REVIEW Minimal Residual Disease in Myeloma: Are We There Yet? Andrew J Hart,1 Madan H Jagasia,1 Annette S Kim,2 Claudio A Mosse,2 Bipin N Savani,1 Adetola Kassim1 Measurement of minimal residual disease is routine in diseases such as chronic myelogenous leukemia, precursor B cell acute lymphoblastic leukemia, and acute promyelocytic leukemia because it provides important prognostic information However, the role of minimal residual disease testing has not been widely adopted in multiple myeloma (MM), with other parameters such as the International Staging System (ISS) and cytogenetic analysis primarily guiding therapy and determination of prognosis Until recently, achieving a complete response (CR), as defined by the International Myeloma Working Group (IMWG) criteria, was rare in patients with MM The use of novel agents with or without autologous peripheral blood stem cell transplantation (auto-PBSCT) has significantly increased CR rates, thus increasing overall survival (OS) rates The majority of patients with MM have persistent levels of residual disease that are below the sensitivity of bone marrow (BM) morphology, protein electrophoresis with immunofixation, and light chain quantitation even after attaining CR and will eventually relapse Measurement of minimal residual disease by more sensitive methods, and the use of these methods as a tool for predicting patient outcomes and guiding therapeutic decisions, has thus become more relevant Methods available for monitoring minimal residual disease in MM include PCR and multiparameter flow cytometry (MFC), both of which have been shown to be valuable in other hematologic malignancies; however, neither has become a standard of care in MM Here, we review current evidence for using minimal residual disease measurement for risk assessment in MM as well as incorporating pretreatment factors and posttreatment minimal residual disease monitoring as a prognostic tool for therapeutic decisions, and we outline challenges to developing uniform criteria for minimal residual disease monitoring Biol Blood Marrow Transplant 18: 1790-1799 (2012) Ó 2012 American Society for Blood and Marrow Transplantation Published by Elsevier Inc All rights reserved KEY WORDS: Minimal residual disease, Multiple myeloma, Flow cytometry, PCR INTRODUCTION Multiple myeloma (MM) is a very heterogeneous disease with protean manifestations, as well as an assortment of genetic and molecular alterations, making prognostic determination at diagnosis quite challeng- From the 1Department of Medicine, Division of Hematology/ Oncology, Section of Hematology and Stem Cell Transplant; and 2Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee Financial disclosure: See Acknowledgment on page 1797 Correspondence and reprint requests: Adetola Kassim, MD, Vanderbilt University Division of Hematology/Oncology, 1301 Medical Center Drive, Suite 3927, Nashville, TN 37232 (e-mail: adetola.kassim@vanderbilt.edu) Received March 20, 2012; accepted May 3, 2012 Ó 2012 American Society for Blood and Marrow Transplantation Published by Elsevier Inc All rights reserved 1083-8791/$36.00 doi:10.1016/j.bbmt.2012.05.009 1790 ing In 1975, Durie and Salmon [1] described an MM staging system using features such as tumor cell mass, the presence of end-organ damage, osteolytic bone lesions, and elevated serum Ig levels More recently, the International Staging System (ISS) was developed that describes disease burden based on b2-microglobulin and serum albumin levels, with both having prognostic significance at diagnosis [2] Cytogenetic abnormalities including 13q deletion and detection of t(4;14), t(14;16), and del17p by fluorescence in situ hybridization (FISH) have been shown to predict a less favorable survival, and the International Myeloma Working Group (IMWG) has proposed a new classification system based on molecular and cytogenetic criteria [3] Gene expression profiling has also recently been used to determine high-risk populations but is not available for widespread use [4,5] The use of these molecular and cytogenetic signatures to direct treatment, in the context of other staging parameters, variable disease manifestations, and expanding therapeutic options, is still being validated Biol Blood Marrow Transplant 18:1790-1799, 2012 The IMWG response criteria are based on (1) serum and urine M-protein by electrophoresis and immunofixation (IFX), (2) percentage of plasma cells on bone marrow (BM) biopsy, and (3) serum free light chains (sFLCs) [6] The importance of achieving a complete response (CR), with an associated benefit in overall survival (OS), has been well-documented, although data also show that development of CR has the most significant benefit in only a small, high-risk