4FISH-IF, a four-color dual-gene FISH combined with p63 immunofluorescence to evaluate NKX3.1 and MYC status in prostate cancer

Abstract

NKX3.1 allelic loss and MYC amplification are common events during prostate cancer progression and have been recognized as potential prognostic factors in prostate cancer after radical prostatectomy or precision radiotherapy. We have developed a 4FISH-IF assay (a dual-gene fluorescence in situ hybridization combined with immunofluorescence) to measure both NKX3.1 and MYC status on the same slide. The 4FISH-IF assay contains four probes complementary to chromosome 8 centromere, 8p telomere, 8p21, and 8q24, as well as an antibody targeting the basal cell marker p63 visualized by immunofluorescence. The major advantages of the 4FISH-IF include the distinction between benign and malignant glands directly on the 4FISH-IF slide and the control of truncation artifact. Importantly, this specialized and innovative combined multiprobe and immunofluorescence technique can be performed on diagnostic biopsy specimens, increasing its clinical relevance. Moreover, the assay can be easily performed in a standard clinical molecular pathology laboratory. Globally, the use of 4FISH-IF decreases analytic time, increases confidence in obtained results, and maintains the tissue morphology of the diagnostic specimen.

Keywords: MYC; NKX3.1; biopsy; fluorescence in situ hybridization; immunofluorescence; prostate cancer.

Figure 1.

Metaphase spread showing 8p telomere (green), 8p21 (red), 8p centromere (aqua), and 8q24 (gold) in chromosome 8. Scale bar: 3 µm.

Figure 2.

(A) A biopsy specimen showing the p63-positive basal cells (arrow) surrounding the benign glands and the p63-negative cancer focus (arrowhead). Texas Red and DAPI channels are shown after thresholds adjustments. Scale bar: 30 µm. (B) p63-positive basal cells (now showing the addition of red and gold due to the fluorescence in both channels, thick arrow) surrounding benign luminal cells with two signals of each probe (thin arrow) and p63-negative cancer cells (arrowhead) with more variable number if signals were due to truncation artifact and NKX3.1 deletion. FISH probes for 8p telomere, 8p centromere, NKX3.1, and MYC are shown in green, aqua, red, and gold, respectively. SpectrumAqua, FITC, Texas Red, SpectrumGold, and DAPI channels are shown after thresholding adjustments. Scale bar: 4.5 µm.

Figure 3.

(A) A high-power view of a case with NKX3.1 deletion, in which a cell with two chromosome 8 centromeres (aqua), two chromosome 8p telomeres (green), two 8q24 loci (MYC, gold), and one 8p21 locus (NKX3.1, red) can be seen. SpectrumAqua, FITC, Texas Red, SpectrumGold, and DAPI channels are shown after thresholds adjustments. Scale bar: 2 µm. (B) A high-power view of a case with both NKX3.1 deletion (left) and MYC amplification (right). The cell on the right has three chromosome 8 centromeres (aqua), four chromosome 8p telomeres (green), three 8q24 loci (MYC, gold), and two 8p21 loci (NKX3.1, red). SpectrumAqua, FITC, Texas Red, SpectrumGold, and DAPI channels are shown after thresholds adjustments. Scale bar: 2.0 µm.