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Clinical Chemistry 0: clinchem.2008.116632v1, 2009; 10.1373/clinchem.2008.116632
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Received on September 4, 2008
Accepted on April 3, 2009

Cancer Diagnostics

Validation of a Phosphoprotein Array Assay for Characterization of Human Tyrosine Kinase Receptor Downstream Signaling in Breast Cancer

Fadila Chergui 1, Anne-Sophie Chrétien 1, Sanae Bouali 1, Carole Ramacci 1, Marie Rouyer 1, Thierry Bastogne 2, Pascal Genin 3, Agnès Leroux 3, Jean-Louis Merlin 1*

1 Unité de Biologie des Tumeurs, and EA << Predicther >> Nancy Université, Centre Alexis Vautrin, Avenue de Bourgogne, 54511 Vandoeuvre-lès-Nancy, France
2 UMR7039 CNRS-UHP-INPL, Faculté des Sciences, 54506 Vandoeuvre-lès-Nancy Cedex, France
3 Laboratoire d'Anatomie Pathologique, and EA << Predicther >> Nancy Université, Centre Alexis Vautrin, Avenue de Bourgogne, 54511 Vandoeuvre-lès-Nancy, France

* To whom correspondence should be addressed. E-mail: jl.merlin{at}nancy.fnclcc.fr.

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® 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 -3{beta}, phosphorylated P70S6 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.




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Copyright © 2009 by the American Association for Clinical Chemistry.