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Reply to Rare Versus Artifactual EGFR Mutations.

EGFR-tyrosine kinase inhibitor (TKI) sensitivity of theses rare mutations differ from classic EGFR mutant. As a matter of fact, they are similar to EGFR wildtype patients. Lohinai et al.’s results are in line with previous report. Rare EGFR mutations had a high incidence, almost the same as classic and synonymous (5%, 6%, and 4%; respectively). They were also associated with smoking and poor response to EGFR-TKI as opposed to classic mutations. In addition, of the 49 rare mutations, 20 were never described before (not registered in the Catalogue of Somatic Mutations in Cancer [COSMIC] database). We must be aware that the diagnostic of advanced non–small-cell lung cancer is often made by biopsy rather than resected specimen, and these rare mutations were all noted from samples of DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue. Artifacts can easily be observed when sequencing multiple polymerase chain reaction (PCR) amplification products of very small amounts of DNA, particularly if the DNA is isolated from paraffin-embedded tissue. Therefore, the hypothesis that several of these rare mutations might be actually PCR-amplification artifacts must be discussed. Akbari et al. performed direct sequencing of the PCR-amplified coding region of the uracil-DNA glycosylase gene using DNA isolated from FFPE tissue specimens from patients with gastric cancer (only one mutation in the uracil-DNA glycosylase gene in human cancer had been previously reported in a sporadic human glioblastoma). In nearly 35% of the samples, they detected base substitutions, which, after further investigation, proved to be PCR artifacts. They also demonstrated that very low concentration of DNA template in PCR mix can give rise to false base substitutions. Marchetti et al. identified 45 rare EGFR mutations in 70 samples of lung tumor DNA extracted from FFPE tissue, and they were all found to be artifacts. This was confirmed because they also found the same mutations in multiple amplifications of DNA extracted from FFPE of normal tissue (50 patients’ lymph nodes without neoplasm). In addition, series whose fresh-frozen tissue was used do not observed rare EGFR mutations. There are some hypotheses for the occurrence of artifactual mutations. For instance, base damages and largescale DNA fragmentation caused by the chemical preparation of FFPE samples might result in cytosine deamination. Thereby the taq DNA polymerase would insert an adenosine instead of a guanosine resulting in C → T and G → A transitions (so-called “a-rule”). Moreover, degraded PCR products allowed the taq DNA polymerase performs a “jump” from a damaged template to another to continue the extension. Some strategies were reported to prevent artifactual mutations. Routine application of microdissection and use of fresh-frozen tissue to enrich tumorcell DNA are one option. Also, if small amounts of DNA extract from FFPE were inevitable, addition of uracil-Nglycosylase to the DNA template before PCR amplification and the examination of multiple amplifications are imperative. Because analysis of EGFR gene is mandatory for decision regarding EGFR-TKI use, the correct interpretation of EGFR exons 18 to 21 sequencing and its genetic alterations is crucial to select patients whom would benefit from treatment. Therefore, these data preclude the indiscriminate use of EGFR-TKI in patients harboring EGFR rare mutations and drive us to a more careful molecular analysis to identify molecular artifacts.