Clinical investigation of EGFR mutation detection by pyrosequencing in lung cancer patients

Abstract

Abstract. Direct sequencing is the standard method for the detection of epidermal growth factor receptor (EGFR) mutations in lung cancer, however, its relatively low sensitivity limits its clinical use. Pyrosequencing is a bioluminometric, real-time non-electrophoretic DNA sequencing technique with a number of advantages compared with direct sequencing, including higher sensitivity, speed, automation and costeffectiveness. Clinical specimens from 202 lung cancer patients were analyzed for EGFR mutations in exons 18, 19, 20 and 21 using the pyrosequencing method following genomic DNA extraction from paraffin-embedded tissue specimens. All clinical data and tumor specimens were obtained from the Konkuk University Hospital (Korea) between July 2006 and December 2008. The results and clinical responses to EGFR-tyrosine kinase inhibitors (TKIs) were compared. Overall, EGFR mutation-positive rate was 26.7% (54/202). Activating EGFR mutations were observed more frequently in female (52.1 vs. 13.0%), non-smoking (47.8 vs. 15.8%) and adenocarcinoma (35.2 vs. 5.2%) patients. However, significant numbers of EGFR mutation-positive patients were identified as male, former or current smokers and non-adenocarcinoma patients. The combinations of favorable clinicopathological factors, including female, non-smoking and adenocarcinoma, were not identified to significantly increase the positive EGFR mutation rate (female, 52.1%; female and non-smoker, 52.6%; female, non-smoker and adenocarcinoma, 51.9%). The present findings indicate that EGFR mutation analysis is a highly useful method for the prediction of response to EGFR-TKI and the use of favorable clinicopathological factors to perform this analysis is not suitable. Exon 19 deletion was the most common mutation (63.6%) and exon 21 L858R substitution was measured at 32.7%. The exon 20 T790M mutation was identified in 1 patient prior to EGFR-TKI treatment. EGFR mutation status is associated with response to EGFR-TKI and the overall response rate in patients who have the activating EGFR mutation was 82.4 vs. 5.9% in patients with a wild-type EGFR. The present study demonstrates that EGFR mutations analyzed by the pyrosequencing method are well correlated with clinicopathological parameters and that this method may be useful in the clinical practice.

A sensitive and practical method to detect the T790M mutation in the epidermal growth factor receptor

“…Based on the mimic human genomic DNA panel that consisted of serially diluted plasmids containing T790M mutation, the present study results revealed that the ACB-ARMS PCR assay can detect 0.01% mutant alleles in total genomic DNA, which corresponds to <10 copies. This method is comparable to PNA-clamping PCR (14,15) or the BEAMing assay (20), and is more sensitive than any other documented method (Table III) (2)(3)(4)(5)(9)(10)(11)(12)(13)(14)(15)(17)(18)(19)22,23). Regarding specificity, the purine/purine and pyrimidine/pyrimidine mismatches are considerably more selective than the purine/pyrimidine mismatches (24).…”

The role of p300 in the tumor progression of oral squamous cell carcinoma

“…The tumor areas were separated from stroma under a microscope using sterilized scalpels. DNA extraction buffer solution consisting of 50 mM Tris buffer (pH 8.3), 1 mM EDTA (pH 8.0), 5% Tween‐20, 200 μg/ml proteinase K, and 10% resin was added as previously described . After incubation at 56°C for 90 min, tubes containing tissue and DNA extraction buffer solution were heated to 100°C for 10 min, followed by centrifugation at 14 000 g for 10 min at 4°C.…”

Frequency of EGFR Mutations in 907 Lung Adenocarcioma Patients of Indian Ethnicity

“…It occurs at the rate of 10–15% in North Americans and Europeans [3], [4], [10], 19% in African-Americans [11], 26–30% in various East Asian series including Chinese [12], Koreans [13], Japanese [14], and as compiled in Table 1 (adapted from [15]). Frequency of EGFR mutations in India however remains sparsely explored.…”