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Coustan-Smith article

"Immunological detection of minimal residual disease in children with acute lymphoblastic leukaemia." (13)

Coustan-Smith's group is based at St Jude’s. They used multiparameter immunological detection by flow cytometry to measure MRD. The principle behind this assay is that leukemia cells display certain surface, cytoplasmic and nuclear leukocyte antigens, or proteins, which normal cells do not. Monoclonal antibodies to these antigens will attach to them when they see them. So, they tag a fluorescent dye to a monoclonal antibody, incubate it with a sample of bone marrow aspirate from a leukemia patient, and then monitor the cells by flow cytometry for the attachment of fluorescent dyes to the cells. If the cells fluoresce, they have leukemia antigens.

Their MRD detection system is not PCR-based nor is it as sensitive as the PCR assay. However, it is a much easier method to do in a clinical lab setting. They studied a large number of patients and their results are definitely worth reviewing. They claim the method is capable of detecting one leukemic cell among10,000 normal cells.

In each test of bone marrow aspirate, Coustan-Smith et al used the antigen-antibody reaction to monitor 10,000 lymphoid cells for the marker combinations in the table below. Cells positive for more than one marker were considered positive for leukemia.

Marker Combinations
early lymphoid leukemia-associated
TdT CD3 (cytoplasmic or surface)
TdT and CD34 IgM (cytoplasmic)
CD10 KORSA3544
CD19 and CD34 or CD10 CD13
CD19 and CD34 or CD10 CD33
CD19 and CD34 or CD10 CD65
CD19 and CD34 CD21
CD34 and CD10 CD56

The advantage of this type of flow cytometry immunological detection of MRD is the speed and ease with which it can be done. Clinical laboratories are already set up to do this type of analysis. The authors report that while they tested 290 children newly diagnosed with ALL for immunophenotypes, the marker combinations used were only applicable to 166 (57.2%) of the patients. 8 others could not be studied (5 did not achieve remission and 3 did not have enough sample). Therefore, MRD in all leukemia patients cannot be studied by their method.

Bone marrow samples were taken at weeks 6, 14, 32, 56, 120. At each time point, the detection of MRD was significantly associated with a greater likelihood of treatment failure and of leukemic relapse, as summarized in the table below.

time of testing MRD cumulative incidence of
leukemia relapse after 3 years(%)
end of remission induction positive 32.5
negative 7.5
week 14 positive 42.1
negative 6.6
week 32 positive 75.0
negative 8.0
week 56 positive 50.0
negative 6.6

In addition, 65 patients were studied at the end of treatment: none of these had detectable residual disease. Still, 4 of these patients relapsed at 7-15 months after the negative test. (One of these 4 had positive tests throughout the first year of therapy, while two had phenotypic switches resulting in loss of leukemia-specific markers.)

Discussion

According to the authors, the absence of MRD at the end of therapy does not guarantee a durable remission. Immunological investigation of MRD provides clinically meaningful information that could be used to improve risk assessment strategies and treatment selection in the management of ALL in children. They suggest that patients with detectable leukemic cells at the end of remission induction should be closely monitored for disease resurgence. Alternative treatment should be considered for patients with persistent disease during early continuation therapy.

In an accompanying response to the above article in the same journal, Farahat et al (14) claim that they can detect MRD in all cases of B-lineage childhood leukemia by their own combination of markers, which consists of three monoclonal antibodies -- TdT, CD10, and CD19. They exploited known differences in antigen expression between precursor B-lineage ALL and normal bone-marrow B-cell precursors. Although normal B-cell precursors express strong TdT and weak CD10 and CD19, leukemic B cells express weak TdT and strong CD10 and CD19.

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