Isis imaging software

DNA fluorescence in situ hybridization (DNA-FISH) analysis was performed on paraffin sections using a three-colour probe designed to detect copy-number changes of chromosome 2, 9 and 10. The bacterial artificial chromosome clones used in the probe mix were based on sequencing data (minimal region of gain/loss) and are as follows: 9qC (RP23-248H6, RP23-340D4, RP23-60M16; labelled with Spectrum Red) and 2qC (RP23-332C13, RP23-186P20, RP23-435A5; labelled with Spectrum Green). All RP11 clones were purchased from the Roswell Park Cancer Institute Genomics Shared Resource. Probe labelling, hybridization, post-hybridization washing and fluorescence detection were performed according to procedures established at the Molecular Cytogenetics Core Facility. Slides were scanned using a Zeiss Axioplan 2i epifluorescence microscope (Carl Zeiss Microscopy) equipped with Isis imaging software (MetaSystems). The entire section was first scanned through 63× to assess signal pattern. Corresponding H&E and/or immunostained slides were used to identify regions of premalignant or cancer morphology (foci of adenocarcinoma). Regions selected for analysis were imaged through the depth of the tissue (compressed stack of 12 z-sections at 0.5 μm intervals) and, for each case, within each representative region at least 50 discrete nuclei were scored.

Imaging was performed using a Zeiss Axioplan 2 microscope with ×20, ×63, or ×100 objectives (Plan-NEOFLUAR ×20/0.5, Plan-APOCHROMAT ×63/1.4 Oil, Plan-NEOFLUAR ×100/1.3 Oil) and Meta Systems ISIS imaging software. ImageJ was used to process micrographs, which included cropping of images and minimal adjustment of signal intensity. All images of any experiment were processed in the same way.

ROS1 and RET FISH assays were performed on 4-μm-thick sections of FFPE tissue blocks using ZytoLight SPEC ROS1 and RET dual color break-apart probes according to the manufacturer’s instructions (ZytoVision, Bremerhaven, Germany). The design of the probes is depicted in Figure 1: the 5′ orange and 3′ green probes hybridized proximal and distal to the kinase domain, respectively. The slides were analyzed by 2 experienced cytogeneticists (FD and FC) by using a fluorescence microscope (Axioskop2, Axio Imager Z2, Zeiss, Göttingen, Germany) and Isis imaging software (Metasystems, Altlussheim, Germany). Per case, at least 100 non-overlapping tumor nuclei were examined. A sample was considered positive for rearrangement if at least 15% of the nuclei showed split signals or isolated 3′ signals. Isolated 5′ signals were thought to result from the deletion of exons encoding the kinase domain and were considered negative. Gene copy number per nucleus was recorded as follows: one copy, two copies, low copy number gain (3 to 6 copies), high copy number gain (7 to 10 copies) and amplification (> 10 copies or innumerable clusters).