The role of multiparametric flow cytometry in the detection of minimal residual disease in acute leukaemia

Pathology (December 2015) 47(7), pp. 609–621

REVIEW

The role of multiparametric flow cytometry in the detection of minimal residual disease in acute leukaemia DENISE LEE1,2, GEORGE GRIGORIADIS3,4,5,6

AND

DAVID WESTERMAN1,2

1Peter MacCallum Cancer Centre, 2University of Melbourne, 3Department of Clinical Haematology, Monash and Alfred Health, 4Alfred Pathology Service, 5Southern Clinical School, Monash University, and 6Centre for Cancer Research, MIMR-PHI of Medical Research, Melbourne, Vic, Australia

Summary Flow cytometry is the most accessible method for minimal residual disease (MRD) detection due to its availability in most haematological centres. Using a precise combination of different antibodies, immunophenotypic detection of MRD in acute leukaemia can be performed by identifying abnormal combinations or expressions of antigens on malignant cells at diagnosis, during and post treatment. These abnormal phenotypes, referred to as leukaemia-associated immunophenotypes (LAIPs) are either absent or expressed at low frequency in normal bone marrow (BM) cells and are used to monitor the behaviour and quantitate the amount of residual disease following treatment. In paediatric acute lymphoblastic leukaemia (ALL), the level of MRD by multiparametric flow cytometry (MPFC) during therapy is recognised as an important predictor of outcome. Although less extensively studied, adult ALL and adult and paediatric acute myeloid leukaemia (AML) have also demonstrated similar findings. The challenge now is incorporating this information for riskstratification so that therapy can be tailored individually and ultimately improve outcome while also limiting treatmentrelated toxicity. In this review we will elaborate on the current and future role of MPFC in MRD in acute leukaemia while also addressing its limitations. Key words: Acute lymphoblastic leukaemia, acute myeloid leukaemia, minimal residual disease. Received 13 November 2014, revised 10 August, accepted 14 August 2015

INTRODUCTION Minimal residual disease (MRD) refers to residual leukaemic cells in the setting of morphological remission (<5% leukaemic blasts on morphology from the nucleated differential count). These cells are undetectable by standard microscopy and their identification relies on more sensitive techniques including molecular studies and flow cytometry. Quantitation and characterisation of MRD by multiparametric flow cytometry (MPFC) has been demonstrated to be of prognostic benefit in select circumstances such as paediatric acute lymphoblastic leukaemia (ALL)1 and adult acute myeloid leukaemia (AML). Improving risk stratification is of particular importance given that despite aggressive therapy, only approximately 24% of adult AML and 40% of adult ALL patients achieve sustained remission.2,3 Cementing a role for MRD detection in Print ISSN 0031-3025/Online ISSN 1465-3931 Copyright DOI: 10.1097/PAT.0000000000000319

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prognostication, surveillance and management will be invaluable in the armamentarium against acute leukaemia. MPFC is a powerful and efficient tool in MRD monitoring. It is easily accessible, relatively economical and applicable in up to 80% of patients with AML4 and 90% with ALL,5 where the majority of patients at diagnosis will have an aberrant immunophenotype. Several single-centre trials have shown that the MRD burden after induction and/or consolidation for both AML and ALL strongly correlate with relapse-free survival (RFS) and overall survival (OS).6–11 Despite what seem obvious benefits, MPFC in MRD monitoring is yet to be universally adopted. Several critical factors hinder its utilisation including inadequate standardisation among laboratories impeding reproducibility of results, lack of precise MRD thresholds and uncertainty regarding appropriate time-points for sequential monitoring. In addition, the expertise and training required for MRD analysis should not be underestimated.12 Molecular monitoring of MRD by polymerase chain reaction (PCR) is generally more sensitive than MPFC by one log, although above the threshold of 0.01% in ALL and 0.1% in AML, the results are relatively comparable.13–16 PCR monitoring is only applicable in patients who express a molecular rearrangement or mutation, which equates to 40-60% of AML (childhood and elderly) and >90% of ALL. Several studies have demonstrated that MRD monitoring of fusion genes (PML-RARA, RUNX1-RUNX1T1, CBFB-MYH11) by realtime quantitative PCR (RT-qPCR) provides prognostic information17–19 and monitoring of PML-RARA in acute promyelocytic leukaemia (APL) is well-established for guiding therapy.20,21 Mutated NPM1 is present in 30% of all AML cases and has proven to be a clinically significant MRD marker, predictive of relapse and survival.22–24 Next-generation sequencing (NGS) technology provides an alternative platform for molecular MRD detection, enabling a high-throughput of samples while also abrogating many of the limitations of MPFC and RT-PCR. Salipante et al.25 observed that the sensitivity of detection for NPM1 by NGS methods was 0.001%, more than a log greater than for MPFC, but comparable to RT-PCR. NGS also eliminated the requirement for multiple allele-specific probes or prior knowledge of the NPM1 mutation subtype. While there is no doubt molecular MRD, particularly in the context of NGS technology, is an informative marker of relapse, a detailed discussion on the comparison of methodologies for MRD assessment in acute leukaemia is beyond the scope of this review; we refer the readers to Grimwade and Freeman26 and Bruggemann et al.27 Our review will focus on

2015 Royal College of Pathologists of Australasia. All rights reserved.

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the current and future role of MPFC in MRD in acute leukaemia while also addressing its limitations.

METHODOLOGIES IN MRD ANALYSIS IN AML Leukaemia associated immunophenotypes (LAIPs) A LAIP is a unique and specific combination of antigens identified on leukaemic cells at diagnosis, making them ideal for sequential MRD monitoring. It should consist of a progenitor antigen (CD34, CD117, HLA-DR for AML; CD34, TdT for ALL), a lineage-specific antigen (CD3/19 for T/B cells respectively and MPO for myeloid cells) and aberrantly expressed antigen(s). Further qualitative information can be gained from aberrant physical characteristics with side- and forward-scatter properties.28 The LAIP may be present on all or a subset of leukaemic cells and should be absent or present only in low frequency on normal bone marrow cells.10 Multiple LAIPs10,29 are often identified and all should be followed during treatment to maximise specificity and accuracy. Figure 1 is an example of MRD analysis in AML. Up to 0.5% of normal bone marrow cells may express a LAIP, compromising specificity29 and resulting in inaccurate quantification of MRD, particularly at lower levels of detection (<0.01%).30 Attaining more events and following multiple LAIPs may mitigate this, although Feller et al.10 found no difference in RFS irrespective of whether background normal LAIP expression was corrected for MRD frequency or not. At diagnosis, 73–88% of cases had malignant myeloblasts expressing an abnormal phenotype differentiating them from normal, immature cells.28,31,32 If present, aberrant antigen expression can be helpful in exposing and highlighting small populations that may otherwise have been missed. Aberrant expression may be due to either one or a combination of the following: (1) asynchronous antigen expression, (2) cross-lineage antigen expression, and (3) antigen over/under-expression.7,28,33,34 LAIPS commonly undergo phenotypic shifts at relapse in AML, increasing false negativity.33,35,36 Baer et al.36 showed that 91% of AML patients expressed different immunophenotypes between diagnosis and relapse due to loss or gain of either antigen expression or discrete leukaemic populations. By increasing their antibody-panel combinations to nine panels (utilising three colours), they were able to identify relapse in all cases (136 patients), abrogating the loss in sensitivity. Ossenkoppele et al.30 and Feller et al.10 agreed with this approach, recommending identification of multiple LAIPS at diagnosis. Since the publication of these studies, the majority of laboratories would now be performing their assessments on 6-colour flow cytometry, and we anticipate this will improve our sensitivity of MRD detection even further. The degree of immunophenotypic shift between diagnosis and relapse can also potentially affect our sensitivity of MRD detection. This was assessed by Baer et al.,36 who found that shifting immunophenotypes was not predictive of outcome, demonstrating similar disease-free survival (DFS) between patients with and without antigen changes ( p ¼ 0.39). This observation should be interpreted cautiously, however, as only 9% of patients demonstrated phenotypic stability between diagnosis and relapse. It is also noteworthy to mention, while there is firm evidence that immunophenotypic shifts may compromise our ability to detect MRD at relapse, little is published regarding this

