Bing-Ching Ho

Pretreatment Epidermal Growth Factor Receptor (EGFR) T790M Mutation Predicts Shorter EGFR Tyrosine Kinase Inhibitor Response Duration in Patients With Non–Small-Cell Lung Cancer

PURPOSE Patients with non-small-cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR)-activating mutations have excellent response to EGFR tyrosine kinase inhibitors (TKIs), but T790M mutation accounts for most TKI drug resistance. This study used highly sensitive methods to detect T790M before and after TKI therapy and investigated the association of T790M and its mutation frequencies with clinical outcome. PATIENTS AND METHODS Direct sequencing, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and next-generation sequencing (NGS) were used to assess T790M in the following two cohorts of patients with NSCLC: TKI-naive patients (n = 107) and TKI-treated patients (n = 85). Results were correlated with TKI treatment response and survival. RESULTS MALDI-TOF MS was highly sensitive in detecting and quantifying the frequency of EGFR-activating mutations and T790M (detection limits, 0.4% to 2.2%). MALDI-TOF MS identified more T790M than direct sequencing in TKI-naive patients with NSCLC (27 of 107 patients, 25.2% v three of 107 patients, 2.8%, respectively; P < .001) and in TKI-treated patients (before TKI: 23 of 73 patients, 31.5% v two of 73 patients, 2.7%, respectively; P < .001; and after TKI: 10 of 12 patients, 83.3% v four of 12 patients, 33.3%, respectively; P = .0143). The EGFR mutations and their frequencies were confirmed by NGS. T790M was an independent predictor of decreased progression-free survival (PFS) in patients with NSCLC who received TKI treatment (P < .05, multivariate Cox regression). CONCLUSION T790M may not be a rare event before or after TKI therapy in patients with NSCLC with EGFR-activating mutations. The pretreatment T790M mutation was associated with shorter PFS with EGFR TKI therapy in patients with NSCLC.

Target identification of microRNAs expressed highly in human embryonic stem cells

MicroRNAs (miRNAs) are noncoding RNAs of approximately 22 nucleotides in length that negatively regulate the post‐transcriptional expression by translational repression and/or destabilization of protein‐coding mRNAs. The impact of miRNAs on protein output was recently shown that although some targets were repressed without detectable changes in mRNA levels, those translationally repressed by more than a third also displayed detectable mRNA destabilization, and, for the more highly repressed targets, mRNA destabilization usually comprised the major component of repression. Thus, comparative profilings of miRNAs and mRNAs from the same samples of different cell types may identify the putative targets of miRNAs. In this investigation, both miRNA and mRNA profiles from the undifferentiated human embryonic stem cell line hES‐T3 (T3ES), hES‐T3 derived embryoid bodies (T3EB), and hES‐T3 differentiated fibroblast‐like cells (T3DF) were compared, and 58 genes were found to be targets of four hES cell‐specific miRNAs miR‐302d, miR‐372, miR‐200c and/or miR‐367 by inverse expression levels (highly negative correlation) of miRNAs to their target mRNAs. Approximately half of these 58 targets are involved in gene transcription. Three common target genes TRPS1, KLF13 and MBNL2 of three highly expressed miRNAs miR‐302d, miR‐372, and miR‐200c were identified, and the target sites of both miR‐302d and miR‐372 in the 3′UTR of TRPS1, KLF13, and MBNL2 genes were confirmed by the luciferase assay. The highly expressed mRNAs and miRNA target mRNAs involved in KEGG pathways among T3ES, T3EB, and T3DF cells were also compared, and the expression levels of target mRNAs predicted by abundantly expressed miRNAs were found to be three‐ to sixfold lower than those of non‐target mRNAs involved in the same signaling pathways. J. Cell. Biochem. 106: 1020–1030, 2009.

Inhibition of miR-146a prevents enterovirus-induced death by restoring the production of type I interferon

There are no antivirals or vaccines available to treat Enterovirus 71 (EV71) infections. Although the type I interferon response, elicited upon virus infection, is critical to establishing host antiviral innate immunity, EV71 fails to induce this response efficiently. Here we provide new insights into potential anti-EV71 therapy by showing that neutralization of EV71-induced miR-146a prevents death in mice by restarting the production of type I interferon. EV71 infection upregulates miR-146a, which targets IRAK1 and TRAF6 involved in TLR signalling and type I interferon production. We further identify AP1 as being responsible for the EV71-induced expression of miR-146a. Surprisingly, knocking out miR-146a or neutralizing virus-induced miR-146a by specific antagomiR restores expressions of IRAK1 and TRAF6, augments IFNβ production, inhibits viral propagation and improves survival in the mouse model. Our results suggest that enterovirus-induced miR-146a facilitates viral pathogenesis by suppressing IFN production and provide a clue to developing preventive and therapeutic strategies for enterovirus infections.

