Il-Chi Chang

Comparison of IHC, FISH and RT-PCR methods for detection of ALK rearrangements in 312 non-small cell lung cancer patients in Taiwan.

Background Recently Echinoderm microtubule-associated protein-like 4- anaplastic lymphoma kinase (EML4-ALK) fusion gene has become an important biomarker for ALK tyrosine kinase inhibitor (crizotinib) treatment in NSCLC. However, the best detection method and the significance of EML4-ALK variant types remain uncertain. Methods Reverse transcriptase-polymerase chain reaction (RT-PCR), fluorescence in Situ hybridization (FISH) and Immunohistochemical (IHC) stain were performed on tumor tissues of 312 NSCLC patients for detection of ALK rearrangements. Mutation analyses for EGFR and KRAS genes were also performed. Results Thirteen of the 312 patients (4.17%) had ALK rearrangements detected by RT-PCR. If RT-PCR data was used as the gold standard, FISH tests had a low sensitivity (58.33%), but very good specificity (99.32%). IHC stain had better sensitivity (91.67%) than FISH, but lower specificity (79.52%), when the cut off was IHC2+. All of the 8 patients with high abundance of EML4-ALK positive cells in tumor tissues (assessed by the signal intensities of the RT-PCR product), were also have high expression of ALK protein (IHC3+), and positive for FISH, except one failed in FISH. Variants 3a+3b (4/5, 80%) of EML4-ALK fusion gene were more common to have high abundance of EML4-ALK positive cells in tumor tissues than variant 1 (1/3, 33.3%). Meta-analysis of the published data of 2273 NSCLC patients revealed that variant 3 (23/44, 52.3%) was the most common type in Chinese population, while variant 1 (28/37, 75.7%) was most common in Caucasian. Conclusions Among the three detection methods, RT-PCR could detect not only the presence of EML4-ALK fusion gene and their variant types, but also the abundance of EML4-ALK positive cells in NSCLC tumor tissues. The latter two factors might affect the treatment response to anti-ALK inhibitor. Including RT-PCR as a diagnostic test for ALK inhibitor treatment in the prospective clinical trials is recommended.

Phenotypic Changes in Mitochondrion-Rich Cells and Responses of Na+/K+-ATPase in Gills of Tilapia Exposed to Deionized Water

Abstract The aim of this study was to illustrate the phenotypic modification of mitochondrion-rich (MR) cells and Na+/K+-ATPase (NKA) responses, including relative protein abundance, specific activity, and immunolocalization in gills of euryhaline tilapia exposed to deionized water (DW) for one week. The plasma osmolality was not significantly different between tilapia of the local fresh water (LFW) group and DW group. Remodeling of MR cells occurred in DW-exposed fish. After transfer to DW for one week, the relative percentage of subtype-I (wavy-convex) MR cells with apical size ranging from 3 to 9 μm increased and eventually became the dominant MR cell subtype. In DW tilapia gills, relative percentages of lamellar NKA immunoreactive (NKIR) cells among total NKIR cells increased to 29% and led to significant increases in the number of NKIR cells. In addition, the relative protein abundance and specific activity of NKA were significantly higher in gills of the DW-exposed fish. Our study concluded that tilapia require the development of subtype-I MR cells, the presence of lamellar NKIR cells, and enhancement of NKA protein abundance and activity in gills to deal with the challenge of an ion-deficient environment.

The Hepatitis Viral Status in Patients With Hepatocellular Carcinoma: a Study of 3843 Patients From Taiwan Liver Cancer Network.

