Table 2.
Results of determination of allelic frequencies of each reference materials by different methods.
| Method | Mutation | Variant allelic frequency (%) | ||||
|---|---|---|---|---|---|---|
| Plasma-Q0 | Plasma-Q1 | Plasma-Q2 | FFPE-Q1 | FFPE-Q2 | ||
| ddPCR | 19del | 0.35 ± 0.13 | 0.85 ± 0.26 | 1.31 ± 0.40 | 1.18 ± 0.14 | 5.09 ±0.37 |
| L858R | 0.53 ± 0.16 | 1.20 ± 0.43 | 1.89 ± 0.20 | 1.61 ± 0.36 | 7.19 ± 0.71 | |
| T790M | 0.03 ± 0.06 | 0.61 ± 0.05 | 1.43 ± 0.36 | 1.70 ± 0.29 | 7.30 ± 0.37 | |
| ARMS-PCR | 19del | 0.32 ± 0.09 | 0.49 ± 0.09 | 0.65 ±0.09 | 0.61 ± 0.06 | 3.10 ± 0.42 |
| L858R | 0.27 ± 0.09 | 0.93 ± 0.44 | 1.94 ± 0.65 | 1.25 ± 0.18 | 5.26 ± 1.23 | |
| T790M | 0.02 ± 0.02 | 0.23 ± 0.08 | 0.50 ± 0.14 | 0.66 ± 0.08 | 3.31 ± 0.50 | |
| NGS | ||||||
| Participant #1 | 19del | 0.58 | 1.55 | 2.17 | 1.34 | 5.59 |
| L858R | 1.02 | 1.85 | 3.43 | 2.38 | 7.33 | |
| T790M | 0.82 | 1.90 | 4.25 | 1.91 | 7.55 | |
| Participant #2 | 19del | †ND | 0.23 | 0.96 | 1.46 | 4.42 |
| L858R | 0.65 | 0.94 | 2.01 | 1.44 | 6.84 | |
| T790M | 0.24 | 0.60 | 1.51 | 2.04 | 7.09 | |
| *Participant #3 | 19del | 0.78 | 0.60 | 1.45 | / | / |
| L858R | 0.57 | 1.53 | 3.25 | / | / | |
| T790M | 0.71 | 0.92 | 2.75 | / | / | |
| Participant #4 | 19del | 0.20 | 0.55 | 1.70 | 1.24 | 3.64 |
| L858R | 1.79 | 3.10 | 2.48 | 1.85 | 7.66 | |
| T790M | 0.23 | 1.59 | 4.81 | 2.19 | 7.05 | |
| Participant #5 | 19del | 0.20 | 0.46 | 0.77 | 0.89 | 2.62 |
| L858R | 0.40 | 1.02 | 2.09 | 1.60 | 6.38 | |
| T790M | 0.23 | 0.49 | 1.31 | 1.48 | 5.18 | |
| Participant #6 | 19del | 1.09 | 1.04 | 0.49 | 1.55 | 3.04 |
| L858R | 0.86 | 1.64 | 6.09 | 1.51 | 5.35 | |
| T790M | 0.14 | 2.42 | 6.70 | 1.94 | 7.30 | |
| Participant #7 | 19del | †ND | 0.04 | †ND | 0.57 | 1.99 |
| L858R | 1.30 | 2.19 | 2.64 | 1.57 | 8.20 | |
| T790M | 0.51 | 1.07 | 1.81 | 2.10 | 7.85 | |
| Participant #8 | 19del | †ND | †ND | †ND | †ND | 3.64 |
| L858R | 0.26 | 0.72 | 0.96 | 1.18 | 5.02 | |
| T790M | 0.22 | 0.34 | 1.00 | 1.43 | 5.79 | |
* Participant #3 didn't develop appropriate NGS assay to deal with FFPE tissue samples.
† All ND results were confirmed by repeated tests.
The results of the ddPCR and ARMS-PCR are shown as mean ± standard deviation from four times repeated measurements. Every reference material was treated as real clinical sample that was detected by the participants using their own developed NGS-based cancer IVDs. All data are expressed as percentage (%) of allelic frequencies of the EGFR mutations.
Abbreviations: ddPCR, droplet digital polymerase chain reaction; ARMS-PCR, amplification refractory mutation system polymerase chain reaction; NGS, next-generation sequencing; del, deletion; T, threonine; M, methionine; L, leucine; R, arginine; FFPE, formalin-fixed, paraffin-embedded; ND, not detected.