Is This the Time to Introduce Minimal Residual Disease in Multiple ...

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Is This the Time to Introduce Minimal Residual Disease in Multiple

Myeloma Clinical Practice?

Bruno Paiva1, Noemi Puig2, Ramón García-Sanz2, and Jesús F. San Miguel1;

on behalf of the Grupo Español de Mieloma (GEM)/Programa para el Estudio

de la Terapéutica en Hemopatías Malignas (PETHEMA) cooperative study

groups

1Clinica Universidad de Navarra, Centro de Investigacion Medica Aplicada

(CIMA), Pamplona, 2Hospital Universitario de Salamanca, Instituto de

Investigaion Biomedica de Salamanca, IBMCC (USAL-CSIC), Salamanca,

Spain

Corresponding Author: Jesús F. San Miguel, Clinica Universidad de Navarra,

Centro de Investigacion Medica Aplicada (CIMA), Av. Pio XII 36, 31008

Pamplona, Spain; E-mail: [email protected]

Running Title: State of the Art: MRD Testing for Myeloma

Disclosure of Potential Conflicts of Interest

B. Paiva reports receiving speakers bureau honoraria from Becton, Dickinson

and Company, Binding Site, Celgene, Janssen, and Millennium

Pharmaceuticals. J.F. San Miguel is a consultant/advisory board member for

Bristol-Myers Squibb, Celgene, Janssen, Merck, Millennium Pharmaceuticals,

Novartis, and Onyx Pharmaceuticals. No potential conflicts of interest were

disclosed by the other authors.

Research. on April 13, 2018. © 2015 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 9, 2015; DOI: 10.1158/1078-0432.CCR-14-2841

http://clincancerres.aacrjournals.org/

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Abstract

Increasing therapeutic options and prolonged survival in multiple myeloma (MM)

have raised interest on the concept of depth of response and its importance to

predict patients’ outcome. While the efficacy of current treatment approaches

has greatly improved in the last decade, the definition of complete response

(CR) remains unaltered and continues to use conventional serological and

morphological techniques. That notwithstanding, there is growing interest in

minimal residual disease (MRD) monitoring, which has emerged in recent years

as one of the most relevant prognostic factors in MM. MRD can be assessed

both inside (e.g., immunophenotypic and molecular techniques) and outside the

bone marrow (e.g., PET-CT). Here, we focus on flow- and molecular-based

assays by which different cooperative groups have demonstrated the efficacy of

MRD assessment to predict outcomes even among patients in CR, and

irrespectively of disease risk. Although, further standardization is still required,

the time has come to implement MRD monitoring in prospective clinical trials as

a sensitive tool to evaluate treatment efficacy and for risk-adapted treatment,

particularly in the consolidation and maintenance settings. Here we present a

comprehensive and critical review on the methodological aspects, specific

characteristics, as well as clinical significance of MRD monitoring by flow

cytometry, PCR and next generation sequencing.




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Introduction

In the last decades, important changes have been introduced in the treatment of

multiple myeloma (MM) patients, resulting in unprecedented complete remission

(CR) rates and prolonged progression-free (PFS) and overall survival (OS) (1-

3). In 2006 the International Myeloma Working Group proposed a new CR

definition, and introduced the normalization of serum free light-chains (sFLC)

and absence of bone marrow (BM) clonal plasma cells (PCs) by

immunohistochemistry/immunofluorescence as additional requirements to

define stringent CR (4). Recently, Kapoor et al. (5) demonstrated the added

prognostic value of the stringent over conventional CR criteria, even though

other groups have failed to show additional value of sFLC assessment among

CR patients (6, 7). However, the sensitivity of

immunohistochemistry/immunofluorescence is rather low (10-2) due to the

recovery of normal PCs after therapy that normalize kappa/lambda ratios (8).

Thus, it becomes evident that current criteria used to assess response lag

behind the extraordinary evolution in the treatment of MM patients, and more

sensitive techniques are needed to detect true minimal residual disease (MRD)

for new CR definitions. This would contribute to improve monitoring of treatment

efficacy as well as to avoid over and under treatment, particularly during

consolidation and maintenance.