phenomenon between induction and consolidation therapy in the AML literature. Inclusion of human myeloid inhibitory C-type lectin (hMICL) and CD123 into the MRD panel may circumvent the problem of false negative results due to immunophenotypic shifts. Recently, Roug et al.37 described the addition of hMICL and CD123 as a universal marker in combination with a CD45/ CD34/CD117 backbone. hMICL is found in 90% of AML samples38 and CD123 is widely expressed but not exclusive to AML. Larsen et al.38 first demonstrated conservation of hMICL expression in paired diagnostic and relapsed AML samples, highlighting its potential as a stable LAIP. In a prospective study of 69 patients, Roug et al.37 showed that this unique antibody panel was applicable to >90% of AML patients with sensitivities of detection ranging from 102 to 104. They also demonstrated that patients with post-induction levels of hMICL/CD123 above the median in regenerating BM had a shorter RFS ( p < 0.001). The evolution of a ‘universal’ MRD marker is growing, with a similar approach being investigated by the EuroFlow Consortium to use single-tube disease-oriented panels for MRD detection over patient-specific antibody combinations.39 This concept of a standardised MRD marker panel will be pivotal in establishing consistency in MRD analysis. ‘Different from normal’ The ‘different from normal’ approach to MRD analysis overcomes the problem of MRD false negatives due to immunophenotypic shifts. Identification of MRD is reliant on comparing patterns of normal antigenic expression through all stages of maturation against the sample of interest. Populations are considered abnormal if they are 0.5 decades away from the position of the corresponding normal cells.8,40 Another advantage to this approach compared to LAIP identification is that MRD analysis is still possible despite not having an available diagnostic LAIP, which may occur when samples are sent to a centralised laboratory for evaluation. This method of analysis necessitates expert knowledge of normal haematopoietic maturation patterns, however, which may be a major limiting factor in its widespread implementation in laboratories. Achieving standardisation in MRD analysis when applying this approach is also more complex, as the determination of ‘normal’ patterns can be somewhat subjective. Each laboratory should establish normal expression with their antibody panel using a minimum of 20 patient/donor samples. We recommend both methods be routinely used when performing MRD analysis. However, this approach significantly increases the time spent per evaluation, therefore workflow and efficiency requirements of diagnostic laboratories should be considered before a laboratory offers MRD testing.

STANDARDISATION Definition of a LAIP One of the key difficulties in standardising MPFC-based MRD monitoring stems from a lack of consensus regarding the true definition of a LAIP. Different groups adopt conflicting criteria resulting in variably reported LAIPs. According to Feller et al.10 and Terwijn et al.41 a LAIP was considered valid only if expressed on at least 10% of leukaemic blasts. Alternatively, Buccisano et al.42 defined a LAIP to be expressed on at

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FLOW CYTOMETRY IN ACUTE LEUKAEMIA Diagnostic AML

[Singlets]

103

[WBCs]

103

[CD45 progenitor gate]

103

CD117+

Viable cells

102

101

102 CD117 PC5

CD45 V500-A

102 SSC-A

611

101 CD45 progenitor gate

100

100

0

0

101 2 0

200

400 600 FSC-A [CD117+]

800

0

1000

10

1

10 SSC-A [CD11b-]

2

10

10

3

102

103

102

102

102

1 102 101 HLA-DR APC-H7

103

CD33 V450

103

5

101 SSC-A

[CD13+ dim]

103

101

100

0

103

CD13 PE

CD11b APC

0

–2 0

101

101 CD13+ dim

2

100

0 0

0.2 0

–5 –2 –4 –2

0

2

4

101 CD34 PE-Cy7

102

–4 –2

103

0

2

4 101 CD34 PE-Cy7

102

–1

103

0

MRD AML [WBCs]

[Singlets]

[CD45 progenitor gate]

103

103

103

CD117+

Viable cells 2

10

101

100

CD117 PC5

102 CD45 V500

SSC-A

102

101

101

CD45 progenitor gate

100

2

0 –0.5

0

0 0

0

200

400 600 FSC-A

800

1

0

10 SSC-A

10

1000 Gate All

2

10

10

3

–2

Number

[CD11b-] [CD117+] 10

101 5

101

101 100

100

MRD

0

–2

0

101 2 CD34 PE-Cy7

102

103

0

CD13+ DIM

0.2 0

–5

103

102 CD33 V450

CD13 PE

CD11b APC

CD11B-

102

103

102

102

101 SSC-A

[CD13+ dim] HLA-DR APC-H7 / CD33 V450

103

3

100

0

390,895

–1 –2

0

101 2 CD34 PE-Cy7

102

0

1

101 102 HLA-DR APC-47

103

103 Gate number All 1,152 MRD 1,152

Fig. 1 Diagnostic AML: Initial sequential gating strategy to include viable cells only (not shown): event number vs time, FSC-A vs FSC-H and FSC-A vs SSC-A. LAIP comprises abnormal antigen expression compared to normal blasts (CD34, dim CD13þ, partial HLA-DR). MRD AML: A sequential gating strategy is used in combination with physical characteristics (SSC-A vs FSC-A) to identify minimal residual disease (MRD). MRD is discriminated from promyelocytes (shown in purple) by their physical characteristics, high side scatter, HLA-DR positivity and bright CD13 expression, compared with dim CD13 expression on the leukaemic blasts. Normal blast expression (arrow) is confirmed by comparing to blasts from a healthy control. MRD ¼ 0.29% (MRD events/total WBCs  100). Black, leukaemic myeloblasts/MRD; purple, promyelocytes. WBC, white blood cells.

