Table 1.
Specifications for the EGFR mutation tests that can be used in the clinical practice
| Criterion |
| Kits used for EGFR mutation test are required to detect the type of mutations described in the Mutations section (see below) from the samples with a ratio of the cancer cells of 1%. To attain this, the kits are required to pass the assay described in the Assay section. |
| Mutations |
| Mandatory† |
| E746‐A750del (2235–2249delGGAATTAAGAGAAGC) |
| E746‐A750del (2236–2250delGAATTAAGAGAAGCA) |
| L858R |
| G719S |
| T790M |
| Recommended† |
| L747‐S752del P753S (2240–2257delTAAGAGAAGCAACATCTC) |
| L747‐E749del A750P (2239–2247delTTAAGAGAA, 2248G > C) |
| G719A |
| G719C |
| L861Q |
| Assay |
| Mutations that occur at the same position are usually detected at similar sensitivity. Therefore, only a single exon 19 deletion is included in the assay. The assay uses plasmid constructs each containing Del E746–A750 (2235–2249delGGAATTAAGAGAAGC), L858R, G719S, or T790M. Each plasmid DNA is mixed with normal human genomic DNA (10 ng/μL) to make the Assay Samples by achieving a copy number ratio of 1–200 of mutant EGFR sequence to normal EGFR sequence (Fig. S1). This simulates the test conditions in which the ratio of cancer cells to normal cells is 1–100 (Fig. 2). For the assay, 100 Assay Samples comprising 20 samples for each of the four mutants, and 20 Assay Samples containing only the normal human genomic DNA (10 ng/μL), are set up. There are randomized, and then investigated. This test is expected to correctly identify both presence and type of mutations in 95% of the samples (Fig. S2). Use of more than 5 ng of DNA from each Assay Sample is mandatory. Because the copy number of the mutant EGFR gene sequence conforms to a binomial distribution, use of <5 ng DNA causes significant sampling errors (see Fig. 5). |