What are the recommended diagnostic methods for detecting mutations, amplifications, and translocations in non-small cell lung cancer (NSCLC) patients, specifically comparing Polymerase Chain Reaction (PCR) and Next-Generation Sequencing (NGS) for mutations, Fluorescence In Situ Hybridization (FISH) and NGS for amplifications, and FISH and NGS for translocations?

Recommended Diagnostic Methods for NSCLC Molecular Testing

For mutations, use NGS or multiplex PCR panels; for amplifications, use NGS with fold-change analysis (validated cutoff ≥2.0) or FISH; for translocations, use RNA-based NGS as the preferred method over FISH, though FISH remains FDA-approved and widely validated. 1, 2

Mutations: NGS or Multiplex PCR (Not Single-Gene PCR)

Avoid sequential single-gene PCR testing—it depletes scarce tissue and misses actionable targets. 1

• DNA-based NGS panels are the preferred approach for detecting point mutations in EGFR (exons 18-21), KRAS (including G12C), BRAF (V600E), HER2, PIK3CA, and TP53 simultaneously 1, 2
• NGS requires sensitivity of ≥1% variant allele frequency to detect all actionable mutations occurring with this frequency 1
• Multiplex PCR systems (Sequenom MassARRAY, SNaPshot) can detect >50 point mutations including EGFR sensitizing and resistance mutations, serving as an alternative when NGS is unavailable 1
• Direct Sanger sequencing is acceptable but less sensitive than NGS or multiplex methods 1

Critical pitfall: DNA-based assays are less sensitive for MET exon 14 skipping alterations, which involve intronic regions 1

Amplifications: NGS Fold-Change Analysis or FISH

NGS can accurately detect gene amplifications using coverage fold-change analysis, potentially replacing FISH in many cases. 3

• NGS fold-change ≥2.0 effectively distinguishes amplified from non-amplified cases for MET and HER2, with strong correlation to FISH metrics (Spearman's ρ = 0.847 for gene copy number) 3
• FISH-negative cases show NGS fold-change of 0.57-1.95, while FISH-positive cases range from 2.11-25.08 3
• FISH remains the validated standard for MET amplification detection when NGS is not available or requires confirmation 1
• NGS panels must include validated bioinformatics pipelines for accurate copy number calling 2

Key advantage: NGS detects amplifications without additional tissue consumption beyond mutation testing 4

Translocations: RNA-Based NGS Preferred Over FISH

RNA-based NGS is strongly preferred over DNA-based methods or FISH for fusion detection, though FISH remains FDA-approved for ALK. 1, 2, 5

Why RNA-NGS is Superior:

• RNA-based assays detect all fusion partners simultaneously without requiring knowledge of specific partner genes 1, 2
• DNA-based NGS may miss fusions with large intronic breakpoints or low-abundance variants 6
• RNA-NGS detects ALK, ROS1, RET, NTRK1-3, and NRG1 fusions in a single assay 2, 5
• Concordance rates with FISH are high: 97% for ALK, 100% for RET, 99.5% for ROS1 5

FISH Remains Valid:

• ALK FISH (break-apart probe) is FDA-approved and required before crizotinib treatment in the US 1
• FISH can confirm equivocal RNA-NGS results 1
• In Europe, validated ALK tests beyond FISH are acceptable 1

Alternative Methods:

• ALK IHC (antibodies 5A4 or D5F3) can screen for rearrangements with sensitivity/specificity approaching 100% compared to FISH, but methodology is not fully standardized 1
• RT-PCR approaches exist but have limitations: fusion-partner-specific methods achieve ~90% sensitivity, while fusion-partner-independent methods theoretically reach 100% 1

Critical limitation: RNA-NGS has higher failure rates (up to 12%) than DNA-based testing, requiring low threshold for reflex to FISH when inadequate 6, 5

NGS does NOT provide chromosome numbers for translocations—this is correct. FISH provides chromosomal localization information that NGS cannot determine. 1

Practical Implementation Algorithm

1. Obtain adequate tissue (≥20-30% tumor cellularity) at diagnosis 1, 7
2. Order comprehensive NGS panel combining DNA-based sequencing (mutations, amplifications) with RNA-based sequencing (fusions) 1, 2
3. For amplifications: Use NGS fold-change ≥2.0 as cutoff; confirm borderline cases with FISH if clinically critical 3
4. For translocations: Accept RNA-NGS results when adequate; reflex failed cases to FISH immediately 5
5. Turnaround time must allow results before first-line therapy initiation 2

Common pitfall: Some NGS panels detect only mutations, not fusions or copy number variations—verify panel capabilities before ordering 1