Oncomine tumor mutational load assay

Comprehensive genomic profiling (CGP) via targeted NGS was performed at the LMU Munich Institute of Pathology. Patients with externally performed CGP were registered and discussed within our MTB in select cases. Starting in 2016, in-house panel sequencing was performed with Oncomine™ Focus Assay, a 52-gene panel, which was continuously replaced by the 161-gene panel Oncomine™ Comprehensive Assay (Thermo Fisher, Darmstadt, Germany) and the TruSight Oncology (TSO) 500™ Assay (Illumina, San Diego, USA). All panels allow simultaneous RNA and DNA sequencing with detection of insertions/deletions (indels), gene fusions, single-nucleotide variants (SNV), and copy number variations (CNV). Additionally, tumor mutational burden (TMB) was evaluated in select cases (Oncomine™ Tumor Mutational Load Assay, ThermoFisher Scientific). CGP was either performed on FFPE tumor tissue or liquid biopsies (e.g., cerebrospinal fluid, blood, or ascites). Material from primary tumor, metastases, or locally recurrent tumor was used. Young patients with rare sarcomas were referred to the National Center for Tumor Diseases (NCT) and German Cancer Consortium (DKTK) Molecularly Aided Stratification for Tumor Eradication (MASTER) program for whole genome/exome and transcriptome analysis in select cases (Horak et al. 2017 (link), 2021 (link)).

Tumor DNA was extracted from manually microdissected 5-µm FFPE sections using the QIAamp Gene Read DNA FFPE kit (Qiagen, Hilden, Germany). Targeted NGS was performed using Oncomine Tumor Mutational Load Assay (Thermo Fisher, Waltham, MA, USA) covering 1.65 megabases of 409 genes with known cancer associations according to the manufacturer’s instructions. Data were processed using the Ion Torrent Suite software (version 5.8.0) and the Variant Caller plugin as previously described [24 (link)]. The Oncomine TML workflow 5.10 on Ion reporter software was adopted to calculate the TMB, considering only nonsynonymous somatic variants not included in the 1000 Genome Project, 5000Exomes Global, and ExAC.

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)].