Following the development of targeted therapies against EGFR and HER2, two 
members of the human epidermal receptor (HER) family of receptor tyrosine 
kinases, much interest has been focused on their expression in tumors. However, 
knowing the expression levels of individual receptors may not be sufficient to 
predict drug response. Here, we describe the development of antibody-based 
time-resolved Förster resonance energy transfer (TR-FRET) assays for the 
comprehensive analysis not only of EGFR and HER2 expression in tumor 
cryosections, but also of their activation through quantification of HER homo- 
or heterodimers. First, EGFR and HER2 expression levels were quantified in 18 
breast tumors and the results were compared with those obtained by using 
reference methods. The EGFR number per cell determined by TR-FRET was 
significantly correlated with EGFR mRNA copy number (P<0.0001). Moreover, our 
method detected HER2 overexpression with 100% specificity and sensibility, as 
confirmed by the standard IHC, FISH and qPCR analyses. EGFR and HER2 
dimerization was then assessed, using as controls xenograft tumors from cell 
lines with known dimer expression profiles. Our results show that quantification 
of HER dimerization provides information about receptor activation that cannot 
be obtained by quantification of single receptors. Quantifying HER expression 
and dimerization by TR-FRET assays might help identifying novel clinical markers 
for optimizing patients' treatment in oncology.