Current techniques to monitor MRD can be divided into those measuring

extramedullary vs. those detecting intramedullary disease. Magnetic resonance

imaging (MRI) and 18-fluoro-deoxyglucose positron emission tomography-

computerized tomography (PET/CT) have emerged as promising tools to

measure extramedullary MRD. In particular, PET/CT has shown to be of




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prognostic value as soon as at day 7 of induction therapy (9) and more

importantly, among patients in conventional CR after HDT/ASCT (10). For

detection of intramedullary disease, both multiparameter flow cytometry

immunophenotyping and molecular assessment of immunoglobulin

rearrangements are the pivotal techniques. The present review focus on current

state-of-the-art intramedullary MRD monitoring in MM.

Methodical Characterization of MRD Techniques

Multiparameter flow cytometry (MFC)

Simultaneous assessment of CD38 and CD138 represents the best marker

combination for specific identification of PC (11-14). In the event of anti-CD38

and/or anti-CD138 therapies, other antigens such as CD229, CD319 or CD54

could be alternative markers for PC identification. Phenotypically aberrant clonal

PCs typically show: i) underexpression of CD19, CD27, CD38, and/or CD45; ii)

overexpression of CD56; iii) asynchronous expression of CD117 (15-24).

Accordingly, these antigens are consensually used for the clinical study of BM

PCs (25) and at least one (8-color) combination of such markers is highly

recommended for MM flow-MRD monitoring. No single parameter reliably

distinguishes clonal from normal PCs; however, a multiparameter approach that

evaluates all markers used in a single tube can readily identify PC aberrant

phenotypes, provided a sufficient number of cellular events are evaluated in the

flow cytometer (26).

Flow-based MRD monitoring has benefited from polychromatic

cytometers that allow simultaneous assessment of ≥8-markers at the single-cell

level which, coupled with novel software, results in more accurate discrimination

of clonal vs. normal PCs (27). The principal component analysis (PCA) of




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unified data files allows automatic multidimensional separation of all cellular

clusters present in a sample. PCA-based interpretation of flow cytometric data

facilitates the development of normal and tumor reference libraries, automated

separation of populations within a sample, and prospective detection and

tracking of any aberrant cell population. Such strategy is likely the method of

choice for accurate and semi-automated flow-MRD monitoring.

Allele specific oligonucleotide polymerase chain reaction (ASO-PCR)

PCR-based detection of MM MRD relies on the identification of persistent tumor

cells through the amplification of the immunoglobulin heavy-chain (IGHV) gene

rearrangement (28). Consensus primers binding to the less variable regions of

the IGHV rearrangement (FR o framework) were initially used for the

identification of residual disease. This approach amplifies the monoclonal IGHV

rearrangement but also those from normal B-cells and thus, the sensitivity

reached (10-1 - 10-2) was insufficient for MRD detection. Afterwards, specific

primers (ASO: allelic specific oligonucleotide) complementary to the most

variable regions of the IGVH rearrangement (CDRs or complementary

determinant regions) were used. The limit of detection with this approach was

optimal (10-4-10-6) but the results obtained were merely qualitative (29, 30). The

subsequent step was the implementation of RQ-PCR strategies that allow the

exact quantification of tumor cells in real-time. This method is based on the use

of an ASO primer (usually complementary to the CDR3 region) together with a

consensus primer (frequently complementary to the JH region) and a

fluorescent probe to monitor the amplification.

Next generation sequencing (NGS)




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In recent years, sequencing technologies that quickly perform millions of reads

of DNA fragments have emerged. This technology not only allows to distinguish

normal from tumor DNA, but also to detect previously known tumor-specific

sequences within normal DNA fragments (i.e., MRD monitoring). Current NGS

methods include: i) pyrosequencing, based on the luminometric detection of the

pyrophosphate released when individual nucleotides are added to DNA

templates from an emulsion PCR; ii) multiplex sequencing-by-synthesis

technology, that rely on light signals emitted during the re-synthesis of small

DNA fragments previously produced by bridge amplification; and iii) ion

semiconductor sequencing, that detects hydrogen ions liberated during DNA

polymerization Using these techniques rearranged B-cell receptor (BCR) and T-

cell receptor (TCR) genes can be deeply sequenced (31-35). These methods

use a consensus PCR to amplify all possible BCR or TCR rearrangements

which, at diagnosis, allow identifying and sequencing monoclonal

rearrangements (35). After therapy, monoclonal rearrangements can be

investigated among thousands of normal cells through several millions reads,

providing high specificity and sensitivity for MRD detection of BCR and TCR

genes.

