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02-10-2016 | Treatment | Book chapter | Article

Multiple Myeloma Minimal Residual Disease

Authors: Bruno Paiva, Ramón García-Sanz, Jesús F. San Miguel

Publisher: Springer International Publishing

Abstract

Assessment of minimal residual disease (MRD) is becoming standard diagnostic care for potentially curable neoplasms such as some acute leukemias as well as chronic myeloid and lymphocytic leukemia. Although multiple myeloma (MM) remains as an incurable disease, around half of the patients achieve complete remission (CR), and recent data suggests increasing rates of curability with “total-therapy-like” programs. This landscape is likely to be improved with the advent of new antibodies and small molecules. Therefore, conventional serological and morphological techniques have become suboptimal for sensitive evaluation of highly effective treatment strategies. Although, existing data suggests that MRD could be used as a biomarker to evaluate treatment efficacy, help on therapeutic decisions, and act as surrogate for overall survival, the role of MRD in MM is still a matter of extensive debate. Here, we review the different levels of remission used to define depth of response in MM and their clinical significance, as well as the prognostic value and unique characteristics of MRD detection using immunophenotypic, molecular, and imaging techniques.
Key facts
The higher efficacy of new treatment strategies for MM demand the incorporation of highly sensitive techniques to monitor treatment efficacy
MRD could be used as a more potent surrogate biomarker for survival than standard CR
We need to understand the pros and cons of the different MRD techniques
The time has come to incorporate highly sensitive, cost-effective, readily available, and standardized MRD techniques into clinical trials to assess its role in therapeutic decisions

1 The Definition of Complete Response in MM: An Historical Overview

Changes in the level of the serum paraprotein and/or urinary light-chain excretion form the basis of assessing the response to therapy and monitoring the progress of MM. Response criteria were first developed by the Committee of the Chronic Leukemia and Myeloma Task Force (CLMTF) of the U.S. National Cancer Institute in 1968 and were reviewed by the same group in 1973. The main response parameter was a reduction in the paraprotein of at least 50 %. In 1972 the Southwest Cancer Chemotherapy Study Group, now the Southwest Oncology Group (SWOG), defined ‘objective response’ as a reduction of at least 75 % in the calculated serum paraprotein synthetic rate (rather than paraprotein concentration) and/or a decrease of at least 90 % in urinary light-chain excretion, sustained for at least 2 months [1].
Neither the CLMTF nor the SWOG response criteria include a definition of complete response (CR) since it was rarely observed with existing treatments. With the introduction of new regimens such as VAD (vincristine, adriamycin, and dexamethasone) and high-dose melphalan (140 mg/m2) followed by autologous stem cell support (ASCT), measurable paraprotein disappeared in a significant proportion of patients and criteria for complete remission were proposed based on the absence of detectable paraprotein in serum or urine together with a normal number of plasma cells (PCs) in the marrow (i.e., <4–5 %); nevertheless, the initial definition had no consensus on whether the absence of paraprotein should be based on routine electrophoresis alone, or combined with more sensitive methods such as immunofixation [2]. The current definition of CR was introduced by Blade et al. on behalf of the European blood and marrow transplantation (EBMT) more than 15 years ago: negative immunofixation in serum and urine, disappearance of any soft tissue plasmacytomas and <5 % PCs in bone marrow (BM) [2]. The prognostic value of CR has extensively been validated both in transplant-candidate [35] and elderly patients [68]. A correlation between deeper quality of responses and better outcomes has also been described in the relapse/refractory setting [3]. As expected, different groups have also shown that more important than achieving CR is to maintain it, since those patients that relapse from CR early-on consistently show a dismal outcome [4, 5]. Interestingly, long follow-up observations show that only 1 out of 4 patients in CR remain progression-free at 10 years [6, 7]. All these data together implies that CR is indeed a strong prognostic marker and a clinically relevant end point, but also that similarly to other hematological malignancies, response criteria in MM can be further improved.
Already in 2006, the International Myeloma Working Group (IMWG) highlighted the need for a new definition of CR, and introduced normalization of serum free light-chains (sFLC) and absence of clonal PCs in BM biopsies by immunohistochemistry (IHC) and/or immunofluorescence, as additional requirements to define more stringent CR criteria [8]. Since then, only one large study was able to show the superiority of the stringent over conventional CR criteria to define patients’ outcomes [9], while other groups failed to demonstrate the utility of the sFLC assay among immunofixation-negative patients [1012], maybe because the latter groups did not include simultaneous assessment of PC clonality in BM biopsies. Importantly, the vast majority of CR patients after therapy show recovery of normal PCs that exceeds the percentage of clonal PCs, implying that more sensitive clonality markers are needed such as the clonotypic immunoglobulin (Ig) gene sequences or immunophenoyping. In addition, it has been suggested that the sFLC might be replaced by the heavy-light format [13] and become merely a surrogate for recovery of the immune system rather than an MRD monitoring tool [14]. Overall, it becomes clear that the definition of CR would benefit from an improvement that matches the rapid evolution observed in MM treatment. Such improvement can only be achieved by highly sensitive technologies able to detect MRD at very low levels and accordingly, the notion of immunophenotypic and molecular CR have been slowly integrated into the response criteria in MM [15].

