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

Hdl Handle:
http://hdl.handle.net/10147/136358
Title:
A Technical Assessment of the Utility of Reverse Phase Protein Arrays for the Study of the Functional Proteome in Non-microdissected Human Breast Cancers.
Authors:
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
Affiliation:
Department of Medical Oncology, Beaumont Hospital, Dublin, Ireland.
Citation:
A Technical Assessment of the Utility of Reverse Phase Protein Arrays for the Study of the Functional Proteome in Non-microdissected Human Breast Cancers. 2010, 6 (4):129-151notClin Proteomics
Journal:
Clinical proteomics
Issue Date:
Dec-2010
URI:
http://hdl.handle.net/10147/136358
DOI:
10.1007/s12014-010-9055-y
PubMed ID:
21691416
Additional Links:
http://www.ncbi.nlm.nih.gov/pubmed/21691416
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.
Item Type:
Article
Language:
en
ISSN:
1559-0275

Full metadata record

DC FieldValue Language
dc.contributor.authorHennessy, Bryan Ten
dc.contributor.authorLu, Yilingen
dc.contributor.authorGonzalez-Angulo, Ana Mariaen
dc.contributor.authorCarey, Mark Sen
dc.contributor.authorMyhre, Simenen
dc.contributor.authorJu, Zhenlinen
dc.contributor.authorDavies, Michael Aen
dc.contributor.authorLiu, Wenbinen
dc.contributor.authorCoombes, Kevinen
dc.contributor.authorMeric-Bernstam, Fundaen
dc.contributor.authorBedrosian, Isabelleen
dc.contributor.authorMcGahren, Mollianneen
dc.contributor.authorAgarwal, Roshanen
dc.contributor.authorZhang, Fanen
dc.contributor.authorOvergaard, Jensen
dc.contributor.authorAlsner, Janen
dc.contributor.authorNeve, Richard Men
dc.contributor.authorKuo, Wen-Linen
dc.contributor.authorGray, Joe Wen
dc.contributor.authorBorresen-Dale, Anne-Liseen
dc.contributor.authorMills, Gordon Ben
dc.date.accessioned2011-07-19T14:17:00Z-
dc.date.available2011-07-19T14:17:00Z-
dc.date.issued2010-12-
dc.identifier.citationA Technical Assessment of the Utility of Reverse Phase Protein Arrays for the Study of the Functional Proteome in Non-microdissected Human Breast Cancers. 2010, 6 (4):129-151notClin Proteomicsen
dc.identifier.issn1559-0275-
dc.identifier.pmid21691416-
dc.identifier.doi10.1007/s12014-010-9055-y-
dc.identifier.urihttp://hdl.handle.net/10147/136358-
dc.description.abstractINTRODUCTION: 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.-
dc.languageENG-
dc.language.isoenen
dc.relation.urlhttp://www.ncbi.nlm.nih.gov/pubmed/21691416en
dc.titleA Technical Assessment of the Utility of Reverse Phase Protein Arrays for the Study of the Functional Proteome in Non-microdissected Human Breast Cancers.en
dc.typeArticleen
dc.contributor.departmentDepartment of Medical Oncology, Beaumont Hospital, Dublin, Ireland.en
dc.identifier.journalClinical proteomicsen
dc.description.provinceLeinster-

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