HuR cytoplasmic expression is associated with increased cyclin A expression and inferior disease-free survival in patients with gastrointestinal stromal tumours (GISTs).

AIMS: HuR is an RNA-binding protein that post-transcriptionally modulates the expression of various target genes involved in carcinogenesis, such as CCNA2, which encodes cyclin A. The aim of this study was to evaluate the significance of HuR expression and subcellular localization in a large cohort of gastrointestinal stromal tumours (GISTs). METHODS AND RESULTS: HuR immunostaining was assessable for nuclear and cytoplasmic expression in 341 cases on tissue microarrays of primary GISTs, of which 318, 296 and 193 cases were also characterized for Ki67 labelling, cyclin A immunoexpression, and KIT and PDGFRA receptor tyrosine kinase (RTK) genotypes, respectively. The results of HuR nuclear and cytoplasmic expression were correlated with disease-free survival (DFS) and clinicopathological, immunohistochemical and RTK genotypic variables. HuR cytoplasmic expression was present in 42% of primary GISTs, and was significantly related to epithelioid histology, larger tumour size, NIH risk category, and nuclear expression of Ki67 and cyclin A. Importantly, HuR cytoplasmic expression (P < 0.001) and cyclin A overexpression (P < 0.001) were strongly associated with worse DFS. Both variables remained independently predictive of adverse outcome [P = 0.020 and risk ratio (RR) 2.605 for cytoplasmic HuR; P = 0.026 and RR 2.763 for cyclin A]. CONCLUSIONS: HuR cytoplasmic expression not only correlates with adverse prognosticators and cyclin A overexpression, but also independently predicts worse DFS, indicating a causative role in conferring tumour aggressiveness.

Alpha-methylacyl coenzyme A racemase overexpression in gallbladder carcinoma confers an independent prognostic indicator.

AIMS: Increased ß-oxidation of branched-chain fatty acids provides an additional metabolic advantage for cancer cells thereby enhancing tumour development and progression. Alpha-methylacyl coenzyme A racemase (AMACR) is an enzyme essential for the catabolism of branched-chain fatty acids that allows their subsequent ß-oxidation and thus plays an important role in generating biological energy. However, the expression of AMACR has never been systemically investigated in gallbladder carcinoma. This study evaluated the expression status, associations with clinicopathological variables and prognostic implications of AMACR in a well-defined cohort of gallbladder carcinoma and confirmed their expression status in gallbladder carcinoma cells. METHODS: AMACR immunostaining was assessable in 89 cases on tissue microarrays of gallbladder carcinoma, and it was correlated with clinicopathological factors and patient survival. In three gallbladder carcinoma cell lines and one non-tumorigenic cholangiocyte, AMACR mRNA expression was measured by real-time reverse transcription PCR and the endogenous expression of AMACR protein was analysed by western blotting. RESULTS: AMACR overexpression was significantly associated with an advanced primary tumour status (p=0.027) and American Joint Committee on Cancer stage (p=0.027), an increased histological grade (p=0.002) and vascular invasion (p=0.017). Importantly, AMACR overexpression independently predicted worse disease-specific survival (p=0.0452, RR 1.887). Expression levels of AMACR mRNA and total protein in various cells were comparable. The abundance of AMACR expression increased in tumour cells and was even higher in the metastatic cell line. CONCLUSIONS: In primary gallbladder carcinoma, AMACR overexpression was correlated with important prognosticators and independently portended worse outcomes, highlighting the potential prognostic and therapeutic utility of AMACR in gallbladder carcinoma.

Homozygous deletion of MTAP gene as a poor prognosticator in gastrointestinal stromal tumors.

PURPOSE: Chromosome 9 is frequently deleted in high-risk gastrointestinal stromal tumors (GISTs), whereas its specific tumor suppressor genes (TSGs) are less understood. We did an integrative study of MTAP gene at 9p21 to analyze its implication in GISTs. EXPERIMENTAL DESIGN: To search TSGs on chromosome 9, we used ultrahigh-resolution array comparative genomic hybridization to profile DNA copy number alterations of 22 GISTs, with special attention to MTAP gene. MTAP immunoexpression was assessable for 306 independent GISTs on tissue microarrays, with 146 cases analyzed for MTAP homozygous deletion, 181 for mutations of KIT and PDGFRA receptor tyrosine kinase genes, and 7 for MTAP hypermethylation. RESULTS: Array comparative genomic hybridization identified 11 candidate TSGs on 9p and six on 9q. MTAP and/or CDKN2A/CDKN2B at 9p21.3 were deleted in one intermediate-risk (11%) and seven high-risk (70%) GISTs with two cases homozygously codeleted at both loci. MTAP homozygous deletion, present in 25 of 146 cases, was highly associated with larger size and higher mitotic rate, Ki-67 index, and risk level (all P < 0.01) but not with receptor tyrosine kinase genotypes. Whereas MTAP homozygous deletion correlated with MTAP protein loss (P < 0.001), 7 of 30 GISTs without MTAP expression did not show homozygous deletion, including three MTAP-hypermethylated cases. MTAP homozygous deletion was univariately predictive of decreased disease-free survival (P < 0.0001) and remained multivariately independent (P = 0.0369, hazard ratio = 2.166), together with high-risk category (P < 0.0001), Ki-67 index >5% (P = 0.0106), and nongastric location (P = 0.0416). CONCLUSIONS: MTAP homozygous deletion, the predominant mechanism to deplete protein expression, is present in 17% of GISTs. It correlates with important prognosticators and independently predicts worse outcomes, highlighting the role in disease progression.

