A Technical Assessment of the Utility of Reverse Phase Protein Arrays for the Study of the Functional Proteome in Non-microdissected Human Breast Cancers

Springer Science and Business Media LLC - 2010
Bryan T. Hennessy1,2, Yiling Lu3, Ana M. González-Angulo1, Mark Carey3, Simen Myhre4,5, Ju Zhang6, Michael A. Davies7, Wenbin Liu6, Kevin R. Coombes6, Funda Meric‐Bernstam8, Isabelle Bedrosian8, Mollianne J. McGahren3, Roshan Agarwal3, Fan Zhang3, Jens Overgaard9, Jan Alsner9, Richard M. Neve10, Wen Lin Kuo10, Joe W. Gray11, Anne Lise Børresen-Dale5,4, Gordon B. Mills1
1Kleberg Center for Molecular Markers, The University of Texas M. D. Anderson Cancer Center (MDACC), Houston, USA
2Department of Medical Oncology, Beaumont Hospital, Dublin, Ireland
3Department of Systems Biology, The University of Texas M. D. Anderson Cancer Center (MDACC), Houston, USA
4Faculty Division, The Norwegian Radium Hospital, Faculty of Medicine, University of Oslo, Oslo, Norway
5Department of Genetics, Institute for Cancer Research, Norwegian Radium Hospital, Rikshospitalet University Hospital, Oslo, Norway
6Department of Bioinformatics and Computational Biology, The University of Texas M. D. Anderson Cancer Center (MDACC), Houston, USA
7Department of Melanoma Medical Oncology, The University of Texas M. D. Anderson Cancer Center (MDACC), Houston, USA
8Department of Surgical Oncology, The University of Texas M. D. Anderson Cancer Center (MDACC), Houston, USA
9Department of Experimental Clinical Oncology, Aarhus University Hospital, Aarhus, Denmark
10Lawrence Berkeley National Laboratory, Berkeley, USA
11Lawrence-Berkeley National Laboratory, Berkeley, USA

Tóm tắt

Abstract Introduction The lack of large panels of validated antibodies, tissue handling variability, and intratumoral heterogeneity potentially hamper comprehensive study of the functional proteome in non-microdissected solid tumors. The purpose of this study was to address these concerns and to demonstrate clinical utility for the functional analysis of proteins in non-microdissected breast tumors using reverse phase protein arrays (RPPA). Methods Herein, 82 antibodies that recognize kinase and steroid signaling proteins and effectors were validated for RPPA. Intraslide and interslide coefficients of variability were <15%. Multiple sites in non-microdissected breast tumors were analyzed using RPPA after intervals of up to 24 h on the benchtop at room temperature following surgical resection. Results Twenty-one of 82 total and phosphoproteins demonstrated time-dependent instability at room temperature with most variability occurring at later time points between 6 and 24 h. However, the 82-protein functional proteomic “fingerprint” was robust in most tumors even when maintained at room temperature for 24 h before freezing. In repeat samples from each tumor, intratumoral protein levels were markedly less variable than intertumoral levels. Indeed, an independent analysis of prognostic biomarkers in tissue from multiple tumor sites accurately and reproducibly predicted patient outcomes. Significant correlations were observed between RPPA and immunohistochemistry. However, RPPA demonstrated a superior dynamic range. Classification of 128 breast cancers using RPPA identified six subgroups with markedly different patient outcomes that demonstrated a significant correlation with breast cancer subtypes identified by transcriptional profiling. Conclusion Thus, the robustness of RPPA and stability of the functional proteomic “fingerprint” facilitate the study of the functional proteome in non-microdissected breast tumors.

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