Abstract P6-05-13: ESR1 gene amplification in breast cancer: influence of RNAse treatment on FISH results
Authors: Moelans CB , Holst F , Hellwinkel O , Simon R , van Diest PJ 
Published: August 2014

Abstract
Introduction The frequency of estrogen receptor 1 (ESR1) amplification in breast cancer has long been controversial. High frequencies of amplification were more likely to be reported when measured by FISH compared to other methods such as qPCR, CGH and multiplex-ligation dependent probe amplification (MLPA). It was recently reported that HSR-like FISH clusters were RNAse sensitive which could lead to overestimation of ESR1 amplification frequency by FISH. We therefore compared copy numbers by FISH without and with RNAse treatment with MLPA in 53 tumor samples from 24 tumors with ESR1 copy number increase by FISH.
Methods Seventy five tumour areas (for each tumor with increased ESR1 copy number at least one area with the highest amplified copy number and cell density by FISH (Zytovysion)) from 38 breast cancers were laser microdissected and subjected to MLPA (kit P078-B1). Twenty four of these tumors (15 amplified and 9 gained; 53 areas) were also treated with RNAse A to compare copy numbers without and with RNAse treatment (blinded).
Results RNAse treatment removed cloudy clusters but did not significantly change ESR1 gene copy numbers (p = 0.47; average copy number was 4.60 pre- and 4.44 post-RNAse). However, CEP6 copy numbers were slightly higher after RNAse treatment (p = 0.09; average copy number 2.17 pre- and 2.23 post-RNAse) leading to a significant decrease in ESR1/CEP6 ratios (p = 0.006; average ratio 2.16 pre- and 1.98 post-RNAse). 12/15 FISH-amplified cases were still amplified after RNAse treatment, whereas three cases were re-classified as gains (cut-off ratio 1.3). 7/9 FISH-gained cases remained gained after RNAse treatment, one case was reclassified as amplified and the other case as non-amplified. Spearman rho correlation between MLPA and FISH prior and after RNAse treatment was 0.57 and 0.56, respectively (both p &lt; 0.0001). Even after RNAse treatment, only 9/13 amplifications and 6/24 gains could be confirmed by MLPA as either gains or amplifications. The correlation between FISH with or without RNAse was 0.68 (p &lt; 0.0001).
Considerable heterogeneity was seen between different areas of the same tumor block and between tumor blocks. Of all samples with multiple tumor blocks and/or areas analyzed, 25% (5/20) showed a different amplification status by MLPA between or within blocks, and 39% (7/18) and 67% (8/12) by FISH without and with RNAse, respectively. The average copy number ratio variation between/within tumor blocks was 0.55/0.56 for FISH +/− RNAse, and 0.25 for MLPA. Also, mosaic heterogeneity (cell-to-cell variation) was frequently observed by FISH (present in 60% of the samples in this study).
Conclusion Cloudy clusters by FISH are RNAse sensitive and could lead to overestimation of copy numbers. However, after RNAse treatment, ESR1 copy numbers remain increased. These copy number increases could at least in some cases be confirmed by MLPA, suggesting that they are DNA-based. ESR1 copy number increases seem to occur quite frequently in breast cancer, but the levels are in most cases not high enough to designate the sample as “amplified” by all techniques. PCR-based methods could be influenced by heterogeneity or contamination with normal cells and reported results depend on the cut-offs used and on the definition of amplification.
Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-05-13.