Standardized MRD quantification in European ALL trials: Proceedings of the Second International Symposium on MRD assessment in Kiel, Germany, 18–20 September 2008

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Abstract

Assessment of minimal residual disease (MRD) has acquired a prominent position in European treatment protocols for patients with acute lymphoblastic leukemia (ALL), on the basis of its high prognostic value for predicting outcome and the possibilities for implementation of MRD diagnostics in treatment stratification. Therefore, there is an increasing need for standardization of methodologies and harmonization of terminology. For this purpose, a panel of representatives of all major European study groups on childhood and adult ALL and of international experts on PCR- and flow cytometry-based MRD assessment was built in the context of the Second International Symposium on MRD assessment in Kiel, Germany, 18–20 September 2008. The panel summarized the current state of MRD diagnostics in ALL and developed recommendations on the minimal technical requirements that should be fulfilled before implementation of MRD diagnostics into clinical trials. Finally, a common terminology for a standard description of MRD response and monitoring was established defining the terms ‘complete MRD response’, ‘MRD persistence’ and ‘MRD reappearance’. The proposed MRD terminology may allow a refined and standardized assessment of response to treatment in adult and childhood ALL, and provides a sound basis for the comparison of MRD results between different treatment protocols.

Introduction

Several studies have shown that detection of minimal residual disease (MRD) in childhood and adult acute lymphoblastic leukemia (ALL) is an independent risk parameter of high clinical relevance, both in de novo and relapsed ALL, as well as in ALL patients undergoing stem cell transplantation.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 Consequently, MRD diagnostics is currently incorporated in most European ALL treatment protocols as a tool for stratification. Flow cytometric and molecular methods that allow sensitive detection and specific quantification of residual leukemic cells have been developed.28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 Standardization of methodologies and definitions of common MRD terms become increasingly important, not only to ensure comparability of MRD data between different MRD laboratories within a single MRD-based treatment protocol but also to provide a sound basis for the comparison of MRD results between different treatment protocols, including those that evaluate the effectiveness of new agents.

Therefore, a Consensus Development Workshop on MRD assessment in ALL was convened within the Second International Symposium on MRD assessment in Kiel, Germany, 18–20 September 2008. The conclusions of this Workshop were endorsed by several European study groups (SG) on ALL, which assess MRD within their clinical treatment protocol (see Table 1).

Table 1 European study groups participating in the development of recommendation for MRD assessment

Organization and consensus process

The Workshop was organized by MK, MS and JJMvD in cooperation with the EWALL (European Working Group for Adult Acute Lymphoblastic Leukemia), the ELN (European Leukemia Net) and the International Berlin–Frankfurt–Münster Study Group (I-BFM-SG). Representatives of all major European study groups on childhood and adult ALL (for participating groups, see Table 1), as well as representatives of the major European Concerted Actions on standardization and optimization of PCR- and flow cytometry (FCM)-based MRD assessment in ALL (ESG-MRD-ALL, EuroFlow, I-BFM-ALL-FLOW-MRD Network), were invited to work on recommendations related to MRD assessment within clinical European ALL trials. MS, GEJ-S, JS, AvS, JJMvD, ABi, YB, ABa, KS, NGol, NGök, OGO, AKF, J-MR, EAM, RF and AR were asked to complete a questionnaire on clinical and technical details regarding the current application of MRD diagnostics within the respective clinical trial in preparation for the meeting, and to briefly summarize their views on the subject during the conference. Five experts (AS, TR, MD, VA and HP), selected for their expertise regarding technical details of MRD analysis in ALL, summarized the data and the current state of the art of major MRD techniques. The subsequent round table discussion with all active participants aimed at recommendations on the minimum technical requirements of MRD assessment within clinical European ALL trials and on a corporate definition of technical MRD terms describing results for MRD measurements. Representatives of the ESG-MRD-ALL (JJMvD, OGO, HP), EuroFlow (JJMvD) and the I-BFM-ALL-FLOW-MRD Network (MD) participated in the panel. An advisory committee (comprising MB, AS, TR, MK, MS, JJMvD) supported and continued the process to end up with a written summary document of the current state of the art of MRD assessment in ALL and a recommendation on minimal technical requirements and MRD terminology. MB and AS were primarily responsible for writing the paragraphs on immunoglobulin (Ig)/T-cell receptor (TCR)-based MRD quantification, HP and OGO prepared the chapters on PCR analysis of BCR-ABL transcripts in ALL and MD and JJMvD wrote the paragraphs on FCM-based MRD assessment. Approval of the participants was sought at different steps of the draft report; issues for which no consensus was attained are marked as controversial.

