Is this the time to introduce minimal residual disease in multiple myeloma clinical practice?

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

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

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Clinica 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.

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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) (13). 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 tomographycomputerized 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 Tcell 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 wellstandardized 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 PCRbased 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 ASORQ-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 (10-5) 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 (10fold 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 ASOPCR 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-tohead 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 nextgeneration 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 MRDpositivity 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). References 1.

Mateos MV, San Miguel JF. How should we treat newly diagnosed

multiple myeloma patients? Hematology Am Soc Hematol Educ Program 2013;2013:488-95. 2.

McCarthy PL, Hahn T. Strategies for induction, autologous hematopoietic

stem cell transplantation, consolidation, and maintenance for transplantationeligible multiple myeloma patients. Hematology Am Soc Hematol Educ Program 2013;2013:496-503. 3.

Kumar SK, Dispenzieri A, Lacy MQ, Gertz MA, Buadi FK, Pandey S, et

al. Continued improvement in survival in multiple myeloma: changes in early mortality and outcomes in older patients. Leukemia 2014;28:1122-8. 4.

Durie BG, Harousseau JL, Miguel JS, Blade J, Barlogie B, Anderson K,

et al. International uniform response criteria for multiple myeloma. Leukemia 2006;20:1467-73.

18



5.

Kapoor P, Kumar SK, Dispenzieri A, Lacy MQ, Buadi F, Dingli D, et al.

Importance of achieving stringent complete response after autologous stem-cell transplantation in multiple myeloma. J Clin Oncol 2013;31:4529-35. 6.

de Larrea CF, Cibeira MT, Elena M, Arostegui JI, Rosinol L, Rovira M, et

al. Abnormal serum free light chain ratio in patients with multiple myeloma in complete remission has strong association with the presence of oligoclonal bands: implications for stringent complete remission definition. Blood 2009;114:4954-6. 7.

Paiva B, Martinez-Lopez J, Vidriales MB, Mateos MV, Montalban MA,

Fernandez-Redondo E, et al. Comparison of immunofixation, serum free light chain, and immunophenotyping for response evaluation and prognostication in multiple myeloma. J Clin Oncol 2011;29:1627-33. 8.

San-Miguel JF, Paiva B, Gutiérrez NC. New tools for diagnosis and

monitoring of multiple myeloma. Am Soc Clin Oncol Educ Book 2013. doi: 10.1200/EdBook_AM.2013.33.e313. 9.

Usmani SZ, Mitchell A, Waheed S, Crowley J, Hoering A, Petty N, et al.

Prognostic implications of serial 18-fluoro-deoxyglucose emission tomography in multiple myeloma treated with total therapy 3. Blood 2013;121:1819-23. 10.

Zamagni E, Patriarca F, Nanni C, Zannetti B, Englaro E, Pezzi A, et al.

Prognostic relevance of 18-F FDG PET/CT in newly diagnosed multiple myeloma patients treated with up-front autologous transplantation. Blood 2011;118:5989-95. 11.

Almeida J, Orfao A, Ocqueteau M, Mateo G, Corral M, Caballero MD, et

al. High-sensitive immunophenotyping and DNA ploidy studies for the

19



investigation of minimal residual disease in multiple myeloma. Br J Haematol 1999;107:121-31. 12.

Garcia-Sanz R, Orfao A, Gonzalez M, Tabernero MD, Blade J, Moro MJ,

et al. Primary plasma cell leukemia: clinical, immunophenotypic, DNA ploidy, and cytogenetic characteristics. Blood 1999;93:1032-7. 13.

Lin P, Owens R, Tricot G, Wilson CS. Flow cytometric immunophenotypic

analysis of 306 cases of multiple myeloma. Am J Clin Pathol 2004;121:482-8. 14.

Rawstron AC, Barrans SL, Blythe D, English A, Richards SJ, Fenton JA,

et al. In multiple myeloma, only a single stage of neoplastic plasma cell differentiation can be identified by VLA-5 and CD45 expression. Br J Haematol 2001;113:794-802. 15.