AbstractHepatocellular carcinoma (HCC) is the leading cancer death in Taiwan. Chronic viral hepatitis infections have long been considered as the most important risk factors for HCC in Taiwan. The previously published reports were either carried out by individual investigators with small patient numbers or by large endemic studies with limited viral marker data. Through collaboration with 5 medical centers across Taiwan, Taiwan liver cancer network (TLCN) was established in 2005. All participating centers followed a standard protocol to recruit liver cancer patients along with their biosamples and clinical data. In addition, detailed viral marker analysis for hepatitis B virus (HBV) and hepatitis C virus (HCV) were also performed. This study included 3843 HCC patients with available blood samples in TLCN (recruited from November 2005 to April 2011). There were 2153 (56.02%) patients associated with HBV (HBV group); 969 (25.21%) with HCV (HCV group); 310 (8.07%) with both HBV and HCV (HBV+HCV group); and 411 (10.69%) were negative for both HBV and HCV (non-B non-C group). Two hundred two of the 2463 HBV patients (8.20%) were HBsAg(-), but HBV DNA (+). The age, gender, cirrhosis, viral titers, and viral genotypes were all significantly different between the above 4 groups of patients. The median age of the HBV group was the youngest, and the cirrhotic rate was lowest in the non-B non-C group (only 25%). This is the largest detailed viral hepatitis marker study for HCC patients in the English literatures. Our study provided novel data on the interaction of HBV and HCV in the HCC patients and also confirmed that the HCC database of TLCN is highly representative for Taiwan and an important resource for HCC research.

EGFR over-expression in non-small cell lung cancers harboring EGFR mutations is associated with marked down-regulation of CD82

Epidermal growth factor receptor (EGFR) gene mutations are strongly associated with lung adenocarcinoma and favorable response to EGFR tyrosine kinase inhibitor. The mutated EGFR proteins (EGFRs) are hyper-phosphorylated and refractory to receptor down-regulation. To address the discrepancy between hyper-phosphorylation and lack of down-regulation of mutant EGFRs, we have examined the expression of EGFR negative regulators in non-small cell lung cancer (NSCLC) cell lines. We found that NSCLC cell lines expressing mutant EGFRs often had low expression of various negative regulators for EGFR. Among them, tumor suppressor CD82 was up-regulated by wild type (WT) EGFR but down-regulated by mutant EGFRs. Reconstitution of CD82 exerted stronger suppressive effects on mutant EGFRs than on WT EGFR. Active exportation of CD82 through the exosome was one of the mechanisms involved in achieving the overall CD82 down-regulation in mutant EGFR-expressing lung cancer cell lines. Over-expression of mutant EGFR protein frequently occurred in the lung cancer tissues of mutant EGFR-transgenic mice and also associated with CD82 down-regulation. Immunoblot analyses on the tumor tissues from 23 lung adenocarcinoma patients (12 with WT EGFR, and 11 with mutant EGFRs) also identified significantly stronger down-regulation of CD82 in tumors with mutant EGFRs than WT. Our data indicate that CD82 down-regulation could be a critical step involved in the EGFR over-expression and the stronger tumorigenic activity triggered by EGFR mutations. Up-regulation of the CD82 level may become a promising new treatment strategy for lung adenocarcinoma.

Identification of Transforming Hepatitis B Virus S Gene Nonsense Mutations Derived from Freely Replicative Viruses in Hepatocellular Carcinoma

Background & Aims The correlation between chronic hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC) has been well-established. But the roles of viral factor remain uncertain. Only HBV X gene and nonsense mutations of S gene (C-terminal truncation of HBV surface protein) have been demonstrated to have transforming activity. Whether they play a significant role in hepatocarcinogenesis is still uncertain. Methods Twenty-five HBV-related HCC patients were positive for hepatitis B core antigen (HBcAg) in the cancerous parts of their HCC liver tissues by immunohistochemistry studies, and had available tissue for whole HBV genome sequence analysis. The results were compared with 25 gender and age-matched HBcAg negative HCCs. Plasmids encoding HBV S gene nonsense mutations identified from HBcAg (+) HCC tissue were constructed to investigate their cell proliferation, transformation activity and the oncogenic potentials by xenograft study and in vivo migration assay. Results HBcAg (+) HCC patients were significantly associated with cirrhosis and small tumor size (≦2 cm) when compared with HBcAg (−) HCC patients. Southern blot analyses revealed freely replicative forms of HBV in the cancerous parts of HBcAg(+) HCC. Three nonsense mutations of S gene (sL95*, sW182*, and sL216*) were identified in the HBcAg(+) HCC tumor tissues. sW182* and sL216* were recurrently found in the 25 HBcAg (−) HCC tumor tissue, too. Functional studies of the above 3 non-sense mutations all demonstrated higher cell proliferation activities and transformation abilities than wild type S, especially sW182*. Tumorigenicity analysis by xenograft experiments and in vitro migration assay showed potent oncogenic activity of sW182* mutant. Conclusions This study has demonstrated potent oncogenic activity of nonsense mutations of HBV S gene, suggesting they may play an important role in hepatocarcinogenesis.

