Table 1.
Average genotyping score, assigned score criteria and error rates for every case analysed by the pilot scheme participants
| Sample | A | B | C | D | E | F | G | H | I | J | A-J |
|---|---|---|---|---|---|---|---|---|---|---|---|
| Variant status | 5% KRAS c.35G>C; p.(G12A) | 1% KRAS c.35G>C; p.(G12A) | 5% NRAS c.182A>G; p.(Q61R) | 1% NRAS c.182A>G; p.(Q61R) | KRAS/NRAS Wild-type | 5% EGFR c.2235_2249del15; p.E746_A750del | 1% EGFR c.2235_2249del15; p.E746_A750del | 5% EGFR c.2573T>G; p.(L858R) and c.2369C>T; p.(T790M) | 1% EGFR c.2573T>G; p.(L858R) and c.2369C>T; p.(T790M) | EGFR Wild-type | Overall |
| Average score per sample on 2 points | 1.5 | 1.3 | 1.7 | 1.5 | 2.0 | 1.7 | 1.7 | 1.9 | 1.6 | 1.9 | 1.7 |
| Score criteria | # obtained scores (%) | ||||||||||
| Correct (2 points) | 11 (35.5%) | 7 (22.6%) | 12 (38.7%) | 8 (25.8%) | 23 (74.2%) | 5 (16.1%) | 4 (12.9%) | 29 (93.5%) | 18 (58.1%) | 30 (96.8%) | 147 (47.4%) |
| Correct but unspecified (2 points) | 5 (16.1%) | 3 (9.7%) | 4 (12.9%) | 2 (6.5%) | 0 (0.0%) | 16 (51.6%) | 16 (51.6%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 46 (14.8%) |
| False-negative ≤ LOD (2 points)a | 0 (0.0%) | 5 (16.1%) | 0 (0.0%) | 4 (12.9%) | 0 (0.0%) | 0 (0.0%) | 2 (6.5%) | 0 (0.0%) | 6 (19.4%) | 0 (0.0%) | 17 (5.5%) |
| False-negative but variant not tested for (2 points)a | 0 (0.0%) | 0 (0.0%) | 3 (9.7%) | 3 (9.7%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 6 (1.9%) |
| False-negative >/unknown LOD (0 points)a | 1 (3.2%) | 5 (16.1%) | 3 (9.7%) | 4 (12.9%) | 0 (0.0%) | 0 (0.0%) | 1 (3.2%) | 0 (0.0%) | 3 (9.7%) | 0 (0.0%) | 17 (5.5%) |
| False-positive (0 points)a | 2 (6.5%) | 2 (6.5%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 1 (3.2%) | 0 (0.0%) | 0 (0.0%) | 1 (3.2%) | 6 (1.9%) |
| Incorrect mutation detected with therapeutic implications (0 points)a | 0 (0.0%) | 0 (0.0%) | 1 (3.2%) | 1 (3.2%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 2 (0.6%) |
| Incorrect mutation detected without therapeutic implications (2 points) | 3 (9.7%) | 1 (3.2%) | 0 (0.0%) | 1 (3.2%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 5 (1.6%) |
| One variant missed in double mutation sample (0 points)a | N/A | N/A | N/A | N/A | N/A | N/A | N/A | 2 (6.5%) | 4 (12.9%) | N/A | 6 (1.9%) |
| Mutation described incorrectly (1 point) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 10 (32.3%) | 6 (19.4%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 16 (5.2%) |
| Technical failure (not scored) | 1 (3.2%) | 0 (0.0%) | 0 (0.0%)° | 0 (0.0%)° | 0 (0.0%) | 0 (0.0%) | 1 (3.2%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 2 (0.6%) |
| Not tested (not scored) | 8 (25.8%) | 8 (25.8%) | 8 (25.8%) | 8 (25.8%) | 8 (25.8%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 40 (12.9%) |
| Total scored (n = 31) | 22 (71.0%) | 23 (74.2%) | 23 (74.2%) | 23 (74.2%) | 23 (74.2%) | 31 (100.0%) | 30 (96.8%) | 31 (100.0%) | 31 (100.0%) | 31 (100.0%) | 268 (86.5%) |
| Error rate | # obtained scores (%) | ||||||||||
| Total with implication on therapy decisiona | 3 (13.6%) | 12 (52.2%) | 7 (30.4%) | 12 (52.2%) | 0 (0.0%) | 0 (0.0%) | 4 (13.3%) | 2 (6.5%) | 13 (41.9%) | 1 (3.2%) | 54 (20.1%) |
| Gene not tested/No method info given | 0 | 0 | 3 | 3 | 0 | 0 | 0 | 0 | 0 | 0 | 6 |
| NGS | 2 | 7 | 3 | 7 | 0 | 0 | 1 | 1 | 7 | 1 | 29 |
| Commercial Kit | 0 | 3 | 1 | 1 | 0 | 0 | 0 | 0 | 1 | 0 | 6 |
| LDT | 1 | 1 | 0 | 0 | 0 | 0 | 1 | 0 | 1 | 0 | 4 |
| BEAMing | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| ddPCR | 0 | 1 | 0 | 1 | 0 | 0 | 2 | 1 | 4 | 0 | 9 |
N/A Not applicable
aValues were used to calculate the error rates. °One partial technical failure for NRAS, only correct KRAS WT status was assessed on these cases. Reference sequence at time of scoring: EGFR: NM_005228.4 or LRG_304t1; KRAS: NM_033360.3 or NM_004985.4; NRAS: NM_002524.4 or LRG_92t1. Methods are ranked from least to more sensitive techniques as reported in literature: NGS 1–3%, commercial kit 0.1%, BEAMing 0.01%, ddPCR 0.001% [28]. For the LDT, a LOD of 0.1% was reported by the laboratory. Abbreviations: BEAMing Beads, emulsification, amplification, and magnetics, ddPCR Droplet digital polymerase chain reaction, LDT Laboratory-developed test, NGS Next-generation sequencing