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Breast Cancer Research

Open Access

Employing multiplex immunofluorescence to quantify Her2 and phosphorylated Her2 in formalin-fixed, paraffin-embedded breast tumor specimens

  • MJ Donovan1, 2,
  • P Puig2,
  • N Erill2,
  • M Clayton1,
  • S Hamann1,
  • F Khan1,
  • D Powell1,
  • J Costa1, 2, 3,
  • C Cordon-Cardo1, 2, 4 and
  • J Baselga5
Breast Cancer Research200911(Suppl 1):P30

Published: 23 June 2009


TrastuzumabFeature SelectionHer2 StatusBreast Tumor CellHer2 Overexpression


Her2 overexpression is a predictor for response to trastuzumab; however, only one-third of patients initially respond and the majority progress within 1 year. Recently, levels of the phosphorylated form of Her2 (pHer2) in hormone receptor-positive, primary tumors was determined to be an independent predictor for poor disease-free and overall survival, suggesting that additional variables may be important for determining outcome. We sought to develop a multiplex immuno-fluorescent (IF) quantitative assay for Her2 that included pHer2 and to compare the initial results with traditional methods of evaluating Her2 levels in formalin-fixed, paraffin-embedded (FFPE) breast tissue specimens.


We developed a Her2 multiplex IF assay (Her2mplex) composed of AE1/AE3 (cytokeratin), TAB250 (Her2-ECD), c-erb B2 (Her2), and Her2 pY1248 (pHer2) utilizing breast tumor cell lines (BTC: MCF7, T47D and SKBR3) that differentially express Her2 and pHer2. The assay was applied to a 37-patient tissue microarray (TMA) with known Her2 status (imunohistochemical (IHC) HercepTest). Image analysis generated quantitative IF intensity features that were evaluated univariately, and then multivariately with feature selection to discriminate Her2 (2+) from Her2 (3+). The AUC was used to estimate performance.


We developed baseline intensity thresholds from the component biomarkers in the Her2mplex (TAB250 Her2, c-erb B2 and phosphorylated Her2) utilizing a series of BTC: MCF7, T47D and SKBR3. IF images were acquired from 72 TMA cores, and 52 cores (26 patients) had appropriate tumor content (>50%) and quality for analysis. Both c-erB B2 and pHer2 were significantly associated with IHC data and were selected (AUC = 0.96, sensitivity = 0.91, specificity = 1) to discriminate Her2 (2+) from Her2 (3+).


Quantitation of Her2 and pHer2 by IF is feasible in FFPE breast tumor specimens. The results correlate with IHC protein data and also provide a quantitative strategy for the development of a standardized assay useful for tumor phenotyping, patient stratification and therapeutic indication.

Authors’ Affiliations

Aureon Laboratories, Yonkers, USA
, Althia, Spain
Yale University School of Medicine, New Haven, USA
Columbia University, New York, USA
Vall d'Hebron University Hospital, Barcelona, Spain


© BioMed Central Ltd. 2009