A technical assessment of the utility of reverse phase protein arrays for the study of the functional proteome in non-microdissected human breast cancers

Bryan T. Hennessy, Yiling Lu, Ana Maria Gonzalez-Angulo, Mark S. Carey, Simen Myhre, Zhenlin Ju, Michael A. Davies, Wenbin Liu, Kevin Coombes, Funda Meric-Bernstam, Isabelle Bedrosian, Mollianne McGahren, Roshan Agarwal, Fan Zhang, Jens Overgaard, Jan Alsner, Richard M. Neve, Wen Lin Kuo, Joe W. Gray, Anne Lise Borresen-DaleGordon B. Mills

Research output: Contribution to journalArticle

145 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)129-151
Number of pages23
JournalClinical Proteomics
Volume6
Issue number4
DOIs
StatePublished - Dec 1 2010

Keywords

  • Breast cancer
  • Functional proteome
  • Kinase signaling
  • RPPA
  • Steroid signaling

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Clinical Biochemistry

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    Hennessy, B. T., Lu, Y., Gonzalez-Angulo, A. M., Carey, M. S., Myhre, S., Ju, Z., Davies, M. A., Liu, W., Coombes, K., Meric-Bernstam, F., Bedrosian, I., McGahren, M., Agarwal, R., Zhang, F., Overgaard, J., Alsner, J., Neve, R. M., Kuo, W. L., Gray, J. W., ... Mills, G. B. (2010). A technical assessment of the utility of reverse phase protein arrays for the study of the functional proteome in non-microdissected human breast cancers. Clinical Proteomics, 6(4), 129-151. https://doi.org/10.1007/s12014-010-9055-y