Over thirty years ago, scientists at the National Cancer Institute started to explore ways of utilizing the patient’s immune system to fight cancer. Their work laid the foundation for what is now an encouraging cancer treatment that doesn’t necessitate radiation or surgery.
It is anticipated that the global immunotherapy market, including CAR T-cell therapy, checkpoint inhibitors, adoptive cell therapies, monoclonal antibodies and other immunotherapies, will experience a growth of up to 10.1% CAGR between 2020 and 2028.1
To develop and deliver these emerging immunotherapies, patients provide blood samples for clinical trial sponsors. Each sample provided is priceless, as each one offers a plethora of biological data that benefits therapy development.
Global logistics and sturdy sample transportation are key to ensuring samples are intact upon arrival to the lab. The vast scientific expertise contained within those labs guarantees the highest-quality analysis and reporting.
A central lab with an international footprint offers both the efficient transportation arrangement and scientific rigor needed to release the crucial information that patients’ biological samples contain.
Flow cytometry (FCM) is the principal method scientists rely on to monitor immune responses in clinical trials, mainly because of its power, speed and capacity to offer a detailed view of a disease’s macro-environment.
With an enhanced focus on personalized medicine, such as immunotherapy, there’s significant demand for FCM. It is principally used to monitor changes in immune cell phenotype, track therapeutic cells and identify and monitor abnormal cells.
The benefits of FCM over other methods
Researchers acquire information on immune cell subsets, memory phenotype, activation, regulatory exhaustion and more using a single tube of blood,.
Flow cytometers can measure over 35,000 events per second (and counting).
Current technology can simultaneously measure 40 or more cellular markers.
Researchers can acquire relative percentages and discover the absolute count and number of receptors in a cell.
FCM’s high-parameter capacity generates powerful data in a limited time frame. The information acquired from FCM advises the diagnosis, treatment and monitoring of residual or relapsed disease.
While its use principally centers on oncology, researchers also utilize it for immunology, rare disease and infectious disease research. Researchers look to FCM for the following:
- Cell cycle analysis to determine each phase of the cell cycle
- Drug occupancy of cellular target markers
- Characterize and/or quantify proteins, cellular activation, proliferation, exhaustion and apoptosis status
- Immunophenotyping to find the presence or absence of antigens without yielding sensitivity. The most commonly used applications include identifying immune cells, detecting and analyzing immune cells’ subsets or statuses, and identifying leukemic and therapeutic cells.
- Immunophenotyping panels for blood and bone marrow aspirate (BMA) are appropriate for patient follow-up and evaluating recovery and remission in lymphoma, myeloma and leukemia clinical trials.
- Report characteristics of cell populations with greater sensitivity, with frequencies as low as 1 in 10,000 cells, critical for detecting minimum/measurable residual disease (MRD).
Flow cytometry in biomarker research
Flow cytometry has become extremely valuable for biomarker research and development because it offers comprehensive information on single cells in a heterogeneous population.
FCM offers researchers the data to help identify potential biomarkers to better grasp why certain patients show resistance to a therapy. This helps sponsors find suitable patients for clinical trials and helps clinicians formulate patient-specific treatment plans for participants.
Moreover, FCM assays accurately measure biomarkers with good reproducibility to ensure consistency of measurement both within and between patients as well as identifying new biomarkers.
FCM data are employed to make clinical decisions around efficacy, safety and pharmacodynamics. More complex panels offer information for exploratory assessment throughout clinical trials.
The importance of validation
All biomarkers must go through a process of advanced validation to guarantee interpretable data within multiple time points, within large patient cohorts and tested at various locations across the world. Data emerging from the validation offers guidance for interpreting clinical data.
A fit-for-purpose approach
To secure the quality of FCM assay performance across preclinical and clinical applications, a number of scientific organizations developed guidance for FCM instrumentation and method validation.
Cerba Research and other laboratories used this guidance - as well as GLP and GcLP guidelines - to establish strategies to show that a method is suitable and ‘fit-for-purpose’ within the context of use.2
Under this approach:
- The data must be accurate and reliable (Fit)
- The data is used for decision-making throughout drug development (Purpose)
- The validation requirements are specific to the stage of drug development, with attention toward the intended use of the biomarker data and any applicable regulatory requirements (Fit-for-Purpose)
With a standardized process such as fit-for-purpose, sponsors ensure they possess the validation data required to present to regulatory authorities to indicate that such validations were performed.
The FDA and/or other regulatory agencies usually request validation reports to assess the assay performance for the intended purpose.2,3 Fit-for-purpose method validation begins with assay development or optimization and transitions through to validation and implementation.
Sponsors should review the data to evaluate whether the assay requires any change or improvement for future expansion of the trial, and if so, the cycle starts over again.
Validation parameters for new assays include assessments of the following:
- Carry-over (especially for high-sensitivity assays) - when assays transfer to another lab, these parameters are assessed again, with the exception of sensitivity and stability.
- Inter-operator variability
- Inter-instrument variability
- Post-stained stability (for larger labs)
This guarantees consistency among instrument settings and other parameters.