Advantages and Disadvantages of MRD Techniques

MFC

An increasingly higher number of flow cytometry laboratories use digital

instruments that provide simultaneous assessment of more parameters per tube

(≥8), as well as a superior event acquisition and analysis of a greater cell

numbers than previously seen with 4-color instruments. The availability of digital

cytometers coupled with novel sample preparation methods allow for fast- and




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cost-effective measurement of millions of leukocytes. Consequently, while

previous MFC studies defined MRD as the presence of a discrete population of

clonal PCs equal or above the 0.01% limit of detection, nowadays such

threshold stands at 0.001% (10-5). Because clonal PCs are readily distinguished

from normal PCs on the basis of their aberrant phenotypes, flow-MRD is

applicable to virtually all patients and does not require patients’ diagnostic

phenotypic profile. Furthermore, the flow-MRD assays incorporates a quality

check of the whole sample cellularity (i.e., B-cell precursors, erythroblasts, etc.),

which is critical to ensure sample quality since hemodiluted BM aspirates can

lead to false-negative results. Even though hemodilution can be assessed with

a microscope - providing that the sample in which hemodilution is being

assessed is exactly the same than that where MRD will be assessed -, the

possibility for MFC to simultaneously analyze MRD and hemodilution

automatically in the same sample is more accurate and particularly attractive.

Extensive expertise in MFC analysis requirement and the lack of a well-

standardized flow-MRD method have been pointed as important disadvantages

of MFC immunophenotyping. However, automated identification and

characterization of cell populations, coupled with reference databases in the

context of full technical standardization have now emerged as the probable

solution for these problems. There is now the unmet need for different MFC

groups to adopt single, standardized and validated antibody panels, sample

processing and cell-analysis methods such as those being developed by the

EuroFlow consortium for MFC to become a universal and fully standardized

option for MRD assessment. Another potential limitation of MFC could be to

ignore potential MM cancer stem cells outside the PC compartment (i.e.,




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memory B-cells) (36). However, recent investigations conducted with optimized

molecular assessment of clonal VDJ sequences among FACS-sorted peripheral

blood B-cell subsets revealed that such clonotypic cells are either absent, or

present below the detection limits (10-6) (37).

Regarding the potential impact of genetic heterogeneity and clonal tiding

after treatment on the feasibility of MFC to detect MRD, it should be highlighted

that the multidimensional approach of current flow cytometry

immunophenotyping not only allows to detect clonal heterogeneity in

approximately 30% of newly-diagnosed patients, but also to monitor all different

phenotypic sub-clones throughout patients’ treatment, thereby assessing

potential (phenotypical) clonal selection upon therapy. Nevertheless, it should

be noted that according to the experience of the Medical Research Council

(MRC) and the Grupo Español de Mieloma (GEM) groups based on large

patient cohorts, there are no major antigenic shifts for consensus markers (e.g.,

CD19, CD38, CD45, or CD56) used to monitor MRD (GEM; unpublished data).

Accordingly, potential clonal evolution throughout the course of treatment does

not influence the efficacy of MFC-based MRD assessment.

ASO-PCR

PCR approaches for MRD detection do not require an immediate sample

processing since they are unaffected by pre-analytical biases such as loss of

viable cells over time (38, 39). Furthermore, the MRD target used is based on

the uniqueness of clonal IGHV rearrangements which leads to a high sensitivity,

adequate for MRD detection (10-5 -10-6) (40, 41). PCR strategies have passed

an exhaustive process of validation and standardization for MRD testing in

different hematological malignancies, which make them readily available and




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reproducible among different centers (42, 43). However, these approaches

require diagnostic samples with high tumor load to identify patient-specific

clonotypic sequences (44, 45). Moreover, MM is a post-germinal center

neoplasia characterized by a high rate of somatic hypermutations both in the

heavy- and light-chain immunoglobulin genes (46-48). Such mutations prevent

the annealing of consensus primers, limit clonal detection, sequencing success

rate and overall ASO performance, forcing the use of specific primers/probes

instead (49). Conversely, it has been recently reported that the use of CD138+

positively selected BM PCs may significantly increase the applicability of PCR-

based MRD studies in MM (50). Nevertheless, the technique remains costly,

laborious, and accordingly, difficult to implement into routine clinical practice.

