Iscan system with the

FFPE tumor tissue from a representative block with or without microdissection was used for nucleic acids extraction for NGS or microarray-based CN analysis, respectively. Saliva was collected for normal matched control. All samples were sequenced at Tempus Labs (Chicago, IL), by using a 596-gene panel (Additional file 1: Table S1), as previously described [8 (link)]. The limit of detection of the assay is 5% variant allele fraction (VAF), with sensitivity of 99.1% for single nucleotide variants. Variant analysis and interpretation was performed by using Tempus proprietary software, ClinVar (National Institutes of Health, Bethesda, MD), Catalogue of Somatic Mutations in Cancer (COSMIC) and VarSome [16 (link)]. The final somatic VAF was adjusted depending on the tumor cell content of the sample. Loss of heterozygosity (LOH) was called for clonal mutations with gene locus CN loss. TMB represents the number of single nucleotide protein-altering mutations per million base pairs. Graphs were plotted by using GraphPad Prism (Version 8.3.1, GraphPad Software, La Jolla, CA). Microarray-based chromosome CN analysis was performed by using the IScan® System with the CytoSNP-850 K v1.1 BeadChip (Illumina, San Diego, CA), and GenomeStudio (Illumina) and Nexus, (Version 9.0, BioDiscovery, Inc., El Segundo, CA) softwares, as previously described [8 (link)].

Microarray-based chromosome analysis of copy number was performed using the IScan^® System with the CytoSNP-850K v1.1 BeadChip (Illumina) and analyzed using GenomeStudio (Illumina) and Nexus, version 9.0 (BioDiscovery, Inc, El Segundo, CA, USA) software. Copy number changes, i.e., clonal changes in less than 100% of cells including deletion, duplication, loss of heterozygosity and ploidy, were determined by using the signal intensity determined by the log² ratio along with the B-allele frequency, as described [43 (link)].