Dahl IM, Rasmussen T, Kauric G, Husebekk A. Differential expression of

CD56 and CD44 in the evolution of extramedullary myeloma. Br J Haematol 2002;116:273-7. 16.

Davies FE, Forsyth PD, Rawstron AC, Owen RG, Pratt G, Evans PA, et

al. The impact of attaining a minimal disease state after high-dose melphalan and autologous transplantation for multiple myeloma. Br J Haematol 2001;112:814-9. 17.

Mateo MG, San MI, Orfao de MA. Immunophenotyping of plasma cells in

multiple myeloma. Methods Mol Med 2005;113:5-24. 18.

Nowakowski GS, Witzig TE, Dingli D, Tracz MJ, Gertz MA, Lacy MQ, et

al. Circulating plasma cells detected by flow cytometry as a predictor of survival in 302 patients with newly diagnosed multiple myeloma. Blood 2005;106:22769.

20



19.

Perez-Andres M, Almeida J, Martin-Ayuso M, Moro MJ, Martin-Nunez G,

Galende J, et al. Clonal plasma cells from monoclonal gammopathy of undetermined significance, multiple myeloma and plasma cell leukemia show different expression profiles of molecules involved in the interaction with the immunological bone marrow microenvironment. Leukemia 2005;19:449-55. 20.

Rawstron AC, Davies FE, DasGupta R, Ashcroft AJ, Patmore R, Drayson

MT, et al. Flow cytometric disease monitoring in multiple myeloma: the relationship between normal and neoplastic plasma cells predicts outcome after transplantation. Blood 2002;100:3095-100. 21.

Robillard N, Pellat-Deceunynck C, Bataille R. Phenotypic

characterization of the human myeloma cell growth fraction. Blood 2005;105:4845-8. 22.

San Miguel JF, Almeida J, Mateo G, Blade J, Lopez-Berges C, Caballero

D, et al. Immunophenotypic evaluation of the plasma cell compartment in multiple myeloma: a tool for comparing the efficacy of different treatment strategies and predicting outcome. Blood 2002;99:1853-6. 23.

San Miguel JF, Gutierrez NC, Mateo G, Orfao A. Conventional

diagnostics in multiple myeloma. Eur J Cancer 2006;42:1510-9. 24.

Moreau P, Robillard N, Jego G, Pellat C, Le Gouill S, Thoumi S, et al.

Lack of CD27 in myeloma delineates different presentation and outcome. Br J Haematol 2006;132:168-70. 25.

Rawstron AC, Orfao A, Beksac M, Bezdickova L, Brooimans RA,

Bumbea H, et al. Report of the European Myeloma Network on multiparametric flow cytometry in multiple myeloma and related disorders. Haematologica 2008;93:431-8.

21



26.

Paiva B, Almeida J, Perez-Andres M, Mateo G, Lopez A, Rasillo A, et al.

Utility of flow cytometry immunophenotyping in multiple myeloma and other clonal plasma cell-related disorders. Cytometry B Clin Cytom 2010;78:239-52. 27.

van Dongen JJ, Lhermitte L, Bottcher S, Almeida J, van der Velden VH,

Flores-Montero J, et al. EuroFlow antibody panels for standardized ndimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes. Leukemia 2012;26:1908-75. 28.

Davies FE, Rawstron AC, Owen RG, Morgan GJ. Minimal residual

disease monitoring in multiple myeloma. Best Pract Res Clin Haematol 2002;15:197-222. 29.

Martinelli G, Terragna C, Lemoli RM, Cavo M, Benni M, Motta MR, et al.

Clinical and molecular follow-up by amplification of the CDR-III IgH region in multiple myeloma patients after autologous transplantation of hematopoietic CD34+ stem cells. Haematologica 1999;84:397-404. 30.

Owen RG, Goulden NJ, Oakhill A, Shiach C, Evans PA, Potter MN, et al.

