Table 1.
Studies of EGFR mutation testing methods using ‘standard’ tissue samples collected from patients with lung cancer
| Reference | Mutation testing method assessed (and comparator method) | Activating mutations assessed | No. of tissue samples | Tissue sample preparation | Macro- or micro-dissected? | Ethnicity of study population | Mutation frequency (vs that with comparator method)* | Reported Se, Sp, PPV, and NPV relative to comparator |
|---|---|---|---|---|---|---|---|---|
| Screening methods | ||||||||
| Sueoka et al20 | dHPLC (vs direct sequencing) | Exons 18–21 | 97 (including 16 PLE samples) | Frozen | NR | Japanese | Any mutation: 34 (35%) (vs 33 (34%)) | NR |
| Jänne et al21 | DNA endonuclease (SURVEYOR) and HPLC (vs direct sequencing) | Exons 18–21 | 160 (more samples were analysed with SURVEYOR/HPLC only) | FFPE/frozen | Macro-dissected (91/117 FFPE samples only) | NR (study performed in USA) | Any mutation: 58 (36%) (vs 51 (32%)) | Se, 100%; Sp, 87%; PPV, 74%; NPV, 100% |
| Do et al22 | HRMA (vs direct sequencing) | Exons 18–21 | 200 | FFPE | Micro-dissected | NR (study performed in Australia) | Any mutation: 118 (59%) (vs 73 (37%)) | Se, 100%; Sp, 90% |
| Takano et al23 | HRMA (vs direct sequencing)† | Exon 19 deletions and exon 21 point mutation (L858R) | 66 (more samples were analysed with HRMA only) | FFPE/methanol-fixed | Micro-dissected (samples for sequencing only) | East Asian patients | Any screened mutation: 34 (52%) for FFPE and 36 (55%) for methanol-fixed (vs 37 (56%)) | FFPE: Se, 92%; Sp, 100%; PPV, 100%; NPV, 90% Methanol-fixed: Se, 97%; Sp, 100%; PPV, 100%; NPV, 97% |
| Borràs et al24 | HRMA (vs direct sequencing) | Exons 19–21 | 36 | FFPE | Macro-dissected | NR (study performed in Spain) | E746–A750: 1 (2.8%) (vs 1 (2.8%)) E746–T751insA: 1 (2.8%) (vs 1 (2.8%)) L858R: 1 (2.8%) (vs 1 (2.8%)) P848L: 1 (2.8%)(vs 1 (2.8%)) |
NR |
| Querings et al25 | Massively parallel sequencing (vs direct sequencing and pyrosequencing) | Exons 18–21 | 24 (including 3 cytology samples) | FFPE/frozen | NR | NR (study performed in Germany) | Any mutation: 14 (58.3%) (vs 12 (50.0%) for pyrosequencing and 9 (37.5%) for direct sequencing) | Se, 100% (vs 89% for pyrosequencing and 67% for direct sequencing) |
| Targeted methods | ||||||||
| Endo et al26 | TaqMan PCR (vs direct sequencing) | 13 mutations across exons 18–21 | 94 (more samples were analysed with TaqMan PCR only) | FFPE | NR | NR (study performed in Japan) | Any screened mutation: 27 (28%) (vs 26 (28%)) | NR |
| Yatabe et al27 | Cycleave PCR (exon 21 (L858R)) or fragment analysis (exon 19 deletion) (vs direct sequencing) | Exon 19 deletion (E746_A750) and exon 21 point mutation (L858R) | 195 | FFPE/frozen | Macro-dissected | NR (study performed in Japan) | E746_A750: 38 (19%) (vs 39 (20%)) L858R:33 (17%) (vs 32 (16%)) |
NR |
| Ohnishi et al28 | Mutation-specific PCR (vs direct sequencing) | Exon 19 deletion (E746_A750) and exon 21 point mutation (L858R) | 62 | Frozen | NR | NR (study performed in Japan) | E746_A750: 8 (13%) (vs 8 (13%)) L858R:14 (23%) (vs 11 (18%)) |
NR |
| Asano et al29 | Mutant-enriched PCR (vs non-enriched PCR and direct sequencing) | Exon 19 deletions and exon 21 point mutation (L858R) | 108 | Frozen | NR | NR (study performed in Japan) | Exon 19 deletions: 17 (16%) (vs 16 (15%) for both non-enriched PCR and direct sequencing) L858R: 20 (19%) vs (17 (16%) for both non-enriched PCR and direct sequencing) |
NR |
| Otani et al30 | Mutant-enriched PCR (vs non-enriched PCR and direct sequencing) | Exon 19 deletions and exon 21 point mutation (L858R) | 26 | Frozen | NR | NR (study performed in Japan) | Exon 19 deletions: 3 (12%) (vs 3 (11%) for both non-enriched PCR and direct sequencing) L858R: 11 (42%) (vs 6 (23%) for both non-enriched PCR and direct sequencing) |