Cysteine-rich protein 2 alters p130Cas localization and inhibits vascular smooth muscle cell migration

AIMS Cysteine-rich protein (CRP) 2, a member of the LIM-only CRP family that contains two LIM domains, is expressed in vascular smooth muscle cells (VSMCs) of blood vessels and functions to repress VSMC migration and vascular remodelling. The goal of this study was to define the molecular mechanisms by which CRP2 regulates VSMC migration. METHODS AND RESULTS Transfection of VSMCs with CRP2-EGFP constructs revealed that CRP2 associated with the actin cytoskeleton. In response to chemoattractant stimulation, Csrp2 (mouse CRP2 gene symbol)-deficient (Csrp2(-/-)) VSMCs exhibited increased lamellipodia formation. Re-introduction of CRP2 abrogated the enhanced lamellipodia formation and migration of Csrp2(-/-) VSMCs following chemoattractant stimulation. Mammalian 2-hybrid and co-immunoprecipitation assays demonstrated that CRP2 interacts with p130Cas, a scaffold protein important for lamellipodia formation and cell motility. Immunofluorescence staining showed that CRP2 colocalized with phospho-p130Cas at focal adhesions (FAs)/terminal ends of stress fibres in non-migrating cells. Interestingly, in migrating cells phospho-p130Cas localized to the leading edge of lamellipodia and FAs, whereas CRP2 was restricted to FAs and stress fibres. Furthermore, we demonstrated that p130Cas expression and phosphorylation promote neointima formation following arterial injury. CONCLUSION These studies demonstrate that CRP2 sequesters p130Cas at FAs, thereby reducing lamellipodia formation and blunting VSMC migration.

Carboxypeptidase E is a prediction marker for tumor recurrence in early-stage hepatocellular carcinoma

Tumor recurrence and metastasis are the major causes of death for hepatocellular carcinoma (HCC) patients who are able to receive curative resection. Identifying the predicting biomarkers for tumor recurrence would improve their survival. RNA extracted from fresh frozen tumors and adjacent non-tumor liver tissues of 120 HCC patients were obtained from Taiwan Liver Cancer Network (TLCN) in year 2010 for determination of the carboxypeptidase E (CPE) expression level (including its splicing mutant CPE-ΔN) in the tumor tissue (T) and paired non-tumor liver tissue (N) by real-time quantitative polymerase chain reaction. All patients were male, had chronic hepatitis B virus infection, were in the early pathology stage, and received curative resection. The T/N ratio of the CPE expression level was correlated with the updated survival data from TLCN in 2015. The CPE expression level in the 120 HCC patients was divided into three groups according to the T/N ratio: <1, ≥1 and ≤2, and >2, respectively. By multivariate analyses, the recurrence-free survival (RFS) was only significantly associated with the pathology stage and the CPE expression level. For overall survival (OS), only the CPE expression level was the significant prognostic factor. The CPE expression level was also significantly correlated with the tumor recurrence for both stage I (p = 0.0106) and stage II patients (p = 0.0006). The CPE mRNA expression level in HCC can be a useful biomarker for predicting tumor recurrence in HCC patients who are in the early pathology stage and able to receive curative resection.

Correlation of anaplastic lymphoma kinase overexpression and the EML4-ALK fusion gene in non-small cell lung cancer by immunohistochemical study.

