Harnessing the Power of Flow Cytometry in Biomarker-Driven Clinical Trials

Flow cytometry has become invaluable in biomarker-driven clinical trials due to its multiparametric nature, which can provide highly detailed information on any single cell in a heterogeneous population. Because of its versatility, flow cytometry is the primary method used to monitor immune responses in both the preclinical and clinical settings, allowing researchers to visualize immune cells and their interactions in disease or after drug treatment, thereby facilitating biomarker discovery and validation and guiding clinical decisions.

Advantages and Key Benefits of Flow Cytometry

Advantages of flow cytometry over other cell analysis methods include:

• High throughput: Flow cytometers can measure >35,000 events per second, allowing researchers to narrow down the biomarker profile and the targeted cell population, accelerating drug development.
• Multiparameter: Current technology can measure >40 fluorophores simultaneously. Surface, intracellular, and intranuclear information can be analyzed at the single-cell level.
• Comprehensiveness: Researchers can obtain information on the heterogeneity and cell differentiation of the immune cell subsets, phenotype, activation, and proliferation.
• Quantification: In addition to providing relative percentages, flow cytometry can provide absolute count and number of receptors in a cell.
• Ability to quantify receptor occupancy: Flow cytometry-based receptor occupancy assays quantify the occupancy of drug-target receptors by measuring the levels of free and occupied receptors after drug treatment and at the single-cell level.

Applications of Flow Cytometry in Biomarker-Driven Trials

There are various potential applications for flow cytometry in biomarker-driven clinical trials, a few of which are enumerated below.

Immunophenotyping to Investigate Immune System Response After Drug Treatment

Immunophenotyping is valuable when transitioning from preclinical research to early-phase clinical development. For targeted therapies, immunophenotyping can be used to demonstrate that the drug induces the activation or depletion of the target cell population while leaving other cell populations unaffected. Flow cytometry can also help support safety data and dosage information in early-phase clinical studies, for example, by showing how drug treatment affects proinflammatory cytokine production, cell activation, or proliferation. Flow cytometry may even be useful for identifying surrogate biomarkers. For instance, to evaluate the immune response to a drug designed to treat solid tumors, which are difficult to analyze, it may be beneficial to identify downstream hematopoietic biomarkers or evaluate non-specific responses in the periphery.

Monitoring Receptor Occupancy with Biologics

Frequently used to generate pharmacodynamic (PD) biomarker data in preclinical and clinical studies, receptor occupancy (RO) assays are designed to quantify binding of therapeutics to cell surface targets. RO is commonly measured by flow cytometry, using one or a combination of three basic formats: free receptor measurement, total receptor measurement, or direct assessment of bound receptor. Since the staining occurs at the single-cell level, the RO is key to determining the saturation and half-maximal active concentration (EC50) of the drug on the target cell population and verifying any collateral effects, such as cleavage or internalization of the receptor, survival of the cells, or activation of unwanted cell populations.

Quantifying Antigen-Specific Responses or Cell Activation

Using tetramer technologies, flow cytometry enables characterization and quantification of antigen-specific responses. Using phospho-protein staining, which measures protein phosphorylation at the cellular level, provides insight into the cell signaling pathway after drug treatment to help determine the activation status of the cells.

Quantifying Rare Cell Populations

In scenarios, such as measuring minimal residual disease (MRD) or evaluating turnover and bioavailability of CAR-T cells, where the cell(s) of interest have a low frequency, flow cytometry can be useful for quantifying these rare populations. While not as sensitive as other techniques such as polymerase chain reaction (PCR), flow cytometry has the advantage of being able to simultaneously measure other cells on the CAR-T cells.

Validation of Flow Cytometry Assays

Biomarkers used in flow cytometry panels should be validated. In clinical studies, all biomarkers must undergo advanced validation to ensure that data are interpretable and reproducible within the expected timeframe, within large patient cohorts, and across different labs. Validation requirements are specific to the stage of drug development and the intended use of the biomarker data.

It is also essential to eliminate sources of variation that may impact flow cytometric analysis by ensuring the following are consistent across all labs:

• Sample preparation
• Sample processing
• Instrument configuration and settings
• Data analysis process
• Standard operating procedure for the assay
• Operator training

Example of Flow Cytometry Application: Monitoring T Regulatory Cells

Regulatory T (Treg) cells are a subpopulation of CD4+ T cells that suppress abnormal or excessive immune responses to self and non-self-antigens to maintain immune homeostasis. In cancer, Treg cells are involved in tumor development and progression by inhibiting antitumor immunity through several immune-suppressive mechanisms. High infiltration by Treg cells is associated with poor survival in various types of cancer. Therefore, strategies for depleting Treg cells and controlling their functions to increase antitumor immune responses are actively being studied in the cancer immunotherapy field.
Dysregulation in Treg cell type, frequency, or function is implicated in a variety of non-oncologic conditions, including autoimmune diseases. Since Treg cells are versatile and heterogeneous, the ability to identify, quantify, and characterize these cells is critical for elucidating their complex role in immunity and their potential as therapeutic targets. Among different Treg cell types, thymus-derived, natural Treg cells require a panel design and gating strategy that identifies CD4+ CD25+ CD127- Foxp3+ cells.

Panel Design

Sample Gating Strategy

Once the panel design and gating strategy have been defined, the assay must be validated to demonstrate that it is fit for purpose within the context of use, including intra-assay, inter-assay, inter-operator variability as shown in Figure 4. Sample aging and staining stability might also be included to determine the cutoff for sample testing and sample acquisition, respectively.