2 The Relationship Between Depth of Response and Survival: Rationale for Implementing MRD Monitoring in MM

At present it is clear that in MM there is a direct correlation between the depth of response, particularly CR, and prolonged progression-free survival (PFS) as well as overall survival (OS). This has been demonstrated in many different individual studies [1621], and confirmed in meta-analyses among transplant-eligible and non-transplant-eligible patients [2224]. It has also been demonstrated among newly diagnosed high risk [25, 26] and relapse/refractory MM patients [3, 27]. Albeit the overwhelming amount of data supporting the concept that “the deepest the response, the longer the survival,” there is also evidence betraying such correlation: (i) patients in CR with early relapses and dismal survival [4, 5]; (ii) different CR rates that do not translate into different outcomes [28]; (iii) similar CR rates associated with different survival [29]; or (iv) some patients failing to achieve CR who show excellent outcome (those with an MGUS-like signature at baseline) [30]. Regarding the latter subset, it should be noted that MGUS-like patients in which indeed CR is not a pre-requisite to achieve long-term disease control represent <10 % of the whole MM population [30, 31]; for the vast majority of patients, higher CR rates were indeed needed to prolong survival [7]. Moreover, most of the controversial results described above concerning the value of CR may be (at least partially) related to either (i) heterogeneity in the consolidation or maintenance treatment used in one of the treatment arms but not in the other after response evaluation which may further affect tumor reduction, or (ii) different CR quality reached after different regimens [7], combined with the relatively limited sensitivity of current methods to define CR [15]. Altogether, these observations do not challenge the importance of achieving CR in MM, but unravel the need for further standardization and optimization of MRD detection. Recent data by Rawstron et al. [32] points out that quantitative assessment of tumor load with a cut-off of 10−4 (using multiparameter flow cytometry; MFC) would be more informative than a positive versus negative categorization, suggesting that a lower cut-off provided by more sensitive assays (e.g., NGS or high-sensitive MFC) will likely improve outcome prediction further. This has already been confirmed by Martinez-Lopez et al. using NGS [33], who identified three groups of patients with different TTP: patients with high (<10−3), intermediate (10−3–10−5), and low (>10−5) MRD levels showed significantly different TTP: 27, 48, and 80 months, respectively. Accordingly, these data highlight that beyond CR the deepest the level of MRD eradication the better survival, and that 10−5 should be currently considered as the target cut-off level for definition of an improved response category and MRD-negativity. This concept has also been reinforced by data obtained with parallel approaches achieving sensitivity levels beyond 10−5 [34].