NR |
| Ellison et al31 | ARMS (vs direct sequencing) | Exon 19 deletion (E746_A750) and exon 21 point mutation (L858R) | 215 | FFPE | Macro-dissected | NR | E746_A750: 9 (4%) (vs 4 (2%)) L858R: 9 (4%) (vs 4 (2%)) |
NR |
| Zhao et al32 | Mutant-enriched ARMS TaqMan PCR (vs direct sequencing) | Exon 19 deletion (E746_A750) and exon 21 point mutation (L858R) | 31 | FFPE | NR | NR (study performed in China) | E746_A750: 5 (16%) (vs 3 (6%)) L858R: 6 (19%) (vs 5 (16%)) |
NR |
| Naoki et al33 | PCR-Invader (vs DNA sequencing) | Exon 18 point mutations (G719A/C/S), exon 19 deletions, exon 20 point mutation (S768I), exon 21 point mutations (L858R and L861Q) | 49 (plus 4 PLE and 1 PCE) | FFPE (tissue samples only) | Macro-dissected | Japanese | Any of the screened mutations: 28 (52%) (vs 19 (35%)) | NR |
| Kawada et al34 | PCR-RFLP (vs direct sequencing) | Exon 18 point mutation (G719X), exon 19 deletions and exon 21 point mutations (L858R and L861Q) | 91 (plus 14 PLE, 3 PCE and 1 sputum) |
Frozen | NR | NR (study performed in Japan) | Any of the screened mutations: 37 (34%) (vs 36 (33%)) |
NR |
| Molina-Vila et al35 | Length analysis for exon 19 deletions and TaqMan assay for exon 21 point mutation (vs direct sequencing) | Exon 19 deletions and exon 21 point mutations (L858R and L861Q) | 217 (includes 72 cytology samples) | FFPE/fresh | Micro-dissected | NR (study performed in Spain) | Exon 19 deletions: 25 (12%) (vs 25 (12%)) L858R: 11 (5%) (vs 11 (5%)) L861Q: 1 (0.5%) (vs 1 (0.5%)) |
NR |
| Pan et al36 | Length analysis (exon 19 deletions) and PCR-RFLP (exon 21 (L858R)) (vs direct sequencing) | Exon 19 deletions and exon 21 point mutation (L858R) | 39 | NR | NR | NR (study performed in USA) | Exon 19 deletions: 15 (38%) (vs 13 (33%)) L858R: 14 (36%) (vs 12 (31%)) |
NR |
| Ikeda et al37 | In-situ LAMP with ARMS (vs PCR-RFLP) | Exon 21 point mutation (L858R) | 26 | Paraffin-embedded | NR | NR (study performed in Japan) | L858R: 15 (58%) (vs 12 (46%)) | NR |
| Dufort et al38 | Pyrosequencing‡ (vs direct sequencing) | Exon 19 deletions and exon 21 point mutation (L858R) | 58 (more samples were analysed with pyrosequencing only) | FFPE/others | NR | NR (study performed in France) | Exon 19 deletions: 11 (19%) (vs 9 (16%)) L858R: 5 (9%) (vs 4 (7%)) |
NR |
| Han et al39 | PCR-PNA clamp (vs direct sequencing) | Exon 19 deletions, exon 20 insertions, and exon 21 point mutation (L858R and L816Q) | 23 (and 41 pleural effusion samples) | FFPE | No | NR (study performed in South Korea) | Any of the screened mutations: 16 (69.6%) (vs 12 (52.2%)) for adequate biopsy specimens and 12 (52.2%) (vs 12 (52.2%)) for matched surgically resected specimens | NR |
| Yang et al40 | PCR/CCP-based FRET (vs direct sequencing and RT-PCR) | Exon 21 point mutation (L858R) | 48 | FFPE | No | NR (study performed in China) | L858R: 20 (41.7%) (vs 19 (39.6%) for direct sequencing and 21 (43.8% for RT-PCR)) | Se, 95.2%; Sp, 96.3% |
| Hoshi et al41 | SmartAmp (vs direct sequencing) | Exon 18 point mutation (G719S), exon 19 deletions and exon 21 point mutation (L858R) | 45 | Frozen | NR | NR (study performed in Japan) | G719S: 0 (0%) (vs 0 (0%)) Exon 19 deletions: 5 (11%) (vs 5 (11%)) L858R: 5 (11%) (vs 4 (9%)) |
NR |
| Miyamae et al42 | Conventional and PNA-clamp SmartAmp2 (vs direct sequencing) | Exon 19 deletions and exon 21 point mutation (L858R) | 43 | FFPE and paired frozen | NR | NR (study performed in Japan) | Exon 19 deletions: 18 (42%) (vs 12 (28%) for frozen and FFPE) L858R: 12 (28%) (vs 5 (12%)) for frozen and 11 (26%) (vs 3 (7%)) for FFPE |
NR |
