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MRD Assessment in ALL
Optimizing MRD Assessment in ALL: Why, How, and When

Released: April 22, 2016

Expiration: April 21, 2017

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Case Report
A 32-year-old woman presented with shortness of breath. She was found to have a large mediastinal mass and pleural effusion. Her WBC count was 10,000 cells/µL (50% blasts) with anemia, thrombocytopenia, and 60% bone marrow infiltration by blasts. The blasts were immature T cells by immunophenotyping: negative for B-cell markers, but positive for TdT, CD2, CD7, and CD34. She was diagnosed with T-cell acute lymphoblastic leukemia (ALL), entered a clinical trial, and was treated on a pediatric protocol. Induction included daunorubicin, steroids, vincristine, and pegylated asparaginase. By Day 32, the mass had disappeared, blood counts normalized, and a bone marrow assessment showed normal morphology with no blasts and normal hematopoiesis. We performed a 7-flow cytometry on the bone marrow sample and determined the patient achieved minimal residual disease (MRD) negativity.

Now what should I do with this information? What would you do in your practice?

MRD in ALL
One criteria for complete response (CR) in acute lymphoblastic leukemia (ALL) is a bone marrow that microscopically has less than 5% blast cells. However, morphology is very insensitive in determining residual tumor burden, since morphology is only able to detect a concentration of approximately 1 malignant cell in 100 normal cells, or 1% of cells. Below that level, minute concentrations of surviving ALL cells escape morphological detection. To determine if residual ALL cells remain in bone marrow after treatment, sensitive tests for MRD have been developed, able to detect 1 malignant cell in 10,000 normal cells. Patients achieving MRD levels below this measure, or < 1 in 10-4, are defined as MRD negative. NCCN guidelines state that MRD testing is essential for patient evaluation during the course of patient treatment.

Outcomes
MRD status is the single most important predictive factor in ALL, with an independent and significant relationship between MRD-negative status and better outcomes. MRD positivity after treatment often represents a decision point for switching treatment or choosing a bone marrow transplant (BMT) in order to improve outcome. MRD status may also be used to identify patients who have a poor response to transplantation and unfavorable outcomes. In addition, MRD is now used to compare responses to new drugs, and is becoming a reliable and early surrogate marker for efficacy in clinical trials.

Timing
Typically, MRD status is evaluated after the first induction (ie, 30-35 days after the start of treatment). Several centers also perform an evaluation after the second induction cycle phase about 9 weeks after the start of treatment. MRD positivity in both tests indicates less optimal treatment response with a higher risk of progression for a patient, whereas MRD negativity followed by MRD positivity indicates an intermediate risk, and 2 MRD-negative results indicate a patient at lower risk of progression.

In many cases, MRD could also be measured after consolidation, although the optimal timing for MRD testing in adults is as yet unclear.

Assessment
In general, leukemic cells have characteristics similar to normal cells, making it difficult to distinguish the very few malignant cells that are not evident with a microscope. In principle, detection of MRD exploits known immunophenotypical and genetic variances between leukemia cell and recovering normal bone marrow cell. Several methods should be able to identify a very small population of malignant ALL cells in a background of normal or recovering bone marrow.

Polymerase chain reaction (PCR) detects MRD by amplification of unique leukemia-specific rearrangements of the immunoglobulin gene (B-ALL) or the T-cell receptor (T-ALL). This assay, which can test up to 95% of patients, is the most commonly used MRD test in Europe and is well standardized, but it is time consuming and more expensive. In some patients with known gene translocation, for example, Philadelphia chromosome (Ph) ALL, PCR can detect MRD by amplifying the ABL/BCR fusion transcripts.

Flow cytometry: The use of multicolor flow cytometry with different antigens (usually 7) can identify leukemic cells with a different immunophenotype than normal bone marrow cells. This is the most commonly used MRD test in the United States, and it is faster than PCR, but needs further standardization between labs, especially with adult ALL patients. The bone marrow sample should be at least 2000 μL to have enough cells for flow cytometry or DNA for PCR but should be no more than 5000 μL to avoid dilution with normal cells. The first “bone marrow pull” will usually provide the best sample.

Next‑generation sequencing for MRD uses deep sequencing to identify leukemia-specific mutations and is able to differentiate between leukemic cells and normal bone marrow cells. However, this complex system requires advanced technology and bioinformatics, and few labs have experience at this time. Within a few years, however, this is expected to become a reliable and probably even more sensitive tool to identify MRD.

What If a Patient Is MRD Positive?

Treatment
MRD-positive patients have poor outcomes even when treated with aggressive pediatric-type regimens or with transplantation, although with the latter, they perform better than without transplantation at all.

Novel immunotherapeutics are investigated in CR patients who are MRD positive after chemotherapy to bring them to MRD negative status. In one study, the majority of MRD-positive patients with relapsed ALL, treated with the approved antibody bi-specific T cell engager blinatumomab, were converted to MRD negativity with better outcomes than those who remained MRD positive. Another still experimental approach is using chimeric antigen receptor (CAR) T cells. In one recent report, 83% of patients with relapsed ALL treated with 19-28z CAR T cells achieved CR with MRD negativity. Of note, the 6-month OS for patients was 80% in the MRD-negative CR group vs 43% in the MRD-positive CR group after treatment.

Transplantation
Allogeneic BMT is rarely used in children with ALL due to excellent cure rates with current pediatric chemotherapy. In adults, BMT is more often considered as a post-remission option, especially in those who remain MRD positive. How can transplantation affect outcomes in first remission? In a retrospective comparison of adult patients in first CR receiving an intensive pediatric protocol, MRD-negative patients again had better outcomes than MRD-positive patients. Transplantation benefited MRD-positive patients, but it did not significantly improve outcomes in MRD-negative patients. For MRD-negative patients, transplantation increased the mortality risk for little or no added benefit to using such intensive chemotherapy.

When Can Patients Stop Treatment?
An unanswered question is when are patients who achieve MRD negativity considered as cured? Currently, maintenance therapy is given for approximately 2.5 years from the beginning of the treatment, with most relapses occurring early in the treatment cycle. If a patient is in remission and MRD negative after 3-4 years, the chances of relapse are extremely low. However, there will be a few patients who will relapse even with MRD negativity; ALL is heterogeneous, and occasionally resistant clones will develop.

Case Report (continued)
Patient continued on the pediatric protocol that included several cycles of multi-agent chemotherapy. Encouraged by her MRD-negative status, she was fully adherent with treatment. After consolidation, she has been taking maintenance therapy for the past 8 months and has returned to full-time work. Her bone marrow is tested every 3 months, and she remains MRD negative. She will receive maintenance treatment for 2.5 years, but there is no evidence of disease.

Final Thoughts
In summary, MRD is a key predictive factor for evaluating outcome and should be a very early part of the clinical management of all patients with ALL. In addition, MRD is already a clinical trial early endpoint. Clearly, MRD assessment is critical to the optimal management of patients with ALL.

How do you use MRD data in your clinical practice? Leave a comment and let me know.

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At what time point do you typically test a patient with ALL for the presence of MRD?
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