3 Immunophenotypic CR

Multiparameter flow cytometry (MFC) is particularly well-suited to study biological samples containing PCs, because it allows: (i) simultaneous identification and characterization of normal versus tumors cells at the single-cell-level, (ii) fast evaluation of high-cell numbers (in a few hours), (iii) quantitative assessment of both normal and tumor cells and their corresponding antigen expression levels (e.g., for antibody-based therapy), (iv) combined detection of cell surface and intracellular antigens (e.g., for unequivocal confirmation of clonality within phenotypically aberrant cells), (v) an overview of the whole hematopoiesis through the simultaneous analysis of the different cell lineages [35].
The prognostic value of MFC-based MRD monitoring in MM was introduced in 2002 by the Spanish [36] and British [37] groups; both studies suggesting the utility of monitoring the BM PC compartment among MM patients treated with conventional or high-dose chemotherapy, even if such patients were in CR [37]. This initial positive experience led these groups to implement their corresponding 4- and 6-color flow-MRD methods in large clinical trials. In the PETHEMA/GEM2000 study, flow-MRD was identified as the most relevant prognostic factor in a series of 295 newly diagnosed MM patients receiving HDT/ASCT [38]. MRD-negativity at day 100 after ASCT translated into significantly improved PFS and OS, and the impact of MRD was equally relevant among patients in CR. Similarly, in the intensive-pathway of the MRC Myeloma IX study, MRD-negativity at day 100 after ASCT was predictive of favorable PFS and OS [39]. This outcome advantage was equally demonstrable in patients achieving CR. Furthermore, current data indicate that attaining MRD-negativity is not only relevant for standard but also high-risk patients. In fact, it is important to emphasize that both the PETHEMA/GEM and UK groups have demonstrated that risk assessment by cytogenetics/FISH and flow-MRD monitoring were of independent prognostic value in transplant-eligible patients [38, 39]. Furthermore, it is particularly interesting to observe the benefit of achieving MRD-negativity in high-risk patients, whose outcome becomes similar to that of standard-risk patients [5]. Accordingly, further research on the role of MRD as a surrogate for prolonged OS among high-risk patients is warranted, since it could represent an attractive clinical end point to improve the typical poor prognosis of this patient population. Thus, combined cytogenetic/FISH evaluation at diagnosis plus MRD assessment after HDT/ASCT (day +100), provided powerful risk stratification, which also resulted in a highly effective approach to identify patients with unsustained CR and dismal outcomes [5]. Collectively, these results confirm the superiority of MRD assessment over conventional response criteria to predict outcome in distinct MM genetic subgroups. The effect of maintenance therapy with thalidomide was also assessed in the UK study. Interestingly, MRD-positive patients randomized to the maintenance arm experienced significantly prolonged PFS as compared to the placebo arm; in MRD-negative patients a similar trend was observed [39]. The Spanish myeloma group has also shown that it was possible for elderly patients treated with bortezomib-based induction regimens to achieve MRD-negativity, and that flow-MRD resulted in superior patient prognostication than conventional and stringent CR response criteria [12]. A recent update of this study [40] after a median follow-up >5 years, shows median PFS and OS rates not yet reached for patients in flow-CR after VMP (but not VTP) induction. These results suggest that MRD monitoring is also clinically relevant in elderly patients but MRD-negative cases after two different regimens (VMP and VTP) did not experienced the same outcome. These findings suggest that the level of MRD tumor depletion may have been different between the two regimens, and that the 4-color MFC assay used in this GEM2005 trial was underpowered for ultra-sensitive detection of MRD [40]. The sensitivity of MFC has recently increased due to simultaneous assessment of ≥8 markers and evaluation of greater numbers of cells than what was previously feasible with analogical (4-color) instruments [41]. Thus, the availability of ≥8-color digital flow cytometers coupled to novel sample preparation protocols that allow fast and cost-effective routine evaluation of >5 million nucleated cells, has boosted the sensitivity of modern MFC-based MRD monitoring into that achieved on molecular grounds (≤10−5) (Table 1). It should be noted that current sensitivity of MFC is at least 1-log superior than that of previous MFC analyses (10−4); therefore, ongoing MFC-based MRD monitoring should result in improved patient’ risk stratification versus 4- or 6-color analyses. Accordingly, the Intergroupe Francophone du Myélome has reported on the prognostic value of their 7-color flow-MRD method implemented in a recent phase II study; overall, 68 % of patients achieved MRD-negativity and none of these patients has relapsed so far [42]. Analysis of larger number of cells (i.e., >5 million events) allows visualization of previously undetectable normal PC subsets with more heterogeneous phenotypes, which implies the need for simultaneous evaluation of at least eight parameters and potentially also Kappa and Lambda to improve specificity (and thereby sensitivity). Accordingly, using validated and standardized 8-color panels, clonal PCs are readily and accurately distinguishable from normal PCs according to aberrant phenotypes [35], and their clonality further confirmed by light-chain restriction. Because such analyzes rely on the recognition of aberrant antigenic patterns (i.e., different from normal), flow-MRD is applicable in virtually every MM patient without requiring for patient-specific diagnostic phenotypic profiles (although these are certainly useful). Equally important, the flow-MRD method incorporates a sample quality check of BM cellularity via simultaneous detection of B-cell precursors, erythroblasts, myeloid precursors, and/or mast cells. This information is critical to ensure sample quality and to identify hemodiluted BM aspirates that may lead to false-negative results
Table 1
Summary of the most relevant studies based on multiparameter flow cytometry (MFC), allele-specific-oligonucleotide PCR (ASO-PCR), next-generation sequencing (NGS), whole-body magnetic resonance imaging (WB-MRI), and positron emission tomography-computed tomography (PET/CT) detection of minimal residual disease (MRD) in multiple myeloma (MM)
Method
LOD
Setting
Number of patients
Applicability (%)
MRD-negativity (%)
PFS(MRD− vs. MRD+)
P
OS(MRD− vs. MRD+)
P
Reference
4-color MFC
10−4
CT or ASCT
87
NA
26
60 m versus 34 m
0.02
NA
San Miguel et al. [36]
3-color MFC
10−3–10−4
ASCT
45
94
56
35 m versus 20 m
0.03
76 % versus 64 % at 5-years
0.28
Rawstron et al. [37]
4-color MFC
10−4
ASCT
295
~95
42
71 m versus 37 m
<0.001
NR versus 89 m
0.002
Paiva et al. [38]
4-color MFC
10−4–10−5
Elderly
102
~95
43
90 % versus 35 % at 3-years
<0.001
94 % versus 70 % at 3-years
0.08
Paiva et al. [12]
4-color MFC
10−4–10−5
ASCT
241 (CR)
~95
74
86 % versus 58 % at 3-years
<0.001
94 % versus 80 % at 3-years
0.001
Paiva et al. [5]
6-color MFC
10−4
ASCT
397
NA
62
29 m versus 16 m
<0.001
81 m versus 59 m
0.02
Rawstron et al. [39]
7-color MFC
10−5
ASCT
31
NA
68
100 % versus 30 % at 3-years
NA
NA
Roussel et al. [42]
4-color MFC
10−4
Relapse/refractory
52 (CR)
NA
46
75 m versus 14 m
0.03
NA
Paiva [43]
ASO
10−5
ASCT
ALLO
50
88
27
110 m versus 35 m
<0.005
NA
Martinelli et al. [44]
ASO
10−6
ALLO
70
69
33
100 % versus 0 %1
 
