Table 1.

Comparison of EGFR testing methods

	FISH	WES	RT/PCR	RNAseq	IHC
Cut-off	15% tumor cells with amplification defined as EGFR/CEP7 ratio ≥ 2	Relative copy number EGFR exons (excluding 2-7) compared with chromosome 7 1.3 log increase of EGFR categorized as amplified	Normalized to β-actin ΔCt of β-actin – EGFR used and ΔCt ≥ −5.5 categorized as overexpressed	RPKM > 40 categorized as overexpressed	Indeterminate
Correlation with FISH	NA	Substantial agreement with amplification by FISH	Substantial agreement with amplification by FISH	Highly associated with EGFR RT-PCR	Low specificity to detect amplification
Pros	Widely used methodology Fluorescence allows for more multiplexing as compared with similar techniques such as chromogenic in situ hybridization (CISH)	Highly flexible and can assess many genetic changes in parallel	Multiple assay options	Highly flexible and can assess many targets in parallel	Broadly used, widely available method of protein expression Cost effective Latest automation minimizes human variable Quick turnaround
Cons	Fluorescence fades over time Fluorescence technology more expensive than CISH	Complex process and algorithms with more room for variation Loss of cell and tissue morphology More expensive and longer turnaround time than FISH	Detects mRNA expression as a surrogate for amplification	Detects mRNA expression as a surrogate for amplification More expensive and longer turnaround time than FISH	Not a direct measurement of gene amplification Measures protein expression only Semi-quantitative False positive and false negative cases