Vysis alk break apart fish probe kit

EGFR mutations and ALK fusions were detected by conventional methods in the specimen diagnosed with adenocarcinoma and NSCLC-NOS. EGFR mutations were detected by amplification refractory mutation system–polymerase chain reaction using EGFR 21 Mutations Detection Kit (Amoy Diagnostics, Xiamen, China). DNA was extracted from 10 to 15 unstained formalin-fixed paraffin-embedded (FFPE) sections, each 5 μm thickness, using QIAamp DNA FFPE tissue kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. The concentration of DNA was measured by SMA4000 spectrophotometer (Merinton, Beijing, China). ALK fusion was tested by IHC using VENTANA ALK (D5F3) assay (F. Hoffmann-La Roche, Tucson, AZ, USA). ALK IHC weakly positive samples were confirmed by fluorescence in situ hybridization (FISH) using Vysis ALK Break Apart FISH Probe Kit (Abbott Molecular, Inc., IL, USA).[15 (link)]

Echinoderm microtubule-associated protein-like 4 (EML4)-ALK translocation was assessed using Vysis ALK Break Apart FISH probe kit (Abbott Molecular, Inc., Des Plaines, IL, USA), which is a US Food and Drug Administration-approved test. It is designed to detect ALK in chromosome 2p23 in formalin-fixed tissue with two adjacent probes: One at the 3′ end (orange) and one at the 5′ end (green) of ALK. Pretreatment, protease digestion, overnight probe hybridization, post-washing and DAPI counterstaining were performed on each sample according to the manufacturer's protocol. For each test, ALK-positive NSCLC tissue was used as a positive control. Normal cells and tumor cells without ALK translocation typically exhibit two fusion signals, whereas cells with ALK translocation exhibit a characteristic ALK split pattern. Processed slides were automatically scanned using a BioView™ workstation (BioView; Abbott Molecular, Inc.) and a preliminary interpretation was made by the system.

The ALK genetic status of each patient was evaluated by FISH in representative tumor areas, with an LSI ALK dual-color, break-apart rearrangement probe (Vysis ALK Break Apart FISH Probe Kit, Abbott Molecular Inc.), following the manufacturer's protocol. In each case, 50 non-overlapping nuclei were examined by fluorescence microscope (BX51; Olympus Corporation, Barcelona, Spain). The kit included red (3′) and green (5′) break-apart probes. ALK was determined to be not rearranged when the two signals were adjacent or fused (appearing yellow under an Orange/Green V2 filter) or when there was a single green signal. ALK was considered positive (rearranged) when ≥1 set of red and green signals were ≥2 signal diameters apart, or when a single red signal was present. A sample was considered negative for ALK if <5 cells out of 50 (<10%), and positive when >25 cells out of 50 (>50%), exhibited split ALK 5′ and 3′ probe signals or isolated 3′ signals. With borderline results, when the number of positive cells was 5–25 (10–50%), a second reader evaluated the slide: First and second cell count readings were added together and a percentage out of 100 cells was calculated. If the positive cell percentage was ≥15%, the sample was considered positive.