Zytodot 2c spec met cen7 probe

Analysis of MET amplification was performed by CISH using the ZytoDot 2C SPEC MET/CEN7 Probe and the ZytoDot 2C CISH Implementation Kit (ZytoVision GmbH, Bremerhaven, Germany).
The results of CISH were evaluated by screening the entire tissue sections in order to find, where present, MET‐amplified invasive cancer areas. Subsequently, MET and centromer 7 signals were counted in at least 20 representative adjacent cancer cell nuclei within the invasive region. Forty nuclei were counted when the MET/centromer 7 ratio ranged from 1.8 to 2.2. The presence of CISH clusters was noted and the ratio of MET/centromer 7 signals was calculated. The gene count was calculated by dividing the number of MET gene signals by the number of cancer cell nuclei studied.

The HER2-and MET-status was assessed as previously described [11] (link), [12] (link), [16] (link), [17] (link), [18] (link) using immunohistochemistry [anti-Her2/neu antibody; clone SP3, Thermo Fisher Scientific; Fremont; USA (#MA5-14509); anti-MET antibody; clone SP44; Spring Bioscience; Pleasanton, California, USA (#M3444)] and in situ–hybridization [ZytoDot 2C SPEC HER2/CEN17 Probe (#C-3032-400), ZytoDot 2C SPEC MET/CEN7 Probe (#C-3057-400) and the ZytoDot 2C CISH Implementation Kit (#C-3044-40); ZytoVision GmbH, Bremerhaven, Germany)]. Epstein–Barr virus (EBV)–encoded RNA was detected using the EBER-probe (Novocastra, Leica Microsystems GmbH, Nussloch, Germany; #PB0589) and the BondMax-detection system according to the manufacturer's instructions (Leica Microsystems GmbH). MSI status was assessed by immunostaining using antibodies directed against MLH1 (clone G168-15, BD Biosciences, Heidelberg, Germany; #MA1-25669), PMS2 (clone MRQ-28; Cell Marque Corporation, Rocklin, USA; #288M-16-ASR), MSH2 (clone FE11; Calbiochem, Merck KGaA, Darmstadt, Germany; #MABE284), and MSH6 (clone 44, BD Biosciences; #610919) as well as by comparison of the allelic profiles of the mononucleotide repeat markers BAT-25, BAT-26, NR-21, NR-24, and NR-27 in tumor and corresponding normal tissue in cases with ambiguous immunostaining [16] (link).