A Step Toward Standardized Residual Disease Testing in Acute Myeloid Leukemia

Minimal residual disease (MRD) has emerged as one of the most important predictors of patient outcome in acute leukemias. The residual disease present after chemotherapy can lead to patient relapse, so by measuring MRD, clinicians can stratify patients into low- and high-risk groups to guide treatment.

MRD testing often relies on multiparameter flow cytometry to identify residual disease cells in the bone marrow; however, there is currently no standard approach for measuring MRD in acute myeloid leukemia (AML), a rare and aggressive cancer that arises from immature myeloid cells.

The director of UW Medicine’s hematopathology laboratory and a world leader in clinical flow cytometry, Dr. Brent L. Wood, recently published a protocol for measuring residual disease in AML in Current Protocols in Cytometry—a much needed step toward accurate and reproducible AML MRD testing.

The protocol outlines a Difference-from-Normal (DfN) approach for detecting MRD in AML using multiparameter flow cytometry. The approach is based on identifying leukemic cells that have abnormal surface antigen patterns, or immunophenotypes, compared to normal hematopoietic cells. In general, abnormal immunophenotypic changes include increases or decreases in antigen expression, timing changes of antigen expression (e.g. mature cells expressing stem cell markers), or the presence of antigens that are usually never found on a specific cell type.

Hunting for leukemic stem cells in AML

AML is a heterogeneous disease with a wide range of phenotypes. Dr. Wood’s protocol includes three antibody combinations, each designed to identify cell populations at different stages of hematopoiesis that are known to have abnormalities in AML. All three combinations also include stem cell markers CD34 and CD38, allowing clinicians to look for abnormal hematopoietic stem cell-like cells, called leukemic stem cells.

Scientists believe that these chemotherapy-resistant leukemic stem cells are responsible for relapse and their presence after therapy is linked to negative outcomes. By using MRD to identify high-risk patients early on, clinicians can intervene to try to reduce the risk of relapse and improve outcomes with different or higher intensity treatments, such as a bone marrow transplant.

“That’s one of the roles of AML MRD at the moment—determining who should have a bone marrow transplant and who should not,” says Dr. Wood.

Two different approaches for measuring MRD in AML

In terms of flow cytometry, clinicians can also monitor MRD in AML patients using the leukemia-associated immunophenotype (LAIP) approach, which tends to be more common in Europe. In LAIP, clinicians look for MRD within the same disease populations that were present at diagnosis, with the assumption that disease immunophenotypes do not change after therapy. 

“The attractiveness of LAIP is that it’s more straightforward operationally,” says Dr. Wood, “you run the sample, create the gate, and count things that show up.”

However, changes in immunophenotype after treatment are common in acute leukemias. Therefore, if the disease immunophenotype changes after therapy, using the LAIP approach can produce false negative results, which may, in certain cases, account for why some patients classified as MRD negative still experience relapse.

In contrast, when using the DfN approach, clinicians cast a wider net and continue to look for cell populations with abnormal immunophenotypes whether or not they were present at diagnosis. 

But either way, MRD is not yet an exact science. 

“The truth of the matter is, although MRD testing is very powerful as a prognostic factor—the most prognostic factor we have—it’s not an absolute predictor of relapse,” says Dr. Wood. “There are patients that don’t have detectable MRD that nevertheless relapse, particularly if they’re high risk.”

As the lack of standardized MRD testing poses a challenge for clinicians worldwide, Dr. Wood continues to engage with groups interested in establishing standards, including the National Cancer Institute and the European LeukemiaNet MRD Working Party, which published guidelines in 2018 recommending that MRD become the standard of care for AML. Thus, as MRD plays an ever more important role in guiding therapy in AML patients, a standardized assay will ensure accurate and reproducible results in clinical practice and across future multicenter trials.