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Primers used for ChIP experiments

List of primers used in real-time PCR experiments

Purpose:KIT mutations, the most prevalent genetic event in gastrointestinal stromal tumors (GIST), are associated with malignant features and poor prognosis. Aggressive GISTs possess a high propensity to spread to the liver. This study aimed to explore the role of KIT mutations in GIST liver metastasis.Experimental Design: A total of 170 GISTs were used to determine the association between KIT mutations and liver metastasis. Immunohistochemistry was performed to assess the correlation of KIT mutations with CXCR4 and ETV1 expression. Genetic and pharmacologic methods were used to study the regulation of CXCR4 and ETV1 by KIT mutations.Results: Codons 557 and 558 in KIT exon 11 were deletion hot spots in GISTs. KIT exon 11 deletions involving codons 557–558 were highly associated with liver metastasis. Overexpression of mutant KIT with exon 11 codons 557–558 deletion (KIT Δ557–558) increased GIST cell motility and liver metastasis. Mechanistically, overexpression of KIT Δ557–558 in GIST cells increased ETV1 and CXCR4 expression. CXCR4 knockdown counteracted KIT Δ557–558–mediated cell migration. Moreover, KIT Δ557–558–induced CXCR4 expression could be abolished by silencing ETV1. The chromatin immunoprecipitation assay showed that ETV1 directly bound to the CXCR4 promoter. After ERK inhibitor PD325901 treatment, the upregulation of ETV1 by KIT Δ557–558 was prevented. In addition, KIT exon 11 codons 557–558 deletion enhanced CXCL12-mediated GIST cell migration and invasion.Conclusions:KIT exon 11 557–558 deletion upregulates CXCR4 through increased binding of ETV1 to the CXCR4 promoter in GIST cells, which thus promotes liver metastasis. These findings highlighted the potential therapeutic targets for metastatic GISTs. Clin Cancer Res; 22(14); 3477–87. ©2016 AACR.

KIT and PDGFRα mutation types in primary GISTs with respect to liver metastasis

List of antibodies

CD44 and osteopontin (OPN) are functionally related molecules that, alone or in combination, play miscellaneous biological and pathophysiologic roles. CD44 cleavage, one unique feature of CD44, occurs in human cancers, but its function remains unclear. This study aimed to assess the clinicopathologic significance and mechanism of CD44 cleavage in gastrointestinal stromal tumor (GIST) with respect to OPN and OPN/CD44 interaction.CD44 cleavage was evaluated by immunoblotting in 31 primary GIST tumor specimens with paired normal tissues. OPN/CD44 interaction was examined by in situ proximity ligation assay. The associations of CD44 cleavage activity with clinicopathologic parameters, cyclin D1 expression, beta-catenin expression, OPN expression, and OPN/CD44 interaction were analyzed.CD44 cleavage activity was demonstrated in 87.1% of GIST, in contrast to its absence in normal tissues. Increased CD44 cleavage activity was significantly associated with enhanced mitosis by multivariate analysis, in addition to being related to tumor size, recurrence, high-risk status, and poor survival by univariate analysis. Mitosis was significantly higher in GIST with increased CD44 cleavage activity, which also positively correlated with tumor-specific beta-catenin and cyclin D1 overexpression, indicating a mitotic effect through aberrant cell cycle. Both OPN and OPN/CD44 interactions were significantly associated with CD44 cleavage.Our study demonstrates the clinicopathological significance of CD44 cleavage in GIST. There is a significantly increased mitosis associated with CD44 cleavage in relation to OPN/CD44 interaction and dysregulated cell cycle in GIST.

Background Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumor of the gastrointestinal tract. Surgery remains the mainstay of curative treatment. Recurrence after surgery was frequent and was associated with poor prognosis. In this study, we tried to identify predictors of recurrence in resectable GISTs. Methods Between January 1995 and December 2005, 100 patients undergoing surgical resection for GISTs in 2 hospitals were studied. Results There were 67 gastric and 33 intestinal GISTs. Recurrence was noted in 11 patients (median follow-up of 43 months). Overall 5-year survival was 84%. Multivariate analysis demonstrated that tumor size ≥10 cm was associated with higher recurrence rates ( P = .032) and was the only independent poor prognostic factor for survival ( P = .020). Conclusions We concluded that tumor size ≥10 cm carried both a higher risk of recurrence and worse survival in resectable GISTs and could be considered an indicator for adjuvant therapy.

