, the Japanese Foun dation for Multidisciplinary Treatment of Cancer, the Mitsui Life Social Welfare Foun dation, the Terumo Life Science Foundation, the Sagawa Foundation for Promotion of Cancer Research, and the Pancreas Research Foundation of Japan. - The first two authors contributed equally to this work. ' To whom requests for reprints should be addressed, at Department of Molecular Pathology, Tohoku University School of Medicine, Sendai. 980-8575, Japan. Phone: 81-22-717-8042; Fax: 81-22-717-8047; E-mail: horii@mail.cc.tohoku.ac.jp. 4 The abbreviations used are: CGH, comparative genomic hybridization; FISH, fluo rescence i/i situ hybridization; LOH. loss of heterozygosity; BAC, bacterial artificial chromosome; YAC, yeast artificial chromosome; STS, sequence-tagged site. adenocarcinoma. Herein, we report identification of the two distinct regions of common allelic loss in 12q in human pancreatic cancer. MATERIALS AND METHODS Tissues and Cell Lines. The materials studied were from 40 cases of primary pancreatic ductal adenocarcinoma (23 men and 17 women, ages 31-81 years; mean age = 63.5 years). Thirty-three cases were surgically resected, and 7 were obtained at autopsy. The average survival period after diagnosis was 15.1 months. According to the clinical staging system of the Japan Pancreas Society (14), 2 cases were in stage II, 9 were in stage III, and 28 were in stage IV. A brief explanation of the clinical staging system of the Japan Pancreas Society was included in our previous report (13). In one case, no description of the clinical stage was available. Histológica! diagnoses of the tumors were as follows: 35 tubular adenocarcinomas, 2 papillary adenocarci- nomas, 2 adenosquamous carcinomas, and 1 anaplastic carcinoma. Of the 40 tissues, 37 specimens were formalin-fixed, paraffin-embedded tissues, and 3 were frozen tissues. Tissues were cut to provide 13-15 20-/nm-thick sections for microdissection. These sections were sandwiched between two 3-/j.m-thick sections that were stained with H&E for histopathological examination. Under a microscope, tumor cells were carefully dissected from the 20-/xm-thick sections that had been deparaffmized, hydrated, and stained with hematoxylin. In each sample, normal cells were also collected from the adjacent normal tissue. DNAs were extracted from the dissected tissues, as described previ ously (12). Nineteen pancreatic cancer cell lines, PK-1, PK-8, PK-9, PK-59. PCI-6. PCI-10, PCI-19, PCI-35, PCI-43, PCI-64, PCI-66, PAN02JCK. PAN03JCK, PAN07JCK, PAN09JCK, Panc-1, MIA PaCa-2, SU.86.86, and BxPc-3, were cultured as described (15-24). The primary tumors from which these cell lines derived were not available. Microsatellite Analysis. Allelic imbalances were detected by the micro- satellite analysis method in 40 tissues, as described previously (12). Twenty- four microsatellite markers were used, as listed in Fig. 2, to cover the entire long arm of chromosome 12. Detailed information on the primers and PCR conditions used is available upon request. The primers were end-labeled with [12P]ATP with polynucleotide kinase (New England Biolabs, Beverly. MA) and used for PCR. The amplified products were fractionated in a gel composed of 6% polyacrylamide, 8 M urea, and 32% formamide and then autoradio- graphed. Relative intensities of allele-specific bands were examined by quan titative densitometry using NIH Image software (details of this software are available at: http://cbel.dcrt.nih.gov/~mvivino/ImgEngrHTML/ImgEngr. html). LOH was defined as 50% or more reduction in relative intensity in one allele-specific band, as compared to the band of the normal counterpart. Homozygous alÃ-eles,microsatellite instabilities, and poor yield of PCR prod ucts for each marker were not scored in this study. Construction of Contigs Using BAC and YAC. DNA superpools of YAC and BAC libraries (Research Genetics, Huntsville, AL) were screened by PCR with primers for the microsatellite markers, according to the manufacturer's instructions. BAC and YAC clones that were positive for microsatellite mark ers were purchased from Research Genetics. Agarose plugs of YACs and BACs were prepared and analyzed for their sizes using the CHEF Mapper pulsed-field gel electrophoresis system (Bio-Rad. Hercules. CA), according to the manufacturer's instructions. Total yeast DNA, including YAC, was puri fied by the methods described (25). Cosmid libraries were generated from the DNAs of YACs and screened to select clones that harbor human sequences by colony hybridization with total human DNA as the probe (26). Cosmid clones harboring microsatellite markers were detected by hybridization with end- labeled oligonucleotide primers that were used for PCR amplifications. A 2456 Research. on January 2, 2016. © 1998 American Association for Cancer cancerres.aacrjournals.org Downloaded from