Abstract P2-03-04: Application of digital-PCR technology to determine c-MET copy number variation in paired primary breast cancer and brain metastases

Abstract

Abstract INTRODUCTION: c-MET amplification/overexpression has been associated with treatment failure and progression in many cancers, including breast cancer (BC). c-MET showed amplification by fluorescent in situ hybridization (FISH) in 27% of trastuzumab-treated HER2-positive patients. These patients had a high trastuzumab failure rate and a shorter time to progression. Up to 50% of patients with metastatic HER2-positive disease will develop brain metastases (BM) during their disease course and in approximately one third, brain is the first site of progression. Amplification/copy number variations (CNVs) are mainly assessed by FISH whereas overexpression is assessed by immunohistochemistry (IHC). We present a PCR-based assay (digital-PCR) able to determine CNV in c-MET and HER2 in a cohort of patients with metastatic BC to the brain and demonstrate the correlation of CNV to protein expression. METHODS: DNA was isolated from paraffin-embedded tissues of 23 paired primary BC-BM cases. CNV was analysed by the QuantStudioTM 3D-Digital-PCR (QS3D) and real-time qPCR (both from ThermoFisher Scientific). The breast MCF7, T47D, BT474, AU565, SKBR3 and the gastric MKN45 cell lines were used as controls for the HER2 and c-MET CNV assays. Copy number per diploid genome was calculated using the absolute quantification number of FAM-labelled target and VIC-labelled RNaseP reference multiplied by 2. Cases with ≤2 copies are classified as normal whereas cases with >2 were classified as amplified. The HER2 positivity of the primary BC cases was routinely assessed by IHC. The c-MET protein expression was assessed by IHC using the c-MET(3D4) monoclonal antibody (ThermoFisher Scientific). RESULTS: CNV in c-MET by QS3D digital-PCR was detected in 69.6% of primary BC (ER-/HER2+:2, ER+/HER2+:5, ER+/HER2-:8, Triple-negatives:5, unknown:3) as well as 69.6% of BM, whereas HER2 CNV was observed in 39.1% primary BC and 52.2% BM. In the HER2-positive cases, the prevalence of HER2 CNV was 100% in both primary BC and BM. Within these cases, c-MET CNV was 85.7% in the primary BC and 71.43% in BM. CNVs in both genes were observed in 30.4 % of all primary and 39.1% of BM. The CNV data are presented in Table 1. CNV data for c-MET and HER2 between primary breast cancer (BC) and corresponding brain metastatic (BM) casesc-MET CNVHER2 CNVBCBMBCBM>2: 16>2: 12≤2: 4>2: 9>2: 8≤2: 1≤2: 7>2: 4≤2: 3≤2: 14>2: 4≤2: 10 There was a high concordance between the QS3D and qPCR data with Pearson's R=0.74 (p<0.00001). A significant correlation between HER2 protein expression and CNV was observed (Fisher's exact test p=0.0005). Data will be presented on c-MET protein expression in the pair samples. CONCLUSIONS: The prevalence of CNV is much higher than that reported by immunohistochemistry and FISH in the literature to date, possibly due to the sensitivity of the digital-PCR technology. The high level of c-MET CNV in primary and metastatic BC, and the concurrent CNV in both genes warrants further investigation. It also highlights the potential to use c-MET directed therapy particularly in HER2+ BC and reinforces the potential importance of precise detection methods in both the primary and metastatic setting. Analysis of a larger series is currently on-going. Citation Format: Giannoudis A, Zakaria R, Platt-Higgins A, Syed KAR, Ashton K, Dawson T, Rudland PS, Holcombe C, Jenkinson MD, Palmieri C. Application of digital-PCR technology to determine c-MET copy number variation in paired primary breast cancer and brain metastases [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-03-04.