NA
 
ASO
10−4
ASCT
87
77
35
64 m versus 16 m
0.001
NA
NS
Bakkus [45]
fASO
10−3–10−5
ASCT
ALLO
20
NA
15
60
NA
76 % versus 34 % at 2-years
0.03
Galimberti [46]
ASO
5 × 10−5
ASCT
24
75
29
34 m versus 15 m
0.042
NA
Sarasquete et al. [47]
fASO
10−3-10−4
ASCT
53
91
53
68 % versus 28 %
0.001
86 % versus 68 %
NS
Martinez-Sanchez et al. [48]
RQ
10−4–10−5
ASCT
ALLO
37
86
53
71
70 m versus 19 m
0.003
NR versus NR
0.1
Putkonen et al. [49]
RQ
NESTED
10−6
Consolidation
39
51
18
NR versus 38 m
<0.001
72 % versus 48 % at 8-years
0.041
Ladetto et al. [34], Ferrero [50]
RQ-ASO
10−4–10−5
ASCT
53
78
48
35 m versus 20 m
0.001
70 m versus 45 m
0.04
Korthals (2012)
RQ-ASO
10−5
ASCT
103
42
46
NR versus 31 m
0.002
NR versus 60 m
0.008
Puig et al. [51]
RQ-ASO
4 × 10−6
ASCT
22
100
59
48 m versus 13 m
0.004
NA
Silvennoinen et al. [52]
NGS
10−5
ASCT
Elderly
133
91
27
80 m versus 31 m
<0.001
NR versus 81 m
0.019
Martinez-Lopez et al. [33]
WB-MRI
ASCT
100
23
0.03
100 % (0 focal lesions)
0.001
Hillengass et al. [53]
PET/CT
ASCT
192
65
47 % versus 32 % at 4-years
0.02
79 % versus 66 % at 4-years
0.02
Zamagni et al. [54]
LOD limit of detection; NA not available; PFS progression-free survival; MRD minimal residual disease; OS overall survival; ASCT autologous stem cell transplantation; CR complete response; ALLO allogeneic stem cell transplantation
A potential limitation of MFC is that current strategies could miss hypothetical MM cancer stem cells with more immature phenotypes. However, recent investigations conducted with sensitive ASO-PCR assessment of clonal Ig genes among FACS-sorted peripheral blood B-cell subsets, revealed that such clonotypic cells are either absent, or present below highly sensitive limits of detection [41]. The need for extensive expertise to analyze flow cytometric data, together with the lack of well-standardized flow-MRD methods have been pointed out as additional and perhaps the main limitations of conventional MFC immunophenotyping [55]. However, new software programs have been developed in recent years with improved multidimensional identification and classification of different cell clusters coexisting in a sample (e.g., through principal component analysis and canonical analysis). These tools together with the use of normal and tumor reference databases, would allow for automated detection of normal versus aberrant phenotypic profiles [56]. If such methods become widely adopted, MFC would represent a method of choice for clinically relevant (Table 1), cost-effective yet highly sensitive, standardized MRD monitoring.

