Strategies for Minimal Residual Disease Detection: Current Perspectives

Marker for molecular MRD

Fusion transcript in core binding factor (CBF) leukemias: CBFB-MYH11 and RUNX1-RUNX1T1

Despite the negative impact of MRD positivity in core binding factor (CBF) leukemias on relapse, no effect was clearly demonstrated on overall survival (OS) in multivariate analysis. This could be ascribed to the relatively high response rates of CBF leukemias to salvage therapy. In inv(16) patients, Yin et al¹⁴ demonstrated that after induction chemotherapy, more than ten CBFB-MYH11copies /10⁵ ABL copies in peripheral blood were the most useful prognostic variables for relapse risk on multivariate analysis. MRD, after the end of therapy, was also informative with levels of transcript inferior to ten copies/10⁵ABL copies in peripheral blood (PB) associated with a cumulative incidence of relapse at 5 years in 36% of cases compared to 78% for those not reaching this threshold. During follow-up, the presence of less than 50 copies/10⁵ABL copies of transcript in bone marrow (BM) and ten copies in PB was associated with an OS at 5 years of 100% and 91%, respectively.¹⁴ We should therefore keep in mind that low and stable levels of transcripts may be detectable by PCR for a relatively long period of time after chemotherapy without evidence of relapse.¹⁵

Similarly, for RUNX1-RUNX1T1, Yin et al¹⁵ established that a threshold of less than 100 copies/10⁵ABL copies in PB and less than 500 copies /10⁵ ABL copies in BM was associated with an OS at 5 years of 95% and 94%, respectively. The French group¹⁶ analyzed 94 RUNX1-RUNX1T1 positive patients during follow-up and showed that the molecular remission after the completion of consolidation therapy was not predictive. By contrast, the negativity in PB at the same time point predicts an OS at 4 years of 96% compared to 63% if positive. As for CBFB- MYH11, the MRD negativity at earlier time was not prognostically relevant.¹⁶

NPM1 mutation

The mutation of NPM1 can be target for MRD by qPCR. NPM1 has been found to be mutated in 50%–60% of AML patients with a normal karyotype.²¹

The presence of measurable NPM1 transcripts in PB or in BM after at least two cycles of chemotherapy is associated with a high risk of relapse in many studies.²⁰ Ivey et al¹⁷ reported a 3-year OS of 75% for patients with NPM1 negativity in PB vs 24% for those with positive NPM1. Shayegi et al¹⁸ reported a 3-year OS of 84% for negative patients at the same time point but measured in BM compared to 76% for those with low NPM1 levels (NPM1/ABL <1%) and 45% with those who maintain positive levels with a NPM1/ABL ratio >1%. Similarly, Kronke et al¹⁹ reported a 4-year OS of 90% for those who reached NPM1 negativity in BM after two cycles of chemotherapy compared to 56% for those with NPM1 positivity.

For patients who obtain a NPM1 negativity in PB but remain positive in BM after the end of treatment, the ELN recommendations suggest to closely monitor the mutation in PB and BM every 4 weeks for at least 3 months.⁹

PML-RARA

In acute promyelocytic leukemia, the most significant MRD end point is the achievement of PCR negativity for PML-RARA at the end of consolidation treatment, independently from the therapeutic strategy, all-trans retinoic acid (ATRA) associated with chemotherapy, or ATRA and arsenic trioxide. PML-RARA negativity at the end of consolidation treatment is associated with a low risk of relapse and a high probability of long-term survival.^22,23

As for CBF leukemia, we should consider that measurable levels of PML-RARA during active treatment should not trigger a treatment change. The usefulness of serial PCR-based MRD monitoring during treatment is still under investigation.