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least 50% of blasts. Different still, Olaru et al.43 deemed a LAIP to be significant if it was expressed on at least 1% of the total population (comprising 100,000 CD45 positive events). Denominator Discrepancy amongst authors also exists regarding the denominator used in MRD calculations. The most common denominator used is the total white blood cell (WBC) count. The lysing reagent eliminates the majority of red cells during sample preparation, (although this process can be imperfect) before further exclusion by a CD45 vs side scatter gating strategy. However, in paediatric ALL the denominator is mononuclear cells, which is based on the tradition of using ficoll to extract the mononuclear component. Although both techniques were recognised as sample preparation methods at the Proceedings of the Second International Symposium on MRD assessment,44 the recently published British Committee for Standards in Haematology (BCSH) Guidelines recommend red cell lysis over density centrifugation.45 Number of events to acquire The total number of events recommended for acquisition ranges from 100,00043 to 250,0007,46 to 1  106.41,44 Variability amongst groups regarding what constitutes an ‘event’ also influences sensitivity with some groups aiming for total events, which would include debris and erythroid precursors, and others aiming for total WBC events. Both of these methods are open to errors in enumeration due to peripheral blood contamination in bone marrow aspirate samples.47 Attaining adequate event detection can also be problematic in paucicellular marrows. Techniques to address these limitations include doubling the standard volume of cells, density gradient centrifugation or ‘bulk lysis’.48 Sample quality can also be assessed by ensuring adequate levels of cells that are normally restricted to the marrow such as erythroid, myeloid and B-cell progenitors are present, a technique recommended by the European Myeloma Network when performing myeloma MRD.49 The number of events that defines a leukaemic population also varies significantly between diseases and groups. The IBFM-ALL-FLOW MRD Network recommends a minimum of 30 leukaemic events per tube accompanied by a confirmatory, independent tube.44 The EuroFlow Consortium recommends at least 100 leukaemic cells (as the sum of events in all tubes) in line with their recommendation for MPFC MRD quantitation of multiple myeloma.49 In our laboratory we aim to acquire >500,000–1,000,000 events in AML and ALL. The appropriate absolute cut-off of abnormal leukaemic cells varies on an individual patient basis, but 50–100 abnormal events would usually ensure a sensitivity around 1 in 10,000. Reproducibility The perceived lack of reproducibility of MPFC, particularly across multicentre studies, has so far hindered the progress of standardisation.39,50 Reproducibility is influenced by a large number of complex factors including equipment and reagent variability and stability, software, and data analysis and interpretation. At the heart is a thorough and robust quality framework. The International Council for Standardisation of Haematology (ICSH), the International Clinical Cytometry Society (ICCS) and the BCSH have released independent

Pathology (2015), 47(7), December

practice guidelines in an attempt to address the issues of standardisation and promote inter-laboratory harmonisation.45,51 The recommendations provide guidance on instrument set up, fluorochrome and panel design, sample preparation, data analysis and quality control. It is hoped these guidelines will improve uniformity of practice across laboratories for all diagnostic cases. Notably, MRD analysis was not addressed in any of the aforementioned guidelines. The EuroFlow Consortium published recommendations for MRD markers in antibody panels, but also did not specifically address standardisation of MRD analysis. Feller et al.52 demonstrated that standardisation of LAIPs for MRD analysis is possible in a multicentre setting. This was achieved by defining a standardised antibody panel and standard operating procedures. Newer developments such as automated analysis algorithms may also be helpful in improving uniformity.53

ACUTE MYELOID LEUKAEMIA Prognostic relevance of MRD following induction and consolidation San Miguel et al.9 were the first to demonstrate the prognostic significance of flow-based MRD in AML, identifying two levels of MRD (0.5% and 0.2%, respectively) that correlated with a higher risk of relapse post-induction and post-intensification. In a later study by the same group, the authors showed that post-induction MRD levels could also stratify patients into different risk groups.11 Buccisano et al.42 demonstrated that post-consolidation MRD levels >0.035% correlated with an unfavourable prognosis in terms of relapse rate, OS and RFS ( p < 0.001). In contrast to San Miguel et al.,11 they found that post-induction MRD status bore no influence on outcome, provided MRD negativity was achieved by the end of consolidation.42 Postconsolidation MRD levels proved to be an independent predictor of outcome when compared against karyotype, age and post-induction MRD levels as covariates. Effect of chemotherapy on MRD The type of chemotherapy administered may affect the level of MRD achievable and re-calculation may be necessary for treatment protocols that involve more intensive regimens resulting in greater tumour debulking and potentially less MRD.4,9,42 The findings of Rubnitz et al.54 and Feller et al.10 would contradict this suggestion where treatment regimens did not appear to have any impact on the degree of MRD positivity. Timing The optimal time-point for MRD assessment is still unresolved with some advocating post-induction,8,11,41 post-consolidation,6,42 or both.7,10 Timing is critical when applying riskadapted therapy. Premature evaluation would potentially identify more patients resulting in additional (and unnecessary) intensive therapy. However, deferred evaluation (i.e., postconsolidation) may have limited practical utility. While there is no consensus regarding the optimal time-point for MRD assessment, earlier evaluation (post-induction) is likely to be the most useful so that treatment could be altered if required.1,5,8,11,41,54,55

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FLOW CYTOMETRY IN ACUTE LEUKAEMIA

Also unclear is the frequency of MRD assessments. Although regular monitoring may provide peace of mind, this would involve frequent and possibly unnecessary bone marrow biopsies, which are not without risks. Intimately associated with this question is whether earlier therapy (i.e., in the absence of morphological relapse) ultimately improves survival. We look forward to prospective, clinical trials that may adequately answer these questions. Quality of response The kinetics of blast reduction has been found to be independently associated with OS. The German AML Cooperative Group determined that morphologically detectable blasts midinduction indicated slow clearance and was a negative predictor of event-free survival (EFS), OS and RFS.56 This has been reaffirmed in later trials demonstrating early blast clearance as an independent predictor of survival post-induction.57,58 Maurillo et al.59 disagreed with these findings, suggesting that achievement of MRD negativity by the end of consolidation is more important than achieving early MRD negativity. They demonstrated that 30% of patients who were MRD positive post induction went on to achieve MRD negativity following consolidation, with no significant difference in RFS and OS between MRD negative patients post-induction and consolidation and MRD positive patients post-induction who converted to MRD negative status following consolidation. This would be concordant with the findings of Buccisano et al.42 and Venditti et al.,6 suggesting the kinetics of blast clearance may not be an accurate predictor of survival, which seems counter-intuitive. Alternatively, Rubnitz et al.54 suggested that the depth of response might be more predictive of outcome. Here, a riskadaptive approach incorporating serial MRD assessments (following induction 1 and 2) was applied to guide therapy intensification in a paediatric AML cohort.54 The outcome for patients with low levels of MRD (0.1% to <1%) after induction 1 was similar to patients with undetectable MRD (<0.1%). In contrast, patients with MRD levels >1% had significantly worse 3-year EFS and OS, independent of cytogenetic risk. MRD thresholds Both the number of cells defining the neoplastic population and the total cells evaluated will influence the sensitivity of the MRD assay. Terwijn et al.41 found the maximal sensitivity achievable in most patients was between 0.01% and 0.1% in adult AML patients. They also demonstrated that all MRD levels between 0.01% and 1% provided independent prognostic information for OS and RFS post-induction or consolidation. Ultimately, they adopted a prospective MRD level of 0.1% with the aim of attaining at least 1  106 events. In the first prospective, multicentre study, 389 patients were evaluated by 4-colour flow cytometry following induction and consolidation. They showed that an MRD of 0.1% was predictive of OS and RFS independent of cytogenetics and molecular profiling [hazard ratio (HR) 2.55 for intermediate risk and 4.75 for poor risk; p < 0.001] and white cell count at diagnosis (HR 2.12, p ¼ 0.0026), confirming the prognostic role of MPFC-based MRD. Prior to Terwijn et al.,41 only retrospective studies were available, which were predominantly from single-centre institutions. Most used a threshold of 0.01–0.1%15,40,60,61 but the levels ranged from 0.01% to 0.5%.6,8,9,11,42