group of patients as defined by gene expression profiling [7,8] As CR rates have improved, more rigorous definitions of response have been developed, including stringent CR (sCR) by the IMWG that incorporates sFLCs along with immunohistochemistry and immunofluorescent techniques to establish plasma cell (PC) clonality [6] It has been proposed that the sFLC ratio, which has been shown at diagnosis to be an independent prognostic factor and predict more aggressive disease, be incorporated into the ISS to help improve risk stratification as well [911] The role of sFLC measurement as a minimal residual disease marker will be further discussed below Improving CR rates, with associated increases in OS and event-free survival (EFS), have made the measurement and monitoring of minimal residual disease in MM with more sensitive techniques a relevant pursuit Microscopic BM examination, radiographic imaging, molecular, and flow cytometric techniques have all been used for this purpose Two very sensitive techniques that have been studied with increasing frequency during the past few years are PCR and multiparameter flow cytometry (MFC) However, for a variety of reasons including the heterogeneity of the disease and the technical complexity of some of the techniques, minimal residual disease monitoring with highly sensitive techniques has not become routine clinical practice Here, we review the use of these approaches and outline the challenges to developing uniform and available methods for minimal residual disease measurement in MM Techniques for Assessing Minimal Residual Disease in MM Protein and imaging studies Measurement of serum and urine paraprotein levels with IFX, sFLC and urine free light chains, and morphologic examination of the BM are all widely available methods used to measure disease burden in MM One definition of CR is defined by the IMWG as \5% PCs in the BM with negative serum and urine IFX, and the clinical significance of achieving CR has been well-described [6,7] Data from the Total Therapy trials have demonstrated the importance of CR on long-term outcomes [12] These treatment regimens, though, are rigorous and may not be amenable for use outside of large referral centers A report Minimal Residual Disease in Multiple Myeloma 1791 from the Korean Multiple Myeloma Working Party demonstrated that achieving a CR/near CR (nCR) before autologous peripheral blood stem cell transplantation (auto-PBSCT) significantly increased 2-year OS from 70.9% to 86.6% compared with patients achieving a partial response, providing data that achieving CR has prognostic significance even before high-dose therapy (HDT) and auto-PBSCT [13] As BM biopsies are expensive, time-consuming, and pose some risk to patients, it has been argued that BM examination is not necessary in patients with negative serum and urine electrophoresis and IFX due to the low likelihood of increased PCs under these circumstances [14] However, the independent value of BM examination has been examined in studies Data from Chee et al [15] showed in 92 patients that 14% with negative IFX and 10% of patients with a normal sFLC ratio had $5% BM PCs in the marrow, with clonality demonstrated in 85% of patients with residual PCs [15] In patients who are IFX-negative, they found significantly improved OS from time of IFXnegativity in patients with \5% total PCs compared with those with $5% (6.2 versus 2.3 years; P 01) More recently, Fernandez de Larrea et al [16] showed in 35 patients after auto-PBSCT that the total number of PCs present in patients in CR after auto-PBSCT correlates with progression-free survival (PFS) but not OS [16] There was a nonsignificant difference in median OS in patients with #1.5% PCs versus 5% PCs (median OS not reached versus 9.7 years; P 195) These results demonstrate that microscopic assessment of the BM can have prognostic significance regardless of the status of protein studies, although the sensitivity of morphology alone is crude and limited by the number of cells evaluated as well as sampling variability Imaging by fluorodeoxyglucose-positron emission tomography (PET) has also been shown to have prognostic significance, with a significant improvement in PFS and OS in patients with 100% standardized uptake value reduction compared with \100% standardized uptake value reduction after treatment with thalidomide-dexamethasone and auto-PBSCT in study [17] This held true even among patients otherwise achieving a CR Moreover, they demonstrated a significant improvement in post-relapse OS if the fluorodeoxyglucose-PET was negative versus positive at 36 months Although PET imaging is widely available, not all patients with MM will have PET-avid lesions, and heterogeneity of