Imaging, Diagnosis, Prognosis ASPM Is a Novel Marker for Vascular Invasion, Early Recurrence, and Poor Prognosis of Hepatocellular Carcinoma Shih-Yeh Lin,1Hung-Wei Pan,1Shu-Hsiang Liu,1Yung-Ming Jeng,3 Fu-Chang Hu,2,4 Shian-Yang Peng,5 Po-Lin Lai,3 and Hey-Chi Hsu1, 3 Abstract Purpose: Abnormal spindle-like microcephaly associated (ASPM) plays an important role in neurogenesis and cell proliferation. This study is to elucidate its role in hepatocelllular carcinoma (HCC), particularly early tumor recurrence (ETR) and prognosis. Experimental Design:We used reverse transcription-PCR assays to measure theASPM mRNA levels in 247 HCC and correlated with clinicopathologic and molecular features. Results: ASPM mRNA levels were high in fetal tissues but very low in most adult tissues.ASPM mRNAwas overexpressedin162 HCC (66%)but notinbenignliver tumors.ASPM overexpression correlated with high a-fetoprotein (P =1Â10 -8), high-grade (grade II-IV) HCC (P =2Â 10 -6), high-stage (stage IIIA-IV) HCC (P =1Â10 -8), and importantly ETR (P =1Â10 -8). ETR is the most criticalunfavorableclinicalprognostic factor. Among the various independent histo- pathologic (tumor size, tumor grade and tumor stage) and molecular factors (p53 mutation, high a-fetoprotein, and ASPM overexpression), tumor stage was the most crucialhistologic factor (odds ratio, 14.7; 95% confidence interval, 6.65-33.0; P =1Â10 -8), whereas ASPM overexpres- sion (odds ratio, 6.49; P =1Â10 -8) is the most important molecular factor associated with ETR. ASPM overexpression was associated with vascular invasion and ETR in both p53-mutated (all P values = 1 Â10 -8) and non-p53-mutated HCC (P =1Â10 -8 and 0.00088, respectively). Hence, patients with APSM-overexpressing HCC had lower 5-year survival (P = 0.000001)in both p53-mutated (P = 0.00008) and non-p53-mutated HCC (P = 0.0027). Inlow-stage (stage II) HCC, ASPM overexpression also correlated withhigher ETR (P =0.008). Conclusion: ASPM overexpression is a molecular marker predicting enhanced invasive/meta- static potentialof HCC, higher risk of ETR regardless of p53 mutation status and tumor stage, and hence poor prognosis. Abnormal spindle-like microcephaly associated (ASPM) gene is the mutation of Drosophila asp causes abnormal spindles, frequent human orthologue of the Drosophila abnormal spindle (asp) and polyploid cells, and cytokinesis failure (6), leading to arrest of the most commonly mutated gene of autosomal recessive neuroblasts in metaphase (7, 8) and larval-pupal lethality primary microcephaly (1–5). The homozygous semilethal (6, 9). In human, the defective neurogenesis caused by homozygous mutation of ASPM leads to microcephaly and mental retardation (4, 10). The ASPM gene encodes a large protein of 3,477 amino acids with a NH2-terminal microtu- Authors’ Affiliations: 1Graduate Institute of Pathology, College of Medicine; bule-binding domain, two calponin homology domains, 74 2College of Public Health, National Taiwan University;3Department of Pathology 4 repeated calmodulin-binding isoleucine-glutamine domains, and NationalCenter of Excellence for GeneralClinicalTrialand Research, National and a COOH-terminal region (4, 11, 12). The NH -terminal Taiwan University Hospital; and 5Department of GeneralEducation, NationalTaipei 2 College of Nursing,Taipei,Taiwan, Republic of China microtubule-binding domain is highly conserved in eukaryotes, Received 12/28/07; revised 4/8/08; accepted 4/11/08. suggesting that ASPM may possess the fundamental function in Grant support: National Health Research Institute, Department of Health of the cytokinesis. However, the multidomain feature suggests that Republic of China,Taiwan grant NHRI-EX94-9427NI, and National Science Council ASPM have more diverse function than the Drosophila of the Republic of China,Taiwan grant NSC94-3112-B-002-018. The costs of publication of this article were defrayed in part by the payment of page counterpart. charges. This article must therefore be hereby marked advertisement in accordance Drosophila Asp protein associates with centrosomes and is with 18 U.S.C. Section 1734 solely to indicate this fact. involved in organizing microtubules at the spindle poles in Note: Supplementary data for this article are available at Clinical Cancer Research mitosis and meiosis (6, 8, 9, 11, 13–16). Centrosome defects Online (http://clincancerres.aacrjournals.org/). S-Y.Lin and H-W. Pan contributed equally to this work. cause chromosome misaggregation and aneuploidy, leading to Current address for S-H. Liu: Department of MedicalResearch, Mackay Memorial genetic instability, a major driving force for malignant transfor- Hospital,Danshui,TaipeiCounty,Taiwan,RepublicofChina. mation and tumor progression (17–22). Recently, ASPM is Requests for