Table 1.

Comparison of EGFR testing methods

FISHWESRT/PCRRNAseqIHC
Cutoff15% tumor cells with amplification defined as EGFR/CEP 7 ratio ≥ 2Relative copy number EGFR exons (excluding 2-7) compared with chromosome 7Normalized to β-actinRPKM > 40 categorized as overexpressedIndeterminate
1.3 log increase of EGFR categorized as amplifiedΔCt of β-actinEGFR used and ΔCt ≥ −5.5 categorized as overexpressed
Correlation with FISHNot applicableSubstantial agreement with amplification by FISHSubstantial agreement with amplification by FISHHighly associated with EGFR RT-PCRLow specificity to detect amplification
ProsWidely used methodologyHighly flexible and can assess many genetic changes in parallelMultiple assay optionsHighly flexible and can assess many targets in parallelBroadly used, widely available method of protein expression
Fluorescence allows for more multiplexing as compared with similar techniques such as chromogenic in situ hybridization (CISH)Cost effective
Latest automation minimizes human variable
Quick turnaround
ConsFluorescence fades over timeComplex process and algorithms with more room for variationDetects mRNA expression as a surrogate for amplificationDetects mRNA expression as a surrogate for amplificationNot a direct measurement of gene amplification
Fluorescence technology more expensive than CISHLoss of cell and tissue morphologyMore expensive and longer turnaround time than FISHMeasures protein expression only
More expensive and longer turnaround time than FISHSemi-quantitative
False positive and false negative cases