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Wulfkuhle, J.D.* ; Berg, D.* ; Wolff, C.* ; Langer, R.* ; Tran, K.* ; Illi, J.* ; Espina, V.* ; Pierobon, M.* ; Deng, J.* ; Demichele, A.* ; Walch, A.K. ; Bronger, H.* ; Becker, I.* ; Waldhör, C.* ; Höfler, H.* ; Esserman, L.* ; Liotta, L.A.* ; Becker, K.F.* ; Petricoin, E.F.*

Molecular analysis of HER2 signaling in human breast cancer by functional protein pathway activation mapping.

Clin. Cancer Res. 18, 6426-6435 (2012)

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PURPOSE: Targeting of the HER2 protein in human breast cancer represents a major advance in oncology but relies on measurements of total HER2 protein and not HER2 signaling network activation. We used reverse-phase protein microarrays (RPMA) to measure total and phosphorylated HER2 in the context of HER family signaling to understand correlations between phosphorylated and total levels of HER2 and downstream signaling activity.EXPERIMENTAL DESIGN: Three independent study sets, comprising a total of 415 individual patient samples from flash-frozen core biopsy samples and formalin-fixed and paraffin-embedded (FFPE) surgical and core samples, were analyzed via RPMA. The phosphorylation and total levels of the HER receptor family proteins and downstream signaling molecules were measured in laser capture microdissected (LCM) enriched tumor epithelium from 127 frozen pretreatment core biopsy samples and whole-tissue lysates from 288 FFPE samples and these results were compared with FISH and immunohistochemistry (IHC).RESULTS: RPMA measurements of total HER2 were highly concordant (>90% all sets) with FISH and/or IHC data, as was phosphorylation of HER2 in the FISH/IHC(+) population. Phosphorylation analysis of HER family signaling identified HER2 activation in some FISH/IHC(-) tumors and, identical to that seen with FISH/IHC(+) tumors, the HER2 activation was concordant with EGF receptor (EGFR) and HER3 phosphorylation and downstream signaling endpoint activation.CONCLUSIONS: Molecular profiling of HER2 signaling of a large cohort of human breast cancer specimens using a quantitative and sensitive functional pathway activation mapping technique reveals IHC(-)/FISH(-)/pHER2(+) tumors with HER2 pathway activation independent of total HER2 levels and functional signaling through HER3 and EGFR.

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