Summary of MRD techniques in ALL

PCR analysis of Ig and TCR gene rearrangements

The junctional regions of rearranged Ig and TCR genes are fingerprint-like sequences that can be used as clone-specific MRD-PCR targets in the vast majority of precursor-B-ALL and T-ALL. In addition, genomic break points of some leukemia-specific translocations, such as MLL gene rearrangements or SIL–TAL1 fusion genes, represent alternative DNA targets; however, they are applicable only to a minority of patients.41, 42 Clone-specific Ig/TCR PCR generally reaches sensitivities of 10−4–10−5. These high sensitivities require the precise identification of junctional region sequences of Ig and TCR genes in each ALL, because these sequences are required to design clone-specific oligonucleotides. Subsequently, sensitivity testing has to be conducted through serial dilution of DNA obtained at diagnosis to assess the sensitivity of individual assays.43, 44

Usage of this technique has its roots in the early 1990s, starting with semi-quantitative approaches estimating the amount of PCR products after PCR, for example, by ethidium bromide staining of electrophoreses or by dot blotting and hybridization of PCR products. These multistep approaches required extensive optimization of each PCR assay and post-PCR manipulation. An early simplified approach with limited sensitivity (10−3) based on competitive PCR and Genescan analysis allowed the detection of patients with high levels of residual blasts.45 The implementation of real-time quantitative (RQ)-PCR techniques was a major advance, allowing for quantification during the early exponential phase of PCR amplification and eliminating variations during later post-exponential phases of the PCR reaction and during post-PCR manipulation of samples.28, 39, 40, 46, 47

Right from the start, standardization of RQ-PCR analysis and data interpretation was a major aim of European collaborations. On the basis of the experience of the MRD Task Force of the I-BFM-SG (starting in the early 1990s), the European SG on MRD detection in ALL (ESG-MRD-ALL) was established in 2002 and now consists of 35 MRD-PCR laboratories spread across Europe, Israel, Singapore and Australia, which perform quality-control (QC) programs every 6 months, developing and evaluating new MRD strategies and techniques, and developing guidelines for the interpretation and reporting of RQ-PCR-based MRD data.38 One of the many contributions of the ESG-MRD-ALL group was to define the concept of quantitative range, which corresponds to the levels of MRD that can be reliably detected and quantified both with regard to the technique used and the number of residual disease targets identified (see below).

Besides the high degree of standardization, a major advantage of these RQ-PCR techniques is the usage of DNA as the analytical sample that is extremely stable even in case of long shipping times. In addition, the method is applicable to the vast majority of precursor B-ALL and T-ALL. Sensitivity can be determined exactly for every junctional region target and currently tends to be 0.5–1.0 log higher than while using flow cytometric MRD assessment.48, 49, 50

However, a main disadvantage of using Ig/TCR gene rearrangements as MRD targets in ALL is the occurrence of continuing rearrangements during the disease course, potentially leading to false-negative PCR results.46, 51, 52, 53 Moreover, follow-up of subclones existing at initial diagnosis may lead to overestimation or underestimation of MRD values. Therefore, preferably two Ig/TCR gene targets should be used for reliable MRD detection. Also, false positivity cannot be completely excluded, as massive regeneration of normal lymphoid progenitors might lead to (very) low levels of nonspecific amplification.54, 55 In addition, the method needs the initial characterization of the leukemic Ig/TCR rearrangements with a panel of different PCR and sequencing reactions and is restricted to experienced molecular hematology laboratories because of the complexity of the analyses (see Table 2).

