452 chapter 11 CNA pattern verification To verify the GH type CNA patterns and appurtenant genotypes that are distinguished using the GWLOH v2 panel, CNA-LOH analysis using the GWLOH panel was performed on four representative historical cases (three OCA and one anaplastic thyroid carcinoma with oncocytic changes, not included in the study cohort) in which LAIR analysis was previously performed according to the methods as previously described by Corver et al. [649]. These included two cases with GH type CNA with suspected endoreduplication of the near-haploid genome (i.e., AA genotype) and two cases with GH type CNA without endoreduplication (i.e., A0 genotype). For each of these historical cases, the findings of the GWLOH panel were consistent with the findings of the LAIR analysis (Figure 3, Supplementary figure 1). In addition, multiparameter DNA flow cytometry was performed according to the methods as described by Corver et al. [649, 688], with minor modifications, on FFPE material of two cases from the study cohort: one OCA case (Table 2, case 29) with GH type CNA (Figure 4) and one OA case (Table 2, case 9) with RCI type CNA (Figure 5). In short, 2 mm tissue punches, taken from the same tissue block as used for the GWLOH panel, were transferred to a recipient paraffin block. Sixtymicron sections were taken, deparaffinized, heat-treated and dissociated using a mixture of collagen I Figure 4. An 85 mm widely invasive oncocytic thyroid carcinoma with GH type CNA (Table 2, case 29). A and B. Haematoxylin and eosin-stained sections (A, 0.5x, 2mm, and B, 40×, 50 μmm) from a formalin-fixed, paraffin-embedded specimen of the thyroid tumour. Punches from the same tissue block were used for CNALOH analysis using the GWLOH panel and for multiparameter DNA content flow cytometry. C. Results of the GWLOH panel (tumour cell percentage at least 80%) showed GH type CNA, with losses of chromosomes 1, 2, 3, 4, 6, 8, 9, 10, 11, 14, 15, 16. 18. 21, and 22, some heterogenicity, and possible endoreduplication. D-I. Results of the multiparameter DNA content flow cytometry. D. Keratin (FITC) vs. vimentin (APC) fluorescence. Different populations R1 (vimentin-positive, keratin negative) and R2 (vimentin-positive and keratin-positive) can be observed. 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 left of the green G0G1peak (the DNA-diploid internal reference), clearly indicating loss of DNA and thus likely representing a nearhaploid carcinoma population. I. The MFI of the G0G1-population of R1 calculated by WinList 3D was linked to ModFit LT allowing accurate calculation of the DNA index of the major G0G1-peak of R2. The DNA index was 0.7, confirming DNA near-haploidy and the GH type CNA pattern that was observed using the GWLOH panel. Although endoreduplication was deemed possible on the GWLOH panel, flow cytometry results indicated that no endoreduplication was present (i.e., A0 genotype). On somatic mutation and fusion analysis, no driver mutations were identified. This tumour was diagnosed as molecularly malignant. 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 (deepred). 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.
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