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.
abstract: BACKGROUND
  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.
  
  
  CONCLUSIONS
  Thus, the robustness of RPPA and stability of the functional proteomic "fingerprint" facilitate the study of the functional proteome in non-microdissected breast tumors.
authors.affiliation: Department of Medical Oncology, Beaumont Hospital, Dublin, Ireland.
authors.forename: Bryan T
authors.initials: BT
authors.lastname: Hennessy
authors.forename: Yiling
authors.initials: Y
authors.lastname: Lu
authors.forename: Ana Maria
authors.initials: AM
authors.lastname: Gonzalez-Angulo
authors.forename: Mark S
authors.initials: MS
authors.lastname: Carey
authors.forename: Simen
authors.initials: S
authors.lastname: Myhre
authors.forename: Zhenlin
authors.initials: Z
authors.lastname: Ju
authors.forename: Michael A
authors.initials: MA
authors.lastname: Davies
authors.forename: Wenbin
authors.initials: W
authors.lastname: Liu
authors.forename: Kevin
authors.initials: K
authors.lastname: Coombes
authors.forename: Funda
authors.initials: F
authors.lastname: Meric-Bernstam
authors.forename: Isabelle
authors.initials: I
authors.lastname: Bedrosian
authors.forename: Mollianne
authors.initials: M
authors.lastname: McGahren
authors.forename: Roshan
authors.initials: R
authors.lastname: Agarwal
authors.forename: Fan
authors.initials: F
authors.lastname: Zhang
authors.forename: Jens
authors.initials: J
authors.lastname: Overgaard
authors.forename: Jan
authors.initials: J
authors.lastname: Alsner
authors.forename: Richard M
authors.initials: RM
authors.lastname: Neve
authors.forename: Wen-Lin
authors.initials: WL
authors.lastname: Kuo
authors.forename: Joe W
authors.initials: JW
authors.lastname: Gray
authors.forename: Anne-Lise
authors.initials: AL
authors.lastname: Borresen-Dale
authors.forename: Gordon B
authors.initials: GB
authors.lastname: Mills