Genomic profiling and assessment of tumor mutational burden
tumor
TUMOR, SOMATIC MUTATIONS
We conducted comprehensive genomic profiling using WES on paired samples from 15 patients, including FFPE tissue and peripheral blood specimens. Genomic DNA was extracted from FFPE slides containing a minimum of 20% tumor content and fragmented to approximately 250 base pairs using sonication. A DNA library was then generated using the KAPA Hyper Prep Kit from KAPA Biosystems. The capture of exonic regions was performed using the Agilent SureSelect approach, following the manufacturer’s instructions (Agilent, Santa Clara, CA). Paired-end sequencing was carried out on the HiSeq2500 next-generation sequencing instrument from Illumina (San Diego, CA), resulting in 100-base reads from each end of the DNA fragments. Various genomic alterations, including single base substitutions (SNVs), short and long insertions/deletions (INDELs), CNVs, and gene rearrangements and fusions, were analyzed. The TMB was assessed by analyzing somatic mutations to estimate the overall mutational load in the tumor samples.