613

Kern et al.62 applied the degree of reduction of the leukaemic cells between diagnosis and follow-up as an MRD threshold. This was defined as a logarithmic difference (LD), calculated as the logarithm of the ratio of the percentage of LAIP positive cells at diagnosis over the percentage of LAIP positive cells detected at follow-up. By separating the LD to the 75th percentile, two risk groups were identified with a 2 year RFS of 83.3% vs 25.7%, p ¼ 0.0034 and OS of 85.7% vs 51.4%, p ¼ 0.0507. The LD post-consolidation was prognostic for OS in a univariate analysis ( p ¼ 0.005) only. On multivariate analysis, the prognostic significance of the LD was independent of cytogenetics for RFS after induction [relative risk (RR) ¼ 0.348; p ¼ 0.006] and consolidation therapy (RR ¼ 0.397; p ¼ 0.006). Several groups have applied statistical methods to determine prognostically significant MRD thresholds. Buccisano et al.42 established a threshold level of 0.035% after evaluating the trend of standardised log-rank statistics using RFS and OS as dependent variables and the values of residual leukaemic cells post induction and consolidation as independent variables. Al-Mawali et al.46 applied a receiver operating characteristic analysis to calculate an MRD value of 0.15% demonstrating this level to be predictive of relapse at both post-induction ( p ¼ 0.05) and post-consolidation ( p ¼ 0.009). While there is no doubt that MRD following therapy is associated with a poor outcome, the wide variability in thresholds makes it difficult to translate into clinical practice (see Table 1). Also unclear is whether MRD-directed therapy influences disease outcome. Some guidance is provided for ALL trials. Bruggemann et al.44 recommend individual trials define their MRD thresholds given this will be dependent on their methodology of determination, the intensity of treatment, the anticipated outcome of the patients and the therapeutic objectives (treatment intensification vs de-escalation). To our knowledge an equivalent AML guideline is not available, but it would be surprising if the same principles did not also apply. Peripheral blood vs bone marrow in AML The majority of trials investigating MPFC in AML MRD are performed on bone marrow samples. One obvious reason for this would be the superior cellularity and presumed leukaemic cell representation (and hence, sensitivity) of a marrow sample as opposed to peripheral blood (PB). There are limited data on the validity of PB MRD monitoring in AML with only two trials published to date. These include Maurillo et al.63 who analysed 50 and 48 pairs of BM and PB samples post-induction and consolidation, respectively. The MRD threshold in PB that correlated with outcome was 0.015%, similar to standard MRD thresholds for BM evaluation. Concordance (r ¼ 0.86 and 0.82, respectively, p < 0.001) between the two samples following induction and consolidation suggests that PB MRD monitoring could be integrated with BM in the assessment of AML. A more recent trial involving 29 adult patients by Zhong et al.64 showed that day 8 PB MRD of 0.01% was an early predictor of therapeutic efficacy following induction chemotherapy. Correlative BM samples were not performed however, precluding any conclusions regarding the sensitivity of PB to BM MRD.

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Table 1

Comparison between adult AML studies

Reference

Patients, BM n or PB

Assessment time-point

San Miguel et al.9

53

BM

End of induction and intensification

0.5% post-induction Yes 0.2% postintensification

San Miguel et al.11 126

BM

End of induction

0.01–1%

Yes

Buccisano et al.42

100

BM

End of induction and consolidation

0.035%

Only after postconsolidation

Maurillo et al.59

142

BM

Venditti et al.6

56

BM

Terwijn et al.41

517

BM

Post-induction and 0.035% post-consolidation Post-induction and 0.035% post-consolidation After cycle 1 and 2 of 0.1% chemotherapy and post-consolidation

Kern et al.7

1054

BM

Post-induction and post-consolidation

75th percentile of LD

Post-induction and consolidation for RFS

Kern et al.62

106

BM

Day 16 (induction)

LD¼2.11

Yes for EFS and RFS

54

BM

0.15%

Post-induction only

0.015%

Post-consolidation for RFS only

Al-Mawali et al.46 Maurillo et al.

63

50

Post-induction and consolidation PB and Post-induction and BM consolidation

Threshold

MRD independent risk factor on multivariate analysis? Co-variates

Post-consolidation only Post-consolidation only Assessed for post-cycle 2 chemotherapy only

Survival parameter

WBC count; RFS platelet count; OS haemoglobin; no. of blasts on morphology; age; MDR1 phenotype Cytogenetics; RFS no. of cycles to achieve CR; OS WBC count Cytogenetics; RFS MDR1 phenotype; OS age; WBC count Cytogenetics; RFS MDR1 phenotype OS Cytogenetics; RFS MDR1 phenotype OS Cytogenetics; RFS AML type; OS achievement of CR after cycle 2; WBC count; age; consolidation treatment Cytogenetics; RFS WBC count; OS no. of blasts on morphology Cytogenetics; RFS presence of FLT3-LM; EFS secondary AML OS WBC count; RFS FAB subtype OS WBC count; RFS MDR1 phenotype OS

AML, acute myeloid leukaemia; BM, bone marrow; EFS, event free survival; FAB, French-American-British classification; FLT3-LM, length mutations of the FLT3 gene; LD, log difference; MDR1, multidrug resistant-1; OS, overall survival; PB, peripheral blood; RFS, relapse-free survival; WBC, white blood cell count at diagnosis.

Box 1: Key points in adult AML  The most common denominator for MRD calculations is the total WBC count.  The optimal timing of MRD assessments remains unresolved with discrepancy between the value of achieving early (mid and end of induction) vs late (end of consolidation) MRD negativity.  Recommended MRD threshold is between 0.1% and 0.01%.  The effect of the intensity of the chemotherapy regimen on the MRD level achievable is controversial with some groups demonstrating negligible effect and others requiring recalibration of MRD levels depending on the therapy.  Bone marrow MRD assessments are preferred over peripheral blood, given the limited data on the latter.