NGS

The greatest advantage 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 (35, 51). Moreover, NGS can identify both stable and dynamic

aspects of the BCR rearrangement, including potential clonal tiding (52).

However, the presence of subclonality in diagnostic samples is typically below

7% of all patients, and virtually impossible to be distinguished from biallellic

rearrangements (53). Furthermore, the main clonal rearrangement is usually

stable from diagnosis to relapse (B. Puig et al.; submitted for publication).

Therefore, since clonal Ig gene sequences remain stable and because current

MRD monitoring by ASO-PCR or NGS does not focus on gene mutations or

copy number abnormalities, molecular techniques are not influenced by genetic

heterogeneity and clonal tiding throughout patients’ treatment. Other

advantages that NGS offers are superior sensitivity, potential scalability and




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less methodological complexity. In particular, the latest advantage would be

crucial in MM since it removes the need of standard curve construction, which is

the main reason of ASO-PCR failure in MM (49). However, there are also some

disadvantages. Molecular-based approaches cannot distinguish hemodiluted

from good quality samples. Albeit the applicability of NGS is superior to that of

ASO-PCR, still 10% of patients will be missed during the initial PCR step (51).

In addition, MRD quantitation is only approximate, because the efficacy of

amplification is highly variable depending on the specific sequence of the

rearrangement (54). Since only B-cells have rearranged immunoglobulins,

NGS-based MRD monitoring will only quantify B-cells; accordingly, global MRD

quantitation requires the addition of an artificial internal control for such

quantification. Finally, NGS is a labor-intensive and expensive technology, and

it is yet not commonly available for clinical practice.

Clinical Results with (Intramedullary) MRD Techniques

MFC

The prognostic value of MFC-based MRD monitoring in MM was introduced in

2002 by San Miguel (55) and Rawstron (20); 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 (20).

This initial positive experience led the Spanish and UK 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

uniform treatment including HDT/SCT (56). MRD negativity at day 100 after

ASCT translated to significantly improved PFS and OS, and the impact of MRD




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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 (57). This outcome advantage was equally

demonstrable in patients achieving CR. Since in MM there has been extensive

debate on whether attaining deep levels of remission (i.e., CR) would be critical

to all patients or in turn is particularly relevant for patients with high-risk disease,

it is important to emphasize that both the PETHEMA/GEM and UK groups have

demonstrated that risk assessment by FISH and flow-MRD monitoring were of

independent prognostic value in transplant-eligible patients (56, 57).

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 (25). 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 overall 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 (25).

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 (57).

Further analyses by the PETHEMA/GEM have shown that combining the




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prognostic information of cytogenetics at diagnosis plus MRD assessment at

day 100 after HDT/ASCT resulted in a highly effective approach to identify

patients with unsustained CR and dismal outcomes (2-years median OS): those

with baseline high-risk cytogenetics plus persistent MRD after ASCT (58). 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 (7). More recently, 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 (59).

Overall, 68% of patients achieved MRD negativity and none of these patients

relapsed. Thus, it is plausible to assume that albeit the already well-established

link between patients’ flow-MRD status and survival, the true clinical utility of

flow-based MRD assessment is likely to be significantly improved with more

sensitive (10-5) and multidimensional (≥8-colors) immunophenotyping. Table 1

summarizes the most relevant flow-based MRD studies reported in MM.

ASO-PCR

A considerable number of studies have explored the value of PCR-based MRD

monitoring in MM (Table 2) (60-66). Although initial observations lacked clinical

value, additional studies performed in patients undergoing autologous or

allogeneic SCT unraveled the prognostic value of reaching molecular

remissions (Table 2) (39-41, 49, 50, 65, 67-72) Using non-quantitative

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.




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Furthermore, Lipinski et al., in a retrospective study performed in 13 patients

undergoing ASCT suggested the potential value of ASO-PCR monitoring to

predict progression (73), and this notion of MRD reappearance heralding

relapse has been recently confirmed by the GIMEMA group (72).

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) (41). 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 also OS (71). Puig et al., in a recent study that included 103

patients undergoing ASCT also found 10-4 as the most significant cutoff level,

distinguishing two subgroups with different PFS and, when applied to patients in

conventional CR, also different OS (50). 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 (70). 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 vs. 48% for those with positive MRD (72).

