Increasing therapeutic options and prolonged survival in multiple myeloma have raised interest in the concept of depth of response and its importance to predict patients' outcomes. Although the efficacy of current treatment approaches has greatly improved in the past decade, the definition of complete response (CR) remains unaltered and continues to use conventional serological and morphologic 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 multiple myeloma. 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 methodologic aspects, specific characteristics, and clinical significance of MRD monitoring by flow cytometry, PCR, and next-generation sequencing. Clin Cancer Res; 21(9); 2001–8. ©2015 AACR.

In the past few decades, important changes have been introduced in the treatment of multiple myeloma, resulting in unprecedented complete response (CR) rates and prolonged progression-free survival (PFS) and overall survival (OS; refs. 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 clonal plasma cells (PC) by immunohistochemistry/immunofluorescence as additional requirements to define stringent CR (4). Recently, Kapoor and colleagues (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 κ/λ ratios (8). Thus, it becomes evident that current criteria used to assess response lag behind the extraordinary evolution in the treatment of multiple myeloma patients, and more sensitive techniques are needed to detect true minimal residual disease (MRD) for new CR definitions. This change would help to improve monitoring of treatment efficacy and to avoid overtreatment and undertreatment, particularly during consolidation and maintenance.

Current techniques to monitor MRD can be divided into those measuring extramedullary versus those detecting intramedullary disease. MRI and 18-fluoro-deoxyglucose PET/CT have emerged as promising tools to measure extramedullary MRD. In particular, PET/CT has been shown to be of prognostic value as soon as day 7 of induction therapy (9) and more importantly, among patients in conventional CR after high-dose therapy/autologous stem cell transplantation (HDT/ASCT; ref. 10). For detection of intramedullary disease, both multiparameter flow cytometry (MFC) immunophenotyping and molecular assessment of immunoglobulin rearrangements are the pivotal techniques. The present review focuses on current state-of-the-art intramedullary MRD monitoring in multiple myeloma.

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 bone marrow PCs (25), and at least one (8-color) combination of such markers is highly recommended for multiple myeloma 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 versus normal PCs (27). The principal component analysis (PCA) of 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 a strategy is likely the method of choice for accurate and semiautomated flow-MRD monitoring.

Allele-specific oligonucleotide PCR (ASO-PCR)

PCR-based detection of multiple myeloma 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 framework regions of the IGHV (which are less variable than the complementary determinant regions, CDR) 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) complementary to the most variable regions of the IGVH rearrangement (CDR 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 real-time quantitative 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 joining region) and a fluorescent probe to monitor the amplification.

Next-generation sequencing (NGS)

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, which relies on light signals emitted during the re-synthesis of small DNA fragments previously produced by bridge amplification; and (iii) ion semiconductor sequencing, which 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.

Multiparameter flow cytometry

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 allows for fast and cost-effective measurement of millions of leukocytes. Consequently, although 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 least 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 incorporate a quality check of the whole sample cellularity (i.e., B-cell precursors, erythroblasts, etc.), which is critical to ensure sample quality since hemodiluted bone marrow 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 as that in which 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. The requirement for extensive expertise in MFC analysis and the lack of a well-standardized flow-MRD method have been mentioned 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 multiple myeloma cancer stem cells outside the PC compartment (i.e., memory B cells; ref. 36). However, recent investigations conducted with optimized molecular assessment of clonal variable, joining, and diversity (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; ref. 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 detection of clonal heterogeneity in approximately 30% of newly diagnosed patients, but also makes it possible to monitor all the different phenotypic subclones throughout a patient's 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.

Allele-specific oligonucleotide PCR

PCR approaches for MRD detection do not require an immediate sample processing because they are unaffected by preanalytical 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; refs. 40, 41). PCR strategies have passed an exhaustive process of validation and standardization for MRD testing in different hematologic malignancies, which make them readily available and 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, multiple myeloma is a postgerminal 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 bone marrow PCs may significantly increase the applicability of PCR-based MRD studies in multiple myeloma (50). Nevertheless, the technique remains costly, laborious, and accordingly, difficult to implement into routine clinical practice.