visual criteria and poor interobserver reproducibility can be a problem with interpretation of data from these imaging studies As CR rates have improved, more sensitive techniques to measure the depth of response have been investigated The sFLC ratio has been shown to have prognostic significance at the time of diagnosis [9] Using this ratio to monitor disease status during 1792 A J Hart et al treatment has also been examined Singhal et al [18] demonstrated a high degree of discordance between the serum IFX and the sFLC ratio Seventy-nine percent of patients with a positive serum IFX had a normal sFLC ratio, whereas only 6% of those with negative serum IFX had an abnormal sFLC ratio, resulting in a sensitivity and specificity for the sFLC ratio compared with the serum IFX of 66% and 69%, respectively [18] Similarly, Giarin et al [19] showed discordance between sFLC ratios and IFX in patients who received transplantation, although when using a method that assesses total sFLC concentration (free and bound), the concordance with IFX was better De Larrea et al [20] reported that an abnormal sFLC ratio is frequently (73% of CR patients) due to presence of oligoclonal bands as part of normal immune reconstitution after auto-PBSCT and is actually associated with a good prognosis The major value of sFLC assessment is in patients with light chain onlyMM, due to lack of sensitivity of electrophoretic methods to detect sFLCs [21] Conversely, secretory MM with fully assembled Igs may have normal sFLC ratios in 5% of cases [22] Thus, although the sFLC ratio is now included in the definition of sCR by the IMWG, it remains controversial how to incorporate sFLC measurement into minimal residual disease monitoring in MM Similarly, other novel methods to quantitate heavy chain Ig ratios are currently lacking sufficient published data to support their routine use in therapy monitoring at this time Minimal residual disease detection by PCR Molecular monitoring of disease by MFC and PCR has been commonly used in chronic myelogenous leukemia, acute lymphoblastic leukemia, and acute promyelocytic leukemia to help determine prognosis and guide therapy [23-25] Several PCR techniques have been described for use in minimal residual disease monitoring in MM Use of PCR allows the amplification of even single cells, providing an exquisitely sensitive method to detect minimal residual disease However, minimal residual disease testing in myeloma by PCR has proved challenging for several reasons The Ig heavy and light chain (IGH, IGK, and IGL) loci have significant somatic hypermutation (SHM), with an average 92.2% homology to the germline sequence for IGH sequences, 93.9% for IGK, and 93.4% for IGL [26] For this reason, standard primers designed against the framework regions of the Ig genes occasionally fail to bind to the patient template DNA sufficiently well to result in amplification As a result of SHM at the binding sites of commercial primers, researchers have attempted to develop highly sensitive molecular assays for individual Ig rearrangements through the design of allele-specific oligonucleotide (ASO) primers, or primers based upon the patient’s individual Biol Blood Marrow Transplant 18:1790-1799, 2012 sequence that is a product of both the specific rearrangement as well as any somatic hypermutation [27] PCR or even quantitative PCR can then be performed using these primers, allowing highly sensitive monitoring of the patient’s clone Table summarizes data available from several studies examining the role of PCR monitoring for minimal residual disease in MM Most early studies used ASO-PCR methods [28-34] However, in these studies done before the era of more effective therapy regimens, molecular minimal residual disease measurement by qualitative PCR was found not to be useful, as nearly all patients were positive for residual disease at the molecular level aside from some molecular remissions after PBSCT An early small case series demonstrated persistent molecular remissions using qualitative, non–ASO-PCR in small numbers of patients, suggesting the possibility of cure, particularly with allogeneic SCT (allo-SCT) [35] Corradini et al [31] described molecular remission with a qualitative PCR approach using ASO primers in significantly more patients after allo-SCT compared to auto-PBSCT [31] Several studies have also demonstrated the prognostic significance of minimal residual disease negativity by PCR after allo-SCT [32,36-38] More recently, semi-quantitative fluorescent and real-time quantitative PCR (RQ-PCR) techniques, primarily using ASO primers, were introduced to correlate the level of the clone with clinical disease manifestations and outcomes [39-43] These methods have allowed