NGS

Since NGS-based MRD monitoring is still a relatively recent approach, there is

yet few data in MM. 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. The applicability of NGS-based




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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 (51). Importantly, Martinez-Lopez identified three groups

of patients with different TTP: patients with high (<10-3), intermediate (10-3 to 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 (51). Similar results are also now being observed

with more sensitive MFC (GEM; unpublished observations).

Discordant Results between (Intramedullary) MRD Techniques

MFC vs. ASO-PCR

Three studies have directly compared MFC and ASO-PCR as alternative

methods for MRD detection in MM. In 2005, Sarasquete et al. quantified MRD

levels in 24 patients at day 100 after HDT/ASCT using both techniques, and

observed discordant results in 6 out of the 24 cases; all of them negative by

MFC but positive by ASO-RQ-PCR (39). Discordances were attributed to the

limit of MRD detection by each technique, since the median number of clonal

PCs in PCR+MFC- cases was 0.014%. A second study by Lioznov et al.,

performed on 69 samples from 13 patients undergoing allogeneic SCT, found a

very high correlation between both techniques. Virtually all results were

concordant, and both techniques had similar applicability and prognostic value

(74). More recently, Puig et al. compared the results of MRD assessment by

MFC and ASO-PCR in 103 cases undergoing HDT/ASCT, and only observed

18 discordant results (11 cases were PCR+MFC- while the other 7 were MFC+

but PCR-). No differences in survival were observed between the discordant

groups of patients (49).




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MFC vs. NGS

Only one study has compared MFC vs. NGS for MRD monitoring in MM (51).

From a total of 99 patients, 82 had concordant results (60 double-positive and

22 double-negative), twelve were MFC–NGS+ and five were MFC+NGS–.

Discordances are likely related to differences in sensitivity and specificity, which

theoretically favor NGS over 4-color MFC. However, it cannot be excluded that

certain clonal rearrangements could be under-amplified against polyclonal

rearrangements and, if present in very low numbers (typical situation in MRD

samples), these could remain undetectable. Noteworthy, the time-to

progression of MRD- patients by NGS was slightly better than NGS+MFC- cases

(P = .05). However, this study was a retrospective comparison and the MFC

approach was a conventional 4-color staining of a limited number of cells (10-

fold less compared to current MFC studies) (51).

ASO-PCR vs. NGS

Only two studies have compared ASO-PCR vs. NGS. Ladetto et al. has

reported preliminary data on 10 MM patients; 8 were evaluable by RQ-PCR, 8

by NGS and 6 by both methods (75). Among total follow-up samples, 20

discordances were recorded: 12 qualitative and 8 quantitative discordances. In

9 samples, RQ-PCR yielded a positive or 1 log higher result compared to NGS,

while the opposite occurred in 11 cases. Overall, major discordance rates in

MM were comparable to ALL and MCL, while minor and quantitative

discordances appeared slightly more frequent (75). Martinez-Lopez et al.

performed a similar comparison in 46 patients, and observed a concordance

rate of 85% (51). Thus, further studies are warranted to elucidate the clinical




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significance of discordant results from two molecular techniques that, albeit

methodologically different, have similar sensitivity.

The ideal MRD test should fulfill a minimum of several relevant

characteristics: i) high applicability, ii) high sensitivity, iii) readiness and rapid

turnaround, iv) feasible in low sample volumes, v) reproducibility, and, vi) of

clinical utility. Because the cost of MRD assessment is significantly lower as

compared to the cost of some drugs its importance is less relevant; however, in

the event of long-term follow-up sequential analysis (similarly to what is done in

CML or ALL), total cost becomes significantly higher and therefore important to

evaluate. While we wait for further results on the comparison between ASO-

PCR and NGS, the higher applicability of the latter would favor its incorporation

in clinical trials. Unfortunately, data on prospective comparison between the

next-generation MFC vs. NGS is not yet available, and knowledge on head-to-

head applicability and sensitivity are missing. Altogether, this precludes us to

indicate at present, a favorite methodology to be implemented in clinical trials.

For routine clinical practice, we would support at this moment the use of next-

generation MFC given its wide availability, cost-effectiveness, and

independence of previous collection of a diagnostic sample.

