[Analysis of signalling pathways in formalin-fixed breast cancer tissues].
OBJECTIVE
The aim of our study was to develop and optimize methods for relative and absolute protein quantifications in formalin-fixed and paraffin-embedded (FFPE) tissues with special emphasis on HER mediated pathways in breast cancer.


METHODS
Using a recently developed technology for extraction of full-length proteins from FFPE tissues, we evaluated >50 commercial antibodies for specificity using Western blots and protein microarrays. Purified HER receptor proteins were used to determine absolute protein concentrations.


RESULTS
We confirmed specificity of 23 commercially available phosphospecific and non-phosphospecific antibodies using Western blots with protein extracts from cell lines and tissue extracts from breast cancer patients. Spiking known amounts of purified HER receptor proteins in HER receptor negative tissue extracts allowed us to precisely measure abundances of HER-receptors.


CONCLUSIONS
Our results will provide a basis for the development of diagnostic techniques for the quantitative analysis of deregulated HER receptors and downstream signalling proteins in typical clinical tissues.
Berg
D
D
Institut für Pathologie, Technische Universität München, München.
Bronger
H
H
Walch
A
A
Höfler
H
H
Becker
K-F
KF
D014408:Biomarkers, Tumor / Q000235:genetics*; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology*; D045744:Cell Line, Tumor; D005260:Female; D005557:Formaldehyde; D006801:Humans; D016612:Paraffin Embedding; D011237:Predictive Value of Tests; D040081:Protein Array Analysis; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics*; D016707:Tissue Fixation
D014408:Biomarkers, Tumor; D005557:Formaldehyde; D018719:Erb-b2 Receptor Tyrosine Kinases; C508053:ERBB2 protein, human