The presence of EGFR mutations is correlated with a positive therapeutic response to tyrosine kinase inhibitors; therefore, the accurate detection of EGFR mutations is crucial when deciding appropriate therapeutic strategies. Recently, the rapid and sensitive assay smart amplification process version 2 (SmartAmp2) was developed. However, this method can only detect one type of mutation in EGFR exon 19; therefore, we applied the PNA technology to the SmartAmp2 assay to develop PNA-clamp SmartAmp2 for the detection of many types of deletions in EGFR exon 19, in a single reaction. This new assay was evaluated using 172 clinical samples. Thirty-nine (22.7%) samples were found to have deletions by PNA-clamp SmartAmp2; whereas 30 (17.4%) and 38 (22.1%) tumors were found to have deletions by direct sequencing and PNA-enriched sequencing, respectively. Three cases, in which we detected mutations with PNA-clamp SmartAmp2, but not with direct sequencing, were treated with gefitinib, and all cases showed a partial therapeutic response. Using clinical samples, we demonstrated that PNA-clamp SmartAmp2 can detect various types of mutations in EGFR exon 19 in a relatively short time and with high sensitivity. This method detected small amounts of mutant DNA and identified patients for whom clinical information was previously unavailable from other tests. This test may contribute to the administration of efficient therapeutic strategies.
Last. Lucie Benesova(Center for Applied Genomics)H-Index: 15
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There is a growing interest in evaluating molecular markers as predictors of response to new generation of targeted cancer therapies. One of such areas is biological therapy targeting epidermal growth factor receptor gene (EGFR) in lung cancer. The testing of tumor tissue is focused on specific EGFR mutations and EGFR gene amplification, since tumors exhibiting positivity of either of the two marker types are highly sensitive towards the treatment. Although traditional methods of DNA sequencing ...
Last. Jan-Gowth Chang(KMU: Kaohsiung Medical University)H-Index: 49
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Epidermal growth factor receptor (EGFR) kinase domain mutations hyperactivate the kinase and confer kinase addiction of the non-small-cell lung cancer (NSCLC) tumor cells. Almost all of these mutations are located within exons 18-21. The -216 single nucleotide polymorphism in the promoter region is associated with increased EGFR production. We present a method for detecting these common mutations in 81 cases of NSCLC. The protocol is based on the multiplex amplification of promoter region and ex...
Recent evidence indicates that the presence of epidermal growth factor receptor (EGFR) or KRAS mutations in non-small cell lung cancer (NSCLC) can predict the response of the tumor to gefinitib. However, it is difficult to detect these mutations using formalin-fixed, paraffin-embedded (FFPE) tissues because the fixation process and aging can damage the DNA. In this study, we describe our work in adapting the Smart Amplification Process version 2 (SmartAmp2) to detect EGFR or KRAS mutations in DN...
KRAS is an oncogene that can be activated by mutations. Patients with non-small cell lung cancer who have KRAS mutations do not respond to tyrosine kinase inhibitors; therefore, accurate detection of KRAS mutations is important for deciding therapeutic strategies. Although sequencing-related techniques have been frequently used, they are usually too complex, have low sensitivity, and are time-consuming for routine screening in clinical situations. We evaluated peptide nucleic acid (PNA)-clamp sm...
Purpose: Activating mutations within the tyrosine kinase domain of epidermal growth factor receptor (EGFR) are found in approximately 10% to 20% of non–small-cell lung cancer (NSCLC) patients and are associated with response to EGFR inhibitors. The most common NSCLC-associated EGFR mutations are deletions in exon 19 and L858R mutation in exon 21, together accounting for 90% of EGFR mutations. To develop a simple, sensitive, and reliable clinical assay for the identification of EGFR mutations in ...
Background The use of tyrosine kinase inhibitors to target the epidermal growth factor receptor gene (EGFR) in patients with non–small-cell lung cancer is effective but limited by the emergence of drug-resistance mutations. Molecular characterization of circulating tumor cells may provide a strategy for noninvasive serial monitoring of tumor genotypes during treatment. Methods We captured highly purified circulating tumor cells from the blood of patients with non–small-cell lung cancer using a m...
Introduction In this study, we examined whether epidermal growth factor receptor ( EGFR ) mutations were detectable using a polymerase chain reaction-based assay and wash fluid of computed tomography (CT)-guided lung biopsy needles. Methods DNA was extracted from wash fluid of CT-guided biopsy needles of 53 lung tumors (as diagnosed according to the results of the CT-guided biopsies). EGFR mutations, specifically exon19 deletions and exon21 L858R mutations, were examined using a mutant-enriched ...
Purpose: A positive response to gefitinib in non–small cell lung cancer (NSCLC) has been correlated to mutations in epidermal growth factor receptor (EGFR) gene. Previous reports have been based mainly on diagnostic screening by sequencing. However, sequencing is a time-consuming and complicated procedure, not suitable for routine clinical use. Experimental Design: We have developed rapid, simple, and sensitive mutation detection assays based on the SMart Amplification Process (SMAP) and applied...