Table 2 Characteristics of the techniques currently used for MRD detection in ALL

However, mainly because of the high sensitivity and high degree of standardization, RQ-PCR with Ig/TCR gene rearrangements is currently the most widely used technique in recent and ongoing large-scale European clinical MRD studies leading to the greatest published experience on clinical relevance of MRD in ALL for future evidence-based ALL treatment (for details, see Tables 1 and 3).

Table 3 Current status of integration of MRD analysis within European trials on Ph-negative ALL

PCR analysis of BCR-ABL transcripts

The Philadelphia chromosome (Ph) represents a shortened chromosome 22 resulting from a reciprocal translocation between chromosomes 9 and 22, resulting in the formation of a fusion gene involving ABL and BCR genes. This genetic aberration is found in 25% of adult patients and in 5% of children with ALL and is associated with a poor prognosis.34, 37 In 70% of patients with Ph+ ALL, the break point in the BCR gene is located in the first exon of the BCR gene (e1) and is juxtaposed to the second exon of the ABL gene (a2). The resultant minor break-point transcript (e1–a2) encodes a 190-kDa chimeric protein (p190). The second most frequent fusion transcript involves break points in a 5.8-kb region spanning exons 12–16, known as the major break-point cluster region (M-bcr) and is typical of chronic myeloid leukemia (CML) but is also seen in 30% of patients with Ph+ ALL.34, 36, 37, 56

Detection of BCRABL fusion transcripts by reverse transcription PCR is relevant not only for diagnostic purposes but also in particular for quantitative monitoring of MRD.34 Several studies have shown that levels of MRD in the bone marrow (BM) of patients with Ph+ ALL, assessed by RQ-PCR after induction and after consolidation treatment, is a powerful indicator of prognosis.14, 15, 18, 20, 21, 27 This also applies to the posttransplant setting, in which sequential analysis of patients who received allogeneic stem cell transplantation showed that repeated PCR positivity correlated with an increased risk of relapse.15, 20 Introduction of novel treatment concepts for Ph+ ALL including MRD-guided therapy for adult ALL, for example, in protocols of the German Multicenter ALL SG (GMALL), has generated interest in quantitative analysis of this fusion gene.14, 15, 18, 20, 21, 27 Taken together, these data suggest that in Ph+ ALL, quantitative monitoring of residual leukemic cells is an essential diagnostic element for assisting in clinical decision making. Recent data comparing MRD levels obtained by quantitation of the BCR-ABL transcript and Ig/TCR rearrangements show that, in ALL, transcript monitoring might provide additional prognostic information compared with standard Ig/TCR follow-up.57 Indeed, because these markers do not have the same role in oncogenesis, their distribution within the leukemia population may differ, leading to apparent discrepancies in MRD evaluation.

Owing to the enormous expertise of the ESG-MRD-ALL group in standardizing quantitative PCR of TCR and Ig rearrangements in ALL, it was the next logical step to pool the experience of the ESG-MRD-ALL and the EWALL to establish common guidelines for standardization of BCR-ABL quantification.

Advantages of RQ-PCR to evaluate fusion transcripts in Ph+ ALL include high sensitivity, ease and rapidity of analysis and relatively low cost. There are a few pitfalls because of various technical issues that may influence the results of RQ-PCR analysis of BCR-ABL transcripts. Of particular importance is sample quality, which is adversely influenced by the availability of too few cells and long transport times resulting in the degradation of RNA. In addition, lack of standardization of RNA extraction, cDNA synthesis, selection of housekeeping genes and variation in the manner by which RQ-PCR analyses are carried out lead to substantial differences in results and carry the risk of false negativity. From a clinical perspective, the highest possible sensitivity is of paramount importance for Ph+ ALL, but as with any RNA-based logarithmic technique, it carries the risk of false-positive results, for example, because of cross-contamination. Minimal requirements for data interpretation have also not been defined to date, neither has the concept of the quantifiable range been applied.

