1249P - Detection of EGFR T790M resistance mutation: real-time allele-specific PCR versus Sanger sequencing

Date 08 October 2016
Event ESMO 2016 Congress
Session Poster Display
Topics Non-Small Cell Lung Cancer
Presenter Shirlyn Hor
Citation Annals of Oncology (2016) 27 (6): 416-454. 10.1093/annonc/mdw383
Authors S.Y. Hor, K.S. Chan, E.X. Chen, M. Goh, L.L.E. Oon
  • Pathology, Singapore General Hospital, 169856 - Singapore/SG



Lung cancer is the most common cause of death from cancer worldwide, and 85-90% of lung cancers are non-small cell lung cancer (NSCLC). Presence of driver mutations in epidermal growth cell receptor (EGFR) gene in a subset of NSCLC has led to the use of tyrosine kinase inhibitors (TKI) that inhibit the EGFR signalling pathway. Tumours which harbour certain EGFR mutations may exhibit initial response to EGFR-TKIs, but many will develop resistance mutations post-treatment, commonly at amino acid position 790 (T790M) of exon 20. Newer EGFR inhibitors with activity against T790M-mutated NSCLC have recently been made available. Accurate detection of T790M in patients whose disease progressed on initial TKI therapy is thus vital for subsequent management. Here, we evaluated the performance of Sanger sequencing in detecting T790M mutation against a real-time allele-specific PCR.


Ninety-six FFPE samples sent to our laboratory for T790M mutation detection by cobas® EGFR Mutation test (Roche), between July 2014 and March 2016 were included in this study. Archived extracts were used for Sanger sequencing of EGFR exon 20 using an in-house developed protocol.


T790M was detected in 49.5% (47/95) and 47.9% (46/96) of the samples by the cobas® assay and Sanger sequencing respectively. Taking cobas® assay as the gold standard for 95 samples with valid real-time PCR results, Sanger sequencing yielded a sensitivity of 95.7% (45/47) and specificity of 100% (48/48). Of the 3 discordant results, cobas® assay detected T790M in 2 samples (both with tumour contents


Sanger sequencing was generally comparable to, but slightly less sensitive than real-time PCR in detecting T790M. Sanger sequencing may be useful in the event of inconclusive results by the real-time assay. Otherwise, for post-treatment T790M mutation testing, where initial driver mutations had already been identified, Sanger sequencing, which requires samples with higher tumour content, offers little advantage over real-time PCR.

Clinical trial identification

Legal entity responsible for the study

Singapore General Hospital


Singapore General Hospital


All authors have declared no conflicts of interest.