The Wilms’ tumor gene (WT1)

The usefulness of WT1 quantitative assessment, using q-PCR, as a marker for MRD detection in AML has been demonstrated many years ago.^24–30 WT1 is overexpressed in about 80%–90% of the patients.^24,25 The persistence of WT1 overexpression after treatment is always indicative of MRD.^24–30WT1 can thus be considered as the most universal marker of AML. In a European study, it was shown that both the level of WT1 reduction from baseline after induction or consolidation therapy and the clearance of the transcript to normal values are highly predictive of relapse.²⁵ Many studies suggested that the persistence of abnormal values of WT1 after induction or consolidation treatment has an impact on the probability of relapse. An increase of WT1 levels during follow-up always predicts the leukemia recurrence.

Importantly, WT1 is overexpressed independently of the genetic lesion(s) in the leukemic cells. This could generate suspicion because it is evident that WT1 is not specific for one particular leukemic clone. By contrast, considering the recent advances coming from NGS studies that clearly indicate that there is a clonal selection in acute leukemias, the usefulness of a molecular marker able to track all the leukemic clones independently from all genetic lesions is unquestionable.

The main advantages of WT1 assay are that it can be measured in PB, the method has been standardized,²⁵ the assessment of the results is not dependent on human expertise, in contrast to flow cytometric analysis of MRD, and it can detect the emergence of leukemic clones that are genetically or phenotypically different from those detected at diagnosis.

In the recent recommendations of the ELN by Schuurhuis et al,⁹ the authors claimed that WT1 mRNA quantitation should not be used as minimal residual disease marker in AML, due to low sensitivity and specificity, unless no other MRD markers, including flow cytometric ones, are available in the patient. Despite these recommendations, in the last 15 years, many scientific papers have been published showing that WT1 is a reliable marker of MRD that is able to predict relapse with a high level of accuracy.^24–30

The fear of the lack of WT1 sensitivity is mainly based on the fact that there is a background of expression in healthy subjects.²⁵ In addition, the absolute value of WT1 in AML at diagnosis is not always two logs higher than in normal samples.²⁸ This led to a sort of skepticism toward WT1 that, however, cannot be justified by the evidence provided by prospective and retrospective studies.^24,25,28

Additional molecular markers

BCR-ABL

The 2016 WHO diagnostic guidelines included BCR-ABL positive trait as a provisional entity. The vast majority of the patients are characterized by p190 transcript, which is uncommon in chronic myeloid leukemia patients.³¹

Since BCR-ABL positive AML is a rare subtype of leukemia, very little data are available on the outcome and the prognostic value of BCR-ABL-based MRD detection.

IDH1/IDH2

Since the evidence that IDH1/IDH2 genes can be mutated in about 10%–20% of AML cases, many groups are investigating the possibility of using IDH1/2 as a marker for MRD detection.

Until now, it is not clear whether IDH1/IDH2 become negative during remission, and contrasting data are reported in literature.^32,33 There are studies reporting the stability and suitability of IDH as a marker of MRD.^32,34 Recently, Petrova t al³⁵ published the evaluation of MRD in 90 patients, 22% of them with IDH1/IDH2 mutations. They based the assessment on NGS and ddPCR. Many patients presented additional mutations such as NPM1 or MLL-PDT and this allowed clinicians to conclude that IDH1/2 correlated with the treatment response. Despite this, they found that the approach based on IDH1/2 is less sensitive than NPM1 in predicting relapse but more sensitive than MLL-partial tandem duplication (PTD).

Brambati et al explored the possibility of detecting IDH1/2 after transplantation to better identify the risk of relapse.³⁴ They concluded that longitudinal monitoring of these mutations can be extremely useful in the allo-transplantation setting, a context in which these alterations can be considered markers of undesired residual pre-leukemic host hematopoiesis.

Multiparameter flow cytometry

Lots of effort has been devoted in an attempt to obtain the standardization of MFC.³⁶ For years, the detection of the leukemic population has been based on a panel of antibodies targeting early markers such as CD34 and CD117, markers of myeloid-lineage such as CD33, and myeloid differentiation antigens like CD11b, CD13, CD14, CD15 or lymphoid antigens including CD2, CD7, CD19, or CD56.