MicroRNA and Pathogenesis of Enterovirus Infection

There are no currently available specific antiviral therapies for non-polio Enterovirus infections. Although several vaccines have entered clinical trials, the efficacy requires further evaluation, particularly for cross-strain protective activity. Curing patients with viral infections is a public health problem due to antigen alterations and drug resistance caused by the high genomic mutation rate. To conquer these limits in the development of anti-Enterovirus treatments, a comprehensive understanding of the interactions between Enterovirus and host cells is urgently needed. MicroRNA (miRNA) constitutes the biggest family of gene regulators in mammalian cells and regulates almost a half of all human genes. The roles of miRNAs in Enterovirus pathogenesis have recently begun to be noted. In this review, we shed light on recent advances in the understanding of Enterovirus infection-modulated miRNAs. The impacts of altered host miRNAs on cellular processes, including immune escape, apoptosis, signal transduction, shutdown of host protein synthesis and viral replication, are discussed. Finally, miRNA-based medication provides a promising strategy for the development of antiviral therapy.

JAG1 Is Associated with Poor Survival through Inducing Metastasis in Lung Cancer

JAG1 is a Notch ligand that plays a critical role in multiple signaling pathways. However, the functionality of JAG1 in non-small cell lung cancer (NSCLC) has not been investigated thoroughly. By comparison of gene transcripted RNA profiles in the cell line pair with differential invasion ability, we identified JAG1 as a potential metastasis enhancer in lung cancer. Ectopic expression of JAG1 on lung cancer cells enhanced cell migration and invasion as well as metastasis in vitro and in vivo. Conversely, knockdown of JAG1 with siRNA in highly invasive cancer cells led to the reduction of migration and invasion. In clinical analysis, JAG1 mRNA expression was higher in tumors than in adjacent normal tissues in 14 of 20 patients with squamous cell carcinoma (SCC). SCC patients with higher JAG1 transcription had poor overall survival than those with low-transcripted JAG1. Microarray analysis indicated that the enforced JAG1 transcription was associated with an elevated HSPA2 RNA transcription, which played a role in promoting cancer cell migration and invasion. In conclusion, this is the first study that demonstrated that JAG1 might act as a potential prognostic marker and JAG1/HSPA2 axis mediates lung cancer malignancy at least partly.

SPANXA suppresses EMT by inhibiting c-JUN/SNAI2 signaling in lung adenocarcinoma

SPANXA (Sperm Protein Associated with the Nucleus on the X-chromosome, family members A1/A2) acts as a cancer-testis antigen expressed in normal testes, but dysregulated in various tumors. We found that SPANXA is highly expressed in low-invasive CL1-0 cells compared with isogenous high-invasive CL1-5 cells. SPANXA was preferably expressed in tumor tissues and associated with the prolonged survival of lung adenocarcinomas. SPANXA suppressed the invasion and metastasis of lung cancer cells in vitro and in vivo. By the expression microarray and pathway analysis, we found that the SPANXA-altered genes were enriched in the epithelial–mesenchymal transition (EMT) pathway. SPANXA reduced SNAI2 expression resulted in up-regulating E-cadherin. c-JUN acts as the positive-regulator of EMT. Silencing SPANXA increased c-JUN mRNA expression and blockage of c-JUN led to SNAI2 down-regulation. Our results clearly characterized SPANXA as an EMT inhibitor by suppressing c-JUN-SNAI2 axis in lung adenocarcinoma.

