Multiplex Ligation-Dependent Probe Amplification to Detect HER2 Amplification in Breast Cancer: New Insights in Optimal Cut-Off Value

In Volume 31 of Cellular Oncology (2009), we published an article titled “HER2-neu amplification in breast cancer by multiplex ligation-dependent probe amplification in comparison with immunohistochemistry and in situ hybridization” [3]. To analyze our multiplex ligation-dependent probe amplification (MLPA) data we used a cut-off value of 1.5 to discriminate between HER2 non-amplified and low-level amplified patients. This cut-off was at that time empirically established in our lab during routine diagnostic application of MLPA kits for trisomy detection. However, based on recently published data [1,2,4], we now believe that a cut-off value of 1.3 (delta value 0.3) instead of 1.5 is better validated and more closely reflects the amplification status. We therefore re-analyzed our data with 1.3 as a cut-off value. HER2 amplification status by MLPA was normal in 82% of cases, low level amplified in 7% and high level amplified, as before, in 11% of cases. Of all immunohistochemistry (IHC) negative cases, 95% were MLPA normal, and in the group of IHC 1+ cases, 88% were MLPA normal. In these IHC 0 and 1+ cases, 4% and 10% were MLPA low level amplified, respectively. In the IHC 3+ group there was no change in the percentage of MLPA normal and low-level amplified cases. In the IHC 2+ group discrepancies with MLPA were, as expected, most pronounced: 59% was not amplified, 22% low level amplified and 19% amplified. Overall, there was 87.5% agreement between both techniques, which is slightly lower than with the former 1.5 cut-off value (90%). Correlation of MLPA with fluorescence in situ hybridization (FISH, selected cases) and chromogenic in situ hybridization (CISH, consecutive cases) was 73% and 91%, respectively, with corresponding Spearman correlation coefficients of 0.78 and 0.83. None of the