Next-generation sequencing

The greatest advantage of NGS approaches for MRD detection in multiple myeloma is its sensitivity which, without compromising specificity, is estimated to be in the range of 10−5 to 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 (N. Puig and colleagues; accepted for publication). Therefore, because 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 less methodologic complexity. In particular, the latest advantage would be crucial in multiple myeloma because it removes the need of standard curve construction, which is the main reason of ASO-PCR failure in multiple myeloma (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). Because 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.

Multiparameter flow cytometry

The prognostic value of MFC-based MRD monitoring in multiple myeloma was introduced in 2002 by San Miguel and colleagues (22) and Rawstron and colleagues (20), both studies suggesting the utility of monitoring the bone marrow PC compartment among multiple myeloma 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 multiple myeloma patients receiving uniform treatment including HDT/SCT (55). MRD negativity at day 100 after ASCT translated to 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 (56). This outcome advantage was equally demonstrable in patients achieving CR. Because in multiple myeloma 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 (55, 56). 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, because 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 multiple myeloma 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 with the placebo arm; in MRD-negative patients, a similar trend was observed (56). Further analyses by the PETHEMA/GEM have shown that combining the 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-year median OS): those with baseline high-risk cytogenetics plus persistent MRD after ASCT (57). The GEM 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 (58). 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-color) immunophenotyping. Table 1 summarizes the most relevant flow-based MRD studies reported in multiple myeloma.

Table 1.

Summary of the most relevant studies based on MFC detection of MRD in multiple myeloma

ReferenceNSettingMethodLODApplicabImmunophenotypic remissionPFS according to MRDPOS according to MRDP
San Miguel et al., 2002 (22) 87 CT or CT+ASCT 4-color MFC 10−4 NA 26% 60 mo vs. 34 mo 0.02 NA — 
Rawstron et al., 2002 (20) 45 ASCT 3-color MFC 10−3–10−4 94% 56% (25/45) 35 mo vs. 20 mo 0.03 76% vs. 64% at 5 years 0.28 
Paiva et al., 2008 (55) 295 CT+ASCT 4-color MFC 10−4 ∼95% 42% (125/295) 71 mo vs. 37 mo <0.001 NR vs. 89 m 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 (57) 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 
Rawstron et al., 2013 (56) 397 CTD or CVAD + ASCT 6-color MFC 10−4 NA 62% (247/397) 29 mo vs. 16 mo <0.001 81 m vs. 59 mo 0.02 
Roussel et al., 2014 (58) 31 VRD + ASCT + VRD + Len 7-color MFC 10−5 NA 68% (21/31) 100% vs. 30% at 3 years NA NA — 
ReferenceNSettingMethodLODApplicabImmunophenotypic remissionPFS according to MRDPOS according to MRDP
San Miguel et al., 2002 (22) 87 CT or CT+ASCT 4-color MFC 10−4 NA 26% 60 mo vs. 34 mo 0.02 NA — 
Rawstron et al., 2002 (20) 45 ASCT 3-color MFC 10−3–10−4 94% 56% (25/45) 35 mo vs. 20 mo 0.03 76% vs. 64% at 5 years 0.28 
Paiva et al., 2008 (55) 295 CT+ASCT 4-color MFC 10−4 ∼95% 42% (125/295) 71 mo vs. 37 mo <0.001 NR vs. 89 m 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 (57) 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 
Rawstron et al., 2013 (56) 397 CTD or CVAD + ASCT 6-color MFC 10−4 NA 62% (247/397) 29 mo vs. 16 mo <0.001 81 m vs. 59 mo 0.02 
Roussel et al., 2014 (58) 31 VRD + ASCT + VRD + Len 7-color MFC 10−5 NA 68% (21/31) 100% vs. 30% at 3 years NA NA — 

NOTE: Although most of the published results were obtained by former (4–7 colors) and less sensitive (10−4) MFC approaches, the persistence of MRD is consistently associated with a significantly shorter PFS and often OS as compared with MRD-negative patients.

