MRD in ALL, the scientific background

Measurable residual disease (MRD) monitoring is one of the most powerful prognostic tools available in the management of acute lymphoblastic leukemia (ALL). This page provides a scientific overview of MRD — what it is, why it matters clinically, how it is measured, and why standardization is essential. It is intended as an educational resource for clinicians, researchers, and laboratory scientists seeking to understand the principles underpinning EuroMRD's work.

After induction chemotherapy, most patients with ALL achieve a morphological complete remission — meaning that leukemic blasts are no longer detectable by conventional bone marrow examination under the microscope (typically defined as fewer than 5% blasts). However, morphological remission does not mean that all leukemic cells have been eliminated. In many patients, millions of leukemic cells may persist at levels that are undetectable by morphology but remain detectable by highly sensitive molecular techniques. These persisting leukemic cells constitute measurable residual disease.

The term 'measurable residual disease' has largely replaced the older term 'minimal residual disease' in recent years, reflecting a shift in emphasis: MRD is not simply a small amount of disease, but a quantifiable signal that can be used to monitor treatment response, predict relapse risk, and guide clinical decisions.

The clinical relevance of MRD in ALL is supported by extensive evidence from both pediatric and adult treatment protocols. Key findings include:

MRD positivity after induction or consolidation is one of the strongest independent predictors of relapse in ALL, across all age groups and most disease subtypes.

• Patients who achieve MRD negativity - defined as MRD below the detection threshold of a validated assay - have significantly better long-term outcomes than those with detectable MRD.
• MRD kinetics (the rate at which MRD becomes undetectable) provide additional prognostic information beyond a single time-point measurement.
• MRD-guided treatment stratification — escalating therapy in MRD-positive patients and potentially de-escalating in MRD-negative patients - has been incorporated into most major contemporary ALL treatment protocols.
• MRD is increasingly used as a primary or secondary endpoint in clinical trials evaluating new drugs, immunotherapies (e.g., blinatumomab, inotuzumab), and cellular therapies (e.g., CAR-T cells).

RQ-PCR of IG/TCR gene rearrangements

Real-time quantitative PCR (RQ-PCR) targeting clonal rearrangements of immunoglobulin (IG) and T-cell receptor (TR) genes is the established standard for MRD detection in ALL. This approach exploits the fact that each leukemic clone carries a unique, patient-specific rearrangement of IG or TCR genes — a molecular fingerprint that persists throughout the disease course and can be detected with high sensitivity.

At diagnosis, the leukemia-specific IG/TR targets are identified by PCR and sequencing, and patient-specific quantitative standards are established. At follow-up time points, bone marrow or peripheral blood samples are analyzed by RQ-PCR using the patient-specific primers, enabling quantification of residual leukemic cells down to approximately 1 in 10,000 to 1 in 100,000 normal cells (sensitivity of 10⁻⁴ to 10⁻⁵).

EuroMRD has played a central role in the standardization of this approach, developing guidelines for the interpretation of RQ-PCR data that define how results should be categorized, reported, and compared across laboratories.

Digital droplet PCR (ddPCR)

Digital droplet PCR (ddPCR) partitions the PCR reaction into thousands of individual droplets, each containing zero or one template molecule. This enables absolute quantification of target molecules without the need for external standard curves, potentially reducing inter-laboratory variability in quantification. ddPCR also offers improved precision at low target concentrations, which is particularly relevant at the low MRD levels.

EuroMRD is involved in evaluating the concordance of ddPCR and RQ-PCR results for IG/TCR-based MRD detection and in developing recommendations for ddPCR-specific data interpretation. Whether ddPCR-specific thresholds are needed, or whether existing RQ-PCR guidelines can be directly applied, is an active area of investigation within the network.

Next-Generation Sequencing (NGS)

NGS-based MRD detection uses high-throughput sequencing of IG/TR regions to identify and quantify the leukemic clone within a polyclonal background. NGS offers the advantages of very high throughput, the ability to track multiple clonal targets simultaneously, and — in some implementations — very high sensitivity. Commercially available NGS-MRD platforms have been validated in several large clinical trials.

Standardization of NGS-MRD is an active challenge, as the technology involves multiple steps (library preparation, sequencing, bioinformatics) each of which can introduce variability. EuroMRD is participating in efforts to define quality standards and reporting frameworks for NGS-MRD that are consistent with the principles applied to RQ-PCR-based detection.

BCR-ABL1 Quantification in Ph+ALL

In Philadelphia chromosome-positive ALL (Ph+ALL), which accounts for approximately 25–30% of adult ALL cases, MRD monitoring by BCR-ABL1 quantification using RQ-PCR is standard practice. This approach detects and quantifies the BCR-ABL1 fusion transcript that results from the t(9;22) chromosomal translocation. EuroMRD organizes dedicated QA rounds for BCR-ABL1-based MRD detection, separate from those for IG/TCR-based approaches.

MRD is only clinically useful if results generated in one laboratory can be compared to results from another. Without standardization, a patient who tests MRD-negative in one center might test MRD-positive in another — not because of a difference in their disease, but because of differences in laboratory technique, primer design, quantification approach, or result interpretation.

Standardization has three essential components. The first is technical standardization — ensuring that assays are performed according to validated procedures using comparable reagents and equipment. The second is interpretive standardization — ensuring that a given analytical result is reported and categorized consistently, which is the primary focus of the EuroMRD interpretation guidelines. The third is ongoing quality assessment — regularly verifying that a laboratory continues to perform at the required standard through participation in external quality assurance programs.

EuroMRD addresses all three components. The interpretation guidelines define what 'MRD negative', 'MRD positive within the quantitative range', and 'MRD positive below the quantitative range' mean — categories that have direct implications for treatment decisions. The QA program verifies that laboratories applying these guidelines can accurately detect and quantify MRD targets in standardized samples.

A result of 'MRD negative' does not mean that no leukemic cells are present — it means that none were detected above the sensitivity threshold of the assay used. The clinical significance of MRD negativity therefore depends critically on the sensitivity of the assay and the number of cells analyzed.

EuroMRD guidelines require that a result be classified as 'MRD negative' only when the analysis was performed at a sensitivity that renders the result clinically meaningful — typically 10⁻⁴ or better. Results obtained at lower sensitivity, or from samples with insufficient cellularity, should be reported with appropriate qualification. These distinctions are explicitly addressed in the EuroMRD interpretation guidelines.

A related challenge is the category of 'MRD positive, below the quantitative range' — results in which leukemic cells are detected but below the level at which accurate quantification is possible. The clinical significance of such results, and how they should be acted upon, is an area of ongoing investigation. The 2024 EuroMRD publication by Kotrova et al. addressed this gray area by proposing a further subdivision of this category.

MRD is increasingly being accepted by regulatory agencies as a clinically meaningful endpoint in clinical trials of new therapies in ALL. The European Medicines Agency (EMA) and the US Food and Drug Administration (FDA) have both issued guidance on the use of MRD as a trial endpoint. For MRD to be acceptable as such an endpoint, the assay used must be analytically validated, the thresholds must be pre-specified, and inter-laboratory comparability must be demonstrated.

EuroMRD's guidelines and QA program directly support these requirements. Laboratories operating within the EuroMRD framework, applying the EuroMRD interpretation guidelines, and participating in QA rounds are well-positioned to generate MRD data that meets regulatory standards for trial endpoints.