Aims: We compared the ¡¥sensitivity¡¦ and ¡¥specificity¡¦ of new rabbit monoclonal antibody SP3 with those of mouse monoclonal and rabbit polyclonal antibodies to detect HER2 amplification as defined by chromogenic in situ hybridization (CISH). Methods and Results: Serial sections from tissue microarrays (TMAs) containing 84 breast carcinomas were submitted to CISH (Zymed HER2 Spot-Light„¥ kit) and immunohistochemistry, using NeoMarkers SP3 (rabbit monoclonal), DAKO A0485 and DAKO HercepTest (polyclonal), Novocastra NCL-CB11, Cell Marque CM-CB11, and Genentech 4D5 (mouse monoclonal). The best antibody concordance was between SP3 and HercepTest (kappa=0.74). SP3, A0485 and HercepTest detected all HER2 amplified tumours, but were less ¡¥specific¡¦ than mouse monoclonal antibodies. There were 3/38 (7.9%) and 8/38 (21.0%) of non-amplified tumours scored as 3+ using SP3 and A0485, respectively. 3/46 (6.5%) of amplified tumours were negative for NCL-CB11. SP3, HercepTest and A0485 showed no gene amplification on 55%, 62.5% and 92.3% of the 2+ scored tumours, but most of the 2+ scored tumours using monoclonal antibodies were amplified by CISH (80% to 92.3%). Conclusions: SP3 is more ¡¥sensitive¡¦ than mouse monoclonal antibodies for Her2 assessment. However, HercepTest, CB11 and 4D5 show higher ¡¥specificity¡¦ than SP3 for the identification of HER2 gene amplification. Mouse monoclonal antibodies show less Her2 2+ tumours and most of them are amplified by CISH. Word-count: 212
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