reprints: Hey-Chi Hsu, Department of Pathology, National Taiwan shown to express in nearly all transformed human cell lines and University Hospital, Taipei, Taiwan, Republic of China. Phone: 886-2-23123456, ext. 5455; Fax: 886-2-23410876; E-mail: [email protected]. in multiple fetal tissues (23, 24). These findings suggest ASPM F 2008 American Association for Cancer Research. play an important role in cell cycle progression and cell doi:10.1158/1078-0432.CCR-07-5262 proliferation in embryonic development and tumorigenesis. Clin Cancer Res 2008;14(15) August 1, 2008 4814 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2008 American Association for Cancer Research. ASPM and Hepatocellular Carcinoma Using differential display analysis of gene expression profile Statistical analysis. The statistical significance of differences of of hepatocelllular carcinoma (HCC), we found frequent over- selected clinicopathologic features between those with and without m2 expression of ASPM, which is located at chromosome 1q31, a ASPM mRNA overexpression was assessed by tests. For 5-year region with frequent gain in HCC (25, 26). In this study, we survival analysis, person-months were calculated from the date of resection to the date of death or 60 months for those who survived for showed that ASPM was often overexpressed in human HCC >5 years. P values < 0.05 or a 95% confidence interval (95% CI), not and associated with tumor progression, early tumor recurrence including 1, were considered statistically significant. Multivariate (ETR), and poor prognosis. analyses of grade and stage were conducted by fitting multiple logistic regression models (42) and then times to ETR and death were analyzed by fitting multiple Cox’s proportional hazards models (43). Basic Patients and Methods model-fitting techniques for (a) variable selection, (b) goodness-of-fit assessment, and (c) regression diagnostics (including residual analysis, Differential display, reverse transcription-PCR, and definition of ASPM influence analysis, and check of multicollinearity) were used in our overexpression. Differential display was done using anchor primer regression analyses to assure the quality of analysis results (42, 43). HT11C (5¶-AAGCTTTTTTTTTTTC-3¶) and arbitrary primer HAP7 (5¶- Two-tailed P < 0.05 was considered statistically significant. AAGCTTAACGAGG-3¶) from the RNAimage kit (GenHunter) as described (27–31). Reverse transcription-PCR assays in the linear range were used for Results the measurements of ASPM mRNA levels using ribosomal protein S26 ASPM (RPS26; 32) or porphobilinogen deaminase (PBGD) mRNA for internal mRNA expression in fetal and adult tissues and various controls (33) as described (29–31, 34). Primers for ASPM spanning solid types of human cancer. By the differential display analysis, intron 20 to avoid DNA contamination are ASPM-forward (5¶- we identified a 240-bp cDNA, which was often expressed in TAAAAAGACATCGAGCTGCTT-3¶) and ASPM-reverse (5¶-CCTCTCCA- HCC and had sequence identical to human ASPM (Genbank TAATGCCTGAATT-3¶). The primers for RPS26 are RPS26-forward NM_018136; Fig. 1A). Using reverse transcription-PCR at the (5¶-CCGTGCCTCCAAGATGACAAAG-3¶) and RPS26-reverse (5¶- linear range, ASPM mRNA was expressed in relative abundance TGTCTGGTAACGGCAATGCGGCT-3¶) and the primers for PBGD are in most fetal tissues, including the liver (Fig. 1B), and all the ¶ ¶ ¶ ¶ ¶ PBGD-5 (5 -TGTCTGGTAACGGCAATGCGGCT-3 ) and PBGD-3 (5 - cell lines examined (Fig. 1C), but the level was very low in most GGTCCACTTCATTCTTCTCCAG-3¶). Relative ASPM mRNA level was adult tissues (Fig. 1B). determined as the ratio of ASPM to PBGD as described (29–31, 34, 35) and scored as high (ratio z 1.0; 128 cases), medium (0.5 < In clinical tissue samples, ASPM mRNA overexpression was ratio < 1.0; 18 cases), low (ratio < 0.5; 39 cases), trace (ratio < 0.2; found in 162 (66%) of 247 surgically removed, unifocal, 44 cases), and negative (24 cases). In 227 nontumorous livers primary HCC (Fig. 2) but in none of hepatocellular adenomas examined, none had a ratio exceeding 0.5. Hence, a ratio z 0.5 was (4 cases), focal nodular hyperplasia (10 cases), and non- regarded as ASPM overexpression. tumorous livers (227 cases). Patients, histologic study, ETR, follow-up observation, and p53 Correlation of ASPM overexpression with HCC progres- mutation. Between 1983 and 1997, 247 surgically resected, unifocal, sion. To better understand the significance of ASPM in HCC, primary HCC pathologically assessed at the National Taiwan University we correlated its mRNA expression with the major clinicopath- Hospital, as described (36–39), formed the basis of this study. This ologic features. As shown in Table 1, ASPM overexpression was study was executed according to