447 A clinically applicable molecular classification of oncocytic cell thyroid nodules 11 Figure 1. The main CNA patterns These main CNA patterns were observed by CNA-LOH analysis using the custom AmpliSeq™ NGS GWLOH v2 panel (GenomeScan BV, Leiden, The Netherlands), which assesses approximately 1,500 SNPs across all autosomes and the X chromosome. These SNP array plots show the median amplicon read count (A-D, top panel) visualizing the quality of the tested sample, the normalized median amplicon read count (A-D, middle panel) visualizing the relative copy number information, and the VAF (A-D, bottom panel) visualizing any chromosomal imbalances. Results are displayed per chromosome (see horizontal numbering in the top row) and separately for p (blue) and q arm (yellow). Figure 1A displays a normal pattern with no CNA, as observed in nodular hyperplasia with oncocytic cell metaplasia (Table 2, case 3). Figure 1B displays the results of the CNALOH analysis performed on the histology (tumour cell percentage ≥70%) of an OCA in a male patient (Table 2, case 23), with GH type CNA with suspected endoreduplication, characterized by clear loss of heterozygosity (a, bottom panel) and chromosomal losses (B, middle panel) of chromosomes 1-4, 6, 8-9, 11, 14, 15 and 20-22. There was some heterogenicity, indicated by the smaller VAF amplitude of chromosomes 15 and 20 as compared to the other affected chromosomes (B, bottom panel). The extreme VAF amplitude (B, bottom panel) of most affected chromosomes is highly suspect of endoreduplication. This neoplasm was diagnosed as molecularly malignant. This case is further illustrated in the Results section. Figure 1C displays the results of the GWLOH panel performed on the histology (tumour cell percentage ≥70%) of an OA in a male patient (Table 2, case 12), with GH type CNA without endoreduplication, characterized by chromosomal imbalances with less pronounced amplitudes (C, bottom panel) and chromosomal losses (C, middle panel) of affected chromosomes 2, 9, 13, and 22. Limited heterogenicity is possibly observed. This OA was considered molecularly benign. Figure 1D displays a oncocytic cell neoplasm in a female patient (Table 2, case 16). On histopathological assessment, this lesion had a thick capsule and no signs of vascular invasion. Based on a focal interruption of the capsule of equivocal invasive malignant or iatrogenic origin, the neoplasm was considered a minimally invasive OCA. On CNA-LOH analysis, the lesion showed RCI type CNA, characterized by chromosomal imbalances (D, bottom panel) and relative chromosomal gains (D, middle panel) affecting chromosomes 1, 5, 6, 10, 12, 16, 17, 19, 20, and X. As such, molecularly, this lesion is considered benign. This case is further illustrated in the Results section. CNA, copy number alterations. CNA-LOH, copy number alterations and loss of heterozygosity. GH type, genome-haploidization type. GWLOH, genome-wide loss of heterozygosity. OA, oncocytic thyroid adenoma. OCA, oncocytic cell thyroid carcinoma. RCI type, reciprocal chromosomal imbalance type. SNP, single nucleotide polymorphism. VAF, variant allele frequency. d
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