Efficient automated assessment of genetic abnormalities detected by fluorescence in situ hybridization on brush cytology in a Barrett esophagus surveillance population

Abstract

BACKGROUND.Automated assessment of genetic abnormalities detected by fluorescence in situ hybridization (FISH) in brush cytology specimens from patients with Barrett esophagus (BE) may enhance the clinical applicability of this methodology. The objectives of this study were to validate a novel, automated, proprietary system (CytoVison SPOT AX) for the assessment of FISH abnormalities in BE brush cytology and, subsequently, to use this automated method for screening of a BE surveillance cohort.METHODS.FISH with DNA probes for chromosomes 9, 17, and Y, and for the 9p21 (p16), 17q11.2 (Her2/neu), and 17p13.1 (p53) loci was applied on brush cytology specimens from a surveillance cohort of 151 patients with BE. Validation of the automated system was performed by comparison of the automated FISH results with manual scores for the first 60 patients.RESULTS.There was 98% concordance between manual and automated FISH analysis with κ values from 0.49 to 1 for the different probes. The loss of 17p13.1 (p53) was observed in only 5% of patients with no dysplasia (ND) and in 9% of patients with low‐grade dysplasia (LGD) but increased to 46% in patients with high‐grade dysplasia (HGD) (P < .005; Fisher exact test). Chromosomes 9 and 17 were observed in 6% of patients with ND, in 21% of patients with LGD, and in 62% of patients with HGD (P < .05). Ten percent of patients with ND had loss of the Y chromosome, which increased to 27% in patients with HGD (P< .05). The amplification of 17q11.2 (Her2/neu) was detected in 62% of patients with HGD (P < .001).CONCLUSIONS.The current investigation indicated that the CytoVison SPOT AX is an objective, efficient system for the analysis of DNA‐FISH on BE brush cytology and is applicable for analyzing large populations of BE patients. In the current study cohort, the loss of 17p13.1 (p53), Y chromosome loss, and polysomy of chromosomes 17 and 9 were correlated with increasing grade of dysplasia in patients with BE. Cancer 2007. © 2007 American Cancer Society.

Gains and Amplifications of c-myc, EGFR, and 20.q13 Loci in the No Dysplasia–Dysplasia–Adenocarcinoma Sequence of Barrett's Esophagus

“…Dual-color probes were used: CEP 7 SpectrumGreen with LSI EGFR (7p12) SpectrumOrange and LSI 20q13.2 SpectrumOrange with LSI c-myc (8q24.12-q13) SpectrumGreen. DNA-FISH was done according to the manufacturer's instructions provided by Vysis as described previously (29).…”

“…The DNA-FISH technique gives an accurate quantification of the signals and has advantages over comparative genomic hybridization methodologies, particularly in analyzing the alteration of specific genes. DNA-FISH allows also simultaneous evaluation of genetic abnormalities with cellular morphology and can be successfully applied to both tissue section (14,23,26) and brush cytology specimens of Barrett's esophagus (27)(28)(29). DNA-FISH on brush cytology specimens offers many advantages as a method to detect genetic markers, including simplicity, lower cost, and the potential to sample a larger area of the Barrett's esophagus epithelium when compared with taking random biopsies (30,31).…”

Increased phosphorylation on residue S795 of the retinoblastoma protein in esophageal adenocarcinoma

“…Pictures were taken with the Olympus XM10 camera and brightness and contrast adjustments were done with olympus Cell^F software. To define per sample P16 DnA-FISh status we used previously determined frequency cut-off values for P16 gains and losses (24). These values were obtained from counts of 20 normal squamous epithelium cytological patient specimens (100 nuclei evaluated per case) and calculated as the mean percentage of squamous nuclei with either p16 gain or loss plus 3x times standard deviation.…”

“…To detect the loss of the p16 gene locus in the cell lines, DnA FISh was performed as previously described (24). For this experiment the specimens were hybridized with a directly labelled probe mix which contained the SpectrumRed-lSI p16 (9p21) locus specific probe for the 9p21 (P16) region on chromosome 9, obtained from Abbott molecular (Chicago, Il, USA).…”

State‐of‐the‐art FISHing: Automated analysis of cytogenetic aberrations in interphase nuclei

“…Non-dot-like signals and touching or overlapping cells make the manual analysis tiring and difficult which presumably is the reason why high interest in automation and quantitative analysis of HER2 FISH images was shown by several workgroups. HER2 status has also been estimated on brush cytology in a Barrett esophagus surveillance population by Rygiel et al using automated i-FISH and they found perfect agreement with manual scoring (101). In addition to HER2 amplification, automated method has also been introduced for detection of several further cytogenetic abnormalities in solid tumors.…”