450 chapter 11 Finally, the molecular diagnosis is categorized as (likely) benign, uncertain malignant, or malignant. We predefined a (likely) benign molecular diagnosis as limited GH type CNA, with 1-5 affected chromosomes, possible heterogenicity of the alterations, and no signs of endoreduplication, or any RCI type alterations, or no molecular alterations on CNA-LOH, somatic mutation, and fusion analysis. An uncertain malignant diagnosis was predefined as GH type alterations in 6-10 chromosomes or limited GH type alterations in 1-5 chromosomes in combination with an OCA-associated driver mutation. Heterogenicity of the alterations may be present among the affected chromosomes; no or inconclusive signs of endoreduplication are observed. A malignant molecular diagnosis is defined as extensive GH type CNA affecting 11-23 chromosomes, often with limited or without heterogenicity, or GH type alterations in 6-10 chromosomes in combination with an OCA-associated driver mutation, or GH type CNA with any number of affected chromosomes and suspected endoreduplication. Figure 3. CNA pattern verification Verification of the CNA patterns observed during CNA-LOH analysis using the GWLOH v2 NGS panel (F and L) in historical cases on which LAIR analysis (A-E and G-K) was previously performed according to the methods as described by Corver et al. [649]. One OCA (A-F) and one ATC-H (G-L) are presented here. A. The SNP array analysis (iCOG and HumanCytoSNP-12, Illumina, Inc., San Diego, CA, USA) of the OCA in a male patient showed homozygosity and chromosomal losses of chromosomes 1-4, 6, 8-11, 12q, and 13-22. B and H. Keratin (FITC) vs. vimentin (APC) fluorescence. Different populations R1 (vimentin-positive, keratin negative) and R2 (vimentinpositive and keratin-positive) can be identified. C and I. Negative control stained with the secondary reagents and PI only. Note the difference in fluorescence intensities as compared to keratin and vimentin-stained samples (B and H). D and J. Overlay of population R1 and R2. Note that the major G0G1-peak of the histogram is painted red and left of the green G0G1-peak (the DNA-diploid internal reference, minor in D and major in J), indicative for loss of DNA. E and K. The MFI of the G0G1-of R1 was used to accurately calculate the DNA index of the major G0G1-peak of R2. E. The DNA histogram of R2, the keratin-positive, vimentin-positive population showed debris for which the ModFit LT algorithm corrects. The DNA index was 0.7. A-E. Together, these findings indicated the loss of DNA and near-haploidy, corresponding to an A0 genotype. G. The iCOG SNP array of the ATC-H in a female patient showed homozygosity and chromosomal losses of chromosomes 1-6, 8-11, and 13-21. K. The DNA histogram of R2, the keratin-positive, vimentin-positive population showed to be bi-modal due to two cycling populations, with DNA indices of 0.5 (i.e., near-haploid tumour fraction) and 1.1 (i.e., fraction with endoreduplication), respectively. G-K. Together, these findings showed loss of DNA, near-haploidy and endoreduplication of the entire near-haploid population. F and L. On the GWLOH panel, both cases showed chromosomal losses of the affected chromosomes relative to the unaffected chromosomes (middle panels, normalized median amplicon read count). The VAF of the OCA (F, bottom panel) showed a less pronounced amplitude of the SNP segregation than the ATC-H (L, bottom panel), corresponding to GH type CNA without (F) and with (L) endoreduplication, respectively. As such, the CNA patterns observed using the GWLOH panel were consistent with the results of the LAIR analysis. The two other historical cases are presented in Supplementary figure 1. 488 or 633, laser wavelength used for excitation. 530/30, band pass filter used to collect FITC fluorescence (green). 670/14, band pass filter used to collect APC fluorescence (infra-red). >610/20, long pass filter used to collect PI fluorescence (deep-red). APC, allophycocyanin. ATC-H, anaplastic thyroid carcinoma with oncocytic changes. 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. OCA, oncocytic thyroid carcinoma. LAIR, lesser-allele intensity-ratio. MFI, median fluorescence intensity. RCI type, reciprocal chromosomal imbalance type. SNP, single nucleotide polymorphism. VAF, variant allele frequency.
RkJQdWJsaXNoZXIy MTk4NDMw