Sherwood JL, Brown H, Rettino A, et al. Key differences between 13 KRAS mutation detection technologies and their relevance for clinical practice. ESMO Open 2017;2:e000235. 10.1136/esmoopen-2017-000235
Tables 3A and 3B have been amended to reflect the error in the use of reagents by the third-party laboratory (IMGM, Munich) for the ddPCR Q61H assay (see appendix). The therascreen® KRAS RGQ PCR Kit Q61 assay does not test for Q61H; the PrimePCR™ ddPCR™ Mutation Assay was not performed for Q61H due to an error at the participating laboratory. This was identified after publication, as referred to in the Letter to the Editor (http://dx.doi.org/10.1136/esmoopen-2017-000294). A new abbreviation “NP” = “not performed” has been added to explain that no ddPCR assay for codon Q61 was performed.
In the abstract, the corrected sentence now reflects a lower number of total data points (718 instead of 728) because of the removal of 5 data points for ddPCR p.Q61H mutation results for 100 mutant copies input and 5 data points for 50 mutant copies input, respectively. Overall 406/718 data points across all 13 technologies were identified correctly. The digital PCR assay (KRAS PrimePCR™ ddPCR™, Bio-Rad Laboratories) identified 70% (100 copies) and 65% (50 copies) of samples correctly.
The ddPCR results section is corrected to reflect that for codon Q61H, the incorrect PrimePCR™ KRAS mutation assay had been used (p.Q61H c.183A>C instead of p.Q61H c.183A>T assay): The PrimePCR ddPCR KRAS Mutation Assays were able to identify codon 12 and 13 mutations down to 1% with the 100 copy input. However, across both admixture and wild-type control samples the assay identified the incorrect mutation in nine different mutation/allele frequency combinations (see table 3). When performing the p.Q61H assay, a mistake was made and the incorrect reagent detection of c.183A>C instead of c.183A>T, was used. Therefore, table 3 and figure 1 reflect data only for codons 12 and 13.
Table 3.
KRAS mutation detection success by codon, concentration and technology for (A.) 100 mutant copies input, (B.) 50 mutant copies input and (C.) wild-type only.
| (A) | |||||||||||||
| 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 v2 | Trusight® Tumor 15 | PrimePCR ddPCR Mutation Assays KRAS |
ABI3730 Sequencing | ||
| p.G12C | 20 | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | x NMD | ✓ MD 14.3% | ✓ MD 20.5% | ✓ MD 11.1% | x NMD | X 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% | x 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% | x 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% | x NMD | ✓ NMD | |
| p.G13D | 20 | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | x NMD | ✓ MD 13.9% | ✓ MD 14.6% | ✓ MD 10.4% | x 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 | x NMD | ✓ MD 0.4% | ✓ NMD - | ✓ NMD 0.0% | ✓ NMD 0.0% | x NMD | ✓ NMD | |
| p.G12V | 20 | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | x NMD | ✓ MD 30.6% | ✓ MD 39.4% | ✓ MD 27.8% | x NMD | ✓ MD | ✓ NMD |
| 10 | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD 5.9% | ✓ MD 16.2% | ✓ MD 24.5% | ✓ MD 15.6% | ✓ MD 13.6% | x 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% | x NMD | ✓ NMD | |
| p.Q61H | 20 | NA | ✓ MD | ✓ MD | ✓ MD | ✓ MD | x NMD | ✓ MD 6.3% | ✓ MD 7.8% | ✓ MD 1.1% | x 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) | |||||||||||||
| 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 v2 | Trusight® Tumor 15 | PrimePCR ddPCR Mutation Assays KRAS |
ABI3730 Sequencing | ||
| p.G12C | 20 | x NMD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | x NMD | ✓ MD 10.8% | ✓ MD 19.8% | ✓ MD 13.1% | x NMD | ✓ MD | ✓ NMD |
| 10 | ✓ MD | ✓ MD | ✓ MD | ✓ NMD | ✓ MD | x NMD | ✓ MD 5.8% | ✓ MD 6.3% | ✓ MD 7.1% | x 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% | x IMD | ✓ NMD | |
| p.G12D | 20 | x NMD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | x NMD | ✓ MD 22.0% | ✓ MD 16.6% | ✓ MD 23.2% | x 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% | x IMD | ✓ NMD | |
| p.G13D | 20 | x NMD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | x NMD | ✓ MD 13.0% | ✓ MD 6.5% | ✓ MD 15.5% | x NMD | ✓ MD | ✓ NMD |
| 10 | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | x NMD | ✓ MD 6.6% | ✓ MD 6.6% | ✓ MD 6.1% | ✓ MD 2.7% | x 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% | x IMD | ✓ NMD | |
| p.G12V | 20 | x NMD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | x NMD | ✓ MD 34.3% | ✓ MD 46.9% | ✓ MD 29.6% | ✓ MD 26.1% | ✓ MD | ✓ NMD |
| 10 | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | x NMD | ✓ MD 17.3% | ✓ MD 13.9% | ✓ MD 15.3% | x NMD | x IMD | ✓ NMD | |
| 5 | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD | ✓ MD 5.1% | ✓ MD 8.5% | ✓ MD 12.2% | ✓ MD 7.0% | ✓ MD 10.8% | x IMD | ✓ NMD | |
| 1 | ✓ MD | ✓ NMD | ✓ MD | ✓ MD | ✓ MD | ✓ NMD | ✓ MD 1.7% | ✓ NMD – | ✓ MD 1.8% | ✓ MD 1.8% | x 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 | x NMD | ✓ MD 6.0% | ✓ MD 10.3% | ✓ MD 4.5% | x NMD | NP | ✓ NMD |
| 10 | NA | ✓ MD | ✓ MD | ✓ MD | ✓ MD | x NMD | ✓ MD 2.0% | ✓ NMD 2.8% | ✓ MD 3.7% | x 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) | |||||||||||||
| Number 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 v2 | Trusight® Tumor 15 | PrimePCR ddPCR Mutation Assays KRAS |
ABI3730 Sequencing | ||
| 20000 | ✓wt | ✓WT | ✓WT | ✓WT | ✓WT | ✓WT | ✓WT | ✓WT | ✓WT | x NMD | ✓ NMD | ✓WT | |
| 10000 | ✓WT | ✓wt | ✓WT | ✓WT | ✓WT | ✓WT | ✓WT | ✓WT | ✓WT | ✓WT 0.0% | x NMD | ✓WT | |
| 2000 | ✓WT | ✓wt | ✓WT | ✓WT | ✓WT | ✓WT | ✓WT | ✓WT | ✓WT | ✓WT 0.0% | x NMD | ✓WT | |
| 1000 | ✓WT | ✓WT | ✓WT | ✓WT | ✓WT | ✓WT | ✓WT | ✓WT | ✓WT | x NMD | ✓ NMD | ✓WT | |
| 500 | ✓WT | ✓WT | ✓WT | ✓WT | ✓WT | x NMD | ✓WT | ✓WT | ✓WT | x NMD | x NMD | ✓WT | |
| 250 | ✓WT | ✓WT | ✓WT | ✓WT | ✓WT | x NMD | ✓WT | ✓WT | ✓WT | x NMD | x 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; X NMD = analysis unsuccessful, no mutation detected; X IMD = incorrect mutation detected; NA = kit does not assay codon; NP = not performed.
MALDI-TOF, Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry; PCR, polymerase chain reaction.