Molecular profiling of signalling pathways in formalin-fixed and paraffin-embedded cancer tissues

Patient selection for individual cancer therapy using reverse phase protein array. Formalin-fixed and paraffin-embedded (FFPE) material is the major source of tissues in hospitals. After histologic inspection and tissue microdissection a tumour-specific network portrait is determined. The identification of deregulated signalling pathways serves as a basis for optimal patient selection and therapy decision. Recently, it became possible to use FFPE tissue samples to identify deregulated signalling pathways by reverse phase protein array (RPPA). Here protein lysates are immobilised on nitrocellulose-coated glass slides and detected with specific antibodies.

Quantification of protein expression by reverse phase protein array. (A) Purified recombinant human epidermal growth factor receptor 2 (HER2) is arrayed together with the patient samples in a dilution curve on nitrocellulose slides as protein reference. The HER2 protein (2.5pg start concentration) was mixed with 20μl of a HER2-negative patient sample (2mg/ml) before spotting to eliminate potential influences of complex protein mixtures on signal-intensity compared to purified proteins. The slides were incubated with a HER2-specific antibody (DAKO; 1:1000 diluted). No HER2 signal was detected in the HER2-negative patient sample and in the patient sample mixed with purified EGFR. (B) The signal-intensity was plotted against the protein concentration generating a signal-intensity–concentration curve. Since the protein concentration of recombinant HER2 is known, the unknown HER2 concentration in a patient sample can be determined according to the HER2 standard curve. The protein concentration was normalised to total protein. In our example the signal-intensity of the patient sample is 39. Hence according to the HER2 standard curve the HER2 concentration in the patient sample is 0.5pg/nl spot (a spot contains 1nl protein lysate). Prior to spotting total protein concentration was determined by Bradford assay (2mg/m in the undiluted first spot). For HER2 quantification we used the first dilution (1mg/ml). After normalisation the HER2 concentration in the patient sample is 0.5pg/ng total protein (own unpublished data).

Reproducibility and variation of spotting and lysate preparation. Protein extracts from 10 breast cancer patients were prepared and assayed for human epidermal growth factor receptor 2 (HER2) expression using reverse phase protein array (RPPA) technology. Each slide was incubated with a HER2-specific antibody (DAKO; 1:1000 diluted) to determine the HER2 protein expression. Total protein was determined by Sypro Ruby Protein Blot stain. Subsequently protein expression was normalised to total protein. (A) For analysis of intra-sample variation proteins of 10 FFPE breast cancer patient samples were extracted three times independently and arrayed on the same slide (own unpublished data). (B) The inter-array variation is determined by spotting the samples on different slides (own unpublished data).