Detecting measurable residual disease (MRD) in hematological malignancies using flow cytometry

A patient may be in complete remission during or after a cancer treatment, but a few cancer cells may persist in what is known as residual disease. To better inform prognoses, understand treatment decisions, predict a patient’s overall survival or understand the potential for disease relapse, it is important to have the ability to detect and quantify these malignant cells.

In this article, Sriram Ramanan, PhD, lead scientist of flow cytometry, discusses how Labcorp Drug Development employs flow cytometry to support immune monitoring during clinical trials. He shares how flow cytometry can evaluate hematological malignancies, such as leukemia and lymphoma, by detecting measurable residual disease (MRD), which is also referred to as minimal residual disease.

Understanding the importance of detecting MRD

In this figure adapted from Hauwel and Matthes (2014), the significance of MRD detection is illustrated. As a patient’s tumor load drops, they enter cytological remission. However, if a tumor load still exists over time, the cancer may return. Knowing if the MRD is considered high, low or negative can help determine if a patient will achieve complete remission or experience relapse.

Methods for measuring MRD in leukemia and lymphoma

While several methods can support MRD testing, the primary objective is to detect very rare cells. Even one malignant cell might lead to relapse or return of the disease, which highlights the importance of using a highly sensitive platform for MRD testing.

• Microscopy is inexpensive and widely available for studying leukemic cells and chromosomal aberrations, yet its sensitivity is considered too low—at 1 in 20-100 (1%-5%)—to detect rare cells in a sample
• Real-time quantitative polymerase chain reaction (RQ-PCR) and next-generation sequencing (NGS) methods have very high sensitivity—around 1 in 1,000,000 (0.0001%)—but these technologies may not be applicable to all patients as they can be limited to mutable or genetically measurable lesions
• Flow cytometry offers high sensitivity and is widely applicable for MRD testing, given that patients’ cells can be labeled with cluster of differentiation (CD) markers to determine how their expression levels change. Furthermore, when compared to NGS, flow cytometry is less time consuming and comparatively inexpensive

Addressing the challenges of flow cytometry

As a technique that performs quantitative and qualitative measurements of single particles in suspension, flow cytometry is considered a sensitive, readily available method for measuring specific markers. However, like any method, flow cytometry has its own challenges.

• Difficulties with sensitivity: The test sensitivity of flow cytometry is dependent on the number of acquired events, which is defined as single particles/cells detected by the instrument. The more events that are acquired, the higher the sensitivity, which can range from 1 event in 1,000 (0.10%) to 1 event in 100,000 (0.001%). With this variation, it becomes critical to obtain sufficient sample volume to reach sensitivity limits.
• Reliance on sample stability: As with any test, sample stability is important for accurate results. Flow cytometry assays are developed using ambient temperature whole blood or bone marrow samples. For global clinical trials that work with Labcorp Central Laboratory Services, Labcorp aims for an average of 24 hours between sample collection and delivery to ensure stability.
• Evaluating hemodilution: Hemodilution can occur if whole blood is drawn and mixed with the bone marrow aspirate during sample collection. This can potentially lead to an underestimation of MRD and may even lead to false negative results if the assay is close to its detection limits. To determine if the quality of a bone marrow sample is adequate, Labcorp uses flow cytometry panels to analyze sample viability (7-AAD negative) and the presence of mast cells and mature neutrophils (both cellular indicators of hemodilution of bone marrow).

Developing assays using a fit-for-purpose approach

Based on a drug development sponsor’s purpose—whether exploratory or to address primary or secondary endpoints in a clinical trial—the validation approach for a flow cytometry assay can vary. An exploratory assay would undergo assessment, for the precision in the matrix along with stability assessments, while a clinical assay used to guide patient management would also examine the limit of detection and lower limits of quantitation and define other parameters such as normal reference intervals for safety considerations.

Most importantly, for primary and secondary endpoints, the validation would also need two levels of quality control reference material. This helps with longitudinal quality monitoring of the flow cytometry assay’s performance throughout a clinical trial and establishes pass/fail criteria for each batch.

Eliminating common sources of variability

Although flow cytometry is challenging to standardize, it is possible to reduce potential sources of variability and provide consistent results for a clinical trial. At the phase of sample acquisition, standardized acquisition templates and instrumentation settings help unify the acquisition criteria for cellular events throughout the trial and across sites. Additionally, Labcorp’s global network of central laboratories uses the same SOPs, instruments and reagents to support cross laboratory correlation and generate reproducible and comparable data across instruments, operators and sites.

The Labcorp flow cytometry department also incorporates quality control monitoring for certain assays to track their ongoing performance. Periodic external proficiency testing provides another level of assurance that the resulting values are within an acceptable range defined by a third-party proficiency testing center. Finally, at the data analysis stage, centralized data analysis software examines the gating of data followed by a secondary review to identify and reduce potential variability. For MRD applications, these data are then reviewed and approved by a board-certified hematopathologist prior to being released to clinical sites and sponsors.

Following guidelines to support the reliable measurement of MRD

The European LeukemiaNet has provided guidelines for the use of MRD in clinical practice, and the EuroFlow Consortium has published recommendations for developing reliable MRD assays targeting diseases such as acute myeloid leukemia (AML). Similarly, the European Research Initiative on CLL (chronic lymphocytic leukemia) (ERIC) has developed clinical guidance for identifying MRD.

In January 2020, the FDA published guidelines for MRD testing. This guidance is intended to help sponsors who plan to use MRD as a biomarker in clinical trials conducted under an investigational New Drug Application (IND) or to support marketing approval of drugs and biological products for treating specific hematological malignancies.

Following these research-based guidelines and recommendations, Labcorp Central Laboratory Services has developed highly sensitive assays for AML, CLL and multiple myeloma (MM). Expanding on this expertise, the Labcorp flow cytometry team is currently developing a panel for acute lymphocytic leukemia (ALL) to assess MRDs in clinical trials.

With extensive experience developing flow cytometry assays that span cellular enumeration, immunophenotyping and immune functional testing, Labcorp has created more than 300 custom assays to support clinical trials. Learn more about how the use of flow cytometry at Labcorp can help advance novel drug discovery and development.