Validation of a phosphoprotein array assay for characterization of human tyrosine kinase receptor downstream signaling in breast cancer.
BACKGROUND
Human epidermal growth factor receptor (HER) downstream signaling kinases have important effects on tumor response to anti-HER monoclonal antibodies and tyrosine kinase inhibitors. We validated an assay that uses phosphoprotein arrays for measurement of HER downstream signaling functionality in breast carcinomas.


METHODS
Using the Bio-Plex(R) phosphoprotein array (BPA), we performed multiplex immunoanalysis to investigate the expression of phosphorylated epidermal growth factor receptor and phosphorylated HER downstream signaling proteins (phosphorylated protein kinase B, phosphorylated glycogen synthase kinase -3beta, phosphorylated P70 ribosomal protein S6 kinase, and phosphorylated extracellular signal regulated kinase 42/44) in 49 frozen specimens of ductal infiltrating breast carcinoma taken at diagnosis. BPA was cross-validated with Western blot analysis. Sample size, homogenicity, tumor content, protein extraction, and monoclonal antibody detection were in accordance with optimized standard operating procedures.


RESULTS
Linear regression showed significant quantitative correlations between BPA and Western blot, with regression coefficient values of 0.71-0.87 (P < 0.001). BPA intra- and interassay CVs were <17% and 15%, respectively. Compared to limits of detection established by using the mean + 3SD of 10 blanks, large variations of phosphoprotein expression, up to several hundred-fold, were observed among the 49 tumor specimens.


CONCLUSIONS
Our results validate the use of the multiplex phosphoprotein array assay in human clinical tumor specimens. Further prospective evaluation is warranted to investigate the use of HER downstream signaling phosphoproteins as predictive and/or surrogate markers for clinical response to anti-HER targeted therapy.
Chergui
Fadila
F
Unité de Biologie des Tumeurs, Centre Alexis Vautrin, Vandoeuvre-lès-Nancy, France.
Chrétien
Anne-Sophie
AS
Bouali
Sanae
S
Ramacci
Carole
C
Rouyer
Marie
M
Bastogne
Thierry
T
Genin
Pascal
P
Leroux
Agnès
A
Merlin
Jean-Louis
JL
D015153:Blotting, Western; D001943:Breast Neoplasms / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D007150:Immunohistochemistry; D010750:Phosphoproteins / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D015203:Reproducibility of Results; D015398:Signal Transduction
D010750:Phosphoproteins; D020794:Receptor Protein-Tyrosine Kinases