In summary, RQ-PCR of BCR-ABL transcripts has proven invaluable in assessing the efficacy of specific treatment strategies, and specific MRD levels are increasingly being used to drive clinical decisions. The importance of this methodology will be further strengthened by the ongoing, international standardization efforts.

Multiparameter FCM

Leukemic lymphoblasts differ from physiological lymphoid precursors in qualitative (for example, presence of myeloid markers) and quantitative antigen expression patterns. In addition, appearance of immature phenotypes outside their normal tissues (such as the thymus) can be exploited in particular in T-ALL. Such leukemia-associated immunophenotypes are present in the vast majority of ALLs, if a panel of at least 6–8 relevant markers in strategic combinations is used for assessment.32, 58, 59, 60, 61, 62, 63 This approach currently reaches sensitivities of (10−3 to) 10−4, which is about one log less than that of molecular methods, because MRD positivity cannot be determined at the single-cell level by FCM. To be unambiguous, it needs to demonstrate a certain amount of cells with similar (leukemia associated) characteristics (‘cluster’). In analyzing a sample, leukemic cells are then directly quantified in relation to other cells in the specimen without a need for external calibrators.

The use of this technique dates back more than 20 years, starting with two- and three-color approaches. The implementation of more than four-color techniques increased the applicability and sensitivity/specificity of the method, and allowed for simultaneous determination of extensive phenotypic patterns at the single-cell level. Further improvements in multicolor FCM, new antibody panels, innovative fluorochromes, upgraded computerization and new software tools allowing for optimized data acquisition and automated pattern recognition are currently evolving.

In addition to national networks, two international consortiums, the I-BFM-ALL-FLOW-MRD Network (founded in May 2000 as a cooperation between AIEOP (Associazione Italiana Ematologia Oncologia Pediatrica) and BFM) and the EuroFlow Consortium (started in March 2006), were established in Europe to optimize and standardize FCM-based MRD quantification: The I-BFM-ALL-FLOW-MRD Network has established criteria for risk assessment and stratification in pediatric ALL treatment based on BFM-backbone protocols, and aims for joint QC programs to achieve similar data quality among different experienced flow laboratories despite the use of different hardware, analysis software or differences in panel composition.33, 64 The EuroFlow Consortium works on full standardization of instrument setups, sample preparation, immunostaining procedures, fluorochromes and eight-color antibody panels, as well as bioinformatics-assisted expert independent automated analysis of the acquired data.65, 66, 67

The major advantage of FCM is its rapidity, which allows result reporting within 1 day. This is specifically useful when MRD results are required quickly to guide therapy. In addition, FCM allows the simultaneous assessment of cell qualities that are required for emerging targeted therapies in ALL.68, 69 A potential pitfall of the method results from similarities between leukemic lymphoblasts and nonmalignant lymphoid precursors in various phases of regeneration during and after chemotherapy that may lead to false positivity.32, 70 So far, time-point-matched cross-leukemia-lineage BM samples can be used as adequate background controls. In addition, phenotypic shifts occur frequently in MRD cells during induction (compared with patterns at diagnosis) among others because of steroid-induced gene expression modulation.61, 68, 69, 70, 71 Hence, initial phenotypes only serve as orientation and not for planning strict gating strategies in follow-up. Nowadays, expert knowledge of typical time-point-related nonmalignant background and experience with patterns of phenotypic shifts of leukemic cells are essential to distinguish MRD properly.

As FCM and PCR do not lead to completely comparable results, FCM cannot simply substitute current PCR-based MRD risk stratification at the same time points.20, 48, 49, 50, 57 Instead, FCM-based risk definition may be independently reliable and therefore has to be independently validated.

Summary and update of current status of integration of MRD analysis within European ALL trials

The main representatives of all European ALL study groups were asked to complete a questionnaire in preparation of the Kiel Conference. Questions were with regard to the following:

  • The published experience on the clinical relevance of MRD in Ph-negative ALL within previous clinical trials.

