Page 42 - 2019_08-Haematologica-web

N.J. Short and F. Ravandi et al.
Multiparameter flow cytometry
Multiparameter flow cytometry (MFC) uses a panel of fluorochrome-labeled monoclonal antibodies to identify aberrantly expressed antigens on leukemic blasts. This MFC-based MRD analysis may be accomplished through either the tracking of leukemia-associated immunopheno- types in the pretreatment and remission samples or the use of “difference from normal” analysis.22 Leukemia-asso- ciated immunophenotypes consist of the aberrant expres- sion of antigens compared to that on normal myeloid pre- cursors, cross-lineage antigen expression (e.g. expression of lymphoid antigens on myeloblasts), over- or under- expression of antigens normally expressed, and aberrant co-expression of antigens normally found in early or late hematopoietic differentiation.6 In contrast, the “difference from normal” approach is used to detect any differences in the remission immunophenotype compared to the highly stereotypical normal immunophenotype distribution.23 The advantage of “difference from normal” analysis is that it does not require knowledge of the diagnostic immunophenotype; furthermore, it may also be less sus- ceptible to immunophenotypic shifts that can occur as a direct result of therapy or due to a shift in clonal architec- ture.24,25 Workflows that incorporate both of these meth- ods (i.e., a leukemia-associated immunophenotype-based, “difference from normal” approach) may help to further optimize MRD assessment.6 Some studies suggest that addition of leukemia stem cell markers (e.g. CLL1, CD44, CD123, and CD184, among others) to flow MRD anti- body panels may add additional prognostic information to standard MFC-based MRD, particularly by identifying those patients at very high risk of relapse (i.e. those who are positive for both MRD and leukemia stem cell mark- ers).26-28
MFC-based MRD assessment can achieve a sensitivity of 10-3 to 10-5, which is dependent on the number of cells analyzed, gating method, and number of antibody colors used; in most cases, a sensitivity of 10-4 is achieved. Compared to real-time quantitative polymerase chain reaction (RT-qPCR), MFC is significantly faster and less labor-intensive. It also has the advantage of being applica- ble to more than 90% of patients with AML, unlike other methods that rely on specific genetic or molecular tar- gets.22 Despite these advantages, the interpretation of MFC MRD is not standardized in most countries, includ- ing the USA, and requires significant technical expertise on the part of the interpreting pathologist, which can lead to inter-laboratory discordance. Because of the complexity of interpreting flow-based MRD, there is interest in the use of machine-based learning artificial intelligence to reduce the potential for bias or other subjective errors in MRD interpretation. Such artificial intelligence-based algorithms are promising and may result in more clinically significant MRD results, although further validation of this technology is needed.29
Polymerase chain reaction
RT-qPCR can be used to monitor recurrent genomic alterations in certain subtypes of AML. To be a useful marker for PCR-based MRD assessment, the target gene fusion or mutation should be stable throughout the dis- ease course and its presence should reflect true persistent disease (rather than preleukemic subclones). Suitable tar- gets that have been evaluated in large studies include PML-RARA in acute promyelocytic leukemia, CBFB-
MYH11 and RUNX1-RUNX1T1 in core-binding factor AML, and mutant NPM1.15,30-36 In contrast, other mutations may emerge or disappear at the time of relapse (e.g., mutant FLT337,38) and are therefore generally unreliable MRD markers for assessing meaningful “MRD negativity,” although their persistence likely represents residual dis- ease in most cases. One disadvantage of PCR is that appropriate, validated targets are present in less than 50% of patients with AML, and the incidence of these AML subtypes declines substantially with increasing age at diagnosis.1 To overcome this limitation, attempts have been made to track residual disease using markers that are expressed at significantly higher levels in leukemic blasts than in normal hematopoietic cells. For example, several studies have evaluated monitoring levels of WT1 or EVI1 mRNA transcripts over the course of treatment as a mark- er of MRD.39-42 While these targets may provide some prognostic information, they are generally not specific enough for residual leukemia to be used in routine prac- tice.6
PCR-based MRD assessment has the advantage of being highly sensitive (sensitivity ≈ 10-4 to 10-6, depending on the input of RNA/DNA) and is generally better standardized than MFC.19 To further refine relapse risk, ultrasensitive digital droplet PCR technologies have been developed in order to detect low levels of residual gene mutations.43 In contrast to standard PCR, digital droplet PCR does not require calibration standards, and is thus faster and more precise and reproducible.44 Due to its absolute quantifica- tion of DNA copy numbers, it can also provide informa- tion about clonality and subclonality. This technology may have greater sensitivity and be better able to quantify very low levels of MRD than standard PCR22; however, whether this added sensitivity will translate into more accurate prognostic discrimination is yet to be deter- mined.
Next-generation sequencing
Targeted next-generation sequencing (NGS) panels are commonly used at the time of diagnosis to identify prog- nostic gene mutations or mutations that may be therapeu- tically targeted (e.g. FLT3 or IDH1/2 mutations). Several studies have also evaluated re-using similar NGS panels at the time of remission to assess the relationship between decline in mutational burden and clinical outcomes.17,45,46 This methodology relies on a similar principal as PCR regarding the tracking of genomic or molecular targets, although NGS is able to target multiple genes at once, or even the entire genome, if desired.
Although there is understandable excitement about the potential for high-throughput NGS-based MRD monitor- ing in AML, there are several practical considerations that limit its clinical use at present. NGS MRD assessment is relatively expensive, is not standardized, and requires complicated bioinformatics. The interpretation of NGS MRD results is further complicated by the presence of preleukemic clones that may not fully clear even in patients who achieve deep, long-term remissions with chemotherapy, such as mutations associated with clonal hematopoiesis of indeterminate potential (CHIP).47-49 CHIP mutations, particularly DNMT3A, TET2, and ASXL1, commonly persist in patients who do not relapse, suggest- ing that they should not be routinely used as MRD mark- ers.17,46 Furthermore, at present, the sensitivity of NGS for MRD assessment is generally ~1% with most commonly
1534
haematologica | 2019; 104(8)