MRD Testing in Hematologic Malignancies

MRD Testing in Hematologic Malignancies

Minimal residual disease (MRD) refers to the small number of cancer cells that remain in a patient after treatment. In hematologic malignancies—cancers of the blood, bone marrow, and lymphatic system—MRD testing has become a critical tool for assessing treatment response, predicting relapse, and guiding therapy decisions.

What is MRD?

Definition

MRD represents disease burden below the threshold of conventional detection methods:

A patient in “complete remission” by conventional criteria may still harbor millions of malignant cells—enough to cause relapse.

Clinical Significance

MRD status is one of the strongest prognostic factors in hematologic malignancies:

• MRD-negative: Associated with longer progression-free and overall survival
• MRD-positive: Higher risk of relapse, may warrant treatment intensification
• MRD conversion: Changing from negative to positive often precedes clinical relapse by months

Why Immune Receptor Sequencing for MRD?

The Clonal Signature

Most hematologic malignancies arise from a single lymphocyte that has undergone malignant transformation. This cell carries a unique, clonally rearranged immune receptor:

• B cell malignancies: Clonal immunoglobulin heavy chain (IGH) rearrangement
• T cell malignancies: Clonal T cell receptor (TCR) rearrangement

This rearrangement serves as a molecular fingerprint—a tumor-specific marker that can be tracked with extraordinary sensitivity.

Advantages of Sequencing-Based MRD

Current Approaches

Single-Chain Sequencing (Standard)

Most commercial MRD tests sequence only one chain of the immune receptor:

For B cell malignancies:

• Target: IGH (immunoglobulin heavy chain)
• Focus: VDJ rearrangement, particularly CDR3
• Example: Adaptive Biotechnologies clonoSEQ (FDA-cleared)

For T cell malignancies:

• Target: TRB (T cell receptor beta chain)
• Focus: VDJ rearrangement, CDR3 sequence

Workflow:

1. Identify dominant clone at diagnosis (baseline sample)
2. Design or select tracking strategy for that clone
3. Monitor subsequent samples for clone presence/frequency

Limitations of Single-Chain Approaches

While effective, single-chain MRD has important limitations:

1. Clonal Evolution: Malignant cells may acquire secondary rearrangements
2. Ongoing Rearrangement: Some malignancies (especially T-ALL) continue V(D)J recombination
3. Ambiguous Clones: Similar sequences may represent different cells
4. T Cell Lymphomas: Often require paired chain analysis for definitive tracking

Paired Chain MRD Testing

The Case for Paired Chains

In certain clinical scenarios, paired α-β (TCR) or heavy-light (BCR) chain information provides critical advantages:

T Cell Malignancies:

• T cell lymphomas and leukemias are less common but challenging
• Single TRB chain may be shared across unrelated clones
• Paired TCR provides definitive clone identification

Clonal Evolution Detection:

• Malignant clones may undergo secondary rearrangements
• One chain may change while the other remains stable
• Paired analysis distinguishes evolution from new clones

Research Applications:

• Understanding tumor heterogeneity
• Tracking multiple subclones
• Correlating with functional characteristics

IMBERA-seq for MRD

IMBERA-seq’s paired chain capability offers unique advantages for MRD:

Diagnostic Sample:
├── Identify malignant clone (paired α-β or H-L)
├── Characterize any subclones
└── Establish baseline clone frequency

Follow-up Samples:
├── Track primary clone with paired specificity
├── Detect clonal evolution (one chain changes)
├── Identify emergent subclones
└── Quantify MRD with high precision

Disease-Specific Applications

Acute Lymphoblastic Leukemia (ALL)

B-ALL:

• IGH rearrangement tracking is standard
• MRD negativity after induction predicts excellent outcomes
• MRD guides decision for transplant vs. consolidation

T-ALL:

• More challenging due to ongoing rearrangement
• TRB and TRG commonly used
• Paired TCR analysis may improve specificity

Clinical Integration:

• MRD assessment at end of induction (day 29)
• MRD at end of consolidation
• Guides transplant decisions, CAR-T eligibility

Chronic Lymphocytic Leukemia (CLL)

