Table 3.
Studies of EGFR mutation testing methods using cytology samples collected from patients with lung cancer
| Reference | Cytology samples (no. of samples for mutation analysis (fail data if available)) | Method(s) of EGFR mutation testing assessed | Authors’ conclusions on use of cytology samples for EGFR mutation testing |
|---|---|---|---|
| Asano et al29 | Cell-free PLE (n=20), CT-guided needle lung biopsies (n=18) | Mutant-enriched PCR versus non-enriched PCR and direct sequencing | Mutant-enriched PCR detected EGFR alterations that were not identified with a non-enriched assay |
| Fassina et al61 | TTNA samples (n=77) | HRMA versus direct sequencing | HRMA of TTNA samples was accurate, fast, easy, cheap, and reliable for the detection of common EGFR mutations |
| Hlinkova et al62 | Cytological samples obtained by endobronchial brushing (n=53) | HRMA versus direct sequencing (with mutant-enriched PCR if <25% tumour cells) | HRMA in combination with mutant-enriched PCR is a sensitive method for mutation detection in cytology samples |
| Horiike et al53 | Transbronchial FNA (n=93 (10 fails (11%) with direct sequencing; 0 fails with Scorpion ARMS)) | Scorpion ARMS versus direct sequencing | Both methods detected EGFR mutations in transbronchial FNA samples although Scorpion ARMS was more sensitive |
| Kawahara et al63 | PLE (n=21), CSF (n=2), and ascites (n=1) | Immunocytochemistry versus PNA-LNA PCR clamp | EGFR mutations were detected in PLE and CSF with 100% sensitivity using antibodies specific for the exon 19 deletion E746_A750 and the exon 21 point mutation L858R |
| Kimura et al64 | Cell-free PLE (n=43) | Direct sequencing | DNA in PLE can be used to detect EGFR mutations |
| Kimura et al51 | Cell-free PLE (n=24) | Scorpion ARMS versus direct sequencing | DNA in PLE can be used to detect EGFR mutations. Scorpion ARMS was more sensitive than direct sequencing |
| Kozu et al44 | Imprint cytological smears from fresh-cut surface of resected tumour specimens (n=36) | HRMA versus IHC | (Results of cytology sample analyses were combined with those of 541 tissue specimens (see table 2)) |
| Lim et al65 | FNA (n=29) | Whole genome amplification followed by direct sequencing | EGFR mutations were identified using direct sequencing of whole genome-amplified genomic DNA from low-volume FNA samples |
| Lozano et al66 | Primary lung tumour FNA (n=68), metastatic lymph node FNA (n=10), bone metastases FNA (n=3), left adrenal metastasis FNA (n=1), PLE (n=6), PCE (n=1), and bronchoalveolar lavage (n=1) | Direct sequencing | Assessment of EGFR mutation in cytology samples is feasible and comparable with biopsy results |
| Nakajima et al60 | EBUS-TBNA samples from metastatic lymph nodes (n=43) | Loop-hybrid mobility shift assay confirmed by direct sequencing | EGFR mutations can easily be detected in metastatic lymph nodes samples by EBUS-TBNA |
| Oshita et al67 | Cytology samples obtained by transbronchial abrasion (n=52) (2 fails (4%)) | Loop-hybrid mobility shift assay | Assessment of EGFR mutations in cytological samples is feasible and comparable with biopsy results |
| Otani et al30 | Biopsy needle wash fluid (n=26) | Mutant-enriched PCR versus non-enriched PCR versus direct sequencing | EGFR mutations can be detected in the wash fluid of CT-guided biopsy needles |
| Rekhtman et al68 | Transbronchial/transthoracic FNA (n=67), extrathoracic FNA (n=29), PLE (n=29), and bronchial brush/wash (n=3) (2 failures (2%)) | Length analysis and PCR-RFLP | EGFR analysis is feasible in routinely processed cytology samples |
| Savic et al69 | Transbronchial FNA (n=35), PLE (n=16), bronchial washing (n=15), bronchial brushes (n=13), and bronchoalveolar lavage (n=5) | PCR-direct sequencing | EGFR analyses are applicable to cytology specimens |
| Schuurbiers et al54 | EBUS-/EUS-FNA samples (n=35 (8 fails (23%)) | Direct sequencing | Molecular analysis for EGFR mutations can be performed routinely in EBUS-/EUS-FNA samples |
| Soh et al50 | Cell-free PLE (n=61) | Direct sequencing versus mutant-enriched PCR versus non-enriched PCR versus PNA-LNA PCR-clamp | Some discrepancies between the results of the four assays were noted. Mutant-enriched PCR detected the most mutations |
