Minimal Residual Disease Testing: Definition and Performance Characteristics
Minimal residual disease (MRD) testing detects cancer cells that persist below the threshold of conventional morphologic methods, with current standardized assays achieving sensitivities of at least 1×10⁻⁴ (0.01%) and molecular methods reaching 1×10⁻⁶ (0.0001%), demonstrating high concordance rates between methodologies. 1
What is MRD Testing?
MRD refers to the presence of leukemic or cancer cells remaining in the body during or after treatment at levels below what conventional imaging or laboratory tests can detect. 2 Patients who achieve complete remission by morphologic assessment alone (bone marrow blasts <5%) can potentially harbor a large number of residual cancer cells. 1
The term "Measurable" residual disease is now preferred over "Minimal" residual disease as it is more objective when the detection limit is specified. 1, 2
Sensitivity and Specificity of MRD Testing
Sensitivity Thresholds
The sensitivity of MRD testing varies by methodology:
Flow cytometry and PCR methods: Detect leukemic cells at a sensitivity threshold of at least 1×10⁻⁴ (<0.01%) bone marrow mononuclear cells. 1
Advanced PCR/NGS methods: Can detect leukemic cells at sensitivity thresholds of <1×10⁻⁶ (<0.0001%) bone marrow mononuclear cells. 1
Minimum acceptable sensitivity: The minimum sensitivity limit for response assessment is 10⁻⁴, which has been demonstrated to be an independent prognostic factor. 1 Methods not achieving these sensitivity levels are not recommended. 1
Concordance Between Methods
The concordance rate for detecting MRD between flow cytometry and PCR/NGS methods is generally high. 1 However, results may differ by assay method even for assays with identical sensitivity, which is why the specific assay method must always be reported. 1
MRD Detection Methodologies
The most frequently employed methods include:
Multiparameter flow cytometry (≥6-color): Detects leukemia-associated immunophenotypes with rapid turnaround time. 1
Real-time quantitative PCR (RQ-PCR): Detects clonally rearranged immunoglobulin and T-cell receptor genes, capable of quantifying MRD at the 10⁻⁵ level. 1
Reverse transcriptase quantitative PCR (RT-qPCR): Detects fusion genes such as BCR::ABL1. 1
Next-generation sequencing (NGS): Detects fusion genes or clonal rearrangements without requiring patient-specific primers, with emerging higher sensitivity. 1
Important Technical Considerations
Assays require prospective technical validation and must undergo cross-laboratory standardization with external quality assurance procedures. 1 In clinical trials, European Research Initiative in CLL (ERIC)-compliant flow and EuroMRD-compliant RQ-PCR are most common, typically performed at specialized centers. 1
Reporting Standards and Interpretation
Nomenclature
"Undetectable-MRD" (U-MRD) is preferable to "MRD-negative" because disease may be detectable below the reporting threshold. 1, 2
Categorical measures specify the upper limit of disease:
- MRD4 denotes <0.01%/10⁻⁴ disease
- MRD5 denotes <0.001%/10⁻⁵ disease 1
Detectable (d) or undetectable (u) designations provide additional information about whether disease is measurable below the stated threshold. 1
Required Reporting Elements
Every MRD report must specify:
- Assay method (e.g., flow cytometry, PCR, NGS) 1
- Tissue assayed (peripheral blood vs. bone marrow) - MRD levels may differ between tissues from the same patient 1
- Detection limit of the sample, which depends on assay used and sample quality 2
Clinical Significance
MRD is the most important independent prognostic factor in acute lymphoblastic leukemia and chronic lymphocytic leukemia, superseding historically relevant prognostic factors including age, white blood cell count, and cytogenetics. 1, 3 Studies in both children and adults have demonstrated strong correlation between MRD and risk for relapse. 1
Common Pitfalls to Avoid
Sample quality matters: The optimal sample for MRD assessment is the first pull or early pull of bone marrow aspirate. 1
Timing considerations: MRD quantification can be affected by bone marrow aplasia; MRD sent during aplasia may need to be repeated after count recovery. 1
Prior treatment effects: Prior treatment with immunotherapy or hematopoietic cell transplantation can affect interpretation of flow cytometry-based MRD results. 1
Tissue selection: For optimal sensitivity, MRD assessment should generally be performed on bone marrow specimens, as levels of detectable MRD may be 1 to 3 logs lower in peripheral blood. 1