Fully Automated Dual-Color Dual-Hapten Silver In-Situ Hybridization Staining for MYC Amplification: A Diagnostic Tool for Discriminating Secondary Angiosarcoma
J S Ko, S D Billings, A P Fernandez, C P Lanigan, R R Tubbs. Cleveland Clinic, Cleveland, OH
Background: MYC amplification has been shown to selectively occur in secondary cutaneous angiosarcoma. We have tested the ability of automated dual-color dual-hapten silver in-situ hybridization (DDISH) staining to correlate with fluorescence in-situ hybridization (FISH) for MYC amplification and to discriminate secondary angiosarcoma (SAS) from atypical vascular lesions (AVL).
Design: Cases of SAS (n=7) and AVL (n=3) were retrieved and examined by FISH and DDISH. DDISH staining was performed using the Dual Color Open Probe software on a Ventana Benchmark XT automated slide stainer. DNP labeled/repeat depleted MYC and DIGoxigenin labeled Chromosome 8 (CHR8) probes were combined into one Prep Kit Dispenser. Pretreatment conditions were: Extended deparaffinization at 72°C, 3 cycles of cell conditioning with CC2 for 12 minutes at 80°C, and protease digestion with ISH protease 3 for 32 minutes. Denaturation was at 80°C for 12 minutes; hybridization at 44°C for 6 hours was followed by stringency washes (3 at 72°C, 8 minutes). MYC DNP probe was detected with ultraView silver ISH (SISH) DNP Detection Kit silver anti-hapten antibody (20 minutes), and SISH detection (8 minutes). CHR8 DIG probe was detected with ultraView Red ISH DIG Detection Kit; anti-hapten antibody incubated (20 minutes) and Red detection (8 minutes). The slides were counterstained with hematoxylin II for 8 minutes, post counterstained with bluing reagent for 4 minutes, and mounted as permanent slides for bright field microscopy. Metallic black silver (MYC) and reference CHR8 red signals were qualitatively and semi-quantitatively enumerated for tumor nuclei. Small and large clusters of silver signals were recorded as 6 or 12 signals respectively. MYC amplification was defined as MYC/CHR8 ratio >2.0.
Results: Where tissue was available for both DDISH and FISH, all SAS cases demonstrated MYC amplification (7/7=100%) by both DDISH and FISH. All AVL were negative for MYC amplification by both techniques (0/3=0%).
Conclusions: In this cohort of cases, use of DDISH identified all MYC amplified cases, and distinguished SAS from AVL. These data suggest that DDISH staining may be useful in distinguishing SAS from AVL in challenging cases.
Tuesday, March 20, 2012 9:30 AM
Poster Session III # 91, Tuesday Morning