Next-Generation Sequencing
Next generation sequencing was available for 11 patients (14 samples). An institutional DNA sequencing panel assaying 479 cancer-related genes was used.16 Genomic DNA was extracted from peripheral blood and tumor tissue microdissected from fresh frozen paraffin embedded blocks, as previously described. 16Capture-based next-generation sequencing (NGS) was performed at the UCSF Clinical Cancer Genomics Laboratory, using an assay targeting the coding regions of these genes, TERT  promoter, select introns from 40 genes (for detection of gene fusions and other structural variants), and intergenic regions at regular intervals along each chromosome (for chromosomal copy number assessment), altogether with a total sequencing footprint of 2.8 Mb Sequencing libraries were prepared from genomic DNA with target enrichment performed by hybrid capture using a custom oligonucleotide library. Sequencing was performed on an Illumina HiSEquation 2500. Duplicate sequencing reads were removed computationally to allow for accurate allele frequency determination and copy number estimates. The analysis was based on the human reference sequence UCSC build hg19 (NCBI build 37). Single nucleotide variants and small insertions/deletions (indels) were visualized and verified using Integrated Genome Viewer.