Automated brightfield break-apart in situ hybridization (ba-ISH) application: ALK and MALT1 genes as models.
Cancer diagnosis can be a complex process, which takes consideration of histopathological, clinical, immunophenotypic, and genetic features. Since non-random chromosomal translocations are specifically involved in the development of various cancers, the detection of these gene aberrations becomes increasingly important. In recent years, break-apart (or split-signal) fluorescence in situ hybridization (FISH) has emerged as an advantageous technique to detect gene translocations on tissue sections. However, FISH assays are technically challenging and require specialized fluorescence microscopes. Furthermore, the FISH signal is not stable for long term archiving due to photo bleaching. Our objective was to demonstrate the feasibility of brightfield break-apart in situ hybridization (ba-ISH) for anaplastic lymphoma kinase (ALK) and mucosa-associated lymphoid tissue translocation protein 1 (MALT1) genes as models. ALK or MALT1 break-apart probes were labeled with digoxigenin (DIG) or 2,4-dinitrophenyl (DNP) on both sides of a known gene breakpoint region and the hybridization sites were visualized with the combination of alkaline phosphatase (AP)-based blue and red detection. Therefore, normal genes are detected as purple dots by mixing blue and red colors while translocated genes are detected as isolated blue or red dots. Formalin-fixed, paraffin-embedded tonsil was used as control for the co-localized 5' and 3' probes. Gene translocations of ALK or MALT1 were detected as separate blue and red dots on ALCL and MALT lymphoma cases. Thus, ISH analyses of gene translocations can be conducted with a regular light microscope and the long term archiving of break-apart ISH slides can be achieved.
Nitta
Hiroaki
H
Office of Medical Affairs, Ventana Medical Systems, Inc., 1910 E Innovation Park Drive, Tucson, AZ 85755, USA. hiro.nitta@ventana.roche.com
Zhang
Wenjun
W
Kelly
Brian D
BD
Miller
Melanie
M
Pestic-Dragovich
Lidija
L
Bieniarz
Christopher
C
Vasicek
Thomas J
TJ
Marafioti
Teresa
T
Rimsza
Lisa
L
Grogan
Thomas M
TM
D000077548:Anaplastic Lymphoma Kinase; D020169:Caspases / Q000235:genetics*; D015342:DNA Probes; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D018442:Lymphoma, B-Cell, Marginal Zone / Q000235:genetics; D017728:Lymphoma, Large-Cell, Anaplastic / Q000235:genetics; D000074884:Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein; D009363:Neoplasm Proteins / Q000235:genetics*; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D020794:Receptor Protein-Tyrosine Kinases; D014178:Translocation, Genetic / Q000235:genetics
D015342:DNA Probes; D009363:Neoplasm Proteins; C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases; D020169:Caspases; C400109:MALT1 protein, human; D000074884:Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein