Identification of somatic EGFR mutations has been integrated
Identification of somatic EGFR mutations has been integrated with the diagnosis of lung adenocarcinoma in therapeutic protocols . Commercial kits, including the therascreen® EGFR RGQ PCR kit, cobas® EGFR mutation test, and MassARRAY® genotyping, are commonly applied in clinical practice in most hospitals, and the results of such tests are used to guide physicians in treating patients. However, the sensitivity and accuracy of EGFR mutation detection using these methods should be considered and the results cautiously interpreted, as the misidentification or false-negative detection of EGFR mutations would provide incorrect information for the guidance of clinical management. Our previous research showed that RNA-based sequencing was more sensitive and accurate than direct genomic DNA sequencing or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis of genomic DNA . Therefore, the results of RNA-based sequencing for EGFR detection could be viewed as the reference data in this study. From our combined study of NTUH patients and those identified from the literature, eight patients who were previously screened using commercial kits, namely the therascreen® and MassARRAY®, showed misclassification of p.L747P or p.L747S mutations as 19DEL mutations or wild-type EGFR. The misidentification of p.L747P as a 19DEL mutation may stem from the fact that a 2-bp mutations in exon 19 (c.2239_2240delinsCC) or may result from the mispriming of oligonucleotides used for PCR [12,17]. Remarkably, two patients harboring p.L747 P were both detected as L747-P753 > Q of exon 19 in the EGFR gene, which was interpreted as 19DEL, by using method of MassARRAY®. Accordingly, we should carefully check the detailed genetic information of the broad spectrum of 19DEL mutations, and the result of variants on LLY507 747 of exon 19 in-frame deletions by MassARRAY® particularly need further investigation and identification. In addition, the results of direct DNA and cDNA sequencing of the EGFR gene in six patients were compared in the NTUH cohort population. Three of the six (50%) patients were identified as having wild-type EGFR by DNA sequencing, rather than having p.L747P or p.L747S mutant EGFR. Several methodological studies have emphasized that direct DNA sequencing is highly dependent on DNA quality and the enrichment of tumor cells in specimens [34,35]. With poor DNA quality or a low proportion of tumor cells in specimens, patients could be misdiagnosed as having wild-type EGFR rather than a p.L747P mutation.
The above conditions could result in misled clinicians making inappropriate treatment decisions. In the era of personal and precision medicine, the methodological limitations of laboratory methods should be clarified. NGS offers an alternative method for obtaining comprehensive information on the mutational spectrum of NSCLC, revealing rare mutations to better target therapies . For that reason, in patients treated with a first-generation EGFR TKI as a targeted therapy for a 19DEL in EGFR based on the results of commercial kits who develop primary resistance to gefitinib or erlotinib, NGS could be used to recheck these specimens, providing additional information for more personalized therapies. With widespread application of NGS in clinical practice, more patients with EGFR p.L747P or p.L747S mutations are likely to be identified, and these patients should preferentially be treated with afatinib.
The mutation p.T790M is the main mechanism of acquired resistance after EGFR TKI failure and was shown to be associated with common EGFR mutations, especially 19DEL [37,38]. Among our three patients harboring EGFR p.L747P or p.L747S mutations and receiving afatinib, none showed the acquisition of the p.T790M mutation after developing resistance. Nevertheless, more data is required to explore the mechanisms of acquired resistance in this specific population.
Conclusions The uncommon p.L747P and p.L747S EGFR mutations can be misclassified as a 19DEL mutation using commercial kits or wild-type EGFR using DNA sequencing because of poor DNA quality or a small proportion of tumor cells in specimens. Considering the limitations of above methods, we should cautiously interpret the results of EGFR mutations from above methods. In clinical, NGS should be widely used in correctly detecting these mutations and guide physicians making clinical decisions. Moreover, our results suggest that the irreversible inhibitor afatinib may provide improvements in PFS and OS in the treatment of lung adenocarcinoma with EGFR p.L747P or p.L747S mutations.