4 Molecular CR

Rearrangements of germline V, (D), and J gene segments in the Ig gene complexes (IGH, IGK, and IGL) provide each B-cell with specific V(D)J combinations. The random insertion and deletion of nucleotides at the V(D)J junction sites create highly diverse junctional regions, which represent unique “fingerprint-like” sequences, that are different in each B-cell and thus also in each B-cell malignancy. Since the 90s, these junctional regions (to be identified in each individual patient at diagnosis) have therefore been used as individual tumor-specific targets using Ig allele-specific oligonucleotides (ASO) as primers, initially for nested PCR approaches and later for real-time quantitative PCR-based MRD analysis (ASO-PCR). Such Ig targets can be identified and sequenced with standardized technologies in >95 % of lymphoid malignancies and used for the design of junctional region-specific oligonucleotides, to be applied for sensitive PCR-based detection of low frequencies of malignant cells, down to one malignant cell in 104–105 normal cells (10−4–10−5) [57]. This time-consuming but sensitive approach has been highly successful for MRD diagnostics in immature B-lineage malignancies, such as acute lymphoblastic leukemia (ALL) and has also been applied in mature B-cell malignancies such as MM, where its clinical relevance has been consistently demonstrated (Table 1).
Initial observations performed in patients undergoing autologous or allogeneic SCT unraveled the prognostic value of reaching molecular remissions [34, 47, 58, 59, 51, 44, 48, 52, 49]. Using nonquantitative approaches, the percentage of molecular remissions observed after allogeneic SCT was significantly higher as compared to patients undergoing autologous SCT, suggesting a role for this technique to evaluate treatment efficacy. Furthermore, Lipinski et al., in a retrospective study performed in 1ññ3 patients undergoing ASCT suggested the potential value of ASO-PCR monitoring to predict progression [60], and this notion of MRD reappearance heralding relapse has been recently confirmed by the GIMEMA group [61].
Semi-quantitative and quantitative approaches have also been used to predict patients’ outcome according to MRD levels. Korthals et al. in a cohort of 53 patients undergoing ASCT have shown that different MRD levels by ASO-RQ-PCR before ASCT allowed two discriminate two groups of patients with different PFS and OS (0.2 % 2IgH/βactin) [59]. Putkonen et al. in a series of 37 patients undergoing autologous and allogeneic stem cell transplantation defined 0.01 % as the optimal MRD threshold to distinguish two groups of patients with different PFS and OS [49]. Puig et al., in a recent study that included 103 patients undergoing ASCT also found 10−4 as the most significant cut-off level, distinguishing two subgroups with different PFS and, when applied to patients in conventional CR, also different OS [51]. Finally, Ladetto et al. with nested and ASO-RQ-PCR have reported on the significant reduction of residual tumor load after bortezomib, thalidomide and dexamethasone (VTD) consolidation, which translated into prolonged PFS [34]. A recent update of the study showed that MRD monitoring also predicted for different OS: 72 % at 8 years for patients in major MRD response versus 48 % for those with positive MRD [61]. More recent studies have provided similar results [52, 62].
In addition to the well-established clinical value, other advantages of PCR approaches for MRD detection are the bypass for immediate sample processing since it is unaffected by pre-analytical biases such as loss of viable cells over time [47]. This feature makes molecular-based MRD monitoring an attractive approach for studies requiring centralized (or necessarily delayed) analysis. Furthermore, taking advantage of the uniqueness of patient-specific clonal IGHV rearrangements, PCR assays can reach highly sensitive MRD detection levels up to 10−6, although experience from different centers suggests that routine limit of detection stands at 10−5 [34, 51]. Importantly, PCR strategies have gone through an extensive validation and standardization process for MRD testing in different hematological neoplasms, such as acute lymphoblastic leukemia, becoming readily standardized and reproducible among different centers [57, 63], although not yet in MM. In contrast to MFC, PCR-based approaches require diagnostic samples to identify patient-specific clonotypic sequences [64]. Furthermore, the high rate of somatic hypermutations both in the heavy- and light-chain immunoglobulins genes [65] prevent the exact annealing of consensus primers, hamper clonal detection, sequencing success rates, and overall ASO performance [51]. To overcome such limitations, additional targets have been tested (e.g.,: DJH and Kde) [66] and the use of CD138+ positively selected PCs has been shown to significantly increase the applicability of PCR-based MRD monitoring in MM, but still remains in the range of 65–80 % of cases [67]. Accordingly, the technique remains costly, laborious, methodologically complex, and difficult to implement into routine clinical practice.