  • The status of implementation of MRD within ongoing clinical trials on Ph-negative ALL (technical details, MRD-based risk stratification, existence of MRD-based definitions of remission and relapse, post-stratification MRD monitoring).

  • The status of implementation of MRD within ongoing clinical trials on Ph-positive ALL (technical details, MRD-based risk stratification, existence of MRD-based definitions of remission and relapse, post-stratification MRD monitoring).

  • Suggestion for the future of MRD in respective future clinical trials.

Answers to the questionnaire are summarized in Table 3 (Ph-negative ALL) and in Table 4 (Ph-positive ALL).

Table 4 Current status of integration of MRD analysis within European trials on Ph-positive ALL

Recommendations on minimal requirements on MRD techniques in European clinical ALL trials

On the basis of the status quo of MRD techniques and the clinical experience with MRD quantification in clinical ALL trials, the panel agreed on recommendations with regard to minimal requirements on the MRD techniques that should be fulfilled before implementation of the respective technique into clinical trials.

PCR analysis of Ig and TCR gene rearrangements

Owing to the long history of European collaboration with the vast majority of European clinical ALL trials performing QC within the ESG-MRD-ALL, the panel chose to consider the ESG-MRD-ALL recommendations38 as the basis (see Table 5).

Table 5 Recommendations regarding minimal technical requirements for MRD assessment within clinical trials using PCR analysis of Ig and TCR gene rearrangements, PCR analysis of BCR-AB L transcripts or multiparameter flow cytometry

Most published ALL data are generated by BM analysis. However, in T-ALL, the analysis of blood samples seems to produce comparable results,72, 73 although the prognostic relevance of MRD in blood has not been proven independently. A minimum of 1 × E+07 mononuclear cells should be available for initial marker characterization and generation of individual dilution series. For follow-up analysis, (1–5) × E+06 mononuclear cells should suffice. The use of Ficoll is recommended because sensitivity is increased and variability is reduced by excluding granulocytes, and the majority of published molecular data are based on mononuclear cell-MRD analysis.

EDTA anticoagulant is appropriate for PCR analysis. However, heparin may also be used if Ficoll precedes analysis. Shipment time is not a major problem in DNA-based analyses, but should preferably not exceed 24 h.

For primer design and RQ-PCR analysis, standardized and validated procedures should be followed, as recommended by the ESG-MRD-ALL.38 Owing to the possibility of oligoclonality and clonal evolution phenomena, preferably two independent Ig/TCR targets should be used for MRD assessment. However, if this is not achievable despite a complete marker screening, MRD assessment is also feasible using one marker.2

In addition, for correct interpretation of RQ-PCR data, as well as for the definition of quantitative range, sensitivity, MRD positivity and MRD negativity and calculation of MRD levels, the ESG guidelines should be used. Strict precautions should be undertaken to limit the possibility of contamination (for details, see Van der Velden et al38).

Successful participation in external QCs (that is, the ESG-MRD-ALL and associated/approved national networks) is a prerequisite for a laboratory that generates MRD data for clinical decision making.

Owing to the complexity of the analysis, Ig/TCR-based MRD assessment should be restricted to reference laboratories that are recognized by the protocol chairman of the study group. To maintain a sufficiently high experience level, it is highly recommended that a minimal number of newly diagnosed ALL cases (preferably 50) are analyzed per year or that a population of at least 10–12 × 106 inhabitants is covered (or a country, in case of a lower number of inhabitants).

PCR analysis of BCR-ABL transcripts

The highly aggressive biology and often rapid growth kinetics of Ph+ ALL may result in relapse shortly after documentation of a hematologically complete remission. To avoid false-negative results that may delay treatment decisions, molecular in particular BCR-ABL-based, techniques with considerably greater sensitivity for detection of leukemic cells will ultimately guide therapy. Conversely, it is essential not to generate false-positive results, which occur more easily using reverse transcription PCR-based than DNA-based techniques. An exact definition of the minimum accepted quantitative range of the assay is mandatory, and the quantitative range actually achieved for any given analysis has to be reported to ensure that therapeutic interventions are indeed justified. To achieve these goals, the following procedures have been agreed upon, pending revision as the standardization efforts progress.

