title: AMACR amplification and overexpression in primary imatinib-naïve gastrointestinal stromal tumors: a driver of cell proliferation indicating adverse prognosis.
abstract: Non-random gains of chromosome 5p have been observed in clinically aggressive gastrointestinal stromal tumors, whereas the driving oncogenes on 5p remain to be characterized. We used an integrative genomic and functional approach to identify amplified oncogenes on 5p and to evaluate the relevance of AMACR amplification at 5p13.3 and its overexpression in gastrointestinal stromal tumors. Thirty-seven tumor samples, imatinib-sensitive GIST882 cell line, and imatinib-resistant GIST48 cell line were analyzed for DNA imbalances using array-based genomic profiling. Forty-one fresh tumor samples of various risk categories were enriched for pure tumor cells by laser capture microdissection and quantified for AMACR mRNA expression. AMACR-specific fluorescence in situ hybridization and immunohistochemistry were both informative in tissue microarray sections of 350 independent primary gastrointestinal stromal tumors, including 213 cases with confirmed KIT /PDGFRA genotypes. To assess the oncogenic functions of AMACR, GIST882 and GIST48 cell lines were stably silenced against their endogenous AMACR expression. In 59% of cases featuring 5p gains, two major amplicons encompassed discontinuous chromosomal regions that were differentially overrepresented in high-risk cases, including the one harboring the mRNA-upregulated AMACR gene. Gene amplification was detected in 19.7% of cases (69/350) and strongly related to protein overexpression (p<0.001), although 52% of AMACR-overexpressing cases exhibited no amplification. Both gene amplification and protein overexpression were significantly associated with epithelioid histology, larger size, increased mitoses, higher risk levels, and unfavorable genotypes (all p≦0.03). They were also independently predictive of decreased disease-free survival (overexpression, p<0.001; amplification, p=0.020) in the multivariate analysis. Concomitant with downregulated cyclin D1, cyclin E, and CDK4, AMACR knockdown suppressed cell proliferation and induced G1-phase arrest, but did not affect apoptosis in both GIST882 and GIST48 cells. In conclusion, AMACR amplification is a mechanism driving increased mRNA and protein expression and conferring aggressiveness through heightened cell proliferation in gastrointestinal stromal tumors.
authors.affiliation: Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan. National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan. Institute of Clinical Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan. Department of Biotechnology, Southern Taiwan University of Science and Technology, Tainan, Taiwan.
authors.forename: Chien-Feng
authors.initials: CF
authors.lastname: Li
authors.affiliation: National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan. Institute of Clinical Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan. Department of Internal Medicine and Cancer Center, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan. Institutes of Molecular Medicine, National Cheng Kung University, Tainan, Taiwan.
authors.forename: Li-Tzong
authors.initials: LT
authors.lastname: Chen
authors.affiliation: Department of Pathology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan.
authors.forename: Jui
authors.initials: J
authors.lastname: Lan
authors.affiliation: Department of Surgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan.
authors.forename: Fong-Fu
authors.initials: FF
authors.lastname: Chou
authors.affiliation: Department of Tourism Management, Southern Taiwan University of Science and Technology, Tainan, Taiwan. Department of Internal Medicine, Chi-Mei Medical Center, Tainan, Taiwan.
authors.forename: Ching-Yih
authors.initials: CY
authors.lastname: Lin
authors.affiliation: Division of Oncology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan.
authors.forename: Yen-Yang
authors.initials: YY
authors.lastname: Chen
authors.affiliation: Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan.
authors.forename: Tzu-Ju
authors.initials: TJ
authors.lastname: Chen
authors.affiliation: Division of Oncology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan.
authors.forename: Shau-Hsuan
authors.initials: SH
authors.lastname: Li
authors.affiliation: Department of Pathology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan.
authors.forename: Shih-Chen
authors.initials: SC
authors.lastname: Yu
authors.affiliation: Department of Radiation Oncology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan.
authors.forename: Fu-Ming
authors.initials: FM
authors.lastname: Fang
authors.affiliation: Department of Pathology, Changhua Christian Hospital, Changhua, Taiwan.
authors.forename: Hui-Chun
authors.initials: HC
authors.lastname: Tai
authors.affiliation: Department of Pathology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan.
authors.forename: Hsuan-Ying
authors.initials: HY
authors.lastname: Huang
mesh_terms: D001393:Azoles / Q000737:chemistry; D001549:Benzamides / Q000008:administration & dosage*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D055028:Comparative Genomic Hybridization; D019938:Cyclin D1 / Q000378:metabolism; D019927:Cyclin E / Q000378:metabolism; D051358:Cyclin-Dependent Kinase 4 / Q000378:metabolism; D004252:DNA Mutational Analysis; D018572:Disease-Free Survival; D005434:Flow Cytometry; D016193:G1 Phase; D046152:Gastrointestinal Stromal Tumors / Q000188:drug therapy*; D018628:Gene Dosage; D005838:Genotype; D006801:Humans; D000068877:Imatinib Mesylate; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D054833:Isoindoles; D015999:Multivariate Analysis; D015513:Oncogene Proteins / Q000378:metabolism; D016566:Organoselenium Compounds / Q000737:chemistry; D010879:Piperazines / Q000008:administration & dosage*; D011237:Predictive Value of Tests; D011379:Prognosis; D011743:Pyrimidines / Q000008:administration & dosage*; D034622:RNA Interference; D019998:Racemases and Epimerases / Q000235:genetics* / Q000378:metabolism*; D016896:Treatment Outcome; D015854:Up-Regulation
chemical_list: D001393:Azoles; D001549:Benzamides; C530104:CCND1 protein, human; C475820:CCNE1 protein, human; D019927:Cyclin E; D054833:Isoindoles; D015513:Oncogene Proteins; D016566:Organoselenium Compounds; D010879:Piperazines; D011743:Pyrimidines; D019938:Cyclin D1; C042986:ebselen; D000068877:Imatinib Mesylate; C495900:CDK4 protein, human; D051358:Cyclin-Dependent Kinase 4; D019998:Racemases and Epimerases; C108352:alpha-methylacyl-CoA racemase