Sequencing technologies can quickly perform multiple reads of many different DNA fragments and are therefore a natural alternative to overcome some of the limitations of ASO-PCR to monitor MRD in MM. Importantly, this technology allows the detection of previously known tumor-specific sequences within normal DNA fragments (i.e.,: MRD monitoring). Current NGS methods include: (1) pyrosequencing, based on the luminometric detection of the pyrophosphate released when individual nucleotides are added to DNA templates from an emulsion PCR; (2) multiplex sequencing-by-synthesis technology, that rely on light signals emitted during the resynthesis of small DNA fragments previously produced by bridge amplification; and (3) ion semiconductor sequencing, that detects hydrogen ions released during DNA polymerization. Using these techniques, several methods have been developed to sequence rearranged B-cell (BCR) and T-cell receptor (TCR) genes [6872]. These methods use a consensus PCR to amplify all possible BCR or TCR rearrangements which, at diagnosis, allow to identify clonal rearrangements (arbitrarily defined as those above 5 % among the total sequences identified) [72]. After therapy, clonal Ig rearrangements can be traced among thousands of normal Ig genes through several millions reads, providing high-specificity and sensitivity for MRD detection of BCR and TCR clonal sequences.
One of the greatest advantages of NGS approaches for MRD detection in MM is its sensitivity which, without compromising specificity, is estimated to be in the range of 10−5–10−6 [33, 72]. Of note, with NGS it would be possible to detect clonal tiding (i.e., suppression or reemergence of two or more clonal Ig rearrangements following treatment) [73], although subclonality in diagnostic samples is typically below 7 % of all tumor cells patients. Furthermore, in MM the main clonal rearrangement is usually stable from diagnosis to relapse, [74] or if it changes, this problem would not affect a proportion much higher than 5 % of the patients [75]. NGS offers additional advantages, particularly when compared to ASO-PCR, because it is methodologically less complex, and obviates the need to construct dilution standard curves which is the main reason of ASO-PCR failure in MM [51]. Another potential advantage of NGS is the information that it provides about the residual normal B-cell compartment, since it can identify the variability of normal polyclonal B-cells and this information may be of potential prognostic value.
However, there are also some disadvantages. Similarly to ASO-PCR, NGS-based MRD monitoring cannot distinguish hemodiluted from representative BM samples. Albeit the applicability of NGS is superior to that of ASO-PCR, still in around 10 % of patients the clonal rearrangement cannot be identified during the initial PCR step [33, 76]. Similarly to ASO-PCR, the NGS method requires a diagnostic sample to identify the patient-specific clonotypic sequence. In addition, MRD quantitation is only approximate, because the efficacy of amplification is highly variable depending on the specific sequence of the rearrangement [77]. NGS-based MRD monitoring is still centralized on commercial vendors and not yet widely available; if it becomes decentralized, this would require additional validation and standardization within the different centers adopting this technology (similarly to what is being currently done for MFC and ASO-PCR). Finally, NGS is relatively labor-intensive and expensive technology, which are important factors to consider prior to incorporation into routine clinical practice.
Since NGS-based MRD monitoring has only recently been developed, there is yet few clinical data in MM (Table 1). However, the PETHEMA/GEM has already described favorable and promising results in a series of 133 MM patients including both transplant and non-transplant-eligible cases [33]. The applicability of NGS-based MRD monitoring using the LymphoSIGHT® methodology was of 90 %. The median TTP and OS of MRD-negative cases were of 80 months and not reached, respectively [33]. As above mentioned, Martinez-Lopez identified three groups of patients with different TTP: patients with high (<10−3), intermediate (10−3–10−5), and low (>10−5) MRD levels showed significantly different TTP: 27, 48, and 80 months, respectively, which indicates that the deepest the quality of CR, the better the patients outcome [33]. Other studies are providing similar results in MM [78, 79] but these are currently available as abstract, and we should wait for their full publication with all the necessary details.