Ezrin overexpression in gastrointestinal stromal tumors: an independent adverse prognosticator associated with the non-gastric location.

Ezrin, a member of the ezrin-radixin-moesin family, acts as a link between the cell membrane and actin cytoskeleton to integrate cell adhesion-mediated signaling. It implicates tumor progression, metastatic dissemination, and adverse outcomes in several cancer types, including pediatric and adult sarcomas. Although ezrin upregulation was shown by cDNA expression profiling, no study has systematically evaluated the significance of ezrin expression in a large cohort of gastrointestinal stromal tumors (GISTs). Ezrin immunostaining was carried out on tissue microarrays of primary GISTs and assessable in 347 cases, 188 of which were successfully evaluated for mutation variants of KIT and PDGFRA receptor tyrosine kinase (RTK) genes by sequencing with or without screening by denatured high-performance liquid chromatography. These GISTs with known RTK genotypes were dichotomized into two prognostically different groups. The endogenous expression and phosphorylation of ezrin in GIST cell lines were analyzed by western blotting. By immunohistochemistry, ezrin overexpression was present in 66% of GISTs and significantly associated with the non-gastric location (P=0.002) and decreased disease-free survival (P=0.032, univariately). However, it was not related to the National Institute of Health (NIH) risk category, Ki-67 labeling index, RTK genotypes, and other variables. In multivariate analyses, ezrin overexpression remained independently predictive of adverse outcome (P=0.008, risk ratio=2.363), together with Ki-67 labeling index >5% (P<0.001, risk ratio=3.581), high-risk category (P<0.001, risk ratio=2.156), and the non-gastric location (P=0.029, risk ratio=1.899). Despite the variation in the ezrin expression level, phosphorylated ezrin at threonine(567) was only detectable in GIST882 and GIST48 cells, but not in colonic smooth muscle cells. In conclusion, ezrin is frequently overexpressed in GISTs, especially those arising from the non-gastric sites. Given that its impact is independent of the NIH risk category, cell proliferation, and tumor location, ezrin immunoreactivity represents a valuable prognostic adjunct of GISTs, suggesting a causative role in conferring an aggressive phenotype.

Heat shock protein 90 overexpression independently predicts inferior disease-free survival with differential expression of the alpha and beta isoforms in gastrointestinal stromal tumors.

PURPOSE: Most gastrointestinal stromal tumors harbor a mutated KIT or PDGFRA receptor tyrosine kinase (RTK). Heat shock protein 90 (Hsp90) is a chaperone mediating the folding and stabilization of many oncoproteins, including KIT. An Hsp90 inhibitor, 17-AAG, can attenuate KIT activation and proliferation of gastrointestinal stromal tumor cell lines. We further evaluated Hsp90 immunoexpression and the difference between alpha and beta isoforms in gastrointestinal stromal tumor specimens. EXPERIMENTAL DESIGN: Hsp90 immunostain was assessable in 306 cases on tissue microarrays of primary gastrointestinal stromal tumors and correlated with various variables and disease-free survival (DFS). RTK mutation variants, confirmed in 142 cases by sequencing with or without precedent denaturing high pressure liquid chromatography screening, were dichotomized into two prognostically different groups. Differential expression of transcript and protein isoforms was measured by real-time reverse transcription-PCR and Western blotting in 16 and 6 cases, respectively. RESULTS: Hsp90 overexpression (55%) significantly correlated with larger size, nongastric location, higher mitotic count and NIH risk level, Ki-67 overexpression (all P < or = 0.001), and unfavorable RTK genotypes (P = 0.020). It strongly portended inferior DFS univariately (P < 0.0001) and remained independent in multivariate analysis (P = 0.031; risk ratio, 2.44), along with high-risk category, Ki-67 overexpression, and old age. For both mRNA and protein, Hsp90beta was more abundant than Hsp90alpha, whereas the latter was significantly higher in high-risk cases. CONCLUSIONS: Hsp90 overexpression represents a poor prognosticator that correlates with several adverse parameters, highlighting its role in disease progression and alternative therapy for high-risk, imatinib-resistant gastrointestinal stromal tumors. Hsp90alpha seems more relevant to the intrinsic aggressiveness of gastrointestinal stromal tumors, albeit less abundant than Hsp90beta.