455 A clinically applicable molecular classification of oncocytic cell thyroid nodules 11 Figure 5. A 58 mm oncocytic thyroid adenoma with RCI type CNA (Table 2, case 9) A and B. Haematoxylin and eosin-stained sections (A, 5x, and B, 40×) from a formalin-fixed, paraffin-embedded specimen of the tumour. Punches from the same tissue block were used for CNA-LOH analysis using the GWLOH panel and for multiparameter DNA content flow cytometry. C. The results of the GWLOH panel (tumour cell percentage at least 50%) showed chromosomal imbalances of chromosomes 5, 6, 7, 11, 12, 13, 17, 20 and X based on chromosomal copy number gains, corresponding to RCI type CNA. D. Keratin (FITC) vs. vimentin (APC) fluorescence, with R1 (vimentin-positive, keratin negative) and R2 (vimentin-positive and keratin-positive) populations. E. Negative control stained with the secondary reagents and PI only. F. DNA histogram of R1 (green), the vimentin-positive, keratin negative DNA-diploid (internal reference) stromal population. G. DNA histogram of R2 (red), the vimentin-positive, keratin-positive population epithelial cell fraction. H. Overlay of population R1 and R2. Note that the major G0G1-peak of the histogram is painted red and to the right of the green G0G1-peak (the DNA-diploid internal reference), indicative for gain of DNA. I. The MFI of the R1G0G1-population was used to accurately calculate the DNA index of the major G0G1-peak of R2. The DNA index was 1.3, indicating gain of DNA and aneuploidy. As such, the results of the multiparameter DNA content flow cytometry are in accordance with the results of the GWLOH panel. On somatic mutation and fusion analysis, no driver mutations were identified. This tumour was considered molecularly benign. 488 or 633, laser wavelength used for excitation. 530/30, band pass filter used to collect FITC fluorescence (green). 660/20, band pass filter used to collect APC fluorescence (infra-red). >670, long pass filter used to collect PI fluorescence (deep-red). APC, allophycocyanin. CNA, copy number alterations. CNA-LOH, copy number alterations and loss of heterozygosity. FITC, fluorescein isothiocyanate. GH type, genome haploidization type. GWLOH, genome-wide loss of heterozygosity. MFI, median fluorescence intensity. RCI type, reciprocal chromosomal imbalance type. and dispase with the aid of a gentleMACSTM (Miltenyi BioTec, Bergisch Gladbach, Germany). Next, cell suspensions were labelled for keratin (FITC), vimentin (APC) and DNA by propidium iodide (PI). Cells were treated with RNase to improve the resolution of the DNA histograms. Cells were interrogated using a FACSCanto II flow cytometer (BD Biosciences, San Jose, CA) [688]. For the historical cases (Figure 3, Supplementary figure 1), a LSRII flow cytometer (BD Biosciences, San Jose, CA) was used. All multiparameter DNA content flow cytometry data were analysed using ModFitTM 6.0, remotely controlled by WinListTM 3D 10.0 (Verity Software House, Topsham, ME). The median relative fluorescence of the vimentin-positive, keratin-negative G0G1-fraction was used as internal DNA-diploid reference [688, 689]. Statistical analysis Where appropriate, parametric and nonparametric data were compared using the one-way ANOVA, and Mann-Whitney U or Kruskall-Wallis test, respectively. P values from the post-hoc analysis were adjusted by Bonferroni correction for multiple tests. Categorical data were compared using the 2-sided Pearson’s chi-squared or Fisher’s exact test, where appropriate. A p value of < 0.05 was considered statistically significant. All statistical analysis were performed using SPSS Statistics version 27 (IBM Corp., Armonk, NY, USA).
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