5 Available Techniques to Monitor Intramedullary and Extramedullary MRD: Towards an Imaging CR in MM?

The possibility of patchy BM infiltration or extramedullary involvement represents a challenge for both immunophenotypic- and molecular-based MRD detection in single BM aspirates. This highlights the potential value of sensitive imaging techniques to redefine CR both at the intramedullary and extra-medullary levels (Table 2).
Table 2
Individual features of currently available techniques to monitor MRD in MM
Technique
Advantages
Disadvantages
MFC (≥8-color)
• Applicable to virtually all patients
• Availability in individual laboratories
• Reproducibility among centers
• Sensitivity (10−5–10−6)
• Direct quantitation of MRD levels
• Ongoing assessment of sample quality
• Diagnostic sample is important but not mandatory
• Possibility to standardize (e.g., EuroFlow/IMF)
• Turnaround time (2–3 h)
• Less expensive technique
• Limited value in patients with patchy BM infiltration and/or extramedullary disease
• Requires fresh samples (<36-h)
• Requires full implementation of a single, standardized method in multiple individual laboratories for complete standardization
• Detection of clonality restricted to the PC compartment
ASO-PCR
• Highly specific detection of clonality
• Sensitivity (10−5–10−6)
• Detection of all clonal Ig sequences irrespectively of phenotype (i.e., putative CSCs)
• Intermediate availability in experienced individual laboratories
• Reproducibility among centers
• Does not require immediate sample processing
• Acquired experienced in standardization (EuroMRD)
• Limited applicability (~60–70 %)
• Limited value in patients with patchy BM infiltration and/or extramedullary disease
• Lack of ongoing assessment of sample quality
• Requires diagnostic sample
• Turnaround time (3–4 weeks for target identification at baseline and ≥5 days during follow-up)
• Indirect quantitation of MRD levels
• Cost (increased by target identification at baseline)
NGS
• Higher applicability compared to ASO-PCR (~90 %)
• Highly-specific detection of clonality
• Sensitivity (10−6)
• Detection of all clonal Ig sequences irrespectively of phenotype (i.e.: putative CSCs)
• Does not require immediate sample processing
• Easy to standardize if confined to commercial services
• Limited availability to commercial services
• Limited experience on individual laboratories (with consequent lack of reported reproducibility)
• Limited value in patients with patchy BM infiltration and/or extramedullary disease
• Lack of ongoing assessment of sample quality
• Requires diagnostic sample
• Indirect quantitation of MRD levels
PET/CT
• Applicable to virtually all patients
• Sensitivity (4 mm)
• Detection of extramedullary disease
• Not biased by patchy BM infiltration
• Diagnostic imaging is important but not mandatory
• Turnaround time (2–3 h)
• Intermediate availability
Lack of standardization
• Moderate reproducibility at MRD assessment
• Cost
MFC multiparameter flow cytometry; ASO-PCR allele-specific oligonucleotide, polymerase chain reaction; NGS next-generation sequencing; PET/CT positron emission tomography-computed tomography; MRD minimal residual disease; PC plasma cell; CSCs cancer stem cells; EuroFlow see www.​EuroFlow.​org; EuroMRD see www.​EuroMRD.​org
Magnetic resonance imaging (MRI) is the most sensitive noninvasive imaging technique to detect focal lesions in the spine. However, it should be noted that due to treatment-induced necrosis and inflammation, focal lesions may remain hyperintense for several months after therapy in both responding and non-responding patients. This can explain some inconsistencies found between serological CR and MRI-based CR [53, 80]. Consequently, an interval of at least 3 months has been recommended before MRI monitoring [81]. Although comparative studies are lacking, it can be envisioned that similarly to that found for newly diagnosed patients [82], whole-body MRI (WB-MRI) would be more effective than MRI on the axial skeleton to define full BM imaging response.
In contrast to MRD, positron emission tomography-computed tomography (PET/CT) combines the morphological images provided by CT with the imaging data of a particular metabolic process (e.g., fluorodeoxyglucose–FDG—uptake), and it is probably the technique of choice to detect extramedullary disease. Similarly to MRI, it is important to emphasize that for MRD monitoring (which will pay particular attention to FDG uptake rather than lytic bone lesions), both false positive (e.g., coexisting infectious or inflammatory processes) and false-negative results (e.g., quiescent tumor cells) may occur [83]. A recent comparison between WB-MRI and PET/CT in transplant-eligible patients showed that, against conventional response criteria, PET/CT had the lower sensitivity (50 % vs. 80 %) but higher specificity (85 % vs. 38 %) than WB-MRI. While the utility of other MRI-based techniques is still under investigation (e.g., dynamic contrast-enhanced MRI) [84], the current perception is that PET/CT represents the most promising imaging tool to monitor MRD in MM. That notwithstanding, Zamagni et al. reported that post-ASCT, PET/CT monitoring was also an independent prognostic marker for PFS and OS, even among patients in conventional CR [54]. However, given the sensitivity and specificity observed against traditional response criteria, standardization of PET-CT (including response criteria) and comparison with other sensitive BM-based MRD methods is still needed in order to implement imaging monitoring in the clinical setting [83].
NGS approaches have also been tested in peripheral blood as a promise for MRD detection in MM outside the BM. This approach has provided initial successful results in NHL [85] and it has also been proposed for myeloma [86] but no real correlation has been found in a small study were specific myeloma DNA is lost in most patients after two cycles of therapy despite they conserve the monoclonal protein [87].