We developed a rapid single nucleotide polymorphism (SNP) detection system named smart amplification process version 2 (SMAP 2). Because DNA amplification only occurred with a perfect primer match, amplification alone was sufficient to identify the target allele. To achieve the requisite fidelity to support this claim, we used two new and complementary approaches to suppress exponential background DNA amplification that resulted from mispriming events. SMAP 2 is isothermal and achieved SNP detec...
BACKGROUND. Somatic mutations of the epidermal growth factor receptor (EGFR) gene in nonsmall-cell lung cancer (NSCLC) may predict responsiveness to tyrosine kinase inhibitors. These mutations are commonly identified using DNA sequencing methods. Although considered the gold standard, this approach is time-consuming. In addition, this approach requires large diagnostic specimens and a high ratio of tumor-to-normal-tissue DNA for optimal results. The use of denaturing high-performance liquid chro...
// Jian Su 1 , Wenzhao Zhong 2 , Xuchao Zhang 1 , Ying Huang 1 , Honghong Yan 1 , Jinji Yang 2 , Zhongyi Dong 2 , Zhi Xie 1 , Qing Zhou 2 , Xiaosui Huang 1 , Danxia Lu 1 , Wenqing Yan 1 and Yi-Long Wu 2 1 Guangdong Lung Cancer Institute, Guangdong Provincial Key Laboratory of Translational Medicine in Lung Cancer, Medical Research Center of Guangdong General Hospital & Guangdong Academy of Medical Sciences, Guangzhou 510080, China 2 Guangdong Lung Cancer Institute, Guangdong General Hospital & G...
#1Xian-Ying Li(Northwest University (United States))H-Index: 1
#2Chao Zhang(Northwest University (United States))H-Index: 10
Last. Yali Cui(Northwest University (United States))H-Index: 20
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Epidermal growth factor receptor (EGFR) mutations predict better outcomes with EGFR tyrosine kinase inhibitors in patients with non-small cell lung cancer (NSCLC). Most common activating mutations include in-frame deletion in exon 19 and L858R substitution in exon 21, which account for >90% of all EGFR mutations in NSCLC. In this study, a PCR-GoldMag lateral flow assay (PCR-GoldMag LFA) was developed for the visual detection of delE746-A750 and L858R of EGFR mutations. Forty formalin-fixed paraf...
Abstract EGFR exon 19 deletion is an important indicator for tyrosine kinase inhibitor treatment in non-small cell lung cancer. However, detection of exon 19 deletions faces a challenge: there are more than 30 types of mutations reported at the hotspot. Moreover, considering the application in body fluid samples, assays with high sensitivity and specificity are necessary for the detection of rare mutant alleles. Here, we describe a single tube reaction which could detect at least 29 types of exo...
Analyzing EGFR mutations and detecting ALK gene fusion are indispensable when planning to treat pulmonary adenocarcinoma. Malignant pleural effusion (MPE) is a devastating complication of lung cancer and sometimes the only source for mutation analysis. The percentage of tumor cells in the pleural effusion may be low; therefore, mutant enrichment is required for a successful analysis. The EGFR mutation status in MPE was determined using three methods: (1) PCR sequencing of genomic DNA (direct seq...
The MDM2 protein plays an important role in the regulation of cell proliferation and apoptosis via ubiquitination and proteasome-mediated degradation of p53. The genetic polymorphism rs2279744 (c.309T>G) of the MDM2 gene is reportedly associated with susceptibility and/or prognosis in various cancers. In this study, we investigated the risk factors for worse survival in patients with lung adenocarcinoma (AC). We examined the association between c.309T>G and the prognosis of lung cancer by retros...
Molecular-targeted therapy has not been established in non-adenocarcinoma lung cancer (non-AdLC), as no targets that affect the clinical efficacy of molecular-targeted drugs have yet been identified. In this study, we investigated the frequency of genetic variations in discoidin domain receptor 2 (DDR2), v-raf murine sarcoma viral oncogene homolog B1 (BRAF), epidermal growth factor receptor (EGFR) and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) in non-AdLC patients, in order to e...
Epidermal growth factor receptor (EGFR) mutations are generally detected in DNA from samples of tumor tissue obtained during biopsy or surgcial resection, usually in the form of formalin-fixed paraffin-embedded (FFPE) diagnostic blocks, using gene sequencing.
Selection of lung cancer patients for therapy with tyrosine kinase inhibitors directed at EGFR requires the identification of specific EGFR mutations. In most patients with advanced, inoperable lung carcinoma limited tumor samples often represent the only material available for both histologic typing and molecular analysis. We defined a next generation sequencing protocol targeted to EGFR exons 18-21 suitable for the routine diagnosis of such clinical samples. The protocol was validated in an un...
Anti-epidermal growth factor receptor (EGFR) therapy has been tried in triple negative breast cancer (TNBC) patients without evaluation of molecular and clinical predictors in several randomized clinical studies. Only fewer than 20% of metastatic TNBCs showed response to anti-EGFR therapy. In order to increase the overall response rate, first step would be to classify TNBC into good or poor responders according to oncogenic mutation profiles. This study provides the molecular characteristics of ...
