<i>AMACR</i>amplification and overexpression in primary imatinib-naïve gastrointestinal stromal tumors: a driver of cell proliferation indicating adverse prognosis

Abstract

// Chien-Feng Li 1,2,3,4 , Li-Tzong Chen 2,3,5,6 , Jui Lan 7 , Fong-Fu Chou 8 , Ching-Yih Lin 9,10 , Yen-Yang Chen 11 , Tzu-Ju Chen 1 , Shau-Hsuan Li 11 , Shih-Chen Yu 7 , Fu-Ming Fang 12 , Hui-Chun Tai 13 and Hsuan-Ying Huang 7 1 Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan 2 National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan 3 Institute of Clinical Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 4 Department of Biotechnology, Southern Taiwan University of Science and Technology, Tainan, Taiwan 5 Department of Internal Medicine and Cancer Center, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan 6 Institutes of Molecular Medicine, National Cheng Kung University, Tainan, Taiwan 7 Department of Pathology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan 8 Department of Surgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan 9 Department of Tourism Management, Southern Taiwan University of Science and Technology, Tainan, Taiwan 10 Department of Internal Medicine, Chi-Mei Medical Center, Tainan, Taiwan 11 Division of Oncology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan 12 Department of Radiation Oncology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan 13 Department of Pathology, Changhua Christian Hospital, Changhua, Taiwan Correspondence: Hsuan-Ying Huang, email: // Keywords : GIST, 5p, AMACR, amplification, proliferation Received : August 13, 2014 Accepted : October 18, 2014 Published : October 18, 2014 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&lt;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 &lt;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 G 1 -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.