Table 3.

Key parameters for the harmonisation of TMB analysis and workflow

Parameter	Principles
Sample	Sample must provide adequate quantity and quality of DNA Most cancer NGS assays are performed on FFPE tissue (optimal fixation time, 24 hours for surgical specimens and 12 hours for biopsies). Cytology/FNA, plasma, and other types of samples may be acceptable for smaller targeted panels and are in development to be established for use in the future
Methodology	WES is considered the gold standard for measuring TMB because it offers high breadth of coverage compared with gene panels Investigations of smaller gene panels are ongoing for TMB assessment In general, larger panels have been shown to be more accurate than smaller panels. As more data become available, there may be a recommended number of genes to profile for optimised gene profiling/TMB; current recommendations indicate that the area of genome covered should be >0.8 Mb (preferably >1 Mb) Screening for driver mutations (eg, EGFR, BRAF, KRAS) and TMB in one assay is recommended to avoid time delays or processing of multiple samples. Inclusion of driver mutations, rearrangements, etc on the panel can provide additional clinical utility for the assay beyond TMB, but may not necessarily be informative for the TMB calculation Sequencing depth can affect detection of low-frequency variants. ≥200× is recommended
Platforms	Stakeholders should be aware of varying parameters such as runtime and throughput capacity of available sequencers
Germline	Experimental approaches by filtering germline variants with matched normal samples In silico approaches by filtering against germline variant databases
Algorithm	Standardised and robust bioinformatic pipelines should be developed There is a need for internationally available reference standards (ie, sets of tumours with WES data) Variant allele frequency cut-offs should be reported. Cut-offs of ≥5% are recommended
Cut-off	Currently no standard cut-off to designate high TMB and there is a need for harmonisation of tests to establish set thresholds. More standardised cut-offs are anticipated as clinical utility is established for individual tumour types and TMB thresholds are studied in the context of response to immunotherapies TMB thresholds of ≥13 mut/Mb and ≥10 mut/Mb, measured by FoundationOne CDx, have recently been associated with enhanced responses to immune checkpoint inhibitor monotherapy and combination therapy, respectively, in patients with non-small cell lung cancer. The value cut-off ≥10 mut/Mb was established in one study and clinically validated in a separate trial.8 9 In patients with small cell lung cancer, improved responses were seen with a cut-off of ≥248 missense mutations, as measured by WES84
Concordance	Recommendations on validation processes and concordance testing with WES for approved gene panels or other commercial panels without clinical data Need for direct comparison between panels (especially for FDA-approved or authorised panels)
Reporting	Reporting standards for how TMB is defined and calculated, including gene panel number, capture region, control (germline subtraction), types of mutations called, variant calling threshold, and sequence depth Recommend reporting results as mutations per megabase of targeted DNA Recommend accurate reporting of TMB thresholds and highlight associated clinical outcomes Quality assurance on data reporting is also required

BRAF, B-Raf proto-oncogene, serine/threonine kinase; EGFR, epidermal growth factor receptor; FFPE, formalin-fixed, paraffin-embedded; FNA, fine-needle aspiration; KRAS, KRAS proto-oncogene, GTPase; mut/Mb, mutations per megabase; NGS, next-generation sequencing; TMB, tumour mutational burden; WES, whole exome sequencing.