Table 3.
KRAS mutation detection success by codon, concentration and technology
| (A) 100 Mutant copies input | |||||||||||||
| Nominal total copies of WT DNA | Real-time quantitative PCR | MALDI-TOF | Next-generation sequencing | Droplet digital PCR |
Sanger capillary sequencing | ||||||||
| therascreen KRAS RGQ PCR Kit | cobas KRAS Mutation Test | Idylla KRAS Mutation Test (point of care) | iPlex Pro | UltraSEEK | Thunderbolts | Oncomine Focus Assay |
Sentosa SQ NSCLC Panel | Ion AmpliSeq Cancer Hotspot panel V.2 |
TruSight Tumor 15 | PrimePCR ddPCR Mutation Assays KRAS | ABI3730 sequencing |
||
| p.G12C | 20 | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✕ NMD | ✓ MD 14.3% | ✓ MD 20.5% | ✓ MD 11.1% | NMD | ✕ IMD | ✓ NMD |
| 10 | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD 4.7% | ✓ MD 6.6% | ✓ MD 5.7% | ✓ MD 5.7% | ✓ MD 4.8% | ✓ MD | ✓ NMD | |
| 5 | ✓ MD | ✓ NMD | ✓ MD | ✓ MD | ✓ MD | ✓ MD 3.7% | ✓ MD 2.4% | ✓ NMD 3.1% | ✓ MD 2.6% | ✓ MD 2.7% | ✓ MD | ✓ NMD | |
| 1 | ✓ NMD | ✓ NMD | ✓ MD | ✓ MD | ✓ MD | ✓ NMD | ✓ MD 0.5% | ✓ NMD – | ✓ NMD 0.0% | ✓ NMD 0.0% | ✓ MD | ✓ NMD | |
| 0.5 | ✓ NMD | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD 0.3% | ✓ NMD – | ✓ NMD 0.0% | ✓ NMD 0.0% | ✕ NMD | ✓ NMD | |
| p.G12D | 20 | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD 17.2% | ✓ MD 19.6% | ✓ MD 25.6% | ✓ MD 27.5% | ✓ MD 31.1% | ✓ MD | ✓ NMD |
| 10 | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD 11.4% | ✓ MD 11.8% | ✓ MD 14.3% | ✓ MD 13.2% | ✕ NMD | ✓ MD | ✓ NMD | |
| 5 | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD 7.6% | ✓ MD 5.8% | ✓ MD 6.7% | ✓ MD 5.1% | ✓ MD 7.3% | ✓ MD | ✓ NMD | |
| 1 | ✓ NMD | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD | ✓ MD 1.5% | ✓ MD 1.2% | ✓ NMD – | ✓ MD 1.0% | ✓ MD 1.6% | ✓ MD | ✓ NMD | |
| 0.5 | ✓ NMD | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD 0.6 % | ✓ NMD – | ✓ NMD 0.0 % | ✓ NMD 0.0 % | ✕ NMD | ✓ NMD | |
| p.G13D | 20 | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✕ NMD | ✓ MD 13.9% | ✓ MD 14.6% | ✓ MD 10.4% | ✕ NMD | ✓ MD | ✓ NMD |
| 10 | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD 2.7% | ✓ MD 7.4% | ✓ MD 7.7% | ✓ MD 8.6% | ✓ MD 6.4% | ✓ MD | ✓ NMD | |
| 5 | ✓ NMD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD 3.9% | ✓ MD 3.1% | ✓ MD 4.6% | ✓ MD 1.6% | ✓ MD 3.8% | ✓ MD | ✓ NMD | |
| 1 | ✓ NMD | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD 0.9% | ✓ NMD – | ✓ NMD 0.0% | ✓ NMD 0.0% | ✓ MD | ✓ NMD | |
| 0.5 | ✓ NMD | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD | ✕ NMD | ✓ MD 0.4% | ✓ NMD – | ✓ NMD 0.0% | ✓ NMD 0.0% | ✕ NMD | ✓ NMD | |
| p.G12V | 20 | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✕ NMD | ✓ MD 30.6% | ✓ MD 39.4% | ✓ MD 27.8% | NMD | ✓ MD | ✓ NMD |
| 10 | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD 5.9% | ✓ MD 16.2% | ✓ MD 24.5% | ✓ MD 15.6% | ✓ MD 13.6% | ✕ IMD | ✓ NMD | |
| 5 | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD 6.1% | ✓ MD 8.1% | ✓ MD 9.6% | ✓ MD 8.3% | ✓ MD 8.5% | ✓ MD | ✓ NMD | |
