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MRD Testing in ALL
What You Need to Know About MRD Testing in Acute Lymphoblastic Leukemia

Released: August 29, 2018

Expiration: August 28, 2019

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Acute lymphoblastic leukemia (ALL) is a neoplasm of precursor B-cells or T-cells. Most patients with B-ALL present with a leukemic picture and are typically diagnosed on the basis of peripheral blood and/or bone marrow evaluation. Sizeable subsets of patients with T-ALL, and a few with B-ALL, present with a tissue mass termed lymphoblastic lymphoma (LBL), occasionally without a detectable leukemic component. We recognize that ALL and LBL are biologically similar entities, hence the World Health Organization term lymphoblastic leukemia/lymphoma. I prefer to retain the term “acute” in my diagnostic reports since it conveys a fundamental characteristic of this disease.

ALL Workup at Initial Presentation
The diagnostic workup of patients with ALL at presentation should include, in addition to histology, flow cytometry immunophenotyping, conventional cytogenetics and FISH, and molecular profiling for recurrent translocations. Mutation profiling for ALL is also performed at some centers. A baseline immunophenotype is important not only to help establish the diagnosis and identify critical biomarkers but also for subsequent minimal, or preferably, “measurable” residual disease (MRD) evaluation. Similarly, the molecular characteristics of the baseline leukemia in terms of immunoglobulin heavy chain or T-cell receptor rearrangement may also be critical for molecular MRD assessment, as detailed below.

Overview of MRD Testing
When we discuss laboratory techniques for MRD testing in ALL, we are essentially talking about testing platforms that are capable of peering beyond morphology with a higher degree of sensitivity and specificity. At present, such platforms primarily include multiparameter/multicolor flow cytometry and PCR. Flow cytometry is the most commonly used MRD assessment platform for patients with ALL in the United States, whereas molecular testing is the platform of choice in Europe.

By definition, MRD status refers to whether or not residual leukemic cells are identified by immunophenotypic or molecular techniques in a patient who has fewer than 5% blasts in the bone marrow. MRD status is typically assessed at the end of first induction and increasingly also after the second induction cycle. At these time points, MRD status is a direct measure of the effectiveness of therapy, which is why it has emerged as the single most important predictor of outcome in ALL. At the MD Anderson Cancer Center, we assess MRD at every follow-up during remission as well as before and after stem cell transplantation.

Multiparameter Flow Cytometry for Measuring MRD Status
Flow cytometry is a robust laboratory technique that interrogates individual cells and allows their characterization based on size, cytoplasmic complexity, and antigen expression patterns. The antibodies used in flow cytometry to detect antigen expression are tagged with covalently bound fluorochromes that emit “colors” upon laser excitation. Most flow cytometers in use today detect 6 or 8 colors. For diagnostic flow cytometry, we usually acquire approximately 20,000 events (cells) per tube, whereas for MRD assessment, a minimum of 200,000 events are required. A population of aberrant events should consist of at least 20 events to achieve a sensitivity of 0.01% (20/200,000 = 10-4). And, since sensitivity is inversely proportional to the total number of events evaluated—the denominator—the lab often strives to acquire between 500,000 and 2,000,000 events per tube to maximize the sensitivity of MRD testing by flow as much as is practicable. This means that sample adequacy is particularly important, which is why an initial aspirate pull of at least 2 mL is recommended to ensure maximum enrichment for bone marrow elements and avoid the hemodilution that often accompanies subsequent pulls.

MRD assessment by flow cytometry is based on the principle that leukemic cells deviate in their immunophenotypic characteristics from the maturation patterns of normal lymphoid precursors (B-cell precursors are called hematogones). These deviations, or aberrancies, are typically present in blasts at presentation and remain identifiable throughout the disease course in most patients. For this reason, the distinction between leukemic cells and regenerating immature lymphoid cells can be facilitated by knowledge of the baseline immunophenotype of the leukemic cells, although this is not an absolute requirement.

The target sensitivity for flow cytometry MRD assessment is 10-4. In other words, a patient is defined as being MRD negative if no leukemic blasts are detected in a sampling of 10,000 cells. It should be noted that these sensitivity targets may not be achievable in all cases, which is why the MRD report should always state the number of acquired events in order to document the level of sensitivity achieved for the sample in question. Also, we need to keep in mind that making an MRD-positive call can be achieved with an inferior sensitivity level if the blasts clearly express aberrant markers, whereas conversely the best sensitivity is needed to reliably call a sample MRD negative.