Concluding Remarks and Future Direction

Approximately fifteen years after the first published results, MRD detection has

emerged as one of the most relevant prognostic factors in MM. Because MRD

represents the collective end result of all of the cellular mechanisms that

determine a patient response to a given therapy, MRD assessment affords

prognostic information even among patients in CR, and irrespectively of disease

risk. Accordingly, there is increasing interest on MRD monitoring as a sensitive




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tool to evaluate treatment efficacy, to become a surrogate marker for clinically

relevant end-points, and for risk-adapted treatment, particularly in the

consolidation and maintenance setting. However, the patchy pattern of BM

infiltration, typically observed in MM, leads to a certain degree of uncertainty

regarding an MRD-negative result irrespectively of the technique adopted: does

it represent real absence of clonal PCs, or is it due to sampling error? The

possibility of patchy BM infiltration or extramedullary involvement represent a

challenge for both immunophenotypic- and molecular-based MRD detection in

single BM aspirates. This highlights the value of sensitive imaging techniques to

re-define CR both at the intramedullary (e.g., whole body MRI and PET/CT) and

extra-medullary levels (PET/CT). However, standardization of imaging

techniques and comparison with other sensitive BM-based MRD methods are

still lacking. By contrast, the persistence of MRD tumor cells is always an

adverse prognostic feature, even among CR patients, envisioning that for the

time being, it would also be safer to take clinical decisions based on MRD-

positivity than on MRD-negativity. The clinical applicability of MRD in MM has

been prospectively confirmed in two large transplant-based studies (56, 57) and

a relatively smaller (yet prospective) clinical trial on transplant-ineligible MM

patients (7); such results were also retrospectively reproduced on smaller

patient series mostly by using molecular techniques. In all studies, the PFS of

MRD-negative patients at least doubled that of MRD-positive CR patients;

conversely, both MFC and ASO-PCR showed that CR patients with persistent

MRD had significantly inferior OS vs. MRD-negative cases. MDR has also

proven to be relevant in both standard- as well as high-risk patients. Altogether,

these results strongly support the rationale for implementing MRD assessment




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to re-define and improve current CR criteria in MM. The time has come to

implement MRD monitoring in prospective clinical trials and fully establish its

role in the management of MM patients.

Grant Support

This study was supported by the Cooperative Research Thematic Network of

the Red de Cancer (Cancer Network of Excellence) (RD12/0036/0058; to J. San

Miguel); Instituto de Salud Carlos III, Spain, Instituto de Salud Carlos

III/Subdirección General de Investigación Sanitaria (FIS: PI1202311; to R.

Garcia-Sanz; Sara Borrell: CD13/00340; to B. Paiva); Junta de Castilla y León

(HUS412A12-1; to B. Paiva); and Asociación Española Contra el Cáncer

(GCB120981SAN; to J. San Miguel).

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Table 1. Summary of the most relevant studies based on MFC detection of MRD in MM. It should be noted that albeit most of the published

results were obtained by former (4-7 color) and less sensitive (10-4) MFC approaches, the persistence of MRD is consistently associated with a

significantly shorter PFS and often OS as compared to MRD-negative patients.

REFERENCE N SETTING METHOD LOD APPLICAB IMMUNOPHENOTYPIC REMISSION

PFS ACCORDING

TO MRD P

OS ACCORDING

TO MRD P

San Miguel et al., 2002 (22) 87 CT or CT+ASCT

4-color MFC 10-4 NA 26% 60m vs 34m 0.02 NA -

Rawstron et al., 2002 (20) 45 ASCT 3-color MFC 10-3 - 10-4 94% 56% (25/45) 35m vs 20m 0.03 76% vs 64% at

5-years 0.28

Paiva et al., 2008 (56) 295 CT+ASCT 4-color MFC 10-4 ~95% 42% (125/295) 71m vs 37m <0.001 NR vs 89m 0.002

Paiva et al., 2011 (7) 102 VMP or VTP 4-color MFC 10-4 - 10-5 ~95% 43% (44/102) 90% vs 35% at

3-years <0.001 94% vs 70% at 3-years 0.08

Paiva et al., 2012 (58) 241 (CR)