Comparison of fluorescent consensus IgH PCR and allele-specific oligonucleotide probing in the detection of minimal residual disease in childhood ALL. Br J Haematol 1997;97:457-9. 31.

Boyd SD, Gaeta BA, Jackson KJ, Fire AZ, Marshall EL, Merker JD, et al.

Individual variation in the germline Ig gene repertoire inferred from variable region gene rearrangements. J Immunol 2010;184:6986-92. 32.

Freeman JD, Warren RL, Webb JR, Nelson BH, Holt RA. Profiling the T-

cell receptor beta-chain repertoire by massively parallel sequencing. Genome Res 2009;19:1817-24.

22



33.

Robins H, Desmarais C, Matthis J, Livingston R, Andriesen J, Reijonen

H, et al. Ultra-sensitive detection of rare T cell clones. J Immunol Methods 2012;375:14-9. 34.

Logan AC, Zhang B, Narasimhan B, Carlton V, Zheng J, Moorhead M, et

al. Minimal residual disease quantification using consensus primers and highthroughput IGH sequencing predicts post-transplant relapse in chronic lymphocytic leukemia. Leukemia 2013;27:1659-65. 35.

Faham M, Zheng J, Moorhead M, Carlton VE, Stow P, Coustan-Smith E,

et al. Deep-sequencing approach for minimal residual disease detection in acute lymphoblastic leukemia. Blood 2012;120:5173-80. 36.

Huff CA, Matsui W. Multiple myeloma cancer stem cells. J Clin Oncol

2008;26:2895-900. 37.

Thiago LS, Perez-Andres M, Balanzategui A, Sarasquete ME, Paiva B,

Jara-Acevedo M, et al. Circulating clonotypic B cells in multiple myeloma and monoclonal gammopathy of undetermined significance. Haematologica 2014;99:155-62. 38.

Ferrero S, Drandi D, Mantoan B, Ghione P, Omede P, Ladetto M.

Minimal residual disease detection in lymphoma and multiple myeloma: impact on therapeutic paradigms. Hematol Oncol 2011;29:167-76. 39.

Sarasquete ME, Garcia-Sanz R, Gonzalez D, Martinez J, Mateo G,

Martinez P, et al. Minimal residual disease monitoring in multiple myeloma: a comparison between allelic-specific oligonucleotide real-time quantitative polymerase chain reaction and flow cytometry. Haematologica 2005;90:136572.

23



40.

Corradini P, Cavo M, Lokhorst H, Martinelli G, Terragna C, Majolino I, et

al. Molecular remission after myeloablative allogeneic stem cell transplantation predicts a better relapse-free survival in patients with multiple myeloma. Blood 2003;102:1927-9. 41.

Korthals M, Sehnke N, Kronenwett R, Bruns I, Mau J, Zohren F, et al.

The level of minimal residual disease in the bone marrow of patients with multiple myeloma before high-dose therapy and autologous blood stem cell transplantation is an independent predictive parameter. Biol Blood Marrow Transplant 2012;18:423-31 e3. 42.

Langerak AW, Groenen PJ, Bruggemann M, Beldjord K, Bellan C,

Bonello L, et al. EuroClonality/BIOMED-2 guidelines for interpretation and reporting of Ig/TCR clonality testing in suspected lymphoproliferations. Leukemia 2012;26:2159-71. 43.

van der Velden VH, Cazzaniga G, Schrauder A, Hancock J, Bader P,

Panzer-Grumayer ER, et al. Analysis of minimal residual disease by Ig/TCR gene rearrangements: guidelines for interpretation of real-time quantitative PCR data. Leukemia 2007;2:604-11. 44.

Biran N, Ely S, Chari A. Controversies in the assessment of minimal

residual disease in multiple myeloma: clinical significance of minimal residual disease negativity using highly sensitive techniques. Curr Hematol Malig Rep 2014;9:368-78. 45.