| Araki et al43 | PNA-clamp SmartAmp2 (vs direct sequencing, PNA-enriched sequencing, and SmartAmp2) | Exon 19 deletions | 172 | Frozen | No | Asian | Exon 19 deletions: 39 (22.7%) (vs 30 (17.4%) for direct sequencing and 38 (22.1%) for PNA-enriched sequencing and 12 (7.0%) for SmartAmp2) | NR |
| Kozu et al44 | IHC (vs HRMA) | Exon 19 deletions and exon 21 point mutation (L858R) | 577 (including 36 cytological smears) | Frozen or MFPE | No (but tumour regions selected for TMA and IHC analysis) | Japanese | Exon 19 deletions: 59 (10%) (vs 135 (23%)) L858R: 139 (24%) (vs 172 (30%)) |
Exon 19 deletions: Se, 42%; Sp, 100% L858R: Se, 76%; Sp. 98% |
| Brevet et al45 | IHC (vs fragment analysis for exon 19 deletion (mutant-enriched PCR assay for discordant results) or PCR-RFLP for L858R (mass-spectrometry-based DNA analysis for discordant results)) | Exon 19 deletions and exon 21 point mutation (L858R) | 194 | FFPE | Macro-dissected | NR (study performed in USA) | E746_A750: 22 (11%) (vs 20 (10%)) Other exon 19 deletions: 25 (13%) (vs 31 (16%)) L858R: 22 (11%) (vs 21 (11%)) |
E746_A750 deletion: Se, 100% Other exon 19 deletions: Se, 74% E746_A750 deletion and other exon 19 deletions: Sp, 98.8% L858R: Se, 95%; Sp. 99% |
| Ilie et al46 | IHC (vs direct sequencing) | Exon 19 deletions | 61 | FFPE (direct sequencing performed on frozen samples) | No (but tumour regions selected for TMA and IHC analysis) | Caucasian | E746_A750: 12 (20%) (vs 8 (13%)) All exon 19 deletions: 13 (21%) (vs 10 (16%)) |
Se, 23%; NPV, 49% (calculated using results from direct sequencing plus other methods) |
| Kato et al47 | IHC (vs direct sequencing) | Exon 19 deletion (E746_A750) and exon 21 point mutation (L858R) | 70 | NR | No (but tumour regions selected for TMA and IHC analysis) | Japanese | E746_A750: 9 (13%) (vs 11 (16%)) L858R: 11 (16%) (vs 12 (17%)) |
E746_A750: Se, 82%; Sp, 100%; PPV, 100%; NPV, 96.7% L858R : Se, 75%; Sp, 97%; PPV, 82%; NPV, 95% |
| Nakamura et al48 | IHC (vs PNA-LNA PCR clamp/direct sequencing) | Exon 19 deletion (E746_A750) and exon 21 point mutation (L858R) | 20 | FFPE | No | NR (study performed in Japan) | E746_A750: 4 (20%) (vs 3 (15%)) L858R: 4 (20%) (vs 5 (25%)) |
Se, 92%; Sp, 100% |
| Simonetti et al49 | IHC (vs fragment analysis, TaqMan assay, and direct sequencing) | Exon 19 deletions and exon 21 point mutations (L858R and L816Q) | 78 | FFPE | Micro-dissected | Caucasian | E746_A750: 17 (22%) (vs 17 (22%)) Other exon 19 deletions: 3 (4%) (vs 12 (15%)) L858R: 25 (32%) (vs 25 (32%)) L816Q: 0 (0%) (vs 2 (3%)) |
NR |
Only studies identified by our literature search and meeting the criteria described in the Methods are listed.
*In many studies, samples were selected and/or purposely enriched to include a higher number of mutated samples; therefore, mutation frequency data should not be considered representative of the general population.
†HRMA was used as a targeted method in this study.
‡Pyrosequencing tends to be performed in a semi-targeted manner.
ARMS, Amplification Refractory Mutation System; CCP-based FRET, cationic conjugated polymer-based fluorescence resonance energy transfer; dHPLC, denaturing high-performance liquid chromatography; EGFR, epidermal growth factor receptor; FFPE, formalin-fixed paraffin-embedded; HPLC, high-performance liquid chromatography; HRMA, high-resolution melting analysis; IHC, immunohistochemistry; LAMP, loop-mediated isothermal amplification; MFPE, methanol-fixed paraffin-embedded; NPV, negative predictive value; NR, not reported; PCE, pericardial effusion; PCR-RFLP, PCR-restriction fragment length polymorphism; PLE, pleural effusion; PNA, peptide nucleic acid; PNA-LNA, PNA-locked nucleic acid; PPV, positive predictive value; Se, sensitivity; SmartAmp2, smart amplification process V.2; Sp, specificity; TMA, tissue microarray.