AimsHER2testing of invasive breast cancer byin‐situhybridization guides therapy decisions. ProbingHER2and centromere of chromosome 17 (cen17) simultaneously is supposed to reveal both a potentialHER2gene amplification and polysomy 17. However, a considerable number of breast cancer patients with quasi polysomy 17 are considered ‘equivocal’, which is diagnostically meaningless. Moreover, patients with equivocal/false polysomic tumours are prevented from a potentially beneficial anti‐HER2treatment. Here we evaluated theRAI1,D17S122andTP53hybridization markers to indicate true polysomy reliably and to reclassify equivocal samples accurately asHER2‐positive.Methods and resultsSamples with (n= 82) and without (n= 52) increased cen17 copy numbers and 78 evidentlyHER2‐amplified specimens were analysed using dual and triple marker hybridization probes. Selected putative polysomic samples were subjected to array‐based comparative genomic hybridization (aCGH). We found that 37.8% samples with putative polysomy 17 did not show any gain inRAI1,D17S122orTP53. Accordingly, aCGH revealed evidence for the presence ofHER2/cen17 co‐amplification rather than for true polysomy in those cases. Reflex testing using alternate 17p markers reclassified up to 56.3% equivocal cases asHER2‐positive and the combined assessment of a 17p and 17q locus allows the differentiation of true versus false polysomy.ConclusionsAn increased cen17 copy number does not necessarily reflect polysomy, and reflex testing facilitates the reclassification of ‘equivocals’. Nevertheless, the prognostic and predictive value of aHER2/cen17 co‐amplification versusHER2gene amplification alone must still be evaluated prospectively.