A Sensitive Method for Detecting EGFR Mutations in Non-small Cell Lung Cancer Samples with Few Tumor Cells

Molina-Vila, Miguel Ángel; Bertrán-Alamillo, Jordi; Reguart, Noemı́; Tarón, Miquel; Castellà, Eva; Llatjós, Mariona; Costa, Carlota; Mayo, Clara; Pradas, Anna; Queralt, Cristina; Botia, Mónica; Perez-Cano, M.; Carrasco, Esther; Tomàs, M.; Mate, José Luís; Morán, Teresa; Rosell, Rafael

doi:10.1097/jto.0b013e318189f579

Cited by 97 publications

(90 citation statements)

References 35 publications

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“…© 2011 American Association for Cancer clincancerres.aacrjournals.org Downloaded from (10,11,36) However, in the present study, we have found the T790M mutation in 35% of 129 patients at baseline, which is similar to the 38% previously reported in 26 patients (15) and the 40% observed in plasma of 10 patients with activating EGFR mutations in a phase I-II study of advanced NSCLC patients treated with docetaxel plus intercalated erlotinib. (37) The lower frequency of pretreatment T790M mutations (2.7%) found in Asian patients (5) could be due to multiple factors, including the use of a different assay, which may be less sensitive at detecting low levels of mutations.…”

Section: Discussionsupporting

confidence: 56%

Pretreatment EGFR T790M Mutation and BRCA1 mRNA Expression in Erlotinib-Treated Advanced Non–Small-Cell Lung Cancer Patients with EGFR Mutations

Rosell

Molina

Costa

et al. 2011

Clinical Cancer Research

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273

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Purpose: Advanced non-small-cell lung cancer (NSCLC) patients harboring epidermal growth factor receptor (EGFR) mutations (deletion in exon 19 or L858R) show an impressive progression-free survival of 14 months when treated with erlotinib. However, the presence of EGFR mutations can only imperfectly predict outcome. We hypothesized that progression-free survival could be influenced both by the pretreatment EGFR T790M mutation and by components of DNA repair pathways.Experimental Design: We assessed the T790M mutation in pretreatment diagnostic specimens from 129 erlotinib-treated advanced NSCLC patients with EGFR mutations. The expression of eight genes and two proteins involved in DNA repair and four receptor tyrosine kinases was also examined.Results: The EGFR T790M mutation was observed in 45 of 129 patients (35%). Progression-free survival was 12 months in patients with and 18 months in patients without the T790M mutation (P ¼ 0.05). Progression-free survival was 27 months in patients with low BRCA1 mRNA levels, 18 months in those with intermediate levels, and 10 months in those with high levels (P ¼ 0.02). In the multivariate analysis, the presence of the T790M mutation (HR, 4.35; P ¼ 0.001), intermediate BRCA1 levels (HR, 8.19; P < 0.0001), and high BRCA1 levels (HR, 8.46; P < 0.0001) emerged as markers of shorter progression-free survival.Conclusions: Low BRCA1 levels neutralized the negative effect of the T790M mutation and were associated with longer progression-free survival to erlotinib. We advocate baseline assessment of the T790M mutation and BRCA1 expression to predict outcome and provide alternative individualized treatment to patients based on T790M mutations and BRCA1 expression.

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Section: Discussionsupporting

confidence: 56%

Pretreatment EGFR T790M Mutation and BRCA1 mRNA Expression in Erlotinib-Treated Advanced Non–Small-Cell Lung Cancer Patients with EGFR Mutations

Rosell

Molina

Costa

et al. 2011

Clinical Cancer Research

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273

232

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“…EGFR status was assessed by microdissection of tumour cells from paraffin-embedded or fresh specimens, and could be determined in samples containing as few as eight tumour cells [23]. Briefly, it involves four steps.…”

Section: Laboratory Methodsmentioning

confidence: 99%

“…The EGFR mutation screening methodology followed in the present study has been described previously [4,23]. EGFR status was assessed by microdissection of tumour cells from paraffin-embedded or fresh specimens, and could be determined in samples containing as few as eight tumour cells [23].…”

Section: Laboratory Methodsmentioning

confidence: 99%

Endobronchial ultrasound-guided transbronchial needle aspiration for identifying EGFR mutations

García-Olivé¹,

Monsó²,

Andreo³

et al. 2009

European Respiratory Journal

121

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The presence of somatic mutations of the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene in patients with advanced nonsmall cell lung cancer (NSCLC) correlates with a good response to tyrosine kinase inhibitors. The usefulness of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) for the detection of EGFR mutations in cells recovered from malignant mediastinal nodes in patients with NSCLC was assessed.All patients with lung adenocarcinoma or unspecified NSCLC referred for staging with EBUS-TBNA were included. Nodes with a short-axis diameter of .5 mm were sampled, and genomic DNA from metastatic tumour cells was obtained for analysis of exons 19 and 21. The impact of sampling on management was assessed.EGFR gene analysis of the EBUS-TBNA sample was feasible in 26 (72.2%) out of the 36 patients with lymph node metastasis. Somatic mutations of the EGFR gene were detected in tissue obtained through EBUS-TBNA in two (10%) out of 20 patients with metastasic lung adenocarcinoma.Malignant tissue samples obtained by EBUS-TBNA from patients with nodal metastasis of NSCLC are suitable for the detection of EGFR mutations in most cases, and this technique demonstrates mutated neoplastic cells in a tenth of patients with adenocarcinoma.

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“…However, the consensus for EGFR mutation testing in NSCLC, including which case to select and when to test, is not well established, particularly with respect to technical methods of EGFR mutation detection. A variety of methods, including direct sequencing, amplification refractory mutation system, length analysis, denaturing high-performance liquid chromatography and peptide nucleic acid-mediated real-time polymerase chain reaction (PCR) clamping (5)(6)(7)(8)(9)(10)(11), have been proposed as suitable methods for the detection of EGFR mutations. Among these various assays, direct sequencing of amplified DNA products is widely used and is considered the standard method.…”

Section: Introductionmentioning

confidence: 99%

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

Kim

et al. 2012

Oncology Letters

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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.

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A Sensitive Method for Detecting EGFR Mutations in Non-small Cell Lung Cancer Samples with Few Tumor Cells

Abstract: The method presented here eliminates the need for DNA purification and allows for detection of EGFR mutations in samples containing as few as eight cancer cells.

Cited by 97 publications

References 35 publications

Pretreatment EGFR T790M Mutation and BRCA1 mRNA Expression in Erlotinib-Treated Advanced Non–Small-Cell Lung Cancer Patients with EGFR Mutations

Pretreatment EGFR T790M Mutation and BRCA1 mRNA Expression in Erlotinib-Treated Advanced Non–Small-Cell Lung Cancer Patients with EGFR Mutations

Endobronchial ultrasound-guided transbronchial needle aspiration for identifying EGFR mutations

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

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