PAEDIATRIC AML MRD Through a series of sequential trials, paediatric AML MRD analysis has evolved from a prognostic measure to directing therapy. Results from earlier trials that focussed on the prognostic value of MRD led to the concept of MRD-directed therapy. The studies discussed below demonstrate this evolving principle (see Table 2). Coustan-Smith et al.65 demonstrated on multivariate analysis that MRD >0.1% post-induction 1 and 2 was an independent predictor of OS, compared with cytogenetics and white cell

count (WCC) ( p ¼ 0.037, HR 3.79, and p ¼ 0.022, HR 6.15, respectively). These findings and their approach to MRD assessment subsequently informed the risk assignment algorithm in the multicentre AML2002 study, which was discussed earlier.54 Sievers et al.8 demonstrated that patients with MRD >0.5% after remission induction were 4.8 times more likely to relapse ( p < 0.0001) with a mortality rate that was 3.1 times greater ( p < 0.0001). Following consolidation, OS at 3 years was 41% vs 69% for patients with >0.5% vs <0.5% residual blasts. In a multivariate analysis, MRD positivity was the most powerful independent prognostic factor compared with age, gender, ethnicity, presence of hepatosplenomegaly and WCC. In contrast, the MRD-AML-BFM Study Group found that MPFC evidence of MRD did not provide any additional prognostic information to risk factors defined in previous AMLBFM studies [French-American-British (FAB) subtype, cytogenetics, morphologically determined bone marrow blasts <5% at day 15].66 In this study, 542 specimens from 150 children were analysed by 4-colour flow cytometry at four time-points during treatment (mid-induction, pre-second induction, pre-third and fourth consolidation). Their analyses revealed at least three sequential, positive MRD measurements were required before statistical significance could be reached. Patients falling within this group were classified as ‘poor risk’ with a 3-year EFS of 31% ( p ¼ 0.007). No significant difference in EFS was found between children who were negative by flow cytometry at all analysed time-points and those with <3

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FLOW CYTOMETRY IN ACUTE LEUKAEMIA

Table 2

615

Comparison between paediatric AML studies Patients, n

BM or PB

Assessment time-point

Threshold

MRD independent risk factor on multivariate analysis?

Coustan-Smith et al.65 Sievers et al.8

46

BM

Induction 1þ2

0.1%

Yes

252

BM

Consolidation

0.5%

Yes

Langbrake et al.66

150

BM

0.1%

No

Rubnitz et al.54

230

BM

Mid-induction; pre-2nd induction; pre-3rd consolidation; pre-4th consolidation Induction 1þ2

0.1%

Only if MRD 1%

Loken et al.40

340

BM

Induction 1þ2; consolidation

0–<1% 1%

Only for RFS

Reference

Co-variates

Survival parameter

Cytogenetics; WBC Age; gender; ethnicity; presence of hepatosplenomegaly; WBC FAB subtype; cytogenetics; no. of blasts on morphology at day 15

OS

Presence of core-binding factor or FLT3-ITD; age; 11q23 translocations other than t(9;11); M7 without t(1;22) Molecular risk group; WBC

EFS OS

RFS, OS

FFS EFS

RFS OS

BM, bone marrow; EFS, event free survival; FAB, French-American-British classification; FFS, failure free survival; MRD, minimal residual disease; OS, overall survival; PB, peripheral blood; RFS, relapse-free survival; WBC, white blood cell count at diagnosis.

MRD positive time points (73% vs 61%; p ¼ 0.43). Multivariate analysis (incorporating FAB subtype, cytogenetics, and morphologically determined blasts at day 15) also failed to establish MRD as an independent prognosticator. The authors suggest their failure to observe MRD as an independent risk factor was due to their inclusion of morphological blast count (>5% or <5%) on day 15 as a covariate. When they compared MRD against age at diagnosis, leukocyte count at diagnosis and karyotype only, they achieved similar results to their contemporaries. On this basis, the authors concluded the AML-BFM risk classification should be adopted in order to accurately interpret the additional value of MRD analyses. In Loken et al.,40 MRD was prospectively evaluated from a 4-colour cytometer using a ‘different from normal’ approach at post-induction 1 or 2, or at the end of therapy. The 3-year relapse rate for patients with MRD following induction 1 was 60% compared to 29% for patients without MRD ( p < 0.001). The corresponding RFS was 30% and 65%, respectively ( p < 0.001). MRD at the end of induction 2 and end of therapy was similarly predictive of poor outcome. In a multivariate analysis, with cytogenetics and molecular abnormalities as covariates, MRD was an independent predictor of relapse ( p < 0.001) but not OS ( p ¼ 0.06). The findings from this study led to the development of the risk-based therapy algorithm in a subsequent Children’s Oncology Group (COG) trial where patients were allocated to two risk groups, based on cytogenetic, molecular and MRD assessments. Although the results of these studies are based on paediatric AML, they should also be applicable to adult AML while awaiting the outcome from prospective clinical trials.

TRANSPLANTATION The presence of MRD prior to allogeneic haematopoietic stem cell transplantation (HSCT) is an important predictor of relapse and survival, as determined in several retrospective studies.67–70 Walter et al.69 examined the role of pre-transplantation MRD on outcome following allogeneic HSCT. Ten-colour flow cytometry was performed on 99 patients in

first complete remission (CR). Of these, 24 patients were MRD positive (defined as any level of residual disease) and more likely to have abnormal cytogenetics at the time of HSCT. The 2-year OS for MRD positive and MRD negative patients was 30.2% vs 76.6%, respectively, and the risk of relapse at 2 years was 64.9% vs 17.6% for MRD positive and MRD negative patients. Multivariate analysis revealed a hazard ratio of 4.05 for overall mortality and 8.49 for relapse (MRD positive vs MRD negative), confirming MRD positivity pre-HSCT as an independent negative risk factor. The Seattle group published a second study,68 which demonstrated the negative prognostic impact of pre-HSCT MRD is similar in both CR1 and CR2 up to a sensitivity of detection of <0.1%. In this study, the authors determined that any level of MRD was associated with an adverse outcome. However, the authors acknowledged that small numbers limited their ability to prove that the risk of relapse or death increased proportionately with MRD levels. Anthias et al.67 confirmed the findings of Walter et al.,68 Walter et al.69 and also demonstrated a direct relationship between the pre-transplant level of MRD and survival. A retrospective analysis on 88 patients who underwent either a reduced-intensity allograft or myeloablative allograft in CR1, 2 or 3 was performed using 3-colour flow cytometry.67 High-level MRD was defined as 1% and low-level MRD as <1%. OS was 66.8% vs 51% vs 30% for MRD negative, low-level MRD and high-level MRD, respectively ( p ¼ 0.012). Interestingly, no difference in OS was observed between MRD positive and MRD negative patients 3 months post-transplant ( p ¼ 0.064), however given there were only 10 out of 70 patients in the MRD positive group, the study was likely underpowered for this measure. The largest analysis to date is by Appelbaum70 and they measured pre- and post-transplant MRD levels on AML and ALL patients transplanted in either CR1 or CR2 using 10- and 7-colour flow cytometry, respectively, to a detection limit of >103 –104. Post-transplant MRD was assessed additionally by fluorescence in situ hybridisation (FISH). AML patients who were transplanted in CR1 (n ¼ 183) had a 3-year OS of 73% vs 32% (MRD negative vs MRD positive, p values not given).

