ANTICANCER RESEARCH 30: 2705-2712 (2010) Comprehensive Analysis of Genes Involved in the Malignancy of Gastrointestinal Stromal Tumors MIYAKO TSUMURAYA1, HIROYUKI KATO1, KAZUHITO MIYACHI1, KINRO SASAKI1, MASAHIRO TSUBAKI1, KAZUMI AKIMOTO2 and MASAKATSU SUNAGAWA1 1Department of Surgical Oncology (Surgery I), and 2General Medical Institution, Dokkyo Medical University, Tochigi 321-0293, Japan Abstract. Background: During tumorigenesis of gastro- Gastrointestinal stromal tumors (GISTs) are mesenchymal intestinal stromal tumors (GISTs), the most frequent changes tumors that arise in the gastrointestinal tract, and account for are reported to be gain-of-function mutations in the C-KIT about 1% of gastrointestinal tumors (1). During tumorigenesis proto-oncogene. However, we speculated that additional of GISTs, the most frequent changes are reported to be gain- genetic alterations are required for the progression of GISTs. of-function mutations in the C-KIT proto-oncogene. These Patients and Methods: Using 15 cases diagnosed with GISTs, mutations can result in autophosphorylation, namely KIT we searched for novel indicator genes by microarray ligand-independent kinase activity (2). Approximately 95% analyses using an Oligo GEArray® PI3K-AKT Signaling of GISTs express the receptor tyrosine kinase KIT, which is Pathway Microarray Kit. In addition, we analyzed the useful for distinguishing these tumors from other sarcomas mutational status of C-KIT and the proliferation status that develop in the abdomen (3). In recent years, it has been indicated by the Ki-67 index. Results: The tumor established that 75-80% of GISTs harbor mutations in the localizations of the 15 GISTs were as follows: 8 in the KIT gene, and that the resulting KIT mutants play important stomach; 2 in the small intestine; 2 in the mesentery; 1 in roles in the development of these tumors (3). Furthermore, the duodenum; 1 in the rectum; and 1 in liver. Regarding the therapeutic targeting of KIT mutants with the tyrosine kinase C-KIT gene analysis, mutations in exon 11 were detected in inhibitor imatinib (Gleevec, Glivec; Novartis Pharma AG, 11 out of 13 patients. In 1 out of the 13 patients, mutations Basel, Switzerland) has proven to be an effective treatment were detected in both exons 11 and 13. No genetic for patients with advanced unresectable GISTs. However, the abnormalities were identified in 1 patient. The Ki-67 labeling efficacy varies depending on the locations of the mutations indices were significantly lower for the low-risk and (4). Recently, approximately 5% of GISTs were found to be intermediate-risk groups than for the high-risk group characterized by mutations in the related receptor tyrosine (p=0.0440). No specific genes were overexpressed in the kinase platelet-derived growth factor receptor alpha >1% Ki-67 group. Regarding the primary lesion sites, the (PDGFRA) exons 12 and 18 (5, 6). Very recently, Lasota et following 6 genes were overexpressed in tumors in the al. (7) reported PDGFRA exon 14 mutations in 11 of 200 stomach: RBL2, RHOA, SHC1, HSP90AB1, ACTB and GISTs that were negative for mutations in C-KIT exons 9, 11, BAS2C. Conclusion: Gene analysis is currently only useful 13 and 17 and PDGFRA exons 12 and 18. However, for diagnostic assessment and predicting therapeutic effects. approximately 5% of all GISTs do not have detectable However, it may be possible for new malignancy-related mutations in either the C-KIT or PDGFRA genes. factors to be identified by comparing and investigating gene Although KIT gene mutations specific to GISTs have been expression levels and other factors using such analyses. used as important diagnostic indicators, histological factors such as tumor diameter and tumor cell mitosis/density have mostly been used to assess GIST malignancy (8). Therefore, we speculated that additional genetic alterations are required for the progression of GISTs. However, malignancy has not Correspondence to: Hiroyuki Kato, MD, Ph.D., FACS, Department been assessed based on genetic abnormalities. In the present of Surgical Oncology (Surgery I), Dokkyo Medical University, 880 study, we aimed to identify novel indicator genes by Kitakobayashi Mibu-machi, Tochigi 321-0293, Japan. Tel: +81 282 ® 872157, Fax: +81 282866213, e-mail: [email protected] microarray analyses using an Oligo GEArray PI3K-AKT Signaling Pathway Microarray Kit. In addition, we analyzed Key Words: Gastrointestinal stromal tumor, GIST, C-KIT gene, Ki- the mutational status of C-KIT and the proliferation status 67 labeling index, PI3K, ALT. indicated by the Ki-67 index. 