The QuIP Study Attempts to Harmonise the Use of TMB Panels

TMB is actively being studied as a predictive biomarker for identifying patients who might benefit from immunotherapy but the issue of variability between assays has been raised
13 Dec 2019
Pathology/Molecular biology;  Translational research

Findings from a large study illustrating that tumour mutational burden (TMB) could be assessed using different assays across separate centres and provide results that correlated well with the standard reference of whole exome sequencing (WES)were presented at the ESMO Immuno-Oncology Congress 2019 in Geneva, Switzerland (11-14 December).  

Albrecht Stenzinger of the Institute of Pathology, University Hospital Heidelberg, in Heidelberg, Germany presented findings on behalf of the Quality in Pathology (QuIP) study investigators. The study was designed in strategic partnership with the Friends of Cancer Research (FoCR) to assess the performance and specifications of diverse TMB panels in a wet-lab setting.

Data from non-small cell lung cancer (NSCLC) trials suggest that whole exome sequencing (WES) and panel-sequencing may be used to determine TMB (psTMB), and that centralised and decentralised/lab-developed testing models for panel-based measurements are acceptable.

Assays were repeated >20 times on diverse tumour samples representing the full range of TMB to determine the reproducibility of each assay

The investigators analysed 20 FFPE samples from patients with NSCLC, head and neck squamous cell carcinoma (HNSCC), colorectal cancer (CRC), including those with microsatellite instability (MSI) and mutated POLE in 11 major pathology centres. TMB was defined as 2 to 200 muts/Mb and WES data served as the reference standard.

Each tumour sample was tested more than 20 times across several panels and institutions, which resulted in >450 datasets. Using raw sequencing data, processed file formats, and reported TMB values, the research team dissected specifications of each panel result, identified panel-specific requirements, and analysed Pearson correlations of TMB data between assays and labs, and also compared psTMB with WES-derived TMB.

Each of the 6 major panels used had different requirements regarding library preparation (for example, hybridisation versus PCR) and input material (range: 20-200 ng).

The investigators found that tumour cell content, as well as the quality and quantity of DNA were crucial pre-analytic factors that require integration with coverage data, variant allele frequency (VAF) cut-points, and assay-specific features (molecular barcodes) to obtain reliable TMB results. Control of C>T artefacts was particularly important for assays not using deduplication methods.

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Assay-Independent and Assay-Specific Parameters Influence the Accuracy of psTMB Scores.

© Albrecht Stenzinger and Daniel Kazdal.

The QuIP study provides harmonisation of TMB panels

For most of the participating laboratories and tested sequencing panels, strong or moderate correlations between panel-TMB measurements were observed. After bridging of panel-TMB to WES-TMB and using a cutpoint of 199 mutations, panel sequencing and WES agreed in classification of 74.9% of the cases.

Conclusions

The QuIP study demonstrated that different TMB panels can be used under real-world conditions that have overall low variability across sites and demonstrate strong correlations with TMB as determined by WES. The authors commented further that they identified both common and panel-specific parameters that influence TMB results in daily practice.

In conjunction with the FoCR study, these findings will support standardisation of TMB testing, enable the implementation in routine diagnostics, and contribute to the understanding of TMB-related clinical trials.

 

Disclosure

This study was partly sponsored by Bristol-Myers Squibb, FMI, Illumina, Merck Sharp & Dohme, NEO New Oncology, QIAGEN, Roche, and Thermo Fisher Scientific.

Reference

1O - Stenzinger A, Endris V, Budczies J, et al. Harmonization and standardization of panel-based tumour mutational burden (TMB) measurement: Real-world results and recommendations of the QuIP study.

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