Development of a TCR beta repertoire assay for profiling liquid biopsies from NSCLC donors

Aim:The aim of this study was to demonstrate the utility of T-Cell receptor beta(TCRβ)sequencing as a robust method for assessing T-cell repertoire changes in donors with non-small cell lung cancer(NSCLC).We further demonstrated the use of the assay by monitoring repertoire modulation in a defined model antigen system,cytomegalovirus(CMV).Methods:Peripheral blood mononuclear cells from four healthy donors were challenged with a 1-week exposure to whole-cell lysate from CMV-infected cells or CMVpp65495-503 peptide(NLVPMVATV).T-cell repertoire perturbations were assessed using the Oncomine TCR Beta-SR Assay and Ion GeneStudio S5 Plus Sequencer.A pp65 tetramer flow cytometry assay was used as an orthogonal method to assess clonal expansion of a subset of CMV-specific T-cells.For evaluation of the assay in peripheral blood lymphocytes from NSCLC donors,five whole blood specimens were evaluated using the same sequencing workflow.Results:The TCR beta assay identified 6,683-61,936 unique clones from 1-2 million reads per sample,and an average of 80%of the total reads were usable for TCR profiling.In the NSCLC donors,TCR convergence and clonality values were consistent with published results and ranged 0.016-0.033 for convergence and 0.09-0.48 for clonality.In the CMV study,TCR sequencing detected the expansion of a common family of clones in all 4 samples in response to antigen stimulation.This expansion corresponded to an increase in pp65 tetramer staining by flow cytometry.Baseline TCR convergence scores ranged 0.009-0.041 and increased 5-fold in one sample as a result of pp65 antigen stimulation.Conclusion:The results of this study demonstrated the utility of profiling of the TCRβrepertoire in a model system and in donors with NSCLC.Additionally,we demonstrated the correlation between RNA-seq methods and protein-tetramer analysis using flow cytometry.These techniques represent an emerging solution that could complement other liquid and tissue diagnostic assays in the clinic and will be of value in predicting host response/resistance and adverse events to immunotherapies.Prospective clinical studies are on-going in which the developed TCR beta assay will undergo further validation。

Longitudinal monitoring for the emergence of epidermal growth factor C797S resistance mutations in non-small cell lung cancer using blood-based droplet digital PCR

Validation of assays for the C797S mutation as a biomarker for osimertinib resistance is promising in guiding treatment decision-making for multidrug resistant non-small cell lung cancer. A newly developed droplet digital PCR (ddPCR) assay was used to retrospectively evaluate the emergence of the C797S mutation in six remnant plasma samples in this case report. It was found that the detected emergence of C797S clearly correlated with clinical signs of treatment resistance. Had these data been available to aid treatment selection in real time, there would have been hope for recaptured disease response and control instead of treatment cessation. The results of this study show that highly sensitive ddPCR methods can be used for the monitoring of emergent epidermal growth factor somatic variant mutations in circulation.