Cooke F, Bakkus M, Thielemans K, Pico JL, Apperley JF, Samson D.

Use of quantitative ASO-PCR to predict relapse in multiple myeloma. Br J Haematol 1999;105:317-9.

24



46.

Garcia-Sanz R, Lopez-Perez R, Langerak AW, Gonzalez D, Chillon MC,

Balanzategui A, et al. Heteroduplex PCR analysis of rearranged immunoglobulin genes for clonality assessment in multiple myeloma. Haematologica 1999;84:328-35. 47.

Kosmas C, Stamatopoulos K, Stavroyianni N, Zoi K, Belessi C, Viniou N,

et al. Origin and diversification of the clonogenic cell in multiple myeloma: lessons from the immunoglobulin repertoire. Leukemia 2000;14:1718-26. 48.

Sahota SS, Leo R, Hamblin TJ, Stevenson FK. Myeloma VL and VH

gene sequences reveal a complementary imprint of antigen selection in tumor cells. Blood 1997;89:219-26. 49.

Puig N, Sarasquete ME, Balanzategui A, Martinez J, Paiva B, Garcia H,

et al. Critical evaluation of ASO RQ-PCR for minimal residual disease evaluation in multiple myeloma. A comparative analysis with flow cytometry. Leukemia 2014;28:391-7. 50.

Puig N, Sarasquete ME, Alcoceba M, Balanzategui A, Chillon MC,

Sebastian E, et al. The use of CD138 positively selected marrow samples increases the applicability of minimal residual disease assessment by PCR in patients with multiple myeloma. Ann Hematol 2013;92:97-100. 51.

Martinez-Lopez J, Lahuerta JJ, Pepin F, Gonzalez M, Barrio S, Ayala R,

et al. Prognostic value of deep sequencing method for minimal residual disease detection in multiple myeloma. Blood 2014;123:3073-9. 52.

Boyd SD, Marshall EL, Merker JD, Maniar JM, Zhang LN, Sahaf B, et al.

Measurement and clinical monitoring of human lymphocyte clonality by massively parallel VDJ pyrosequencing. Sci Transl Med 2009;1:12ra23.

25



53.

Martinez-Lopez J, Fulciniti M, Barrio S, Carlton V, Moorhead M, Lahuerta

JJ, et al. Deep sequencing reveals oligoclonality at the immunoglobulin locus in multiple myeloma patients [abstract]. In: Proceedings of the 55th ASH Annual Meeting and Exposition; 2013 Dec 7-10; New Orleans, LA. Washington (DC): American Society of Hematology. Abstract nr 401. 54.

van Dongen JJ, Langerak AW, Bruggemann M, Evans PA, Hummel M,

Lavender FL, et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 2003;17:2257-317. 55.

San Miguel JF, Almeida J, Mateo G, Blade J, Lopez-Berges C, Caballero

D, et al. Immunophenotypic evaluation of the plasma cell compartment in multiple myeloma: a tool for comparing the efficacy of different treatment strategies and predicting outcome. Blood 2002;99:1853-6. 56.

Paiva B, Vidriales MB, Cervero J, Mateo G, Perez JJ, Montalban MA, et

al. Multiparameter flow cytometric remission is the most relevant prognostic factor for multiple myeloma patients who undergo autologous stem cell transplantation. Blood 2008;112:4017-23. 57.

Rawstron AC, Child JA, de Tute RM, Davies FE, Gregory WM, Bell SE,

et al. Minimal residual disease assessed by multiparameter flow cytometry in multiple myeloma: impact on outcome in the Medical Research Council Myeloma IX Study. J Clin Oncol 2013;31:2540-7. 58.

Paiva B, Gutierrez NC, Rosinol L, Vidriales MB, Montalban MA, Martinez-

Lopez J, et al. High-risk cytogenetics and persistent minimal residual disease by multiparameter flow cytometry predict unsustained complete response after

26



autologous stem cell transplantation in multiple myeloma. Blood 2012;119:68791. 59.