| Takano et al23 | Bronchial brushing/washing (n=43), PLE (n=40), transbronchial FNA (n=9), PCE (n=8), superficial lymph node FNA (n=7), tumour FNA (n=6), and sputum (n=4) | HRMA versus direct sequencing | Exon 19 deletions and the exon 21 point mutation L858R can likely be detected from archived Papanicolaou-stained cytology slides with sensitivity of ca. 90% and specificity of ca. 100% |
| van Eijk et al56 | EBUS-TBNA/EUS-FNA samples (numerous samples from 43 patients) | Real-time PCR with hydrolysis probes | All mutations detected in matched histological samples were also identified in the cytology samples |
| Yasuda et al70 | ELF (n=23) | PNA-LNA PCR clamp | Sensitivity for detecting mutations in ELF was 58% |
| Zhang et al71 | PLE cells and matched cell-free PLE (n=26) | Mutant-enriched PCR versus direct sequencing | Direct sequencing may miss a significant proportion of mutations in PLE samples. Mutant-enriched PCR may be more reliable |
| Smits et al72 | Cytology and FFPE samples (n=816; 719 samples had interpretable result) | Direct sequencing or HRMA | (Results of cytology sample analyses were combined with those of FFPE specimens) |
| Tsai et al73 | PLE (n=78) | IHC versus direct sequencing |
EGFR mutations were detected in PLE with 71% and 88% sensitivity using antibodies specific for the exon 19 deletion E746_A750 and the exon 21 point mutation L858R, respectively Correlation of TKI response rate with EGFR mutation status was comparable when determined by IHC and direct sequencing (67% vs 72%) |
| Navani et al57 | EBUS-TBNA samples (n=774) | ARMS or MassARRAY | EBUS-TBNA cytology samples are suitable for EGFR analysis |
| Aisner et al74 | Cytology cell blocks, including FNA of primary and metastatic lung lesions and exfoliative cytology specimens (n=42) | PCR-sequencing | Cell block specimens provide an alternative DNA source to surgical specimens for EGFR analysis |
| Zhuang et al55 | CT-guided FNA biopsy (n=43) | Direct sequencing | CT-guided FNA biopsy is a feasible and safe method to provide samples for EGFR analysis |
| Santis et al58 | EBUS-TBNA lymph node samples (n=131; successful analysis of 126 samples) | COLD-PCR | EBUS-TBNA samples provide sufficient tumour material for EGFR mutation analysis COLD-PCR is a robust screening assay for EGFR mutations |
| Malapelle et al75 | LBC (n=42) | Direct sequencing | LBC samples can be used for EGFR mutation analysis; however, direct sequencing requires micro-dissection to provide sufficient sample DNA |
| Betz et al76 | Romanowsky-stained direct cytology smears (n=33) | Direct sequencing | Following micro-dissection, direct smears can be used as a specimen source for EGFR analysis when cell blocks exhibit insufficient cellularity |
| Cho et al77 | Body fluid specimen (n=32: pleural fluids (n=29), CSF (n=1), pericardial (n=1), and ascites (n=1)) | Direct sequencing | Combined direct sequencing and cytological analysis might be clinically useful and sensitive for the detection of EGFR mutations |
| Tsai et al52 | PLE (n=150) | Direct sequencing of cell-derived RNA versus genomic DNA | Sequencing of RNA improves sensitivity for EGFR mutation detection in PLE samples compared with genomic DNA |
| Lozano et al66 | Cytology samples (n=150: Papanicolaou smears (n=120), Fresh/liquid (n=14), cell block (n=10), ThinPrep tests (n=6)) | Direct sequencing | EGFR analysis using cytological samples is feasible and comparable with biopsy results |
| Nakajima et al59 | EBUS-TBNA metastatic lymph node samples (n=156) | PNA-LNA PCR clamp | EBUS-TBNA samples can be used for multi-gene mutational analysis |
Only studies identified by our literature search and meeting the criteria described in the Methods are listed.
ARMS, Amplification Refractory Mutation System; COLD-PCR, coamplification at lower denaturation temperature PCR; CSF, cerebrospinal fluid; EBUS-FNA, endobronchial ultrasound-guided fine needle aspiration; EBUS-TBNA, endobronchial ultrasound-guided transbronchial needle aspiration; EGFR, epidermal growth factor receptor; ELF, epithelial lining fluid; EUS-FNA, trans-oesophageal ultrasound scanning with fine needle aspiration; FFPE, formalin-fixed paraffin-embedded; FNA, fine needle aspirate; HRMA, high-resolution melting analysis; IHC, immunohistochemistry; LBC, liquid-based cytology; MassARRAY, matrix-assisted laser desorption/ionisation mass spectroscopy; PCE, pericardial effusion; PCR-RFLP, PCR-restriction fragment length polymorphism assay; PLE, pleural effusion; PNA-LNA, peptide nucleic acid-locked nucleic acid; TKI, tyrosine kinase inhibitor; TTNA, trans-thoracic needle aspiration.