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616

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Several AML patients transplanted in CR2 had a 3-year OS of 73% vs 44% (MRD negative vs MRD positive). Multivariate analysis (including age at the time of HSCT, CR status, cytogenetics, type of AML, number of chemotherapy cycles prior to HSCT, CR duration before HSCT, pre-transplant peripheral count recovery and conditioning regimen) confirmed pre-transplant MRD to be an independent predictor of outcome following myeloablative HSCT. Post-transplant MRD was measured in a separate but overlapping cohort of 287 AML patients. Up to 100 days post-HSCT, DFS was superior in patients who were MRD negative by MPFC and FISH. It is important to note also that not all patients who were MRD positive, relapsed. Clinical characteristics of MRD positive patients were similar across the four studies. Patients who were MRD positive were more likely to have secondary AML,67–70 poor cytogenetics68–70 and been heavily pre-treated prior to HSCT or failed induction therapy.67 Whether MRD monitoring is therapeutically relevant in the transplant setting has not yet been established. Central to this issue is determining whether MRD positive patients pre- and post-transplant will benefit from either pre-emptive or salvage therapy. Clarification of the optimal timing and frequency of MRD assessments is also paramount. These questions should be resolved in a prospective analysis for accurate interpretation of MRD results in this select population. Box 2: Key points in transplantation  MRD assessments can be applied pre- and post-transplant to risk-stratify patients.  There are no prospective studies to date on whether MRD-directed therapy in the transplant setting has therapeutic benefit.  The optimal timing and frequency of MRD assessments requires clarification in prospective studies.

ACUTE LYMPHOBLASTIC LEUKAEMIA MRD monitoring by MPFC in paediatric ALL is well-established,71–73 with a pivotal role in treatment stratification.44 Several paediatric trials have provided much insight into the clinical relevance, prognostic significance and pitfalls of MPFC MRD in ALL. Leukaemia associated immunophenotype Like AML, immunophenotypic monitoring of ALL relies on the detection of LAIPs identified at diagnosis. The key principle is assessment of antigen expression within the spectrum of normal maturation, which can be complicated by shared antigen expression on malignant blasts and haematogones,74 potentially leading to false negativity and decreasing specificity. This may be abrogated in part by identifying different light scatter and fluorescent characteristics at diagnosis,75,76 which remain constant throughout the initial phases of therapy, irrespective of immunophenotypic modulation.77 Another pitfall to be avoided is the effect of corticosteroids on antigen expression. Dworzak et al.78 demonstrated that CD10 and CD34 expression decreased, and CD20 and CD45 expression increased as a result of corticosteroid administration. However, the effect on CD10 and CD34 was reversible on cessation of steroid therapy. In T-ALL, Roshal et al.79 demonstrated that TdT and CD99 expression decreased during chemotherapy and argued against reliance on these markers for MRD analysis. In contrast

Pathology (2015), 47(7), December

to TDT and CD99, T-cell lineage markers, CD2, CD3, CD4, CD5, CD7 and CD8 remained relatively stable throughout therapy. Figure 2 is an example of MRD analysis in ALL. Different from normal Foster et al.80 demonstrated that LAIPS are not essential for MRD monitoring. In a retrospective review of 116 patients, none of the diagnostic specimens were available for comparison. Residual disease was identified using a ‘different from normal’ approach where abnormal lymphoblasts were defined as having antigen intensities of >0.5 log units away from normal expression. The advantages of a ‘different from normal’ approach are similar to those in AML, namely, MRD analysis is still possible despite the absence of a diagnostic LAIP or sample. The effect of corticosteroid administration on antigen expression still applies, however, and it is important to be mindful of this when applying either approach. Recognition of normal maturation patterns, irrespective of the method adopted, is also important. Given lymphoblasts and haematogones frequently share similar antigenic expressions, dysregulated maturation is often the only indication of residual disease. Paediatric ALL A seminal study by the Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) group highlights the importance of disease kinetics.55 They demonstrated that MPFC MRD on day 15 is an independent prognostic factor and can be used to predict the risk of relapse in childhood ALL. Based on the amount of MPFC detectable MRD present in the bone marrow on day 15, patients were stratified as having standard (<0.1%), intermediate (0.1–<10%) or high (>10%) risks of relapse. Patients in the low or intermediate groups had 5-year cumulative incidences of relapse (CIR) of 7.5% and 17.5%, respectively, and patients in the high-risk group had a CIR of 47.2%. In a multivariate analysis, including response to steroids and the National Cancer Institute criteria as covariates, the authors found that MRD >10% was the most potent prognostic parameter. The prognostic value of pre-transplant MRD has also been demonstrated in paediatric ALL.80,81 Elorza et al.,81 found the 2-year EFS for MRD negative patients was 75% compared to 20% for MRD positive patients ( p ¼ 0.005). However, at the end of the study two out of 10 patients who were MRD positive pre-HSCT remained alive in morphological complete remission (mCR; one was MRD negative and the other MRD positive at 0.03%). Although the numbers are small, this last point importantly highlights that MRD positivity does not inevitably lead to relapse, an observation that was also made by Appelbaum70 in adult AML patients. Sanchez-Garcia et al.82 demonstrated that quantification of MRD at the time of transplant could be used to stratify patients and predict outcome in ALL. The authors found that any level of MRD was associated with poor leukaemia-free survival (LFS), OS and EFS (20.8%, 10.7% and 3.8%, respectively). Patients could be further stratified depending on their level of pre-transplant MRD. OS in patients with low levels of MRD (0.1%) was significantly superior to patients with intermediate-high levels (>0.1%): 28.6% vs 0% ( p ¼ 0.0153). Patients with low-level MRD also had a longer median time to relapse following HSCT compared to patients with intermediate-high levels (8.5 months vs 4.6 months, respectively). Although this

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617

FLOW CYTOMETRY IN ACUTE LEUKAEMIA

Diagnostic ALL [Viable cells] SSC-A / CD19 PE-Cy7

[Viable cells]

WBCs CD19 PE-Cy7

CD45 Pac OR

102

101

103

102

102 CD19+

101

1

100

100

101 SSC-A

102

0 –1 101 SSC-A

102

103

101 5 0

0 –2 0

2

101

102

1

[Viable cells]