• IGH-based MRD is well-established
• Undetectable MRD (less than 10⁻⁴) associated with prolonged remission
• Used to assess response to targeted therapies (venetoclax, BTK inhibitors)

Multiple Myeloma

• Plasma cell malignancy with clonal immunoglobulin
• MRD negativity increasingly important endpoint
• NGS-based testing complementary to flow cytometry

Lymphomas

Diffuse Large B Cell Lymphoma (DLBCL):

• Circulating tumor DNA (ctDNA) emerging as MRD marker
• IGH sequencing from blood possible
• Predicts relapse before clinical/imaging evidence

T Cell Lymphomas:

• Rarer, less standardized MRD approaches
• Paired TCR sequencing particularly valuable
• IMBERA-seq enables comprehensive clone tracking

Technical Considerations

Sample Requirements

Sensitivity Factors

Achieving maximum sensitivity requires:

1. Adequate cell input: More cells = better sensitivity

   • 10⁻⁶ sensitivity requires ~1 million cells minimum

2. Sequencing depth: Sufficient reads to detect rare clones

   • Typically 1-2 million reads per sample

3. Baseline clone characterization: Clear identification of target

4. Bioinformatic precision: Distinguishing true signal from noise

Quality Metrics

Key quality indicators for MRD testing:

• Clone identification rate: % of diagnostic samples with trackable clone
• Assay sensitivity: Limit of detection (typically reported as 10⁻⁵ or 10⁻⁶)
• Specificity: False positive rate in negative controls
• Reproducibility: Concordance between replicates

Clinical Implementation

When to Test

Standard MRD Timepoints:

• End of induction therapy
• End of consolidation
• Pre-transplant
• Post-transplant (day +30, +100, +365)
• Any time point with clinical concern

Surveillance:

• Regular monitoring in high-risk patients
• Triggered by rising disease markers
• Research protocols may specify additional timepoints

Interpreting Results

MRD Negative:

• Clone not detected at assay sensitivity
• Report includes sensitivity achieved
• Does not guarantee cure—very rare cells may persist

MRD Positive:

• Clone detected; report includes quantification
• Trend over time more informative than single value
• Rising MRD often precedes clinical relapse

Clinical Decision-Making

MRD results inform therapy in multiple ways:

Future Directions

Emerging Applications

1. Liquid Biopsy Integration: Combining cell-based and cell-free (ctDNA) MRD
2. Real-Time Monitoring: Point-of-care or rapid turnaround testing
3. Predictive Algorithms: Machine learning to predict relapse risk from MRD kinetics
4. Response-Adapted Therapy: Using MRD to personalize treatment intensity

Paired Chain Advantages

As paired chain sequencing becomes more accessible through technologies like IMBERA-seq:

• Improved specificity for T cell malignancies
• Better detection of clonal evolution
• Enhanced research into tumor heterogeneity
• Potential for functional characterization of malignant clones

Key Concepts Summary

1. MRD is disease below conventional detection limits; its status strongly predicts outcomes in hematologic malignancies

2. Immune receptor sequencing exploits the clonal rearrangement as a tumor-specific marker, achieving 10⁻⁵ to 10⁻⁶ sensitivity

3. Single-chain sequencing (IGH or TRB) is the current standard, with FDA-cleared tests available

4. Paired chain analysis offers advantages for T cell malignancies, clonal evolution detection, and definitive clone identification

5. Clinical integration of MRD guides therapy intensity, transplant decisions, and early intervention for relapse

Related Articles

• Immune Repertoire — Understanding clonality and diversity
• Chain Pairing Methods — Technologies for paired sequencing
• IMBERA-seq Technology — Paired chain sequencing platform
• Clonotype Analysis — Tracking specific clones

References

1. Borowitz MJ, et al. (2017). Minimal residual disease detection in childhood ALL. Journal of Clinical Oncology, 35(23):2693-2701.

2. Thompson PA, Wierda WG. (2016). Eliminating minimal residual disease as a therapeutic endpoint. Blood, 127(3):279-286.

3. Ladetto M, et al. (2014). Next-generation sequencing and real-time quantitative PCR for minimal residual disease detection. Leukemia, 28:1299-1307.