Abbreviations: Applicab, applicability; CT, VBMCP/VBAD; CTD, cyclophosphamide, thalidomide, dexamethasone; CVAD, cyclophosphamide, vincristine, doxorubicin, dexamethasone; Len, lenalidomide; LOD, limit of detection; mo, months; NA, not available; TD, thalidomide, dexamethasone; VMP, bortezomib, melphalan, prednisone; VRD, bortezomib, lenalidomide, dexamethasone; VTD, bortezomib, thalidomide, dexamethasone; VTP, bortezomib, thalidomide, prednisone.

Allele-specific oligonucleotide PCR

A considerable number of studies have explored the value of PCR-based MRD monitoring in multiple myeloma (Table 2; refs. 59–65). 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; refs. 39–41, 49, 50, 64, 66–71) Using nonquantitative approaches, the percentage of molecular remissions observed after allogeneic SCT was significantly higher as compared with patients undergoing autologous SCT, suggesting a role for this technique to evaluate treatment efficacy. Furthermore, Lipinski and colleagues, in a retrospective study performed in 13 patients undergoing ASCT, suggested the potential value of ASO-PCR monitoring to predict progression (72), and this notion of MRD reappearance heralding relapse has been recently confirmed by the GIMEMA group (71).

Table 2.

Summary of the most relevant studies based on PCR detection of MRD in multiple myeloma

ReferenceNSettingMethodLODApplicabMolecular remissionPFS according to MRDPOS according to MRDP
Martinelli et al., 2000 (64) 50 ASCT o ALLO ASO 10−5 88% (44/50) 27% (12/44) 110 mo vs. 35 mo <.005 NA — 
Ladetto et al., 2000 (66) 29 ASCT RT NESTED 10−410−3 66%73%–100% NA NA — NA — 
Corradini et al., 2003 (40) 70 ALLO ASO 10−6 69% 33% (16/48) 100% vs. 0%a  NA — 
Bakkus et al., 2004 (67) 87 ASCT ASO 10−4 77% 35% (21/60)≤ 0.015%65% (39/60) >0.015% 64 mo vs. 16 mo 0.001 NA NS 
Galimberti et al., 2005 (75) 20 ASCT+ALLO fASO 10−3–10−5 NA 15% (3)60% (12) NA 76% vs. 34% at 2 years 0.03 
Sarasquete et al., 2005 (39) 24 ASCT ASO 5 × 10−5–10−5 75% (24/32) 29% (7/24) 34 mo vs. 15 mo 0.042 NA — 
Martinez-Sanchez et al., 2008 (68) 53 ASCT fASO 10−3–10−4 91% 53% (28/53) 68% vs. 28% 0.001 86% vs. 68% NS 
Putkonen et al., 2010 (70) 37 ASCT and ALLO RQ 10−4–10−5 86% 53% (16/30)71% (5/7) 70 mo vs. 19 mo 0.003 Median not reached 0.1 
Ladetto et al., 2010 (69)Ferrero et al., 2014 (71) 39 ASCT+VTD RQ NESTED 10−6 51% 18% NR vs. 38 mo vs. 9 mob <0.001 72% vs. 48% at 8 years 0.041 
Korthals et al., 2012 (41) 53 ASCT RQ-ASO 10−4–10−5 78% 48% (26/53) 35 vs. 20 0.001 70 vs. 45 0.04 
Puig et al., 2014 (49) 103 ASCT RQ-ASO 10−5 42% 46% NR vs. 31 mo 0.002 NR vs. 60 moc 0.008 
ReferenceNSettingMethodLODApplicabMolecular remissionPFS according to MRDPOS according to MRDP
Martinelli et al., 2000 (64) 50 ASCT o ALLO ASO 10−5 88% (44/50) 27% (12/44) 110 mo vs. 35 mo <.005 NA — 
Ladetto et al., 2000 (66) 29 ASCT RT NESTED 10−410−3 66%73%–100% NA NA — NA — 
Corradini et al., 2003 (40) 70 ALLO ASO 10−6 69% 33% (16/48) 100% vs. 0%a  NA — 
Bakkus et al., 2004 (67) 87 ASCT ASO 10−4 77% 35% (21/60)≤ 0.015%65% (39/60) >0.015% 64 mo vs. 16 mo 0.001 NA NS 
Galimberti et al., 2005 (75) 20 ASCT+ALLO fASO 10−3–10−5 NA 15% (3)60% (12) NA 76% vs. 34% at 2 years 0.03 
Sarasquete et al., 2005 (39) 24 ASCT ASO 5 × 10−5–10−5 75% (24/32) 29% (7/24) 34 mo vs. 15 mo 0.042 NA — 
Martinez-Sanchez et al., 2008 (68) 53 ASCT fASO 10−3–10−4 91% 53% (28/53) 68% vs. 28% 0.001 86% vs. 68% NS 
Putkonen et al., 2010 (70) 37 ASCT and ALLO RQ 10−4–10−5 86% 53% (16/30)71% (5/7) 70 mo vs. 19 mo 0.003 Median not reached 0.1 
Ladetto et al., 2010 (69)Ferrero et al., 2014 (71) 39 ASCT+VTD RQ NESTED 10−6 51% 18% NR vs. 38 mo vs. 9 mob <0.001 72% vs. 48% at 8 years 0.041 
Korthals et al., 2012 (41) 53 ASCT RQ-ASO 10−4–10−5 78% 48% (26/53) 35 vs. 20 0.001 70 vs. 45 0.04 
Puig et al., 2014 (49) 103 ASCT RQ-ASO 10−5 42% 46% NR vs. 31 mo 0.002 NR vs. 60 moc 0.008 

