Chromogenic in situ hybridization is a reliable alternative to fluorescence in situ hybridization for diagnostic testing of 1p and 19q loss in paraffin-embedded gliomas

Abstract

Recent studies imply the importance of rapid and reliable diagnostic assessment of 1p/19q status in oligodendroglial tumors. To date, fluorescence in situ hybridization (FISH) is the most commonly applied technique. FISH, however, has several technical shortcomings that are suboptimal for diagnostic applications: results must be viewed in a fluorescence microscope, results are usually evaluated by a single investigator only, and signal fading excludes physical archiving. Also, in gliomas, the distinction of diffusely infiltrating tumor cells from reactively altered normal tissue may be challenging in fluorescence microscopy. Dual-color chromogenic in situ hybridization (CISH) has started to replace FISH in some diagnostic tests performed in pathology. Here, we present the first single institute experience with a side-by-side analysis of 1p/19q FISH and CISH in a series of 42 consecutive gliomas. FISH and CISH produced identical results for 1p and 19q in 93% of cases (n = 39/42). Discrepant results were reevaluated by repeated FISH and a polymerase chain reaction (PCR)-based microsatellite marker analysis for loss of heterozygosity. Reevaluation confirmed CISH data in all three cases. We conclude that CISH is a reliable alternative in 1p/19q testing in paraffin-embedded tissues likely to be more sensitive to detect 1p/19q status than FISH analysis.

Figure 1

Chromogenic in situ hybridization (CISH) and repeated fluorescence in situ hybridization (FISH) analysis in tumor ID56604 (OIII). Upper panel: overview of a tumor area suggestive for an unbalanced tetraploidy by CISH analysis (A), magnification ×200. Insets to the right: CISH analysis (upper panel) and FISH analysis (lower panel) identify a high number of tumor cells harboring three signals for the target probe 1p36 (red in both FISH and CISH) and three to four signals for the control probe 1q25 (green in both FISH and CISH). Magnification ×400, inset in FISH analysis ×1000. Lower panel: overview of a CISH‐identified tumor area with clear evidence of 1p loss (B), magnification ×200. Insets to the right: CISH analysis (upper panel) and FISH analysis (lower panel) in the CISH‐preselected tumor area confirm 1p loss in the majority of tumor cells. Tumor cells show two signals for reference probe 1q25 and one signal for the target probe 1p36 in both the FISH and CISH analysis. Magnification ×400, inset in FISH analysis ×1000.

Figure 2

Validation of chromogenic in situ hybridization (CISH) results by repeated fluorescence in situ hybridization (FISH) and loss of heterozygosity (LOH) analysis in tumor ID56140 (OII). Upper panel: representative high‐magnification illustrations of CISH analysis for 1p (left) and 19q (right). Majority of tumor cells harbor one signal of the target probes on 1p and 19q (red signals) and two signals for the reference probes 1q and 19p (green signals). Magnification ×1000. Middle panel: high‐magnification illustrations of repeated FISH analysis in single tumor cells in CISH‐preselected tumor area. Of note, as in CISH analysis, tumor cells harbor one signal for target probes on 1p and 19q (red) and two signals for reference probes 1q and 19p (green) confirming CISH analysis data. Magnification ×1000. Lower panel: polymerase chain reaction (PCR)‐based microsatellite loss of heterozygosity analysis for 1p (left) and 19q (right) confirms CISH and FISH data of loss of chromosomal material on 1p and 19q. Microsatellite markers D1S1161 and D19S431 amplify a polymorphic chromosomal DNA fragment within the commonly deleted target zone on 1p and 19q. Leu: patient's leukocyte DNA serves as indicator of heterozygosity for the investigated microsatellite marker. Tu: patient's tumor DNA with evidence for loss of one allele on 1p and 19q (*) illustrated by a significant decrease in allele signal intensity. The second allele remains detectable at lower intensity and is not completely lost due to the presence of non‐tumorous cells within the biopsy (leukocytes, astrocytes, microglia, neurons) that are not affected by 1p/19q loss.

Figure 3

Comparison of locus‐specific CytoDot2C and CytoLight probes by Zytovision for 1p (A) and 19q (B). The target regions are depicted in the colors displayed by the respective detection systems. A. The probes for 1p36 deletion (red/orange) map to the smallest region of consistent deletion (SRD) and cover identical regions in chromogenic in situ hybridization (CISH) and fluorescence in situ hybridization (FISH). The reference probes target the same region in 1q25.3 with the CISH probe covering a shorter sequence localized in the centre of the FISH probe target sequence. B. The probes for 19q13 deletion (red/orange) map to the region of common deletion in gliomas at 19q13.32 and cover identical regions in CISH and FISH. The reference probes target the same region in 19p13.3 with the CISH probe covering a shorter sequence localized in the centre of the FISH probe target sequence.