Molecular gene signature and prognosis of non-small cell lung cancer

The current staging system for non–small cell lung cancer (NSCLC) is inadequate for predicting outcome. Risk score, a linear combination of the values for the expression of each gene multiplied by a weighting value which was estimated from univariate Cox proportional hazard regression, can be useful. The aim of this study is to analyze survival-related genes with TaqMan Low-Density Array (TLDA) and risk score to explore gene-signature in lung cancer. A total of 96 NSCLC specimens were collected and randomly assigned to a training (n = 48) or a testing cohort (n = 48). A panel of 219 survival-associated genes from published studies were used to develop a 6-gene risk score. The risk score was used to classify patients into high or low-risk signature and survival analysis was performed. Cox models were used to evaluate independent prognostic factors. A 6-gene signature including ABCC4, ADRBK2, KLHL23, PDS5A, UHRF1 and ZNF551 was identified. The risk score in both training (HR = 3.14, 95% CI: 1.14–8.67, p = 0.03) and testing cohorts (HR = 5.42, 95% CI: 1.56–18.84, p = 0.01) was the independent prognostic factor. In merged public datasets including GSE50081, GSE30219, GSE31210, GSE19188, GSE37745, GSE3141 and GSE31908, the risk score (HR = 1.50, 95% CI: 1.25–1.80, p < 0.0001) was also the independent prognostic factor. The risk score generated from expression of a small number of genes did perform well in predicting overall survival and may be useful in routine clinical practice.

Implementation and Quality Control of Lung Cancer EGFR Genetic Testing by MALDI-TOF Mass Spectrometry in Taiwan Clinical Practice.

Molecular diagnostics in cancer pharmacogenomics is indispensable for making targeted therapy decisions especially in lung cancer. For routine clinical practice, the flexible testing platform and implemented quality system are important for failure rate and turnaround time (TAT) reduction. We established and validated the multiplex EGFR testing by MALDI-TOF MS according to ISO15189 regulation and CLIA recommendation in Taiwan. Totally 8,147 cases from Aug-2011 to Jul-2015 were assayed and statistical characteristics were reported. The intra-run precision of EGFR mutation frequency was CV 2.15% (L858R) and 2.77% (T790M); the inter-run precision was CV 3.50% (L858R) and 2.84% (T790M). Accuracy tests by consensus reference biomaterials showed 100% consistence with datasheet (public database). Both analytical sensitivity and specificity were 100% while taking Sanger sequencing as the gold-standard method for comparison. EGFR mutation frequency of peripheral blood mononuclear cell for reference range determination was 0.002 ± 0.016% (95% CI: 0.000–0.036) (L858R) and 0.292 ± 0.289% (95% CI: 0.000–0.871) (T790M). The average TAT was 4.5 working days and the failure rate was less than 0.1%. In conclusion, this study provides a comprehensive report of lung cancer EGFR mutation detection from platform establishment, method validation to clinical routine practice. It may be a reference model for molecular diagnostics in cancer pharmacogenomics.

Abstract 3075: miRNA-10a* enhances invasiveness via suppression of HDAC5 in lung cancer

Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. Metastasis, the spread process of cancer cells from a primary tumor to seed secondary tumors in distant sites, is one of the big challenges in cancer treatment today. For most patients, when the cancer is detected, metastasis has already occurred. The growing evidences implied that miRNAs play an important role in developmental lineage and different states of cancers as well as cancer invasion. In this study, we would like to establish a metastasis-based prognostic miRNA signature. First, 16 lung adenocarcinoma cells were divided into the high or low-invasive group. The miRNAs whose expression correlated with invasion were identified. Next, we investigated whether the expressions of identified miRNAs are correlated to patients’ survival. Quantitative RT-PCR results showed that the expression of miR-10a* is significantly correlated to the five-year survival in 98 lung adenocarcinoma patients. The inhibition of miR-10a* attenuated the invasion abilities of the highly invasive lung cancer cells. Moreover, we investigated the underlying regulation of miR-10a*. We found that miR-10a* negatively regulated Histone deacetylase 5 (HDAC5) was assayed by Western Blot, RT-PCR and luciferase reporter assay. These evidences suggested that HDAC5 is a direct target of miR-10a*. Besides, inhibition of HDAC5 promoted invasive ability. RNAs derived from two paired groups (pre-miR-10a* and pre-NC; sh-HDAC5 and sh-LacZ) were assayed by microarray and pathway analysis. The analysis result of MetaCore implied that miR-10a* trigger TGF-beta signalings. RT-PCR and Western Blot were utilized to validate the differentially altered expression of certain genes in miR-10a*-mediated pathways. The rescue effect of HDAC5 expression on miR-10a*-mediated phenotypic changes is also evaluated to elucidate the importance of HDAC5 in miR-10a*-mediated invasive ability. Our findings indicated that miR-10a* may play an oncogenic miRNA role in lung cancer. Citation Format: Pin-Yen Hsu, Bing-Ching Ho, Chiou-Ling Cheng, Hsuan-Yu Chen, Sung-Liang Yu, Guan-Tarn Huang. miRNA-10a* enhances invasiveness via suppression of HDAC5 in lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3075. doi:10.1158/1538-7445.AM2015-3075