445 A clinically applicable molecular classification of oncocytic cell thyroid nodules 11 Data collection From the pathology records, we recorded cytopathological and histopathological characteristics. Patient demographics and clinical, radiological, and surgical characteristics were collected from the patient medical records. Thyroid cytopathology was described using the Bethesda classification [18]. The Thyroid Imaging Reporting and Data System (TIRADS) classification was infrequently reported and it was considered inappropriate to retrospectively reassess stored ultrasound captures, as ultrasound is a dynamic technique [394]. Follow-up data was updated until 1 October 2022. Molecular analysis For molecular testing, total nucleic acid (DNA and RNA) was isolated either from formalin-fixed paraffin-embedded (FFPE) histopathology samples using FFPE tissue cores (0.6 mm diameter and variable length), from micro-dissected cytology cell blocks, or from tumour cells scraped off cytology slides [684-686]. NGS was performed on the Ion Torrent GeneStudio™ S5 platform (GenomeScan BV, Leiden, The Netherlands) using custom NGS panels for CNA-LOH analysis, somatic DNA analysis, and gene fusion analysis. CNA-LOH analysis was performed using the custom AmpliSeq™ NGS genome-wide LOH (GWLOH) v2 panel, which assesses LOH and other chromosomal imbalances using 1,500 SNPs, evenly distributed across all autosomes and the X chromosome. This analysis has a transit time of only a few days. The results of the GWLOH panel are visualized using SNP array plots (Figure 1) displaying the median amplicon read count (Figure 1A-D, top panel) visualizing the quality of the tested sample (>2.0 [log10(100) = 2] is considered good quality), the normalized median amplicon read count (middle panel) visualizing the relative copy number information, and the variant allele frequency (VAF) plot (bottom panel) visualizing any chromosomal imbalances including LOH. A VAF of 0.50 indicates heterozygosity; distinct segregation of the SNPs indicates chromosomal imbalance. Somatic mutation analysis was performed using the custom Ampliseq™ Cancer Hotspot v6 panel (Thermo Fisher Scientific, Waltham, MA, USA) or the custom Ampliseq™ NGS ENDO32 v1 panel (Thermo Fisher Scientific), which respectively assess 87 and 27 (thyroid) cancer-related genes, as previously described (details provided in Supplementary Data) [684, 685]. Any alterations classified as (likely) pathogenic (i.e., class 4 or 5, respectively) were reported [687]. Gene fusion analysis was performed using the Archer® FusionPlex CTL v1 or v2 panel (ArcherDX Inc., Boulder, CO, USA), which respectively assess fusions in 16 and 19 genes, as previously described (Supplementary Data) [684-686].
RkJQdWJsaXNoZXIy MTk4NDMw