Zytolight spec fgfr2 cen 10 dual color probe

FISH was performed using a ZytoLight SPEC FGFR2/CEN 10 Dual Color Probe (Z-2122–200, Zytovision, Bremerhaven, Germany). Respective DNA probe sets were applied to 1 μm-thick tumor sections and incubated overnight at 37°C. We counted the hybridization signals in 20 nuclei per sample under a fluorescence microscope. All overlapping nuclei were excluded and only nuclei with a distinct nuclear border were evaluated. The FGFR2 gene was considered amplified when the FGFR2:CEP17 FISH-signal ratio was ≥ 2.0.
IHC was performed for FGFR2 on formalin-fixed, paraffin-embedded, 4-μm-thick tissue sections with a primary mouse monoclonal anti-human FGFR2 antibody (MAB6841, R&D Systems, Minneapolis, MN). The tissue sections were deparaffinized and rehydrated. After antigen retrieval and endogenous peroxidase blocking, the samples were incubated with primary antibody for 15 min. A BOND-MAX autoimmunostainer (Leica Biosystems, Melbourne, Australia) with Bond™ Polymer Refine Detection, DS9800 (Vision Biosystems, Melbourne, Australia), was used according to the manufacturer's protocol.

We used FISH to evaluate the FGFR2 amplification status using the Zytolight SPEC FGFR2/CEN 10 Dual Color Probe (Zytovision, Germany) according to the manufacturer’s protocol. We processed samples as described previously (Loeser et al. 2017 (link)). We scanned tumor tissue for gene copy gains including chromosomal cluster amplifications hot spots using a 63 × objective (DM5500 fluorescent microscope; Leica, Germany). If the signals were distributed homogeneously, then we used random areas to count the signals. We evaluated 20 tumor cells by counting green FGFR2 and orange centromere signals. The reading strategy for detecting amplifications followed the recommendations of an FGFR2/CEN10 ratio > 2.0 or FGFR2 extrachromosomal cluster amplification signals (O’Sullivan et al. 2014 (link)). Because there are still insufficient data on the distribution of FGFR2-amplified tumor cells in GIT adenocarcinomas, we semi-quantitatively documented all amplified tumor cells as a percentage of all tumor cells examined in a sample.