Mononuclear cells obtained by Ficoll separation are commonly accepted as an appropriate sample and are recommended as starting material. BM is the preferred sample source for quantitation of BCR-ABL transcripts, as the sensitivity is generally 0.5–1 log higher than with peripheral blood (PB) samples. The use of RNA stabilizing agents is not performed routinely by most laboratories and is currently not recommended, even more so as stabilizing systems have not been validated for BM. The Qiagen kit system (Qiagen, Hilden, Germany) and the TRIzol reagent (Invitrogen Inc, Carlsbad, CA, USA) are the most frequently used RNA extraction procedures; it is currently being evaluated to determine whether one of these techniques is superior. Both Taqman (Applied Biosystems, Foster City, CA, USA) and LightCycler (Roche Diagnostics, Basel, Switzerland) platforms yield equivalent results. The additional value of nested PCR has not been proven because of the very high risk of contamination associated with this approach. Nearly all laboratories use ABL as the housekeeping gene, based primarily on recommendations by the CML study groups. Nevertheless, comparisons show that beta-glucuronidase (GUS) and β2-microglobulin can be used as well.74 BCR-ABL plasmids are considered to be the appropriate standards by the majority of laboratories. At present, no internationally available reference material is available for use as a control. Analysis should be performed at least in duplicate to ensure reproducibility, although in practice, individual laboratories differ considerably in this regard. There is no consensus and substantial variability regarding the need to perform multiple cDNA syntheses per sample. The most commonly accepted level of sensitivity for a given sample to be considered PCR negative is at least 104 ABL copies. It will be one of the aims of the upcoming ESG/EWALL meetings to establish a reference material for use as control. Quality assurance is also a major goal of the upcoming QC meetings, and will address the technical issues of slope, intercept, quantitative range and standardized interpretation. Subsequently, more precise methodological details will be provided in a separate publication that will include key recommendations for generating reliable quantitative data for BCR-ABL transcripts in Ph+ ALL. Successful participation in these external QCs is highly recommended for a laboratory that generates MRD data for clinical decision making.

Multiparameter FCM

As for molecular methods, most published FCM data have been based on BM analysis (with EDTA or heparin as preservative). Both gradient separation and erythrocyte lysis are used as preparation methods. Ficoll preparation can be mimicked by gating out high side-scatter cells when analyzing data files based originally on total nucleated cell preparations. A minimum of 3(−5) × 106 nucleated cells should be available for initial marker characterization and for follow-up analysis. Initial diagnostic material for identification of the leukemic immunophenotype is highly recommended and should be omitted only for very rare exceptions (for example, early follow-up sample with high-level MRD). Shipment time is an important issue when analyzing vital cells and should optimally not exceed 24 h; however, dead cells should only be excluded from analysis if scatter properties are severely changed and autofluorescence is present.

A minimum of 6–8 relevant markers in strategic combinations are recommended, that is, a constant backbone of 2–4 lineage markers, which allow tracking of the cells of interest in similar ways in all staining combinations, plus additional aberration markers. Directly labeled monoclonal antibodies are preferred throughout. Antibodies should be carefully selected for optimal performance and the choice of fluorochromes should be based on avoiding fluorochrome interactions and the type of marker (or aberration), to allow the best discrimination between normal and malignant cells. Controls for staining should be in accordance with standard FCM procedures.75 Owing to the enormous ongoing innovation in FCM technology, so far no common final recommendations have been reached with regard to minimal technical requirements on degree of standardization, instrumentation and data analysis (for further details, see Table 5).