Two different approaches have been used to measure MRD with MFC: the leukemia-associated immunophenotype (LAIP) approach which characterizes the LAIP at diagnosis and tracks the identified population of blast cells during follow-up.³⁷ A second approach is based on “different from normal” approach (DfN), which is based on the identification of aberrant differentiation/maturation profiles at any time point. The DfN approach has the advantage that it can be applied even in the absence of the immunophenotype at diagnosis and can detect the immunophenotype shifts,³⁸ which is caused by the appearance of new clones. Immunophenotype shifts may emerge from leukemia evolution or clonal selection.³⁹

The use of a large panel of antibodies can overcome the problem of using LAIP rather than DfN, as it is able to cover both the aspects.

Recently, the ELN recommendations suggest the term “LAIP-based DfN approach” for this combination strategy that can provide characterization of leukemic cells at diagnosis, MRD evaluation, and detection of new aberrancies not present at diagnosis.⁹ For all these reasons, the ELN researchers recommend to use at least eight colors.

Differently from what has been demonstrated for qPCR-based MRD assessment, the evaluation of MRD by MFC is not recommended in PB for the lower frequency of leukemic cells.

Another important limitation of MFC was the absence of a commonly accepted threshold of negativity that is able to distinguish between MRD-positive and -negative cases. As 0.1% was found to be relevant in many published studies, the ELN recommends using this threshold. However, an MRD below 0.1 can be consistent with the persistence of residual leukemic cells. Several studies demonstrated the prognostic value of MRD to be below 0.01%,^40,41 showing that this threshold can identify patients with a very good prognosis. Independent validation of these very low levels may be highly relevant in future.

MRD in the setting of allogeneic stem cell transplantation

Many published data support the notion that the presence of MRD immediately prior to allo-HSCT is a strong, independent predictor of post-transplant outcomes in AML.⁴²

The MRD positivity has been defined as one of the stronger predictive factors both in the ablative and non-myeloablative transplants.⁴³

In a large study carried out in 279 AML patients, Zhou et al investigated MRD before and after HSCT.⁴⁴ It was shown that patients with MRD positivity before transplantation have a high relapse risk regardless of whether or not they clear MRD with conditioning chemotherapy.⁴⁴

Studies in NPM1-mutated patients confirmed the impact of MRD pre-transplantation on the OS.⁴⁵ It was shown that only those patients who achieved at least a suboptimal reduction of NPM1 levels after chemotherapy showed an improved OS after HSCT.

Bill et al⁴⁶ analyzed a cohort of 51 NPM1-mutated patients who received HSCT. Mutated NPM1 MRD-positive patients, measured by ddPCR, had higher cumulative incidence of relapse and shorter OS. They demonstrated that NPM1 MRD positivity, measured by ddPCR before allogeneic stem cell transplantation, is associated with worse prognosis independent of other known prognostic markers. Similar results have been described by Kayser et al⁴⁷ in a series of 67 patients. More recently, to extend the evaluation of MRD to all the patients treated with HSCT, an NGS approach has been used in a cohort of 116 patients in complete remission who were treated with HSCT.⁴⁸ The MRD was measured before transplantation, and it was demonstrated, in multivariate analysis, that MRD positivity was an independent negative predictor of relapse.

Finally, many papers reported the value of WT1 monitoring in the setting of HSCT, both before HSCT to assess the quality of the remission and therefore predict the outcome and after HSCT to predict relapse.^48–52 The data reported in this setting are quite strong and led several centers to adopt WT1-based pre-emptive immunotherapy with cyclosporine discontinuation and/or donor lymphocyte infusion in patients with increasing WT1 values after transplantation.^53,54

Finally, although many studies reported that the CBFB-MYH11 fusion transcript can persist at low level in patients during long-term remission after chemotherapy, only a few studies with small samples have addressed the detection of the CBFB-MYH11 fusion transcripts after allo-HSCT.⁵⁵

More recently, Tang et al reported 53 high-risk adult AML patients with inv(16) who received allo-HSCT.⁵⁶ During follow-up, seven patients experienced relapse. All relapses occurred in patients who either did not achieve major molecular response within the first 3 months or who lost major molecular response (MMR) in the first 3 months from transplantation.⁵⁶