0250-7005/2010 $2.00+.40 2705 ANTICANCER RESEARCH 30: 2705-2712 (2010) Table I. Fletcher's GIST malignancy classification system. min. Next, the sections were incubated with an anti-human Ki-67 murine monoclonal antibody (Dako, Carpinteria, CA, USA) as the Risk Tumor size (cm) Mitotic count primary antibody overnight at 4˚C. Subsequent immunohisto- (per 50 HPF) chemical staining steps were performed using an ABC system (Vectastain; Vector Laboratories Inc, Burlingame, CA, USA). Very low risk <2 <5 Briefly, incubation with biotinylated anti-rabbit IgG as the Low risk 2-5 <5 secondary antibody was carried out for 30 min. The chromogen Intermediate risk <5 6-10 utilized was 3,3’-diaminobenzidine tetrahydrochloride, which was 5-10 <5 applied as a 0.02% (w/v) solution containing 0.005% (v/v) H2O2 High risk >5 >5 in 50 mM ammonium acetate/citric acid buffer (pH 6.0). The >10 Any sections were lightly counterstained with hematoxylin. Any >10 HPF: High-power field. TUNEL staining. TUNEL staining was carried out using Apoptag® Plus Peroxidase In Situ Kit (Chemicon, Temecula, CA). Briefly, deparaffinized hydrated esophageal biopsy sections were treated with proteinase K (20 μg/ml) in 0.05 M PBS (pH 7.4) at room temperature for 15 min. After washing with distilled water, the sections were Patients and Methods incubated with 3% H2O2 for 5 min. Each slide was then covered with equilibration buffer at room temperature for 1 h, incubated with Patients and tumor tissues. The study comprised 15 patients (7 men working-strength terminal transferase (TdT) enzyme at 37˚C for 1 h and 8 women) diagnosed with GISTs between July 2005 and July in a humidified chamber, and washed with 0.05 M PBS. The sections 2008, and included 2 patients with recurrent GISTs. The specimens were then incubated with a peroxidase-conjugated anti-digoxigenin excised by surgical resection were histopathologically assessed and antibody at room temperature for 30 min in a humidified chamber, classified into four groups using Fletcher's GIST malignancy washed with 0.05 M PBS and incubated with diaminobenzidine classification system (Table I) (8). (DAB) solution for 6 min. After washing with distilled water, the The risk grades of the GISTs were evaluated according to Fletcher sections were counterstained with Mayer’s hematoxylin (Sigma et al. (8). Tumors of <2 cm in diameter with 0-4 mitoses/50 high Chemical Co, St. Louis, MO, USA) and coverslipped. power fields (HPF) were considered to be very low risk, while tumors of 2-5 cm in diameter with 0-4 mitoses/50 HPF were considered to be Oligo GEArray. RNA was extracted from each frozen specimen using low risk. Tumors of <5 cm in diameter with 6-10 mitoses/50 HPF or an acid guanidinium-phenol-chloroform method. Microarray analyses tumors of 5-10 cm in diameter with 0-4 mitoses/50 HPF were were conducted using an Oligo GEArray® PI3K-AKT Signaling considered to be intermediate risk. Tumors of >5 cm in diameter with Pathway Microarray Kit (SuperArray, Frederick, MD, USA). For each >5 mitoses/50 HPF, tumors of >10 cm in diameter with any number of total RNA sample (2 μg), a TrueLabeling-AMPTRM 2.0 Linear RNA mitoses or tumors with >10 mitoses/50 HPF were considered to be Kit (SuperArray) was used to synthesize cDNA, and an IVT enzyme high risk. Tumors with necrosis were also classified as high risk. mix was used to prepare biotin-labeled cRNA. The samples were hybridized with membranes containing spots for biotin-labeled cRNA C-KIT gene analysis. Genomic DNA was extracted from formalin- and 122 DNAs (Table III) overnight at 60˚C. The hybridized fixed paraffin-embedded tumor tissues using standard proteinase K membranes were placed in a CDP-Star substrate solution for 5 min digestion and phenol/chloroform extraction methods, and used for at room temperature, and luminescent signals were detected using a subsequent molecular analyses. To avoid contamination from normal charge-coupled device camera system. The GEArray® Expression tissues, only tumor tissues were scraped and obtained from each Analysis Suite (SuperArray) was used to analyze the results. Since paraffin section, by reference to serial sections stained with the tumor diameter was smallest in Case 1, the expression levels were hematoxylin and eosin. compared with those in Case 1. The cases were divided into two Exons 9, 11, 13 and 17 of C-KIT were amplified by PCR using groups with respect to their median gene expressions to compare the previously published primer sets (Table II). Each of the amplified risk classifications, primary tumor sites and Ki-67 labeling indices. fragments was purified from a polyacrylamide gel after electrophoresis, and subjected to direct sequencing. All the sequencing Statistical analysis. The Ki-67 labeling indices were calculated as reactions were carried out in both the forward and reverse directions. the percentages of cells with nuclear staining of Ki-67 in 10 HPFs Immunohistochemistry of Ki-67. Resected specimens were fixed (HPF; ×400). The TUNEL indices were calculated as the numbers with 10% (v/v) formaldehyde and embedded in paraffin blocks. of TUNEL-positive cells among more than 1.000 cells counted. The Immunohistochemical staining was performed on sections using a relationships between the Ki-67 labeling indices and other standard avidin-biotin-peroxidase complex (ABC) method.