The detection limit of an FCM MRD assay is partly determined by the minimum number of events that can reliably be used to define a population of neoplastic cells. It has been shown that under certain circumstances, accurate identification of a population using up to 4-color FCM requires at least 20 events.76, 77 No final consensus was reached with regard to the required minimal number of neoplastic cell events for accurate quantification: The EuroFlow Consortium claims a minimum of at least 100 neoplastic cell events for quantification (as the sum of events if the assay consists of several tubes) that are analogous to the consensus on FCM-based MRD quantification in multiple myeloma.78 The I-BFM-ALL-FLOW-MRD Network quantifies MRD once a minimum of 30 neoplastic cell events per tube are acquired with a confirmation of the result by an independent second tube being strongly recommended. However, the number of leukemia-associated events necessary to reliably predict outcome in ALL remains to be determined in prospective clinical studies. To reach the recommended reproducible sensitivity of 1 leukemic blast among 1 × 104 cells (that is, 10−4 level), the minimal number of total events required is 1 × 106. If the assay consists of several individual tubes, then the minimum requirements are the sum, not the average, of the individual tubes.

Standardization based on written guidelines, along with suitable QC schemes, is a prerequisite for optimal usage of FCM for MRD assessment in the clinical setting. Technical guidelines should also include strategies for longitudinal performance evaluation, that is, a QC program for cytometer performance, staining quality and analytical skills of staff involved in MRD assessment. In particular, when several laboratories collaborate, bringing about and maintaining a high degree of similarity in MRD results requires continued exchange of experience and external QC (for example, by blinded inter-laboratory tests with data files and/or MRD samples). When involved in clinical trials, similar prerequisites to laboratories as discussed for molecular methods should be respected for FCM-MRD assessment as well, that is, restriction to recognized reference laboratories with a verifiable high experience level and a minimal number of MRD analyses per year (preferably, analysis of 50 new ALL cases per year, coverage of a population of at least 10–12 × 106 inhabitants or a country, in case of a lower number of inhabitants).

Proposals on definition of MRD terms

Technically, for all methods used, there are three possible outcomes:

  1. 1)

    Quantifiable MRD positivity

  2. 2)

    Low-level MRD positivity being not quantifiable because of the fact that one is dealing with rare events, and therefore the result of a measurement is both dependent on the difference between the arithmetic and geometric mean of its Poisson distribution and the background rate.

  3. 3)

    MRD negativity.

To facilitate comparability of MRD data in various trials, a common terminology for describing ‘MRD key states’ is desirable. Therefore, the panel agreed on a concerted proposal for a common MRD terminology (Table 6), referring to terms that are already established for molecular response definition and monitoring in CML and acute promyelocytic leukemia,79, 80, 81, 82, 83 as these diseases are regarded as model diseases for the implementation of MRD analysis in clinical practice. The proposed definitions are technically oriented toward a universal description of analytical results and are not intended to replace cutoffs for MRD-based treatment stratifications. MRD thresholds for decision making have to be defined within each individual ALL trial as they depend on the method used for determination, the therapy given before MRD follow-up time points, the expected prognosis of the stratified cohort of patients and the treatment aims of the protocol (therapy reduction versus escalation).

Table 6 Proposals for definition of MRD terms in ALL. General remark: Low-level, non-quantifiable MRD positivity does not fit to any of the given MRD terms. This MRD result may be interpreted as a sort of gray area, which should be valued within the respective protocols considering the treatment strategy and the respective time point, for example, as a warning signal in post-remission monitoring

For response assessment, the panel recommends the term ‘complete MRD response’ for MRD negativity, provided that minimal technical requirements are fulfilled. Within the panel, there was an extensive discussion on the usage of the term ‘complete molecular response’ instead of ‘complete MRD response’, as the majority of clinical MRD data are based on molecular MRD quantification. However, a common and comparable terminology for flow- and PCR-based MRD trials was preferred. MRD negativity with insufficient sensitivity is not considered within this definition.

Second, ‘MRD persistence’ was defined as a continuously quantifiable MRD positivity measurable at at least two time points with at least one relevant treatment element in between.