NOTE: 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 with MRD-negative patients.

Abbreviations: ALLO, allogeneic stem cell transplantation; Applicab, applicability; fASO, fluorescent PCR; mo, months; NA, not available; NR, not reached; NS, not statistically significant; RQ, real-time quantitative PCR; VTD, bortezomib (V), thalidomide (T), dexamethasone (D).

aThe cumulative risk of relapse was 0% for PCR-negative patients and 100% for PCR-positive patients.

bMedian remission duration was not reached for patients in major RD response, 38 months for those experiencing MRD reappearance, and 9 months for patients with MRD persistence (P < 0.001).

cAmong patients in CR.

Semiquantitative and quantitative approaches have also been used to predict patients' outcome according to MRD levels. Korthals and colleagues in a cohort of 53 patients undergoing ASCT have shown that different MRD levels by ASO-RQ-PCR before ASCT allowed to discriminate two groups of patients with different PFS and OS (41). Putkonen and colleagues 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 (70). Puig and colleagues, 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 and colleagues 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 (69). 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 (71).

Next-generation sequencing

Because NGS-based MRD monitoring is still a relatively recent approach, there is yet few data in multiple myeloma. However, the PETHEMA/GEM has already described favorable and promising results in a series of 133 multiple myeloma patients, including both transplant and non–transplant-eligible cases. 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 (51). Importantly, Martinez-Lopez and colleagues (51) 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. Similar results are also now being observed with more sensitive MFC (GEM; unpublished observations).

Multiparameter flow cytometry versus allele-specific oligonucleotide PCR

Three studies have directly compared MFC and ASO-PCR as alternative methods for MRD detection in multiple myeloma. In 2005, Sarasquete and colleagues quantified MRD levels in 24 patients at day 100 after HDT/ASCT using both techniques, and observed discordant results in 6 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, because the median number of clonal PCs in PCR+MFC cases was 0.014%. A second study by Lioznov and colleagues, 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 (73). More recently, Puig and colleagues 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 whereas the other 7 were MFC+ but PCR). No differences in survival were observed between the discordant groups of patients (49).

Multiparameter flow cytometry versus next-generation sequencing

Only one study has compared MFC versus NGS for MRD monitoring in multiple myeloma (51). From a total of 99 patients, 82 had concordant results (60 double-positive and 22 double-negative), 12 were MFCNGS+, and 5 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 = 0.05). 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 with current MFC studies; ref. 51).

