Monitoring of minimal residual disease (MRD) is becoming increasingly relevant in a clinical context for patients with leukemias and lymphoma. While conventional methods like cytomorphology are able to assess highly relevant clinical factors like the achievement of complete remission, method such as multiparametric flow cytometry and quantitative real-time PCR allow clinicians to obtain precise information on the amount of residual malignant cells present for patients in complete remission. In many cases, the extent of this MRD is highly correlated with the further progression of the disease and is hence used as a baseline control for risk-adapted therapy.
Molecular determination of MRD
Most patients suffering from acute leukemia (AML and ALL) can now be offered extremely sensitive progress diagnostics for the identification of minimal residual disease (MRD) by means of molecular genetics. Where a certain mutation or fusion gene is identified during diagnostics, it can be used for quantitative measurements and tracking as the disease progresses. This usually involves techniques such as quantitative real-time PCR, nested PCR or next-generation sequencing (NGS) by means of deep amplicon sequencing. The sensitivity of molecular MRD diagnostics depends on the mutation analyzed and the method used; nonetheless, its sensitivity is far superior to cytomorphology and classic cytogenetics.
In many cases, molecular monitoring of MRD is considered a standard for acute leukemias. They include
- acute promyelocyte leukemia WITH PML-RARA rearrangement,
- AML with inv(16)/CBFB-MYH11 or t(8;21)/RUNX1-RUNX1T1
- NPM1-positive AML with NPM1 mutation, and
- Philadelphia, i.e. BCR-ABL1-positive ALL.
Molecular MRD diagnostics based on real-time PCR are also key to treatment control with tyrosine kinase inhibitors for patients with chronic myeloid leukemia (CML).
Moreover, molecular MRD determination is extremely helpful fur indication in the case of allogenic stem cell transplants for acute leukemia patients who have reached cytomorphological remission (for instance if there is an inadequate molecular response, or during molecular remission of the disease).
MRD using immunophenotyping
Leukemia-associated aberrant immunophenotypes (LAIPs) that do not – or very rarely – occur in this constellation on the cells of normal bone marrow can be detected in cases of acute leukemia. They are divided into:
- Cross-lineage expression of lymphatic markers for AML (e.g. CD19-positive AML), i.e. myeloid markers for ALL (e.g. CD33-positive ALL)
- Asynchronous antigen expression (simultaneous expression of immature markers with mature markers, e.g. CD34- and DD11b-positive AML)
- Absent antigen expression (e.g. HLA-DR-negative AML)
- Antigen overexpression (e.g. CD33++CD34++ AML).
Of central importance is the correct identification of an LAIP, so precise knowledge of – and benchmarking with – the immunophenotype in the normal bone marrow is essential for MRD quantification. The LAIP is often characterized by the expression intensity of a particular antigen, which can vary significantly from case to case. It is therefore necessary to define one or several LAIPs for each patient individually. Hence, individual identification of the LAIP at the time of diagnosis is a precondition to enable MRD testing. The use of an extensive panel of antibodies is necessary for detection of an LAIP, especially for AML, in order to identify rare LAIPs and to make them available for downstream progress examinations.