Furthermore, post-remission monitoring for early detection of impending relapses is increasingly being used in different European ALL trials (see Tables 3 and 4). It was extensively discussed in the panel whether any level of quantifiable MRD can be generally defined as ‘MRD relapse’. The GMALL already published data on molecular definition of an impending relapse;19 however, comparable data in childhood ALL are still lacking. Therefore, the following terminology was proposed: ‘MRD reappearance’ as ‘Conversion from MRD negativity to quantifiable MRD positivity’, with confirmation of the result being strongly recommended before drawing clinical consequences. The term ‘MRD reappearance’ can be equated with ‘MRD relapse’ in a specific ALL protocol, if in such a protocol an invariable association has been found between MRD reappearance and (subsequent) hematological relapse.19

Low-level, non-quantifiable MRD positivity does not fit into any of the given MRD terms. However, depending on the clinical setting, it potentially has added value as a sort of gray area, and therefore should be valued within the respective protocols considering the treatment strategy and the respective time point, for example, as a warning signal in post-remission monitoring.

In summary, the proposed MRD terminology may serve as event/end point for treatment evaluation in particular ALL protocols as a refined and standardized assessment of response to treatment. However, it is technically oriented and therefore does not replace the determination of cutoffs and time points for MRD-based treatment stratification within clinical protocols.

Conclusions and future perspectives in MRD assessment in ALL

MRD diagnostics has acquired a prominent position in European treatment protocols for ALL patients, on the basis of its high prognostic value for predicting outcome and the possibilities for implementation of MRD diagnostics in treatment stratification. Despite its technical complexity, PCR-based detection of rearranged Ig/TCR genes (and other leukemia-specific DNA targets, such as MLL gene rearrangements) is currently the most broadly applied MRD technique owing to its high level of standardization, its well-defined quantitative range and good sensitivity, as well as its applicability in the majority of ALL patients. However, this MRD technique cannot easily provide results early after diagnosis, because of its time-consuming nature, mainly caused by the need for Ig/TCR MRD target preparations for each individual patient. Nevertheless, this MRD technique will retain its prominent position in the ALL field until another MRD technique reaches the same level of reliability and reproducibility while providing additional advantages, such as speed and early applicability.

Routine diagnostic application of fusion transcripts as MRD-PCR targets (such as BCR-ABL transcripts) has not (yet) been established, but this approach might have value for specific treatment strategies in Ph+ ALL patients, particularly if early assessment of treatment effectiveness (within 4–6 weeks from diagnosis) is required for treatment intervention. However, for such application, significant investment in standardization and QC is still required.

FCM-based MRD diagnostics has the advantage of instant availability of the obtained results with regard to both remaining ALL cells and normal leukocytes. Furthermore, the recent technical innovations in routine FCM (3 lasers and 8 colors) and the new developments in software for data analysis make this technology increasingly attractive for MRD diagnostics. Whereas FCM has so far suffered from limitations in sensitivity, as well as in treatment-induced phenotypic shifts and difficulties in discriminating ALL cells from regenerating precursor B cells, the new developments might well overcome all these pitfalls. This places FCM in a progressively better position to become the leading MRD technology, if a high degree of standardization can be reached for both technical aspects and data interpretation and if quality assurance programs can guarantee the same level of reliability as that obtained with Ig/TCR-based MRD diagnostics. Particularly in protocols dealing with rare leukemias (for example, infant ALL), the level of centralization obtained with PCR-based techniques may be hard to reach with FCM. Finally, the choice of the MRD methodology largely depends on the aims of the study protocol, resources available and treatment strategy.

The current application of MRD diagnostics has become possible by intensive networking processes and open collaboration between clinical and diagnostic research groups throughout Europe and by the willingness to bring innovative diagnostics into patient care, a process that has taken more than 10 years. Owing to the collaborative attitude of all involved teams, the current key position of MRD diagnostics has been reached. Continuation of such collaboration, high-level standardization and regular QC rounds remain critically important for further improvements in MRD diagnostics.

Conflict of interest

The authors declare no conflict of interest.

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Acknowledgements

This study was supported in part by the European LeukemiaNet.

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Correspondence to M Brüggemann.

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Keywords

  • acute lymphoblastic leukemia
  • ALL
  • minimal residual disease
  • MRD
  • flow cytometry
  • PCR

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