Table 3.
KRAS mutation detection success by codon, concentration and technology
| (A) 100 mutant copies input | |||||||||||||
| Real-time quantitative PCR | MALDI-TOF | Next-generation sequencing | Droplet digital PCR |
Sanger capillary sequencing | |||||||||
| Nominal total copies of WT DNA | thera screen KRAS RGQ PCR Kit |
cobas KRAS Mutation Test | Idylla KRAS Mutation Test (point of care) | iPLEX Pro | UltraSEEK | Thunder Bolts |
Oncomine Focus Assay | Sentosa SQ NSCLC Panel | Ion AmpliSeq Cancer Hotspot Panel v2 | TruSight Tumour 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 | IMD | ✓ NMD |
| 10 | NA | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD | ✓ MD 2.3% | ✓ MD 2.7% | ✓ NMD 2.4% | ✓ MD 3.0% | ✓ MD 4.3% | IMD | ✓ NMD | |
| 5 | NA | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD 1.2% | ✓ NMD 2.5% | ✓ MD 1.8% | ✓ MD 1.5% | IMD | ✓ NMD | |
| 1 | NA | ✓ NMD | ✓ MD | ✓ NMD | ✓ NMD | ✓ NMD | ✓ MD 0.2% | ✓ NMD– | ✓ NMD 0.0% | ✓ NMD 0.0% | IMD | ✓ NMD | |
| 0.5 | NA | ✓ NMD | ✓ MD | ✓ NMD | ✓ NMD | ✓ NMD | ✓ MD 0.1% | ✓ NMD– | ✓ NMD 0.0% | ✓ NMD 0.0% | NMD | ✓ NMD | |
| (B) 50 mutant copies input | |||||||||||||
| Real-time quantitative PCR | MALDI-TOF | Next-generation sequencing | Droplet digital PCR |
Sanger capillary sequencing | |||||||||
| Nominal total copies of WT DNA | thera screen KRAS RGQ PCR Kit |
cobas KRAS Mutation Test | Idylla KRAS Mutation Test (point of care) | iPLEX Pro | UltraSEEK | Thunder Bolts |
Oncomine Focus Assay | Sentosa SQ NSCLC Panel | Ion AmpliSeq Cancer Hotspot Panel v2 | 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 | NMD | ✓ NMD |
| 10 | NA | ✓ MD | ✓ MD | ✓ MD | ✓ MD | NMD | ✓ MD 2.0% | ✓ NMD 2.8% | ✓ MD 3.7% | NMD | IMD | ✓ NMD | |
| 5 | NA | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD | ✓ NMD | ✓ MD 1.7% | ✓ NMD− | ✓ MD 1.6% | ✓ NMD 0.0% | IMD | ✓ NMD | |
| 1 | NA | ✓ NMD | ✓ NMD | ✓ NMD | ✓ NMD | ✓ NMD | ✓ MD 0.4% | ✓ NMD− | ✓ NMD 0.0% | ✓ NMD 0.0% | IMD | ✓ NMD | |
| 0.5 | NA | ✓ NMD | ✓ MD | ✓ NMD | ✓ NMD | ✓ NMD | ✓ MD 0.3% | ✓ NMD− | ✓ NMD 0.0% | ✓ NMD 0.0% | IMD | ✓ NMD | |
| (C) Wild-type only | |||||||||||||
| Real-time quantitative PCR | MALDI-TOF | Next-generation sequencing | Droplet digital PCR |
Sanger capillary sequencing | |||||||||
| Number of copies | thera screen KRAS RGQ PCR Kit |
cobas KRAS Mutation Test | Idylla KRAS Mutation Test (point of care) | iPLEX Pro | UltraSEEK | Thunder Bolts |
Oncomine Focus Assay | Sentosa SQ NSCLC Panel | Ion AmpliSeq Cancer Hotspot Panel v2 | 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.
MALDI-TOF, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.