| 1 | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ NMD | ✓ MD 1.3% | ✓ NMD 1.6% | ✓ MD 1.4% | ✓ MD 1.5% | ✓ MD | ✓ NMD | |
| 0.5 | ✓ MD | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD 1.0% | ✓ NMD – | ✓ NMD 0.0% | ✓ NMD 0.0% | ✕ NMD | ✓ NMD | |
| p.Q61H | 20 | NA | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✕ NMD | ✓ MD 6.3% | ✓ MD 7.8% | ✓ MD 1.1% | ✕ NMD | NP | ✓ NMD |
| 10 | NA | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD | ✓ MD 2.3% | ✓ MD 2.7% | ✓ NMD 2.4% | ✓ MD 3.0% | ✓ MD 4.3% | NP | ✓ NMD | |
| 5 | NA | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD 1.2% | ✓ NMD 2.5% | ✓ MD 1.8% | ✓ MD 1.5% | NP | ✓ NMD | |
| 1 | NA | ✓ NMD | ✓ MD | ✓ NMD | ✓ NMD | ✓ NMD | ✓ MD 0.2% | ✓ NMD – | ✓ NMD 0.0% | ✓ NMD 0.0% | NP | ✓ NMD | |
| 0.5 | NA | ✓ NMD | ✓ MD | ✓ NMD | ✓ NMD | ✓ NMD | ✓ MD 0.1% | ✓ NMD – | ✓ NMD 0.0% | ✓ NMD 0.0% | NP | ✓ NMD | |
| (B) 50 Mutant copies input | |||||||||||||
| Nominal total copies of WT DNA | Real-time quantitative PCR | MALDI-TOF | Next-generation sequencing | Droplet digital PCR |
Sanger capillary sequencing | ||||||||
| therascreen KRAS RGQ PCR Kit | cobas KRAS Mutation Test | Idylla KRAS Mutation Test (point of care) | iPlex Pro | UltraSEEK | Thunderbolts | Oncomine Focus Assay | Sentosa SQ NSCLC Panel | Ion AmpliSeq Cancer Hotspot panel V.2 |
TruSight Tumor 15 | PrimePCR ddPCR Mutation Assays KRAS | ABI3730 sequencing |
||
| p.G12C | 20 | ✕ NMD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✕ NMD | ✓ MD 10.8% | ✓ MD 19.8% | ✓ MD 13.1% | ✕ NMD | ✓ MD | ✓ NMD |
| 10 | ✓ MD | ✓ MD | ✓ MD | ✓ NMD | ✓ MD | ✕ NMD | ✓ MD 5.8% | ✓ MD 6.3% | ✓ MD 7.1% | ✕ NMD | ✓ MD | ✓ NMD | |
| 5 | ✓ NMD | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD | ✓ MD 2.1% | ✓ MD 2.9% | ✓ MD 3.3% | ✓ MD 1.9% | ✓ MD 3.6% | ✓ MD | ✓ NMD | |
| 1 | ✓ MD | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD 0.6% | ✓ NMD – | ✓ NMD 0.0% | ✓ NMD 0.0% | ✓ MD | ✓ NMD | |
| 0.5 | ✓ NMD | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD 0.4% | ✓ NMD – | ✓ NMD 0.0% | ✓ NMD 0.0% | ✕ IMD | ✓ NMD | |
| p.G12D | 20 | ✕ NMD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✕ NMD | ✓ MD 22.0% | ✓ MD 16.6% | ✓ MD 23.2% | ✕ NMD | ✓ MD | ✓ NMD |
| 10 | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD 9.3% | ✓ MD 11.5% | ✓ MD 18.4% | ✓ MD 8.4% | ✓ MD 9.7% | ✓ MD | ✓ NMD | |
| 5 | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD 5.0% | ✓ MD 5.0% | ✓ MD 5.2% | ✓ MD 7.6% | ✓ MD 6.7% | ✓ MD | ✓ NMD | |
| 1 | ✓ NMD | ✓ NMD | ✓ MD | ✓ MD | ✓ MD | ✓ NMD | ✓ MD 1.2% | ✓ NMD 2.2% | ✓ MD 1.9% | ✓ NMD 0.0% | ✓ MD | ✓ NMD | |
| 0.5 | ✓ NMD | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD 0.3% | ✓ NMD – | ✓ MD 1.2% | ✓ NMD 0.0% | ✕ IMD | ✓ NMD | |
| p.G13D | 20 | NMD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✕ NMD | ✓ MD 13.0% | ✓ MD 6.5% | ✓ MD 15.5% | ✕ NMD | ✓ MD | ✓ NMD |
| 10 | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✕ NMD | ✓ MD 6.6% | ✓ MD 6.6% | ✓ MD 6.1% | ✓ MD 2.7% | ✕ IMD | ✓ NMD | |
| 5 | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD 2.8% | ✓ MD 2.9% | ✓ NMD 2.5% | ✓ MD 2.0% | ✓ MD 2.6% | ✓ MD | ✓ NMD | |