Roussel M, Lauwers-Cances V, Robillard N, Hulin C, Leleu X,

Benboubker L, et al. Front-line transplantation program with lenalidomide, bortezomib, and dexamethasone combination as induction and consolidation followed by lenalidomide maintenance in patients with multiple myeloma: a phase II study by the Intergroupe Francophone du Myelome. J Clin Oncol 2014;32:2712-7. 60.

Corradini P, Voena C, Astolfi M, Ladetto M, Tarella C, Boccadoro M, et

al. High-dose sequential chemoradiotherapy in multiple myeloma: residual tumor cells are detectable in bone marrow and peripheral blood cell harvests and after autografting. Blood 1995;85:1596-602. 61.

Bjorkstrand B, Ljungman P, Bird JM, Samson D, Gahrton G. Double

high-dose chemoradiotherapy with autologous stem cell transplantation can induce molecular remissions in multiple myeloma. Bone Marrow Transplant 1995;15:367-71. 62.

Swedin A, Lenhoff S, Olofsson T, Thuresson B, Westin J. Clinical utility

of immunoglobulin heavy chain gene rearrangement identification for tumour cell detection in multiple myeloma. Br J Haematol 1998;103:1145-51. 63.

Corradini P, Voena C, Tarella C, Astolfi M, Ladetto M, Palumbo A, et al.

Molecular and clinical remissions in multiple myeloma: role of autologous and allogeneic transplantation of hematopoietic cells. J Clin Oncol 1999;17:208-15. 64.

Martinelli G, Terragna C, Zamagni E, Ronconi S, Tosi P, Lemoli R, et al.

Polymerase chain reaction-based detection of minimal residual disease in

27



multiple myeloma patients receiving allogeneic stem cell transplantation. Haematologica 2000;85:930-4. 65.

Martinelli G, Terragna C, Zamagni E, Ronconi S, Tosi P, Lemoli RM, et

al. Molecular remission after allogeneic or autologous transplantation of hematopoietic stem cells for multiple myeloma. J Clin Oncol 2000;18:2273-81. 66.

Cavo M, Terragna C, Martinelli G, Ronconi S, Zamagni E, Tosi P, et al.

Molecular monitoring of minimal residual disease in patients in long-term complete remission after allogeneic stem cell transplantation for multiple myeloma. Blood 2000;96:355-7. 67.

Ladetto M, Donovan JW, Harig S, Trojan A, Poor C, Schlossnan R, et al.

Real-Time polymerase chain reaction of immunoglobulin rearrangements for quantitative evaluation of minimal residual disease in multiple myeloma. Biol Blood Marrow Transplant 2000;6:241-53. 68.

Bakkus MH, Bouko Y, Samson D, Apperley JF, Thielemans K, Van

Camp B, et al. Post-transplantation tumour load in bone marrow, as assessed by quantitative ASO-PCR, is a prognostic parameter in multiple myeloma. Br J Haematol 2004;126:665-74. 69.

Martinez-Sanchez P, Montejano L, Sarasquete ME, Garcia-Sanz R,

Fernandez-Redondo E, Ayala R, et al. Evaluation of minimal residual disease in multiple myeloma patients by fluorescent-polymerase chain reaction: the prognostic impact of achieving molecular response. Br J Haematol 2008;142:766-74. 70.

Ladetto M, Pagliano G, Ferrero S, Cavallo F, Drandi D, Santo L, et al.

Major tumor shrinking and persistent molecular remissions after consolidation

28



with bortezomib, thalidomide, and dexamethasone in patients with autografted myeloma. J Clin Oncol 2010;28:2077-84. 71.

Putkonen M, Kairisto V, Juvonen V, Pelliniemi TT, Rauhala A, Itala-

Remes M, et al. Depth of response assessed by quantitative ASO-PCR predicts the outcome after stem cell transplantation in multiple myeloma. Eur J Haematol 2010;85:416-23. 72.