Emerging trends in flow cytometry
While early flow cytometers were only capable of measuring one or two characteristics, or parameters, of a cell, today’s technology can measure 40 or more parameters. High-parameter flow cytometers utilize spectral detection for the simultaneous identification of up to 50 parameters on a single cell.
These tools are customizable to detect multiple cell subsets in a single panel, optimizing the amount of data acquired from each sample. These instruments assure enhanced data quality over traditional instruments, as well as cost and time savings.
It is anticipated that a significant increase in the use of high-parameter flow cytometry will occur over the course of the next few years, especially for exploratory assays. High-parameter devices grant researchers the power to assess numerous combinations of biomarkers using a smaller sample.
As instrumentation has progressed, so has the chemistry, including developments in reagents and fluorochromes. Tools for data analysis — a key component for high-parameter flow cytometry due to the increase in potential subsets — are also emerging.4
Flow cytometry in global clinical trials: points to consider
When organizing an immunotherapy trial, the following aspects of FCM assay development and validation should be considered to ensure all the necessary data required to evaluate a therapy and monitor disease are acquired.
Fresh blood and BMA samples need to be sent to the labs within the stability period (about 72 hours) and analyzed the same day. This rapid turnaround maintains sample integrity and mitigates the risk of data loss. Especially for early research, when speed is key, scientific teams must also assess and review data in short time periods.
Fresh whole blood and BMA remain industry standards. However, a number of projects do require processed whole blood so that researchers can store cells for prolonged periods or conduct batch testing over numerous time points.
In situations such as these, the lab will sequester peripheral blood mononuclear cells (PBMCs) from whole blood and freeze them for testing at a later time. These blood cells are made up of lymphocytes (T, B, NK cells), monocytes and dendritic cells.
These blood cells are a key component in the immune system for combatting infection and adapting to intruders. While it’s simpler to acquire blood and BMA, the stability of samples is limited and therefore must be processed immediately. PBMCs can be frozen and stored for later use.
However, relying on PBMCs introduces an additional processing step to FCM, which increases turnaround time and leads to additional costs. Blood and BMA are more economical and are inclined to align well with other samples pulled for the clinical research project.
Whether to use PBMCs is dependent on the budget, timeline and intended purpose of the data. After PBMC processing has been completed, some populations of interest may not be present. Scientists at central labs can offer support to develop and validate appropriate assays.
Once method validation has been performed, FCM scientists should review the data to ensure it fulfills all the sponsors’ needs. When clinical trials are initiated, centralized data analysis and standardization among labs help make sure consistency and quality is maintained throughout.
Data from all the labs are reviewed by FCM scientists to check for quality and accuracy in analysis.
A central lab with an international network of FCM scientists offers a number of advantages for immunotherapy developers. No matter how large or global the study, a well-coordinated network with standardized equipment and methods, as well as central analysis, means data stays consistent.
The lab performs experiments and tests in the same manner, employing the same instrument settings and the same reporting format. With FCM scientists situated in various locations, experts can work across different time zones, which helps speed-up turnaround time.
A point of contact in the sponsors’ time zone facilitates easier, more effective communication. Partnering with a lab that can offer a global footprint also establishes assurance in an environment that remains uncertain.
Kits and samples can be dispatched from local hubs to sites on the same continent or in the same country, reducing customs requirements and bypassing the need for an expensive premium carrier.
When sponsors need to screen or randomize patients immediately, they can rest assured the lab will receive samples in a timely manner, even in the face of natural disasters or other major disruptions.
In precision medicine, each tube of blood shows a patient’s complete story. Flow cytometry grants researchers the power to read every detail of that story instantly.
When selecting a central lab for the analysis of those samples, team up with an organization that possesses the expertise, technology and facilities to consistently convey all the information needed with great accuracy.
A central lab with an international infrastructure ensures each and every story remains intact, offering sponsors the data needed to develop life-changing therapies for people who demand them.
- Cancer Immunotherapy Market Size to Reach USD 168.48 Billion by 2028 — Reports and Data. BioSpace. July 7, 2021.
- Lee JW, Devanarayan V, Barrett YC, et al. Fit-for-purpose method development and validation for successful biomarker measurement. Pharm Res. 2006;23(2):312-328. doi:10.1007/s11095-005-9045-3.
- Nithianandan Selliah, Steven Eck, Cherie Green, Teri Oldaker, Jennifer Stewart, Alessandra Vitaliti and Virginia Litwin. (2019). Flow Cytometry Method Validation Protocols. Current Protocols in Cytometry. Published Jan 2019.
- Chattopadhyay PK, Hogerkorp CM, Roederer M. A chromatic explosion: the development and future of multiparameter flow cytometry. Immunology. 2008;125(4):441-449. doi:10.1111 /j.1365-2567.2008.02989.
About Cerba Research
For over 35 years, Cerba Research has been setting the industry standard for exemplary clinical trial conduct. Today, across five continents, with a focus on precision medicine, we are changing the paradigm of the central lab’s role in complex clinical research.
From protocol inception through development and to market, our passionate experts deliver the highest quality specialized and personalized laboratory and diagnostic solutions. Partner with us for the most efficient strategy to actualize your biotech and pharmaceutical products sooner and improve the lives of patients worldwide.
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