| 1 | ✓ NMD | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD 0.8% | ✓ NMD – | ✓ NMD 0.0% | ✓ NMD 0.0% | ✓ MD | ✓ NMD | |
| 0.5 | ✓ NMD | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD 0.4% | ✓ NMD – | ✓ NMD 0.0% | ✓ NMD 0.0% | ✕ IMD | ✓ NMD | |
| p.G12V | 20 | ✕ NMD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✕ NMD | ✓ MD 34.3% | ✓ MD 46.9% | ✓ MD 29.6% | ✓ MD 26.1% | ✓ MD | ✓ NMD |
| 10 | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✕ NMD | ✓ MD 17.3% | ✓ MD 13.9% | ✓ MD 15.3% | ✕ NMD | ✕ IMD | ✓ NMD | |
| 5 | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD 5.1% | ✓ MD 8.5% | ✓ MD 12.2% | ✓ MD 7.0% | ✓ MD 10.8% | ✕ IMD | ✓ NMD | |
| 1 | ✓ MD | ✓ NMD | ✓ MD | ✓ MD | ✓ MD | ✓ NMD | ✓ MD 1.7% | ✓ NMD – | ✓ MD 1.8% | ✓ MD 1.8% | ✕ IMD | ✓ NMD | |
| 0.5 | ✓ MD | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD 0.8% | ✓ NMD – | ✓ NMD 0.0% | ✓ NMD 0.0% | ✓ MD | ✓ NMD | |
| p.Q61H | 20 | NA | ✓ MD | ✓ NMD | ✓ MD | ✓ MD | ✕ NMD | ✓ MD 6.0% | ✓ MD 10.3% | ✓ MD 4.5% | ✕ NMD | NP | ✓ NMD |
| 10 | NA | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✕ NMD | ✓ MD 2.0% | ✓ NMD 2.8% | ✓ MD 3.7% | ✕ NMD | NP | ✓ NMD | |
| 5 | NA | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD 1.7% | ✓ NMD – | ✓ MD 1.6% | ✓ NMD 0.0% | NP | ✓ NMD | |
| 1 | NA | ✓ NMD | ✓ NMD | ✓ NMD | ✓ NMD | ✓ NMD | ✓ MD 0.4% | ✓ NMD – | ✓ NMD 0.0% | ✓ NMD 0.0% | NP | ✓ NMD | |
| 0.5 | NA | ✓ NMD | ✓ MD | ✓ NMD | ✓ NMD | ✓ NMD | ✓ MD 0.3% | ✓ NMD – | ✓ NMD 0.0% | ✓ NMD 0.0% | NP | ✓ NMD | |
| (C) Wild-type only | ||||||||||||
| No of copies | Real-time quantitative PCR | MALDI-TOF | Next-generation sequencing | Droplet digital PCR | Sanger capillary sequencing | |||||||
| therascreen KRAS RGQ PCR Kit | cobas KRAS Mutation Test | Idylla KRAS Mutation Test (point of care) | iPlex Pro | UltraSEEK | Thunderbolts | Oncomine Focus Assay |
Sentosa SQ NSCLC Panel | Ion AmpliSeq Cancer Hotspot panel V.2 | TruSight Tumor 15 | PrimePCR ddPCR Mutation Assays KRAS | ABI3730 sequencing |
|
| 20 000 | ✓ WT | ✓ WT | ✓ WT | ✓ WT | ✓ WT | ✓ WT | ✓ WT | ✓ WT | ✓ WT | ✕ NMD | ✓ NMD | ✓ WT |
| 10 000 | ✓ WT | ✓ WT | ✓ WT | ✓ WT | ✓ WT | ✓ WT | ✓ WT | ✓ WT | ✓ WT | ✓ WT 0.0% | ✕ NMD | ✓ WT |
| 2000 | ✓ WT | ✓ WT | ✓ WT | ✓ WT | ✓ WT | ✓ WT | ✓ WT | ✓ WT | ✓ WT | ✓ WT 0.0% | ✕ NMD | ✓ WT |
| 1000 | ✓ WT | ✓ WT | ✓ WT | ✓ WT | ✓ WT | ✓ WT | ✓ WT | ✓ WT | ✓ WT | ✕ NMD | ✓ NMD | ✓ WT |
| 500 | ✓ WT | ✓ WT | ✓ WT | ✓ WT | ✓ WT | ✕ NMD | ✓ WT | ✓ WT | ✓ WT | ✕ NMD | ✕ NMD | ✓ WT |
| 250 | ✓ WT | ✓ WT | ✓ WT | ✓ WT | ✓ WT | ✕ NMD | ✓ WT | ✓ WT | ✓ WT | ✕ NMD | ✕ NMD | ✓ WT |
✓ MD, analysis successful, mutation detected; ✓ NMD, analysis successful, but no mutation detected (in the case of the Sentosa assay, a mutation was detected but deemed to be below the defined cut-off); ✓ WT, analysis successful, wild-type sample; ✕ NMD, analysis unsuccessful, no mutation detected; ✕ IMD, incorrect mutation detected; NA, kit does not assay codon; NP, not performed.
MALDI-TOF, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.