Like all in vitro diagnostic assays, flow cytometry has advantages and limitations. Advantages include a quick turnaround time, low cost, and applicability to all patients with ALL. On the other hand, limitations of flow cytometry include the requirement for fresh material, the need for high-level expertise for performance and interpretation, a moderate sensitivity level in comparison to molecular techniques, and the fact that standardization efforts remain in progress. Furthermore, novel immunotherapy approaches in B-ALL target antigens such as CD19 and CD22, which are the backbone for gating strategies for MRD assessment by flow cytometry.

Molecular Tests for Measuring MRD Status
In B-ALL cases with a recurrent chromosomal translocation having a measurable fusion transcript, the use of quantitative reverse transcriptase PCR provides a highly sensitive means for MRD assessment. The BCR-ABL1 fusion in Ph-positive B-ALL is a classic example, with sensitivity levels that typically approach 10-5 and even higher with newer technologies such as droplet digital PCR. However, beyond Ph-positive B-ALL, this option applies only to a small subset of patients and tapers off significantly in those older than 60 years of age.

Receptor loci on the surface of B-cells and T-cells undergo physiologic combinatorial rearrangements that generate the diverse repertoire that characterizes such receptors and forms the foundation of the adaptive immune system. In malignancies ensuing from these cells and their progenitors, the neoplastic clone is characterized by a clonal rearrangement that is unique to that particular neoplasm. Detecting this clonal arrangement forms the basis of conventional molecular tests for MRD assessment.

In their most basic approach, these tests use consensus primers followed by postamplification analysis by capillary electrophoresis to detect clonality. Although straightforward to perform and interpret, this approach has a sensitivity of only 10-2 to 10-3. A variation on this approach entails the use of a combination of consensus primers and custom-designed primers called allele-specific oligonucleotide (ASO) primers to detect specific sequences present in the original leukemic clone. Coupled with quantitative PCR technology, ASO-PCR achieves a sensitivity of 10-5 with superb specificity.

Next-Generation Sequencing for Measuring MRD Status
High-throughput sequencing (HTS) or next-generation sequencing (NGS) are umbrella terms that refer to molecular technologies that enable massively parallel sequencing. Various NGS technologies are changing the molecular diagnostics landscape and are continuing to evolve and become integrated into clinical laboratories.

Several groups, including Faham and colleagues, described robust NGS-based assays for high-sensitivity MRD detection in B-ALL capable of achieving a sensitivity of approximately 10-6. In addition, in a recent study by Wood and colleagues in pediatric patients with ALL, HTS identified 38.7% more patients than flow cytometry as MRD positive at the threshold of 0.01%, and these patients had poorer outcomes than flow cytometry MRD-negative patients. In the realm of MRD assessment, the inherent genomic structure of immune receptors initially posed challenges that precluded the unleashing of the full potential for monitoring patients with B-ALL or T-ALL. To circumvent those challenges, Carlson and colleagues developed an NGS-based assay approach that optimizes primers for MRD evaluation. This approach led to the development of the primers for MRD evaluation, an approach that has been adapted for and that is being increasingly used in the clinic. NGS-based MRD assays rely on the detection of a dominant clonal sequence in a baseline ALL sample that may consist of peripheral blood, bone marrow, or tissue. Monitoring of MRD status subsequently relies on detecting and quantitating the presence of the clonal sequence in follow-up blood or bone marrow samples.

In addition to its increased sensitivity, NGS-based MRD offers the advantage of being feasible on older samples. However, although flow cytometry is applicable in nearly every patient with B-ALL, NGS-based MRD has a finite failure rate. In the Wood study, 89.0% of patients with B-ALL had a trackable IGH clone, 6.4% had a trackable cross-lineage TRG clone, and 4.6% of patients had neither. In addition, NGS-based MRD assays require significant infrastructure and specialized expertise, and they remain expensive relative to flow cytometry.

Future Directions
Using a cutoff sensitivity of 10-4 is clearly insufficient for optimal risk stratification of patients with ALL. As such, lowering the sensitivity cutoff is becoming imperative.

Flow cytometry ALL MRD panels are being incrementally optimized to increased sensitivity through the use of novel markers that mitigate the need for knowledge of the baseline immunophenotype. In addition, 10-color flow cytometers are gradually making their way into more clinical laboratories. By reducing the number of tubes needed, these advanced flow cytometers allow the acquisition of more events, thus increasing the signal-to-background ratio and improving sensitivity and specificity.

Assessment of MRD by NGS breaks through the built-in sensitivity limitations of flow cytometry. Through emerging guidelines and regulatory routes, standardization of NGS-based clonality assays will likely emerge. Another aspect of MRD that could evolve in the coming years will be the use of peripheral blood cell-free DNA to detect MRD without the need of bone marrow sampling in some patients.

What challenges have you experienced in your clinic when implementing MRD testing for patients with ALL? Share your thoughts below.

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