0000000000198953
AUTHOR
Marij J. P. Welters
Hierarchical modeling for rare event detection and cell subset alignment across flow cytometry samples.
Flow cytometry is the prototypical assay for multi-parameter single cell analysis, and is essential in vaccine and biomarker research for the enumeration of antigen-specific lymphocytes that are often found in extremely low frequencies (0.1% or less). Standard analysis of flow cytometry data relies on visual identification of cell subsets by experts, a process that is subjective and often difficult to reproduce. An alternative and more objective approach is the use of statistical models to identify cell subsets of interest in an automated fashion. Two specific challenges for automated analysis are to detect extremely low frequency event subsets without biasing the estimate by pre-processing…
Managing Multi-center Flow Cytometry Data for Immune Monitoring.
With the recent results of promising cancer vaccines and immunotherapy 1 – 5 , immune monitoring has become increasingly relevant for measuring treatment-induced effects on T cells, and an essential tool for shedding light on the mechanisms responsible for a successful treatment. Flow cytometry is the canonical multi-parameter assay for the fine characterization of single cells in solution, and is ubiquitously used in pre-clinical tumor immunology and in cancer immunotherapy trials. Current state-of-the-art polychromatic flow cytometry involves multi-step, multi-reagent assays followed by sample acquisition on sophisticated instruments capable of capturing up to 20 parameters per cell at a…
Flow Cytometry in Cancer Immunotherapy: Applications, Quality Assurance, and Future
Cancer immunotherapy seeks to elicit or augment the antitumor immune response in a patient in order to enlist the help of the patient’s own immune system for tumor control. In this context, immune monitoring provides evidence of immunogenicity, guides the choice and dosage of antigens, assesses the effects of immune modulators and therapy combinations, and has the potential to reveal early biomarkers of clinical efficacy. In view of their role in the anticancer immune response, the quantity and quality of tumor antigen-specific effector CD4+ and CD8+ T cells are of particular interest, and characterization of regulatory T cells and myeloid-derived suppressor cells is increasingly relevant. …
Development of an RNA-based kit for easy generation of TCR-engineered lymphocytes to control T-cell assay performance.
Cell-based assays to monitor antigen-specific T-cell responses are characterized by their high complexity and should be conducted under controlled conditions to lower multiple possible sources of assay variation. However, the lack of standard reagents makes it difficult to directly compare results generated in one lab over time and across institutions. Therefore TCR-engineered reference samples (TERS) that contain a defined number of antigen-specific T cells and continuously deliver stable results are urgently needed. We successfully established a simple and robust TERS technology that constitutes a useful tool to overcome this issue for commonly used T-cell immuno-assays. To enable users t…
Immunoguiding, the Final Frontier in the Immunotherapy of Cancer
T cells play an important role in cancer. This notion is strongly supported by an enormous number of trials on the clinical impact of tumor-infiltrating T cells and studies consequently showing that therapeutic interventions which are based on transfer, activation and expansion, or de-blocking of tumor-specific T cells, which have met with clinical success. To optimally profit from the flow of newly developed immune-based therapeutics aiming to reinforce the systemic and local tumor-specific T-cell response, it will be required to identify biomarkers that provide the rationale to use a particular therapy, that measure the effect of intervention, and that can predict the outcome of a therape…
Harmonization of Immune Biomarker Assays for Clinical Studies
Assays that measure a patient's immune response play an increasingly important role in the development of immunotherapies. The inherent complexity of these assays and independent protocol development between laboratories result in high data variability and poor reproducibility. Quality control through harmonization--based on integration of laboratory-specific protocols with standard operating procedures and assay performance benchmarks--is one way to overcome these limitations. Harmonization guidelines can be widely implemented to address assay performance variables. This process enables objective interpretation and comparison of data across clinical trial sites and also facilitates the ide…