Automation of manual components and image quantification of direct dual label fluorescence in situ hybridization (FISH) for HER2 gene amplification: A feasibility study.
Determination of HER2 status by fluorescence in situ hybridization (FISH) in breast carcinoma correlates well with response to targeted therapy and prognosis. However, manual time consuming methods and quantification aspects of the procedure may be challenging for some laboratories. We examined the feasibility of automating these components of the FISH assay using a tissue microarray (TMA-118 clinically annotated cases) and a series of 41 whole sections. An in situ hybridization automated staining workstation was used to automate a programmed overnight start, on line baking, deparaffinization, cell conditioning, protease digestion, and prehybridization buffer washing. Dual label probe/target codenaturation/hybridization and stringency washing were done off line. The HER2 and CEP17 spot counts were quantified, and the HER2/CEP17 ratio calculated, via an imaging workstation. Results were benchmarked against manual counts for whole sections, and bright field in situ hybridization [silver in situ hybridization (SISH)] for the TMA. Automated FISH results using whole sections correlated well with manual results: HER2/CEP17 ratio correlation coefficient r = 0.9154, r = 0.8380, P < 0.0001. Correlation between automated and manual TMA FISH results was also excellent, and disease-free survival was significantly shorter (P < 0.001) for the HER2 amplified cases. Automation of the laborious manual prehybridization and image quantification components of FISH using directly labeled probes is feasible. Operational gains and enhanced consistency are inherent in this automated approach to HER2 clinical FISH testing.
Tubbs
Raymond R
RR
Department of Anatomic and Clinical Pathology, The Cleveland Clinic Foundation and the Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH 44195, USA. tubbsr@ccf.org
Pettay
James D
JD
Swain
Eric
E
Roche
Patrick C
PC
Powell
William
W
Hicks
David G
DG
Grogan
Thomas
T
D001331:Automation; D001943:Breast Neoplasms / Q000378:metabolism*; D018270:Carcinoma, Ductal, Breast / Q000378:metabolism*; D005240:Feasibility Studies; D005260:Female; D005784:Gene Amplification; D018734:Genes, erbB-2; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D012815:Signal Processing, Computer-Assisted; D046888:Tissue Array Analysis