2

10 2 10 CD10 PE

–2 0

CD10 PE

MRD ALL

102 20 0

10

102 101 5 0 –5 –20

3

0

0

10

1

2

10 10 CD20 FITC

0

103

103

102

102

20

102

103

3

10

CD38 PerCP-5-5

[CD19+] CD10 PE / CD19 PE-Cy7

[Viable cells] SSC-A / CD19 PE-Cy7

103

CD38 normal maturation

–20

–5

103

103

[CD10+ 19+]

103

Normal CD38 /CD20 expression

CD45 Pac OR

100

CD38 Per CP-5-5

CD45 Pac OR

CD58APC

101

102

102

101

2

CD10 PE

CD10 normal maturation Normal CD58 expression

0

–2

103

[CD10+ 19+]

[CD10+ 19+]

103

1

–1 0

CD10+ 19+

101

0

103

[CD10+ 19+] 103 102

[CD19+]

103

CD19 PE-Cy7

103

[CD19+CD10+] 103

100

1

10

2

101 2

0

100

101

102

100

0

103

102

103

0

100

101

102

103

101

0

102

103

CD10PE

CD10PE Gate number All 288,861 CD19+ 1, 144

[CD19+CD10+] 103

101 1 0 –1

20 0

–20 2

101 102 CD10 PE

103

0

100 101 CD20 FITC

[MRD] 103

CD38 normal maturation

102

CD45 Pac OR

CD38 PacCP-5-5

CD10 normal maturation

102

[CD19+CD10+] 103

CD20 normal maturation

102

103

102

CD45 Pac OR

[CD19+CD10+] 103

0

101 5

–5

–2

SSC-A

SSC-A Gate number All 288,861 WBCs 274,176

101

Normal CD58 expression

0

0

–2

CD45 Pac OR

102 CD58APC

101

CD19 PE-Cy7

CD19 PE-Cy7

CD45 Pac OR

CD19+CD10+ WBCs

102

101 1 0 –1 –20

0 20 102 CD38 PerCP-5-5

103

102

Final MRD

101 1 0 –1 –20

0

20

102

103

CD38 PerCP-5-5 Gate Number 431 All Final MRD 407

Fig. 2 Initial sequential gating strategy comprised of viable cells to include CD45 negative events: event number vs time, FSC-A vs FSC-H and FSC-A vs SSC-A (not shown). Diagnostic ALL: CD19 vs SSC-A highlights CD19þ B cells (shown in red). Back-gating on the CD45 vs SSC-A plot shows the lymphoblast population expressing CD45 negativity. Sequential gating on CD19þ cells highlights the abnormal lymphoblast population expressing bright CD10þ. Final four plots display an outline of normal maturation/antigen expression with the abnormal lymphoblast population demonstrating marked dysmaturation. The bright CD10þ and dysmaturation comprises the LAIP. MRD ALL: An equivalent gating strategy was employed for MRD analysis. The diagnostic LAIP persists, gated on viable cells to include CD45 negative events, with the abnormal lymphoblast population (shown in red) expressing bright CD10þ and dysmaturation. This is distinguished from the normal maturing lymphoblast population (shown in pale blue). MRD is determined by calculating the percentage of abnormal lymphoblasts over total nucleated cells (calculated by boolean gating of WBCs ‘OR’ CD19þ cells). Final MRD ¼ 0.15%. Dark blue, mature lymphocytes; pale blue, haematogones; red, lymphoblasts/MRD.

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Pathology (2015), 47(7), December

LEE et al.

trial incorporated a heterogeneous patient population, it confirms previous similar findings in paediatric ALL and reinforces its relevance in adult ALL (Tables 3 and 4). Peripheral blood vs bone marrow in ALL Coustan-Smith et al.83 compared PB to bone marrow (BM) for MRD monitoring in paediatric ALL. They analysed 719 pairs of PB and BM samples. MRD (defined as 1 leukaemic cell in 10,000) was detected in both samples in 72 pairs. In 67 pairs, only BM MRD was detectable and in the remaining 579 pairs, MRD was undetectable in both PB and BM. Complete concordance between PB and BM was present in 150 paired samples from patients with T-ALL (35 of which were positive), irrespective of when the measurements were made. However, in B-ALL 104 BM samples were positive for MRD, of which only 37 were MRD positive in PB. The 4-year CIR in these patients was 80.8% (BM þ PB positive) compared to 13.3% (BM positive only). Therefore, the authors concluded that PB MRD monitoring could be a useful, alternative measure to BM MRD monitoring in T-ALL and prognostic in B-ALL. The findings of Borowitz et al.1 align with those of CoustanSmith et al.83 by demonstrating that patients with PB MRD positivity at day 8 had a lower 5-year EFS compared to patients who were MRD negative, even if they achieved MRD negativity by the end of induction, providing further evidence that early blast clearance improves survival. Adult ALL Similar to adult AML, clarification is needed regarding the optimal time-point and frequency of MRD assessments. Different groups have also adopted variable MRD thresholds in which to define residual disease, again making it difficult to translate the results into clinical practice. Vidriales et al.84 found MRD <0.03% LAIPþ cells at day þ14 could identify a subset of patients with an extremely superior prognosis: 11 out of 12 patients within this group were relapse free at 5 years. On multivariate analysis, MRD >0.05% at day þ35 was the most significant independent prognostic marker ( p ¼ 0.01) for predicting RFS in adults using age, WBC count at diagnosis, presence of Phþ chromosome and time to achieve mCR as covariates. Interestingly, no difference in RFS was found based on the type of consolidation therapy used (chemotherapy vs transplantation: autologous or allogeneic).

Table 3

In Holowiecki et al.,85 MRD 0.1% after induction therapy for standard and high risk ALL had the highest predictive value for incidence of relapse and LFS (RR ¼ 3.07 and 2.57, respectively) over age >35 years, WBC count >30  109/L and risk status. In Weng et al.,86 MRD was defined as 0.01%. On multivariate analysis, MRD after induction (OR 4.427; p¼0.002) and one consolidation cycle (OR 9.832; p < 0.001) was independently associated with an increased RFS. For OS, only MRD after one consolidation cycle had any predictive value (OR 5.652; p < 0.001). The PETHEMA ALL-AR-03 Trial is the first to demonstrate in an adult cohort, that treatment stratification using MPFC MRD can be successfully applied.87 Here, 326 adults (age 15– 60 years) with high-risk ALL (defined as either age between 30 and 60 years, WBC >30  109/L, t(4;11) or other MLL rearrangement) were assessed for MRD by 4-colour flow cytometry to guide treatment stratification. Patients with MRD levels <0.05% at the end of early consolidation and <10% BM blasts at day 14 of induction were assigned to delayed consolidation and maintenance therapy. Patients falling outside any of these parameters were scheduled to receive allogeneic HSCT. Five-year DFS and OS was 32% and 37% for patients assigned to allogeneic HSCT compared to 55% and 59% for those assigned to receive chemotherapy. Multivariate analysis showed persistent BM MRD (0.1% after induction and 0.05% after early consolidation) to be the only prognostic parameter for DFS and OS. MPFC MRD is also predictive of outcome in the transplanted adult ALL population.70 A retrospective review of 157 patients revealed 61 patients who were MRD positive (>0.1%) pre-HSCT had a 3-year OS of 40% compared to 68% in the MRD negative group ( p ¼ 0.0005, HR 2.39). The probability of relapse in the MRD positive group compared to the MRD negative group was 33% vs 16% ( p ¼ 0.002, HR 3.12). Post-HSCT, 144 patients were analysed. At 84 days post-HSCT, the risk of relapse was higher in all patients with any MRD positivity (HR 3.21). Nineteen patients were in mCR but MRD positive within 50 days post-HSCT. Seven out of 19 patients converted to MRD negativity and remained alive at last contact (726 days postHSCT). This last point raises the importance of determining optimum time-points for MRD analysis following transplantation to prevent unnecessary and toxic salvage therapy

