You're using an outdated browser. Please upgrade to a modern browser for the best experience.
Please note this is an old version of this entry, which may differ significantly from the current revision.
Subjects:
Hematology
Measurable (“minimal”) residual disease (MRD) is defined as the post-therapy persistence of leukemic cells at levels below the morphologic detection limit. Mounting evidence indicates that the presence of MRD is a strong, independent prognostic marker of increased risk of relapse and of shorter survival in patients with acute leukemia compared with patients with a negative MRD test. MRD assessment primarily involves the determination of leukemia-associated immunophenotypic patterns (LAIP) using multiparameter flow cytometry (MCF) and the polymerase chain reaction (PCR)-based evaluation of expression levels of leukemia-related genes (specific reciprocal gene rearrangements and other mutation types). In addition, next-generation sequencing and digital PCR may further enrich current MRD-detection methods. Adding the MRD evaluation to other post-treatment assessments could be of help in guiding the post-remission treatment strategies by identifying patients at high risk of relapse who might benefit from pre-emptive therapy. Several studies have clearly shown that treatment is more effective if at molecular relapse with a low disease burden than at overt relapse.
- allogeneic bone marrow transplantation
- relapse
- acute leukemia
1. Multiparameter Flow Cytometry (MFC)
MFC uses panels of fluorochrome-labeled monoclonal antibodies to identify aberrantly expressed antigens located on leukemic cells. By using combinations of multiple monoclonal antibodies, the sensitivity of MFC is increased to detect 10–3 to 10–5 leukemic cells within the white blood cell compartment [33].
MFC assessment after transplantation (day +100) discriminated different risk populations in AML patients. Overall survival (OS) was 73% vs. 25% after 4 years among patients with low (<10–3) vs. high (≥10–3) MRD at day +100 after transplantation (p = 0002) [34].
MFC for well-defined leukemia-associated immunophenotype patterns (LAIP) has broad applicability (<90%) and high specificity. However, the leukemic phenotype is not necessarily stable over time (e.g., the initial LAIP may be lost in the course of the disease), and there remains limitations of this method due to a lack of comparability and reproducibility among different laboratories, the use of different instruments, fluorophores and operating procedures that require further standardization [35]. Current guidelines recommend to use, in the follow-up of the patient the combined approach of detecting the LAIP and the different from normal (DfN) phenotypes (which individuate aberrant differentiation or maturation phenotype) [36].
2. Real-Time Quantitative PCR (RT-qPCR)
Molecular assessment of MRD can be performed by monitoring mutated genes [29], fusion-gene transcripts [30] and overexpressed genes [31]. The PCR-based techniques applied to the quantitative measurement of these markers currently represent the standard of care with the highest sensitivity (down to 1:106 cells) and specificity [36].
About 30% of patients with normal-karyotype AML have an nucleophosmin-1 (NPM1) gene mutation. Several groups have already demonstrated the predictive value for relapse of a persistent NPM1-mutation in peripheral blood (PB) and bone marrow (BM) in patients in complete remission after conventional chemotherapy [37]. NPM-1 mutation is recommended in clinical practice as MRD-marker [36].
Molecular testing for FLT3 mutations is part of the diagnostic setting at the first diagnosis and offers essential prognostic information. However, it is not routinely used to monitor MRD after HSCT due to its relative instability during the course of the disease [38].
Other mutations (DNMT3A, IDH1/2, TET2, ASXL1), because of their relatively low frequency, have not been studied in larger cohorts and lack broad applicability [38].
In about 20% of patients with AML, distinct fusion genes are approachable for MRD monitoring, with most of them expressed by the core-binding factor (CBF) AML (RUNX1-RUNX1T1 and CBFB-MYH11 fusion genes). Several reports have already demonstrated that MRD positivity detected by quantitative PCR is predictive for imminent relapse in patients with CBF leukemias after conventional therapy [39].
Overexpression of Wilms tumor 1 (WT1) mRNA is present in about 90% of patients with AML and 50% of patients with MDS. Thus, monitoring of WT1 is broadly applicable in a large proportion of AML and MDS patients [40]. Furthermore, WT1 expression is measurable in peripheral blood with even higher sensitivity and specificity than in bone marrow, thereby facilitating the patient’s comfort in contrast to other molecular methods which require a myeloaspirate to gain a comparable sensitivity. However, WT1 lacks specificity and is not routinely recommended for MRD monitoring unless there is no alternative marker, including flow-cytometric markers [36]. When WT1 expression is used for MRD monitoring, a validated ELN assay offers a reproducible cut-off and comparable results among different laboratories [41].
The advantages of real-time quantitative polymerase chain reaction (RT-qPCR) are the broad applicability and the high sensitivity (superior to MFC); it is well standardized and maybe run by a certified laboratory with expertise in RT-qPCR.
However, given the limited available molecular targets, RT-qPCR assessment of MRD is applicable to only 50% of AML cases and less than 35% in older patients (whereas MFC can detect MRD in 90% of patients when a comprehensive antibody panel is used) [42]. Limitations of the RT-qPCR-based assays are their dependence on specific mutations, requiring individual reference standard curves based on target serial dilutions; in addition, the results of RT-qPCR may take multiple days. It is an expensive assay and requires a high level of expertise [43].
3. Digital PCR
Digital droplet PCR (ddPCR) is a biotechnological refinement of the conventional qPCR methods that can quantify and clonally amplify the nucleic acid strands directly. Compared to qPCR, it is more precise, reproducible and accurate. For example, digital PCR can detect a variety of NPM1-mutation subtypes without the need for multiple plasmid standards [44]. A disadvantage of ddPCR is that for each single mutation a specific assay has to be developed [36].
4. Next-Generation DNA Sequencing (NGS)
Next-generation DNA sequencing technologies, which allow parallel and repeated sequencing of millions of small DNA fragments, can be used to evaluate a few genes or an entire genome [45]. The ability of NGS to assay large numbers of mutated genes could be of help in tracing the evolution of malignant clones. Several studies have demonstrated the feasibility of NGS to monitor the mutations for which targeted therapies are available, such as FLT3-ITD [46], IDH1/2 [47] and the mutations having prognostic relevance, such as CEBPA and NPM1 in patients with AML [48]. The NGS may provide further information for relapse, particularly when MRD evaluated by conventional methods is negative. Recently, Heuser and co-workers showed that MRD measured by NGS-based techniques at days +90 and +180, together with MRD measured pre-HSCT, is highly predictive for relapse and of overall survival [49]. However, this technique is time-consuming and costly and is not recommended for MRD measurement outside of clinical trials [36,45].
5. Chimerism Analyses
The analysis of donor/recipient chimerism is the standard practice to monitor donor cell engraftment and can be performed in all patients after HSCT. Chimerism analysis detects the host-derived hematopoiesis on the basis of the genomic differences of highly variable loci between the recipient and the donor. A mixed donor/recipient chimerism does not equate directly to the relapse of the leukemic clone in all cases. However, in malignant disorders such as AML, a decrease of donor chimerism is often associated with disease recurrence [50]. In the absence of other markers, chimerism analysis has been suggested as a surrogate of MRD monitoring. The variant allele-specific qPCR method for detection of small insertion and deletions is more sensible than the short tandem repeats (STR) analysis and it should be preferred for MRD monitoring [51].
This entry is adapted from the peer-reviewed paper 10.3390/jcm11010253
This entry is offline, you can click here to edit this entry!