Ferrero S, Ladetto M, Drandi D, Cavallo F, Genuardi E, Urbano M, et al.

Long-term results of the GIMEMA VEL-03-096 trial in MM patients receiving VTD consolidation after ASCT: MRD kinetics' impact on survival. Leukemia 2014 Jul 6. [Epub ahead of print]. 73.

Lipinski E, Cremer FW, Ho AD, Goldschmidt H, Moos M. Molecular

monitoring of the tumor load predicts progressive disease in patients with multiple myeloma after high-dose therapy with autologous peripheral blood stem cell transplantation. Bone Marrow Transplant 2001;28:957-62. 74.

Lioznov M, Badbaran A, Fehse B, Bacher U, Zander AR, Kroger NM.

Monitoring of minimal residual disease in multiple myeloma after allo-SCT: flow cytometry vs PCR-based techniques. Bone Marrow Transplant 2008;41:913-6. 75.

Ladetto M, Bruggemann M, Monitillo L, Ferrero S, Pepin F, Drandi D, et

al. Next-generation sequencing and real-time quantitative PCR for minimal residual disease detection in B-cell disorders. Leukemia 2014;28:1299-307. 76.

Galimberti S, Benedetti E, Morabito F, Papineschi F, Callea V, Fazzi R,

et al. Prognostic role of minimal residual disease in multiple myeloma patients after non-myeloablative allogeneic transplantation. Leuk Res 2005;29:961-6.

29


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

IMMUNOPHENOTY PIC REMISSION

PFS ACCORDING TO MRD

P

OS ACCORDING TO MRD

P

San Miguel et al., 2002 (22)

87

CT or CT+ASCT

10-4

NA

26%

60m vs 34m

0.02

NA

-

Rawstron et al., 2002 (20)

45

ASCT

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

10-4

~95%

42% (125/295)

71m vs 37m

Minimal residual disease in multiple myeloma: bringing the bench to the bedside.

A CD138-independent strategy to detect minimal residual disease and circulating tumour cells in multiple myeloma.

The current status of minimal residual disease assessment in myeloma.

Minimal Residual Disease Assessment in the Context of Multiple Myeloma Treatment.

The prognostic value of multiparameter flow cytometry minimal residual disease assessment in relapsed multiple myeloma.

Prognostic value of deep sequencing method for minimal residual disease detection in multiple myeloma.

New criteria for response assessment: role of minimal residual disease in multiple myeloma.

Minimal residual disease analysis in myeloma - when, why and where.

Next Generation Flow for highly sensitive and standardized detection of minimal residual disease in multiple myeloma.

Minimal residual disease testing after stem cell transplantation for multiple myeloma.

Minimal residual disease.

Comparative analysis of minimal residual disease detection by multiparameter flow cytometry and enhanced ASO RQ-PCR in multiple myeloma.

Is it time to use minimal residual disease to stratify post-remission treatment for acute myeloid leukemia?

It is a great pleasure to introduce to you the fourth edition of this year.

Minimal residual disease after transplantation or lenalidomide-based consolidation in myeloma patients: a prospective analysis.

Minimal residual disease following autologous stem cell transplant in myeloma: impact on outcome is independent of induction regimen.

Minimal residual disease in myeloma by flow cytometry: independent prediction of survival benefit per log reduction.

Analytical and clinical validation of a novel in-house deep-sequencing method for minimal residual disease monitoring in a phase II trial for multiple myeloma.

Multiple Myeloma, Version 2.2016: Clinical Practice Guidelines in Oncology.

Acting on minimal residual disease in ALL.

Minimal Residual Disease Monitoring in Adult ALL to Determine Therapy.

Clinical value of pre-transplant minimal residual disease in childhood lymphoblastic leukaemia: the results of the French minimal residual disease-guided protocol.

Minimal Residual Disease in NPM1-Mutated AML.

Consensus guidelines for myeloma minimal residual disease sample staining and data acquisition.