CTn or TD or CT/Btz or

VTD +ASCT

4-color MFC 10-4 - 10-5 ~95% 74% (154/241) 86% vs 58% at

3-years <0.001 94% vs 80% at 3-years 0.001

Rawston et al., 2013 (57) 397 CTD or CVAD + ASCT

6-color MFC 10-4 NA 62% (247/397) 29m vs 16m <0.001 81m vs 59m 0.02

Roussel et al., 2014 (59) 31 VRD + ASCT + VRD + Len

7-color MFC 10-5 NA 68% (21/31) 100% vs 30% at

3-years NA NA -

MFC: multiparameter flow cytometry; LOD: limit of detection; NA: not available; PFS: progression free survival; MRD: minimal residual disease;

OS: overall survival; ASCT: autologous stem cell transplantation; CT: VBMCP, VBAD; VMP: bortezomib, melphalan, prednisone; VTP:

bortezomib, thalidomide, prednisone; TD: thalidomide, dexamethasone; VTD: bortezomib, thalidomide, dexamethasone; CTD:

cyclophosphamide, thalidomide, dexamethasone; CVAD: cyclophosphamide, vincristine, doxorubicin, dexamethasone; VRD: bortezomib,

lenalidomide, dexamethasone; Len: lenalidomide; CR: complete response.

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Table 2. Summary of the most relevant studies based on PCR detection of MRD in MM. The low number of cases in most of the series is

probably due to the technical problems related to this technique. However, in all studies the persistence of MRD was associated with a

significantly shorter PFS and often OS as compared to MRD-negative patients.

REFERENCE N SETTING METHOD LOD APPLICAB MOLECULAR REMISSION

PFS ACCORDING

TO MRD P

OS ACCORDING

TO MRD P

Martinelli et al., 2000 (65) 50 ASCT o ALLO ASO 10-5 88% (44/50) 27% (12/44) 110m vs 35m <.005 NA -

Ladetto et al., 2000 (67) 29 ASCT RT NESTED

10-4 10-3

66% 73-100% NA NA - NA -

Corradini et al., 2003 (40) 70 ALLO ASO 10-6 69% 33% (16/48) 100% vs 0%1 NA -

Bakkus et al., 2004 (68) 87 ASCT ASO 10-4 77% 35% (21/60)≤ 0.015% 65% (39/60) >0.015% 64m vs 16m 0.001 NA ns

Galimberti et al., 2005 (76) 20 ASCT+ALLO fASO 10-3-10-5 NA 15% (3)

60% (12) NA - 76% vs 34% at 2ys .03

Sarasquete et al., 2005 (39) 24 ASCT ASO 5x10-5-10-

5 75% (24/32) 29% (7/24) 34m vs 15m .042 NA -

Martínez-Sánchez et al., 2008 (69) 53 ASCT fASO 10-3-10-4 91% 53% (28/53) 68% vs 28% .001 86% vs 68% ns

Putkonen et al., 2010 (71) 37 ASCT&ALLO RQ 10-4-10-5 86% 53% (16/30)

71% (5/7) 70m vs 19m .003 median not reached .1

Ladetto, et al., 2010 (70) Ferrero et al., 2014 (72) 39 ASCT+VTD RQ

NESTED 10-6 51% 18% NR vs 38mo vs 9 ms2 <.001 72% vs 48% at

8 ys .041

Korthals et al., 2012 (41) 53 ASCT RQ-ASO 10-4-10-5 78% 48% (26/53) 35 vs 20 .001 70 vs 45 .04

Puig et al., 2014 (49) 103 ASCT RQ-ASO 10-5 42% 46% NR vs 31mo .002 NR vs 60mo3 .008

PCR: polymerase chain reaction; NA: not available; fASO: fluorescent PCR; LOD: limit of detection; APPLICAB: applicability; PFS: progression

free survival; MRD: minimal residual disease; OS: overall survival; ASCT: autologous stem cell transplantation; ALLO: allogeneic stem cell

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transplantation; ASO: allelic specific oligonucleotide PCR; m: months; ns: non significant; VTD: bortezomib (V), thalidomide(T) y

dexamethasone (D); RQ: real-time quantitative PCR; The cumulative risk of relapse was 0% for PCR-negative patients and 100% for PCR-

positive patients; median remission duration was not reached for patients in major MRD response, 38 months for those experiencing MRD

reappearance and 9 months for patients with MRD persistence (P<0.001); among patients in CR.

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Published OnlineFirst March 9, 2015.Clin Cancer Res Bruno Paiva, Noemi Puig, Ramón García-Sanz, et al. Multiple Myeloma Clinical Practice?Is This the Time to Introduce Minimal Residual Disease in

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