Tumor Mutational Load and TCR Profiling

Four to eight sections of paraffin-embedded tumor tissue, with the tumor region selected by a pathologist, were obtained, and DNA extraction was performed using the ‘QIAamp DNA FFPE Kit GenRead’ kit (QIAGEN, Germantown, MD, USA). The QUBIT 3.0 fluorometer instrument (Thermo Fisher Scientific, Waltham, MA, USA) was used for DNA quantification.
The Oncomine Tumor Mutational Load Assay (Thermo Fisher Scientific, Waltham, MA, USA) was used for next-generation sequencing (NGS) tumor mutational burden (TMB) and mutational profile analyses. Oncomine TCR-Beta-SR assay (Thermo Fisher Scientific, Waltham, MA, USA) was used for NGS analysis of the nucleotide sequence of the CDR3 region coding for the T-cell receptor beta (TCRβ) chain. Libraries were loaded on an Ion 540 chip using the Ion Chef Instrument, sequenced in an Ion GeneStudio S5 System (Thermo Fisher Scientific, Waltham, MA, USA) and analyzed using the Ion Reporter version 5.12 (Thermo Fisher Scientific, Waltham, MA, USA). Shannon’s diversity and evenness were defined and calculated as previously described [11 (link)].