Non-isotopic silver-stained SSCP is more sensitive than automated direct sequencing for the detection of PTEN mutations in a mixture of DNA extracted from normal and tumor cells

Fan, Xing; Furnari, Frank B.; Cavenee, Webster K.; Castresana, Javier S.

doi:10.3892/ijo.18.5.1023

Cited by 31 publications

(27 citation statements)

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“…Direct sequencing seems unable to provide satisfactory results for detection of EGFR mutations in samples containing a mixture of mutated and wild-type DNA. Although direct sequencing has generally been used to detect EGFR mutations, detection by direct sequencing requires at least 30% of the DNA in the sample to be mutated (Bosari et al, 1995;Fan et al, 2001). Small amounts and low percentages of mutated DNA in serum can be missed by direct sequencings.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of epidermal growth factor receptor mutation status in serum DNA as a predictor of response to gefitinib (IRESSA)

et al. 2007

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The aim of this study was to evaluate the usefulness of EGFR mutation status in serum DNA as a means of predicting a benefit from gefitinib (IRESSA) therapy in Japanese patients with non-small cell lung cancer (NSCLC). We obtained pairs of tumour and serum samples from 42 patients treated with gefitinib. EGFR mutation status was determined by a direct sequencing method and by Scorpion Amplification Refractory Mutation System (ARMS) technology. EGFR mutations were detected in the tumour samples of eight patients and in the serum samples of seven patients. EGFR mutation status in the tumours and serum samples was consistent in 39 (92.9%) of the 42 pairs. EGFR mutations were strong correlations between both EGFR mutation status in the tumour samples and serum samples and objective response to gefitinib (Po0.001). Median progression-free survival time was significantly longer in the patients with EGFR mutations than in the patients without EGFR mutations (194 vs 55 days, P ¼ 0.016, in tumour samples; 174 vs 58 days, P ¼ 0.044, in serum samples). The results suggest that it is feasible to use serum DNA to detect EGFR mutation, and that it's potential as a predictor of response to, and survival on gefitinib is worthy of further evaluation.

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Section: Discussionmentioning

confidence: 99%

Evaluation of epidermal growth factor receptor mutation status in serum DNA as a predictor of response to gefitinib (IRESSA)

et al. 2007

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“…Second, direct sequencing may be not able to provide satisfactory results for detection of EGFR mutations in mixed samples of mutated and wild DNA. Although direct sequencing has generally been used to detect EGFR mutations in previous studies, detection of a mutation by this method requires at least 30% of the mutated DNA in a sample (Bosari et al, 1995;Fan et al, 2001). Lung cancers are very heterogeneous, and as normal cells, such as inflammatory cells or mesothelial cells, are contained in the pleural effusion fluid of lung cancer patients, in addition to tumour cells, a small amount of mutated DNA in pleural effusion fluid can be missed by direct sequencing.…”

Section: Discussionmentioning

confidence: 99%

EGFR mutation status in tumour-derived DNA from pleural effusion fluid is a practical basis for predicting the response to gefitinib

et al. 2006

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Epidermal growth factor receptor (EGFR) mutations are strong determinants of tumour response to EGFR tyrosine kinase inhibitors in non-small-cell lung cancer (NSCLC). Pleural effusion is a common complication of lung cancer. In this study, we assessed the feasibility of detection of EGFR mutations in samples of pleural effusion fluid. We obtained 43 samples, which was the cell-free supernatant of pleural fluid, from Japanese NSCLC patients, and examined them for EGFR mutations. The epidermal growth factor receptor mutation status was determined by a direct sequencing method (exons 18 -21 in EGFR). EGFR mutations were detected in 11 cases (E746_A750del in seven cases, E746_T751del insA in one case, L747_T751del in one case, and L858R in two cases). The EGFR mutations were observed more frequently in women and non-smokers. A comparison between the EGFR mutant status and the response to gefitinib in the 27 patients who received gefitinib revealed that all seven patients with partial response and one of the seven patients with stable disease had an EGFR mutation. No EGFR mutations were detected in the patients with progressive disease. The results suggest that DNA in pleural effusion fluid can be used to detect EGFR mutations and that the EGFR mutation status may be useful as a predictor of the response to gefitinib.

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“…These discrepancies may be due to the methodology used in studies. As LH-MSA, which is a modified heteroduplex technique, is able to detect mutations in samples containing as little as 5% mutated DNA, 20 whereas direct sequencing has been reported to require at least 30% of mutated DNA in the sample, 22 LH-MSA is considered to be more sensitive than direct sequencing 20 and to have an advantage especially in analyzing tissues such as atypical adenomatous hyperplasias, which often contains not so many tumor cells as compared with the normal counterparts. As atypical adenomatous hyperplasias and nonmucinous bronchioloalveolar carcinomas probably represent continuum of progression of alveolar intraepithelial neoplasia, differential diagnosis between atypical adenomatous hyperplasias and small-sized nonmucinous bronchioloalveolar carcinomas is often difficult.…”

Section: Discussionmentioning

confidence: 99%

Epidermal growth factor receptor gene mutations in atypical adenomatous hyperplasias of the lung

et al. 2007

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Activating epidermal growth factor receptor (EGFR) gene mutations are frequently detected in lung adenocarcinomas, especially adenocarcinomas with a nonmucinous bronchioloalveolar carcinoma component. EGFR-mutated lung adenocarcinomas respond well to EGFR tyrosine kinase inhibitors. We previously found that most (88%) pure nonmucinous bronchioloalveolar carcinomas (adenocarcinoma in situ) already harbor EGFR mutations, indicating that the mutations are an early genetic event in the pathogenesis. We examined 54 atypical adenomatous hyperplasias, precursor lesions of lung adenocarcinomas, obtained from 28 Japanese patients for the hotspot mutations of EGFR exons 19 and 21 and K-ras codon 12. EGFR mutations were observed in 17 of the 54 (32%) atypical adenomatous hyperplasias examined: Ten and seven atypical adenomatous hyperplasias had deletion mutations at exon 19 or point mutations (L858R) at exon 21, respectively. We did not observe apparent histological differences between atypical adenomatous hyperplasias with and without EGFR mutations. K-ras mutation (G12S) was detected in only one atypical adenomatous hyperplasia. As EGFR mutational frequency of atypical adenomatous hyperplasias was much lower than that of nonmucinous bronchioloalveolar carcinomas, we surmise that EGFR-mutated atypical adenomatous hyperplasias, but not atypical adenomatous hyperplasias with wild-type EGFR, are likely to progress to nonmucinous bronchioloalveolar carcinomas.

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Non-isotopic silver-stained SSCP is more sensitive than automated direct sequencing for the detection of PTEN mutations in a mixture of DNA extracted from normal and tumor cells

Cited by 31 publications

References 0 publications

Evaluation of epidermal growth factor receptor mutation status in serum DNA as a predictor of response to gefitinib (IRESSA)

Evaluation of epidermal growth factor receptor mutation status in serum DNA as a predictor of response to gefitinib (IRESSA)

EGFR mutation status in tumour-derived DNA from pleural effusion fluid is a practical basis for predicting the response to gefitinib

Epidermal growth factor receptor gene mutations in atypical adenomatous hyperplasias of the lung

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