A Technical Assessment of the Utility of Reverse Phase Protein Arrays for the Study of the Functional Proteome in Non-microdissected Human Breast Cancers.
Hennessy, Bryan T Lu, Yiling Gonzalez-Angulo, Ana Maria Carey, Mark S Myhre, Simen Ju, Zhenlin Davies, Michael A Liu, Wenbin Coombes, Kevin Meric-Bernstam, Funda
Hennessy, Bryan T
Lu, Yiling
Gonzalez-Angulo, Ana Maria
Carey, Mark S
Myhre, Simen
Ju, Zhenlin
Davies, Michael A
Liu, Wenbin
Coombes, Kevin
Meric-Bernstam, Funda
Author
Hennessy, Bryan T
Lu, Yiling
Gonzalez-Angulo, Ana Maria
Carey, Mark S
Myhre, Simen
Ju, Zhenlin
Davies, Michael A
Liu, Wenbin
Coombes, Kevin
Meric-Bernstam, Funda
Bedrosian, Isabelle
McGahren, Mollianne
Agarwal, Roshan
Zhang, Fan
Overgaard, Jens
Alsner, Jan
Neve, Richard M
Kuo, Wen-Lin
Gray, Joe W
Borresen-Dale, Anne-Lise
Mills, Gordon B
Lu, Yiling
Gonzalez-Angulo, Ana Maria
Carey, Mark S
Myhre, Simen
Ju, Zhenlin
Davies, Michael A
Liu, Wenbin
Coombes, Kevin
Meric-Bernstam, Funda
Bedrosian, Isabelle
McGahren, Mollianne
Agarwal, Roshan
Zhang, Fan
Overgaard, Jens
Alsner, Jan
Neve, Richard M
Kuo, Wen-Lin
Gray, Joe W
Borresen-Dale, Anne-Lise
Mills, Gordon B
Advisors
Editors
Other Contributors
Date
2010-12
Date Submitted
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Other Subjects
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Planned Date
Start Date
Collaborators
Principal Investigators
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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.
Language
en
ISSN
1559-0275
eISSN
ISBN
DOI
10.1007/s12014-010-9055-y
PMID
21691416