Comparison between paediatric ALL studies

Reference

Patients, n

BM or PB

Assessment time-point

MRD threshold

Key points

Coustan-Smith et al.83

226

PB and BM

Variable

0.01%

Borowitz et al.1

2143

PB

0.01%

Basso et al.55

830

BM

Days 8 & 29 (induction) End of consolidation Day 15 (mid-induction

Foster et al.80

116

BM

0.1%

31

BM

1 month prior to allogeneic HSCT Median 10 days prior to allogeneic HSCT

102 (paediatric and adult)

BM

1 month prior to allogeneic HSCT

0.01%

PB MRD monitoring could be a useful, alternative measure to BM MRD monitoring in T-ALL and prognostic in B-ALL Early blast clearance in peripheral blood may be prognostic for survival Early assessment of MPFC MRD is predictive of relapse and can be applied for risk stratification Persistent disease can be identified using a ‘different from normal’ strategy prior to HSCT MRD positivity prior to HSCT has a RR of 10.5 (1.4–78) ( p < 0.01) for relapse and 4.2 (1.6–10.7) ( p < 0.01) for death Pre-transplant MRD levels can risk-stratify patients and predict outcome

Elorza et al.

81

Sanchez-Garcia et al.82

0.01%

0.01%

BM, bone marrow; HSCT, haematopoietic stem cell transplantation; MPFC, multiparametric flow cytometry; MRD, minimal residual disease; PB, peripheral blood; RR, relative risk.

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FLOW CYTOMETRY IN ACUTE LEUKAEMIA

Table 4

619

Comparison between adult ALL studies

Reference

Patients, n

BM or PB

Assessment time-point

MRD threshold

Key points

102

BM

Day 14 þ D35

0.05%

116

BM

Post-induction and post-consolidation

0.1%

Weng et al.86

125

BM

Post-induction and post-consolidation

0.01%

Ribera et al.87

326

BM

Post-induction and consolidation

0.05%

Appelbaum et al.70

157

BM

Within 1 month of allogeneic HSCT; within 84 days following allogeneic HSCT

0.01%

MRD evaluation during induction can risk-stratify patients and is predictive of outcome Post-induction MRD was predictive for relapse in standard and high risk Ph negative ALL; post-consolidation MRD did not significantly affect relapse rate or LFS MRD status post-induction and consolidation was independently predictive of RFS; only MRD status post-consolidation was predictive of OS MRD post induction and consolidation is prognostic for DFS and OS; MRD post-consolidation can direct and stratify therapy Pre-transplant MRD is predictive of relapse; post-transplant MRD is predictive of relapse

Vidriales et al.84 Holowiecki et al.

85

ALL, acute lymphoblastic leukaemia; BM, bone marrow; DFS, disease-free survival; HSCT, haematopoietic stem cell transplantation; LFS, leukaemia-free survival; MRD, minimal residual disease; OS, overall survival; PB, peripheral blood; RFS, relapse-free survival.

in those patients who convert to MRD negativity at a later time-point. Sanchez et al.88 suggested that MPFC MRD could be used to direct the timing of donor lymphocyte infusions (DLI) following transplantation. In their series (adults and paediatrics), five out of six patients were MRD positive prior to DLI. One out of five patients remained in sustained remission at last follow-up, 1-year post DLI. One patient relapsed at day þ30 and another relapsed at day þ110 (this patient was MRD negative prior to DLI). Three out of five patients failed to respond to DLI and died from progressive disease. Given the small numbers in this series, it is difficult to determine at what time-points MRD analysis should be performed for the purpose of guiding preemptive therapy following transplantation. A larger cohort in a prospective trial would be informative in resolving these questions. Box 3: Key points in ALL  Knowledge of normal maturation patterns is essential for MRD analysis.  Steroids reduce CD34 and CD10 expression transiently in B-ALL.  CD99 and TdTexpression decreases during therapy for T-ALL.  Early assessment of MRD (PB or BM) in paediatric ALL is prognostic of outcome.  The optimal time points for MRD assessment in adult ALL remains unclear.

CONCLUSION MRD detection and monitoring by MPFC is evolving to become a powerful prognostic tool in adult acute leukaemias. Its role has long been established in routine paediatric ALL practice. With several retrospective trials having demonstrated its role in adult AML and ALL, and paediatric AML, we eagerly look forward to testing the principle of risk-directed and individualised therapy through prospective, multicentre clinical studies. Before MRD-directed therapy in the pre and post-transplant setting can be wholly accepted and regarded as fundamental to

leukaemia management, several issues require resolution. The key clinical questions that remain unanswered include: what are the optimal time-points for MRD assessments? How frequently should MRD be performed? Will MRD-directed therapy ultimately improve outcome? Closely associated with these issues is standardisation. Without this, confidence in the accuracy and clinical applicability of MPFC MRD cannot be achieved. Guidelines released by the ICCS, BCSH and the EuroFlow Consortium have helped advance the process of harmonisation, however, notably, MRD evaluation has not been addressed. In order to fulfil this need, it is likely that standardisation will require collaborative efforts including reference data sharing of profiles, analyses and robust LAIPs. A minimum standard for technical expertise, aided in part by automated analysis software, and enforced by quality assurance programs will also help in ensuring and promoting uniformity. With the advent of NGS, our ability to detect MRD will continue to improve. To date, however, only a few molecular markers are considered to be of clinical relevance.89 For the time being, MPFC MRD remains the most widely applicable and established platform, although it is likely that in the future the combination of molecular and MPFC MRD assessments will form the standard of care. Acknowledgements: The authors thank Miles Prince for reviewing the manuscript. Conflicts of interest and sources of funding: Denise Lee is a recipient of the New Investigator Scholarship, Haematology Society of Australia and New Zealand. George Grigoriadis is a Victorian Cancer Agency Clinical Research Fellow. The authors state that there are no conflicts of interest to disclose. Address for correspondence: Dr Denise Lee, Department of Haematopathology, Peter MacCallum Cancer Centre, Locked Bag 1, A’Beckett Street, Melbourne, Vic 8006, Australia. E-mail: [email protected]

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