CD44 is a transmembrane glycoprotein belonging to the cell-adhesion molecule family. It has been identified as being involved in tumor progression and metastasis, and its expression has been found to be of prognostic significance in several human malignancies. The aim of this study was to assess CD44 expression in gastrointestinal stromal tumors (GISTs), the most common mesenchymal tumor of the gastrointestinal tract. Between January 1995 and March 2006, 92 patients undergoing surgical resection for GIST in National Cheng Kung University Hospital were evaluated. To study the significance of CD44 expression, immunohistochemical staining of CD44 in tumor specimens was performed, and the clinicopathological information of patients was reviewed. Fifty-nine of 81 patients (73%) showed positive CD44 expression. Loss of CD44 expression was associated with disease progression (p = 0.019). Kaplan-Meier analysis revealed better progression-free survival among patients with strong CD44 expression (++ and +++) (p = 0.034), absence of disease progression (p

Purpose: KIT mutations, the most prevalent genetic event in gastrointestinal stromal tumors (GIST), are associated with malignant features and poor prognosis. Aggressive GISTs possess a high propensity to spread to the liver. This study aimed to explore the role of KIT mutations in GIST liver metastasis. Experimental Design: A total of 170 GISTs were used to determine the association between KIT mutations and liver metastasis. Immunohistochemistry was performed to assess the correlation of KIT mutations with CXCR4 and ETV1 expression. Genetic and pharmacologic methods were used to study the regulation of CXCR4 and ETV1 by KIT mutations. Results: Codons 557 and 558 in KIT exon 11 were deletion hot spots in GISTs. KIT exon 11 deletions involving codons 557–558 were highly associated with liver metastasis. Overexpression of mutant KIT with exon 11 codons 557–558 deletion (KIT Δ557–558) increased GIST cell motility and liver metastasis. Mechanistically, overexpression of KIT Δ557–558 in GIST cells increased ETV1 and CXCR4 expression. CXCR4 knockdown counteracted KIT Δ557–558–mediated cell migration. Moreover, KIT Δ557–558–induced CXCR4 expression could be abolished by silencing ETV1. The chromatin immunoprecipitation assay showed that ETV1 directly bound to the CXCR4 promoter. After ERK inhibitor PD325901 treatment, the upregulation of ETV1 by KIT Δ557–558 was prevented. In addition, KIT exon 11 codons 557–558 deletion enhanced CXCL12-mediated GIST cell migration and invasion. Conclusions: KIT exon 11 557–558 deletion upregulates CXCR4 through increased binding of ETV1 to the CXCR4 promoter in GIST cells, which thus promotes liver metastasis. These findings highlighted the potential therapeutic targets for metastatic GISTs. Clin Cancer Res; 22(14); 3477–87. ©2016 AACR.

The term gastrointestinal stromal tumor (GIST) in the description of a specific group of gastrointestinal nonepithelial tumors lacking the microscopic evidence of smooth muscle or characteristics of neural immunoreactivity was first introduced by Mazur and Clark (Mazur & Clark, 1983). The common origin of GIST and interstitial cell of Cajal (ICC), the pacemaker cells in the digestive tract, was proposed due to their immunohistochemical and ultrastructural similarities. The definition of GISTs as tumors originating from ICC was further confirmed according to the findings that both GIST and ICC express KIT and that most GISTs have gain-of-function mutations of KIT, the proto-oncogene that encodes a 145 kDa transmembrane tyrosine kinase KIT receptor. Mutation of different exons of the KIT oncogene results in activation of the tyrosine kinase activity of KIT, leading to ligandindependent kinase activity and uncontrolled cell proliferation as well as resistance to apoptosis (Demetri et al., 2002; Hirota et al., 1998; Kindblom et al., 1998; Savage & Antman, 2002). More than 90% of GIST have constitutive activation of the KIT protein as a result of KIT mutation and in GIST without KIT mutations, gain-of-function of platelet derived growth factor receptor α (PDGFRA) are present in about one-third of cases. It was recently proposed that ETV1, one of the ETS family transcription factor, can be a lineage survival factor that cooperates with KIT in gastrointestinal stromal tumours. GIST arises from ICCs with high levels of endogenous ETV1 expression that, when coupled with an activating KIT mutation, drives an oncogenic ETS transcriptional program (Chi et al., 2010; Roberts & Eisenberg, 2002; Rubin et al., 2001). GIST was estimated to occur in about 14.5 cases per million and was the most common mesenchymal tumor of the gastrointestinal tract. The most common locations of GIST are the stomach (50-60%), small intestine (20-30%), colon and rectum (10%), and esophagus (5%). Patients mostly present with nonspecific symptoms and signs (69%) and initial metastasis was noted in about 15-50% of GISTs (DeMatteo et al., 2000; Fletcher et al., 2002; Nilsson et al., 2005; Roberts & Eisenberg, 2002; Shinomura et al., 2005). The accurate diagnosis of GIST should be based on tumor morphology and immunohistochemistry. GIST tumors grossly appear as well-defined submucosal lesion with prominent vasculature and occasional hemorrhage or ulceration (Fig. 1A and 1B.). Under morphologic examination in the immunohistochemical analysis, GIST tumor cells are