6 Conclusions and Future Perspectives: MRD Incorporation into Clinical Trials

So far no clinical trial has randomized MM patients according to their MRD status, in order to investigate the role of MRD to individualize therapy. Overall, the experience of several cooperative groups using different MRD techniques indicates that persistence of MRD is always an adverse prognostic feature (Tables 1 and 2), even among CR patients. Consequently, it would be safer to take clinical decisions based on MRD-positivity rather than on MRD-negativity, since the patchy pattern of BM infiltration typically observed in MM leads to a degree of uncertainty regarding MRD-negative results: does this guarantee absence of tumor cells or is it the result of a nonrepresentative BM sample due to patchy tumor infiltration? Many studies have shown the value of MRD to evaluate the efficacy of specific treatment phases and therefore, to support potential treatment decisions. For example, both the Spanish PETHEMA and the UK MRC study groups have shown that MRD kinetics before and after HDT/ASCT allow identification of chemosensitive versus chemoresistant patients [38, 39]. For the latter, it could be hypothesized that consolidation with alternative therapies would be needed to improve outcomes. Following consolidation physicians face another treatment decision: maintenance versus no maintenance and duration? Ladetto et al. reported PFS rates of 100 % versus 57 % for patients in molecular CR versus MRD-positive cases after consolidation, respectively [34]. Since no maintenance therapy was given in the GIMEMA VEL-03-096 study, one might hypothesize that for those cases failing to reach MRD-negativity despite being in CR/nCR after consolidation, maintenance may represent an effective approach to eradicate MRD levels and improve outcome. Accordingly, Rawstron et al. have shown that one out of four MRD-positive patients randomized to the maintenance arm of the MRC-myeloma IX (intensive) study turned into MRD-negative, and experienced significantly prolonged PFS versus the abstention arm [39]. However, because even MRD-negative patients receiving maintenance continue to show late relapses [39], it may be envisioned that we need to increase the sensitivity of MRD techniques in order to better monitoring “theoretically MRD negative” patients during maintenance therapy; moreover, if treatment decisions are taken according to patients’ MRD status, follow-up MRD studies would also become useful to detect MRD reappearance preceding clinical relapse [61]. This approach is likely to imply serial MRD assessment which, at the moment, would require the need of invasive and inconvenient multiple BM aspirates. Most recently, NGS has been evaluated in PB (i.e., plasma) from MM patients after induction and this would represent an attractive minimally invasive approach. However, preliminary data indicates that clonotypic sequences identified at baseline, become undetectable with just a few cycles of chemotherapy, even among electrophoresis positive patients. Thus, further research is warranted to establish the feasibility of PB (e.g., cell- or free DNA-based) MRD monitoring. Furthermore, our knowledge on clonal tiding (i.e., disappearance of pre-existing or occurrence of new clones), during maintenance or progression-free periods without therapy is very limited if exiting at all, and the concept of clonal tiding should also be taken into consideration while designing such treatment strategies.
The choice of MRD technology for monitoring will depend on how individual centers’ priorities adjust to the specific advantages that each tool has to offer (Table 2). In turn, extensive research is still warranted to determine how to best integrate medullary and extramedullary MRD monitoring. In other hematological malignancies, baseline risk-factors and MRD monitoring have an established and complementary role to individualize treatment. Over the last two decades, several groups have consistently confirmed the added value of MRD in MM, and the time has come to establish the role of baseline risk-factors plus MRD monitoring for tailored therapy. This requires the introduction of standardized, highly sensitive, cost-effective, and broadly available MRD techniques in clinical trials.
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