Allele-specific oligonucleotide PCR versus next-generation sequencing

Only two studies have compared ASO-PCR versus NGS. Ladetto and colleagues have reported preliminary data on 10 multiple myeloma patients; 8 were evaluable by RQ-PCR, 8 by NGS, and 6 by both methods (74). 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 with NGS, whereas the opposite occurred in 11 cases. Overall, major discordance rates in multiple myeloma were comparable to ALL and MCL, whereas minor and quantitative discordances appeared slightly more frequent (74). Martinez-Lopez and colleagues performed a similar comparison in 46 patients and observed a concordance rate of 85% (51). Thus, further studies are warranted to elucidate the clinical 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) feasibility in low sample volumes, (v) reproducibility, and (vi) clinical utility. Because the cost of MRD assessment is significantly lower as compared with 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 chronic myelogenous leukemia or acute lymphoblastic leukemia), total cost becomes significantly higher and therefore important to evaluate. Although 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 versus NGS is not yet available, and knowledge on head-to-head applicability and sensitivity is 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.

Approximately 15 years after the first published results, MRD detection has emerged as one of the most relevant prognostic factors in multiple myeloma. 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 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 bone marrow infiltration, typically observed in multiple myeloma, leads to some 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 bone marrow infiltration or extramedullary involvement represents a challenge for both immunophenotypic- and molecular-based MRD detection in single bone marrow aspirates. This highlights the value of sensitive imaging techniques to redefine CR both at the intramedullary (e.g., whole body MRI and PET/CT) and extramedullary levels (PET/CT). However, standardization of imaging techniques and comparison with other sensitive bone marrow–based MRD methods are still lacking. By contrast, the persistence of MRD 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 multiple myeloma has been prospectively confirmed in two large transplant-based studies (55, 56) and a relatively smaller (yet prospective) clinical trial on transplant-ineligible multiple myeloma 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 versus 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 to redefine and improve current CR criteria in multiple myeloma. The time has come to implement MRD monitoring in prospective clinical trials and fully establish its role in the management of multiple myeloma patients.

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.

This study was supported by the Cooperative Research Thematic Network of the Red de Cancer (Cancer Network of Excellence; RD12/0036/0058; to J.F. San Miguel); the Instituto de Salud Carlos III, Spain, Instituto de Salud Carlos III/Subdirección General de Investigación Sanitaria (FIS: PI1202311; to R. García-Sanz; Sara Borrell: CD13/00340; to B. Paiva); the Junta de Castilla y León (HUS412A12-1; to B. Paiva); and the Asociación Española Contra el Cáncer (GCB120981SAN; to J.F. San Miguel).

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 transplantation-eligible 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
.
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
.
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 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
:
2276
9
.
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
.
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 n-dimensional 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
.
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 high-throughput 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
:
1365
72
.
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
.
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
.
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 and 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.
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
.
56.
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
.
57.
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 autologous stem cell transplantation in multiple myeloma
.
Blood
2012
;
119
:
687
91
.
58.
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
.
59.
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
.
60.
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
.
61.
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
.
62.
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
.
63.
Martinelli
G
,
Terragna
C
,
Zamagni
E
,
Ronconi
S
,
Tosi
P
,
Lemoli
R
, et al
Polymerase chain reaction-based detection of minimal residual disease in multiple myeloma patients receiving allogeneic stem cell transplantation
.
Haematologica
2000
;
85
:
930
4
.
64.
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
.
65.
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
.
66.
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
.
67.
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
.
68.
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
.
69.
Ladetto
M
,
Pagliano
G
,
Ferrero
S
,
Cavallo
F
,
Drandi
D
,
Santo
L
, et al
Major tumor shrinking and persistent molecular remissions after consolidation with bortezomib, thalidomide, and dexamethasone in patients with autografted myeloma
.
J Clin Oncol
2010
;
28
:
2077
84
.
70.
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
.
71.
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
2015
;
29
:
689
95
.
72.
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
.
73.
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
.
74.
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
.
75.
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
.