Clinical evidence demonstrates that treatment with immune checkpoint inhibitor immunotherapy agents can have considerable benefit across multiple tumours. However, there is a need for the development of predictive biomarkers that identify patients who are most likely to respond to immunotherapy. Comprehensive characterisation of tumours using genomic, transcriptomic, and proteomic approaches continues to lead the way in advancing precision medicine. Genetic correlates of response to therapy have been known for some time, but recent clinical evidence has strengthened the significance of high tumour mutational burden (TMB) as a biomarker of response and hence a rational target for immunotherapy. Concordantly, immune checkpoint inhibitors have changed clinical practice for lung cancer and melanoma, which are tumour types with some of the highest mutational burdens. TMB is an implementable approach for molecular biology and/or pathology laboratories that provides a quantitative measure of the total number of mutations in tumour tissue of patients and can be assessed by whole genome, whole exome, or large targeted gene panel sequencing of biopsied material. Currently, TMB assessment is not standardised across research and clinical studies. As a biomarker that affects treatment decisions, it is essential to unify TMB assessment approaches to allow for reliable, comparable results across studies. When implementing TMB measurement assays, it is important to consider factors that may impact the method workflow, the results of the assay, and the interpretation of the data. Such factors include biopsy sample type, sample quality and quantity, genome coverage, sequencing platform, bioinformatic pipeline, and the definitions of the final threshold that determines high TMB. This review outlines the factors for adoption of TMB measurement into clinical practice, providing an understanding of TMB assay considerations throughout the sample journey, and suggests principles to effectively implement TMB assays in a clinical setting to aid and optimise treatment decisions.

The clinical demand for mutation detection within multiple genes from a single tumour sample requires molecular diagnostic laboratories to develop rapid, high-throughput, highly sensitive, accurate and parallel testing within tight budget constraints. To meet this demand, many laboratories employ next-generation sequencing (NGS) based on small amplicons. Building on existing publications and general guidance for the clinical use of NGS and learnings from germline testing, the following guidelines establish consensus standards for somatic diagnostic testing, specifically for identifying and reporting mutations in solid tumours. These guidelines cover the testing strategy, implementation of testing within clinical service, sample requirements, data analysis and reporting of results. In conjunction with appropriate staff training and international standards for laboratory testing, these consensus standards for the use of NGS in molecular pathology of solid tumours will assist laboratories in implementing NGS in clinical services.Electronic supplementary materialThe online version of this article (doi:10.1007/s00428-016-2025-7) contains supplementary material, which is available to authorized users.

Tumor mutation load (TML) has been proposed as a biomarker of patient response to immunotherapy in several studies. TML is usually determined by tumor biopsy DNA (tDNA) whole exome sequencing (WES), therefore TML evaluation is limited by informative biopsy availability. Circulating cell free DNA (cfDNA) provided by liquid biopsy is a surrogate specimen to biopsy for molecular profiling. Nevertheless performing WES on DNA from plasma is technically challenging and the ability to determine tumor mutation load from liquid biopsies remains to be demonstrated. In the current study, WES was performed on cfDNA from 32 metastatic patients of various cancer types included into MOSCATO 01 (NCT01566019) and/or MATCHR (NCT02517892) molecular triage trials. Results from targeted gene sequencing (TGS) and WES performed on cfDNA were compared to results from tumor tissue biopsy. In cfDNA samples, WES mutation detection sensitivity was 92% compared to targeted sequencing (TGS). When comparing cfDNA-WES to tDNA-WES, mutation detection sensitivity was 53%, consistent with previously published prospective study comparing cfDNA-TGS to tDNA-TGS. For samples in which presence of tumor DNA was confirmed in cfDNA, tumor mutation load from liquid biopsy was correlated with tumor biopsy. Taken together, this study demonstrated that liquid biopsy may be applied to determine tumor mutation load. Qualification of liquid biopsy for interpretation is a crucial point to use cfDNA for mutational load estimation.