Minimal Residual Disease by Flow Cytometry: Latest Insights on Validation

Despite effective cancer treatment with innovative cellular and antibody-based therapeutics that eliminate malignant cells, patients who have achieved complete remission may experience relapse. Increasing evidence suggests that the presence of a few persisting cancer cells, known as measurable/minimal residual disease (MRD) in bone marrow (BM) tissue, is a prognostic factor for long term disease outcome in patients with hematological conditions¹. There is now much interest around early use of MRD as a surrogate marker of long-term survival, especially given its potential to accelerate trial reporting and be accepted as a primary/secondary trial endpoint, also potentially supporting accelerated drug approvals. Here, we explore how next-generation flow cytometry (flow’) is integral to evaluating MRD in hematological malignancies.

MRD: role as a surrogate biomarker for long term progression-free survival in clinical trials

MRD as a general measure of remaining tumor burden has multiple potential regulatory and clinical uses as a biomarker with the potential to reflect a patient’s response to treatment, disease progression, or provide prognostic and predictive value to assess the risk of relapse (fig.1).

Figure 1: MRD detection as a prognostic biomarker for progression-free survival (figure adapted from ²).

Negative MRD, or undetectable MRD, post treatment indicates a measurement of <1 in 105 residual malignant tumor cells in a BM specimen. However, can we be confident that MRD is an appropriate prognostic and surrogate biomarker of treatment response in studies in hematologic malignancies)? The robust association between complete response (CR), and progression-free survival (PFS), with a negative MRD, together with the reproducibility of results suggests that we can.

In general, flow cytometry readouts designed for MRD measurements have been proposed for such endpoints in several hemato-malignancies such as Acute Myeloid Leukemia (AML)³, Chronic Lymphocytic Leukemia (CLL)⁴ and Multiple Myeloma (MM)⁵. 

Specifically to MM, The International Myeloma Working Group (IMWG) has defined flow MRD-negative response criteria in multiple myeloma as an “absence of phenotypically aberrant clonal plasma cells by next-generation flow cytometry on bone marrow aspirates using the EuroFlow standard operating procedure for MRD detection (or a validated equivalent method) with a minimum sensitivity of 1 in 105 nucleated cells or higher”5,6 – indicating that CR plus MRD-negative status enhances multiple myeloma prognosis and drug development. In addition, in April 2024, the U.S. Food and Drug Administration (FDA) Oncologic Drugs Advisory Committee (ODAC) unanimously voted in favor of using MRD-negative CR as an early endpoint reasonably likely to predict clinical benefit in multiple myeloma that may be used to support accelerated drug approval.

A EuroFlow/Cytognos-based assay from Labcorp

Our Central Laboratory Services (CLS) offering is the EuroFlow/Cytognos-based flow panel: rigorously validated by CLS globally (Indianapolis, Geneva, Singapore and Shanghai)

EuroFlow Consortium – a multinational strategy

Our validation approach builds on the extensive experience from EuroFlow – a scientific consortium of over 20 individual diagnostic research groups. Collaboratively with Cytognos, Waters Biosciences, they provide a standardized reference platform for flow cytometry techniques7. This includes sample preparation, antibody panel construction and automated identification of plasma cells against reference databases of patient, and control, BM samples. EuroFlow/Cytognos-based tools also allow analysis of plasma cells from a larger total cell density (>107) to generate reproducible, highly sensitive MRD data.

Validation of the EuroFlow/Cytognos-based panel

Validation of this panel included critical assay performance parameters such as robustness, reproducibility, sample stability, and sensitivity measurements such as the limit of detection (LOD) and the lower limit of quantitation (LLOQ) for MRD cut-off criteria. Also, CLS developed a set of quality control (QC) reagents that monitor sample processing, staining and analysis for assay trending and for assuring the validity of the sample run during multiple myeloma clinical trials across several sites.

Ensuring specificity and sensitivity is multifactorial

Assay configuration: This assay is a two-tube/eight-color flow cytometry assay, with intracellular staining. It is composed of 10 markers, including cytoplasmic kappa and lambda chains. The immunophenotypic markers identify and discriminate between normal and abnormal plasma cells.

Precision assessments, gating strategies and custom-developed QCs optimize assay performance while additional validation tests defined sample stability, shipping conditions and lower limits of detection/quantification (LOD/LLOQ). Our intra-assay and inter-assay precision have been evaluated with results indicating mean % coefficient of variations (CVs) within acceptance criteria at 5.9% and 8.8 %CV, respectively. Validation has confirmed that samples are stable up to 48 hours post collection.

Why does MRD by flow cytometry resonate with drug developers?

Flow cytometry is a standard and convenient method of MRD detection in clinical trials. This follows Food and Drug Administration (FDA) approval in 2018 of the first next-generation sequencing (NGS)-based MRD assay for multiple myeloma. NGS assays typically offer higher test sensitivity with increased costs compared to flow – although both flow (non-FDA approved to date) and NGS are acceptable methods, and there is an argument for either technique.

Key advantages of MRD by flow:

• The EuroFlow-based panel achieves IMWG sensitivity requirements for clinical trials, rapid results turnaround and supports cost effectiveness in trials. Using MRD does not require collection and assessment of a baseline comparator sample
• Supports decisions allowing for rapid stratification of study groups based on findings and desired outcomes. For this reason, if MRD testing is conducted after the initial therapy (e.g., autologous stem cell transplant, ASCT), the subsequent induction regimens can be compared
• Provides additional data compared to NGS: phenotypic information from normal and abnormal plasma cells
• Final data analysis and hematopathologist interpretation of findings is undertaken by a centralized data analysis unit and pathology team. This provides the consistency, convenience and reassurance of a secure testing and sample storage facility “under one roof” (fig.2).
• A dedicated pathway is available for processing BM samples (fig.2) – supporting the 48-hour integrity limits for fresh BM tissue as stated in the International Clinical Cytometry Society (ICCS), European Society for Clinical Cell Analysis (ESCCA) guidelines and confirmed by Labcorp internal validation data.
• QC material is utilized to monitor test performance across our global CLS locations (Indianapolis, Geneva, Singapore, Shanghai).
• A QC for bone marrow hemodilution is incorporated into the process, which aids in MRD data interpretation

Figure 2: Flow cytometry workflow for MRD testing

What are the potential challenges of validating and implementing MRD assays by flow and how are they addressed?

There are three main challenges during MRD assay validation and testing. First, the teams must be highly trained in flow techniques and analysis. Second, standardized sample processing and analytical approaches are required globally. Finally, it is paramount to procure sufficient fresh BM samples with sufficient cellularity to assess assay performance and matrix stability during validation and to ensure appropriate testing during clinical conduct.

An important area of focus to monitor laboratory performance after assay implementation, beyond the use of custom-made QCs with every batch, is our enrolment to proficiency testing program with the United Kingdom National External Quality Assessment Services (UK NEQAS) consortium. We compare our MM MRD test with known global standards and against other laboratories. Outcomes from this program confirm assay accuracy and performance and allow us to fine tune our lab operations data analysis, data review and data reporting procedures when necessary.

In summary

Our EuroFlow/Cytognos-based assay has evolved and undergone extensive validation to provide rapid and accurate MRD results. MRD has the potential to be an established endpoint informing trial and treatment decisions at an earlier stage and fast-tracking drug to patient on a global scale. MRD by flow cytometry offers a high-quality, clinically robust resource with future potential as a companion diagnostic for multiple myeloma.  

Contact us to learn more about how our MRD by flow assay can help accelerate your clinical trials.