BACKGROUND Recently the echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion gene with transforming activity was identified in non-small cell lung cancer (NSCLC). In addition, NSCLC patients with the EML4-ALK fusion gene had a dramatic response and longer progression free survival after ALK inhibitor treatment than those without this fusion gene. However, the incidence and clinical and molecular characteristics of the EML4-ALK fusion gene in NSCLC patients of Taiwan are still unclear. METHODS Sixty-four fresh frozen tumor specimens were obtained from the tissue bank of Chang Gung Memorial Hospital for RNA extraction and EML4-ALK fusion gene detection. Paraffin sections of lung tumors from all of these patients were available and were analyzed for ALK protein expression by immunohistochemical (IHC) study. The results were correlated with clinical and molecular biomarkers. RESULTS Three of the 64 tumors (4.7%) had the EML4-ALK fusion gene. Two were adenocarcinomas, and one was adenosquamous carcinoma. Twenty patients with non-squamous cell carcinomas had epidermal growth factor receptor (EGFR) mutations, so the EML4-ALK fusion gene was found in 14.3% of EGFR wild type non-squamous cell carcinomas. Two tumors were variant 3 (3a+3b with 3b predominant) and had strong staining (3+) for ALK by IHC stains. One tumor was variant 1 and had moderate staining (2+) for ALK. None of the ALK wild type tumors had strong staining for ALK. When compared with other clinical and molecular features, only the IHC stain for ALK was significantly correlated with the EML4-ALK fusion gene (p = 0.0002). CONCLUSIONS ALK overexpression detected by IHC study could be a promising detection method for the EML4-ALK fusion gene and is worth further confirmation with more samples.

Overexpression of MutL homolog 1 and MutS homolog 2 proteins have reversed prognostic implications for stage I–II colon cancer patients

Background The outcome of colon cancer patients without lymph node metastasis is heterogeneous. Searching for new prognostic markers is warranted. Methods One hundred twenty stage I–II colon cancer patients who received complete surgical excision during 1995–2004 were selected for this biomarker study. Immunohistochemical method was used to assess p53, epidermal growth factor receptor, MLH1, and MSH2 status. KRAS mutation was examined by direct sequencing. Results Thirty three patients (27.5%) developed metachronous metastasis during follow up. By multivariate analysis, only female gender (p = 0.03), high serum carcinoembryonic antigen (CEA) level (≧5 ng/ml) (p = 0.04), and MLH1 overexpression (p = 0.003) were associated with the metastasis group. The 5-year-survival rate were also significantly lower for female gender (71.7% versus 88.9%, p = 0.025), high CEA level (64.9% versus 92.4%, p < 0.001), and MLH1 overexpression (77.5% versus 94.4%, p = 0.039). In contrast, MSH2 overexpression was associated with better survival, 95.1% versus 75.5% (p = 0.024). Conclusions The reversed prognostic implications in the overexpression of MLH1 and MSH2 for stage I–II colon cancer patients is a novel finding and worthy of further confirmation.

Dysregulation of the TGFBI gene is involved in the oncogenic activity of the nonsense mutation of hepatitis B virus surface gene sW182

The nonsense mutations of the hepatitis B virus (HBV) surface (S) gene have been reported to have oncogenic potential. We have previously identified several transforming nonsense mutations of the HBV S gene from hepatocarcinoma (HCC) patients. Among them, the sW182* mutant (the stop codon for tryptophan 182) showed the most potent oncogenicity in a mouse xenograft model using stably transfected mouse fibroblast cells. This study is aimed at understanding the molecular mechanisms leading to the oncogenic activity of the sW182* mutant. A gene expression microarray in combination with gene set enrichment analysis (GSEA) revealed differentially expressed gene sets in the sW182* cells, including those related to cell-cycle regulation, deoxyribonucleic acid repair, and genome instability. Of the differentially expressed genes, the transforming growth factor-β-induced (TGFBI) gene was further validated to be dysregulated in the sW182* cells. This dysregulation was accompanied by hypermethylation of the TGFBI promoter. The level of cyclin D1, a negatively regulated TGFBI target, was highly elevated in the sW182* mutant cells, which is consistent with the potent oncogenicity. Furthermore, frequent abnormal mitosis and multinucleation were observed in the mutant cells. Exogenous expression of TGFBI alleviated the oncogenic activity of the sW182* cells. In human HBV-related HCC cancerous tissue, expression of TGFBI was downregulated in 25 of the 55 (45%) patients examined, suggesting that TGFBI dysregulation could occur in HBV-related HCC development in some cases. These results suggest that dysregulation of TGFBI is involved in the oncogenic activity of the sW182* mutant of the hepatitis B virus S gene.