Minimal Residual Disease by Flow Cytometry: Latest Insights on Validation

Despite the development of cellular and antibody-based therapeutics that eliminate malignant cells, patients who have achieved complete remission or response may experience relapse. Increasing evidence suggests that the presence of measurable/minimal residual disease (MRD) in bone marrow (BM) tissue is one, if not the strongest, prognostic factor for disease outcome in patients with hematological conditions.¹ There is now much interest around early use of MRD as a surrogate marker of survival, especially given its potential to accelerate trial reporting and be accepted as a primary/secondary trial endpoint. Here, we explore how next-generation flow cytometry (‘flow’) is integral to evaluating MRD.

MRD: role as a surrogate biomarker for survival in clinical trials

MRD as a general measure of tumor burden has multiple potential regulatory and clinical uses as a biomarker with the potential to reflect a patient’s response to treatment or provide prognostic value to assess the risk of relapse. Negative MRD, or undetectable MRD, post treatment indicates a measurement of <1 in 10⁵ residual 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 of multiple myeloma? The robust association between complete response (CR), and progression-free survival (PFS), with a negative MRD, together with the reproducibility of results, and the relevance to the multiple myeloma trial setting, suggests that we can.

Recently, 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”^2,3 – indicating that CR plus MRD-negative status enhances multiple myeloma prognosis and drug development.

A EuroFlow/Cytognos-based assay from Labcorp

Our new Central Laboratory Services (CLS) offering is the EuroFlow/Cytognos-based flow panel: rigorously validated by CLS and supporting MRD use as an inclusion/exclusion criterion, and as a surrogate primary/secondary endpoint marker.

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 and one small/medium-sized enterprise (Cytognos). Collaboratively, they provide a standardized reference platform for flow cytometry techniques. 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 stability, the limits of detection (LOD) and the lower limits 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.

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 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) 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.”
• A dedicated pathway is available for processing BM samples (fig.1) – supporting the 48-hour integrity limits for fresh tissue as stated in the International Clinical Cytometry Society (ICCS) and European Society for Clinical Cell Analysis (ESCCA) guidelines
• QC material is utilized to monitor test performance across our global central labs
• An early QC for bone marrow hemodilution is incorporated into the process, which aids in MRD data interpretation

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

There are three main challenges during MRD assay validation. 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 to assess assay performance and matrix stability.

An important area of focus in 2021 will be proficiency testing with the United Kingdom National External Quality Assessment Services (UK NEQAS) consortium. We compare our tests with known global standards and against other laboratories. This testing allows us to fine tune our lab operations data analysis, data review and data reporting procedures.

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 primary/secondary endpoint, informing trial and treatment decisions at an earlier stage and fast tracking drug to patient on a global scale. MRD by flow 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.