BACKGROUND: The recurrence of hepatocellular carcinoma (HCC) after hepatectomy is a serious event. It has been demonstrated that different ground-glass hepatocyte (GGH) patterns harbor specific hepatitis B virus (HBV) pre-S deletion mutants and represent preneoplastic lesions in chronic HBV infection. In the current study, the authors investigated whether a specific GGH pattern in nontumorous liver tissues was associated with the recurrence of HBV-related HCC after surgery. METHODS: Clinicopathologic data from 82 patients with HBV-related HCC were reviewed. GGH patterns were assessed on hematoxylin and eosin-stained sections. Tissue hepatitis B surface antigen (HBsAg) expression was evaluated by immunohistochemical staining. Serum profiles of pre-S status, viral load, and HBV genotype were determined and correlated with clinical recurrence and survival after surgery. RESULTS: The results indicated that the clustered pattern of GGHs or HBsAg expression was associated significantly with decreased local recurrence-free survival (LRFS) during a mean follow-up of 46.4 months (P

Osteopontin (OPN) is a multifunctional secreted glycophosphoprotein involved in miscellaneous physiologic and pathologic processes and is functionally related to the transmembrane receptor CD44. Although OPN expression has been identified to be associated with poor prognosis in several gastrointestinal malignancies, its expression in gastrointestinal stromal tumor (GIST) has not been thoroughly investigated. This study was designed to evaluate the clinicopathologic significance of OPN expression in patients with resectable GIST.OPN expression was analyzed for its clinicopathologic significance in surgical specimens from 99 patients with resectable GIST by immunohistochemistry. In situ Proximity Ligation Assay was used for examining OPN and CD44 interaction in tumor tissues and GIST cell lines. The in vitro effects of OPN and its interaction with CD44 also were assessed.Increased OPN expression was a significant poor prognostic factor that independently predicted recurrence and poor disease-free survival in patients with resectable GIST. The interaction of OPN and CD44 was confirmed by their significant correlation in both GIST tumor tissues and cell lines by in situ Proximity Ligation Assay analysis. OPN and its interaction with CD44 were related to increased mitosis and significantly enhanced GIST tumor cell proliferation in vitro.Our study identified the clinicopathologic significance and biologic effects of OPN expression in resectable GIST. Increased OPN expression was an independent poor prognostic factor and its interaction with CD44 significantly correlated with increased mitosis as well as in vitro proliferation-promoting effects.

Pseudomyxoma peritonei is a rare disease characterized by diffuse intraperitoneal mucinous tumor and massive mucinous ascites. The mainstay of treatment is surgery in combination with adjuvant therapy.From 1995 to 2002, 8 patients with pathologically confirmed pseudomyxoma peritonei were studied. After surgical debulking, intraoperative intraperitoneal hyperthermia was performed followed by 5 days of postoperative intraperitoneal hyperthermia. Normal saline at 46 C was used for hyperthermia. Patients were followed for clinical data, survival, morbidity, and mortality.The appendix was the origin of pseudomyxoma peritonei in 6 patients, the ovary in 1 patient, and the fallopian tube in another. The pathologic diagnosis was mucinous adenocarcinoma in 4 patients, mucinous cystadenocarcinoma in 3, and mucinous cystadenoma in 1. Associated morbidity and mortality was 12.5% and 0, respectively. The recurrence rate was 62.5%, with a median follow-up of 52.5 months. The estimated 1-, 3-, and 5-year survival rates were 100%, 88%, and 49%, respectively.Surgical debulking followed by 46 C normal saline intraperitoneal hyperthermia resulted in a 5-year survival of 49% with low morbidity and no mortality for the treatment of pseudomyxoma peritonei. We concluded that this is an easy, safe, and efficacious treatment for patients with this rare disease.