very sensitive minimal residual disease monitoring on the order of 1026 and permit trends in levels to be followed rather than binary positive or negative results Using ASO RQ-PCR, Korthals et al [44] showed that low minimal residual disease (\0.2% 2IgH/b-actin) measured before HDT and auto-PBSCT was significantly associated with improved median EFS and OS compared with high minimal residual disease Sensitivity reached 1025 in 88% of cases in this study using Taqman technology on the LightCycler system (Roche, Mannheim, Germany) Ladetto et al [45] documented persistent molecular remissions using either qualitative nested ASO-PCR or ASO RQ-PCR in patients with MM treated with bortezomib, thalidomide, and dexamethasone as consolidation after auto-PBSCT, with no patient in molecular remission relapsing after a median follow-up of 42 months An update to this study showed after a median follow-up of 65 months, a 5-year OS of 100% for patients achieving minimal residual disease negativity; no patient achieving molecular remission has died [46] Thus, PCR is a very sensitive technique that has demonstrated prognostic significance after allo-SCT, auto-PBSCT, and after consolidation therapy with novel agents Unfortunately, ASO-PCR–based Authors No of Patients Bird et al [35] Corradini et al [28] Bj€ orkstrand et al [29] Swedin et al [30] Corradini et al [31] 18 15 36 51 Martinelli et al [32] Martinelli et al [33] 26 229 Method Sensitivity Technical Success Rate Treatment Regimen CR Minimal Residual Disease Negativity Rate PCR (not ASO) ASO nested PCR ASO-PCR ASO semi-nested PCR ASO-PCR NR NR NR 1024-1025 1025-1026 100% 83% 62.5% 67% 84% Allo-BMT Auto-SCT or allo-SCT Auto-SCT Â2 Auto-SCT or allo-SCT Auto-SCT or allo-SCT 100% 50% (6/12) 53% (8/15) 42% (15/36) 71% (36/51) ASO-PCR ASO-PCR 1025 1025 54% 88% (44/50) Allo-SCT Auto-SCT or allo-SCT Allo-SCT Auto-SCT 38% (26/68) Allo 38% (26/68) Auto 22.5% (36/161) 92% (12/13) NR 100% (3/3) 0% (0/12) 80% (4/5) 21% (5/24) 7% (2/27, auto-PBSCT); 50% (7/14, allo-SCT) 50% (7/14) 27% (12/44) Auto-SCT Auto-SCT 53% (50/96) 24% (7/29) Allo-SCT Auto-SCT or allo-SCT Auto-SCT 100% 45% (5/11) 28% (19/67) 100% Allo-SCT Auto-SCT 27% (3/11) 49% (19/39) 76% Auto-SCT 25% nCR before auto-PBSCT 29% nCR after auto-PBSCT 13 29 ASO-PCR Real-time PCR (not ASO) ASO nested PCR 1025-1026 1024 1023-1024 Davies et al [66] Novella et al [43] 96 36 Corradini et al [37] Fenk et al [42] Bakkus et al [41] 70 11 87 PCR (not ASO) PCR (not ASO) ASO nested PCR ASO-PCR ASO real-time PCR ASO-PCR 1024 1024-1026 1024-1025 1026 1024-1026 1024 Raab et al [38] Ladetto et al [45] 11 39 Korthals et al [44] 70 ASO real-time PCR ASO nested Real-time PCR (not ASO) Real-time ASO-PCR 1024-1025 1026 Â 1026 1024-1025 100% 66% 73% (FR1/ASO) 100% (FR3/ASO) NR 67% 69% NR 77% 69% (9/13) NR NR NR 33% (16/48) 27% (3/11) 35% (21/60) #0.015% 65% (39/60) >0.015% 27% (3/11) 15% (6/39) NR 17% (9/53) before auto-PBSCT 21% (8/38) after auto-PBSCT MM indicates multiple myeloma; CR, complete response; ASO, allele-specific oligonucleotide; NR, not reported; Allo-BMT, allogeneic bone marrow transplant; Auto-SCT, autologous stem cell transplant; Allo-SCT, allogeneic stem cell transplant; Auto-PBSCT, autologous peripheral blood stem cell transplant; nCR, near complete response Minimal Residual Disease in Multiple Myeloma Cavo et al [34] Ladetto et al [39] Biol Blood Marrow Transplant 18:1790-1799, 2012 Table Studies Examining PCR for Minimal Residual Disease Monitoring in MM 1793 1794 A J Hart et al Biol Blood Marrow Transplant 18:1790-1799, 2012 methods require sequencing each patient’s individual clone(s) and developing primers specific to the patient’s rearranged and somatically mutated sequence, a process impractical in the general clinical setting However, in 2003, the BIOMED-2 Concerted Action group designed a panel of primers, which have been extensively applied to clonality testing in a number of B cell lymphomas [47-49] Plasma cell neoplasms were not included in the design or in the validation of the primers However, in other B cell malignancies, it was demonstrated that the use of a combination of a panel of IGH and IGK primers could identify a clonal rearrangement in 98% of all cases, including post– germinal center (therefore somatically hypermutated) B cell neoplasms such as many diffuse large B cell lymphomas and marginal zone lymphomas [50] Although plasma cell neoplasms were avoided in these initial studies due to the known complication of SHM, the use of multiple panels of primers against numerous loci of the Ig genes, including nonfunctional rearrangements more common in myeloma, increases the likelihood that at least primer pair will bind and allow amplification Subsequently, Martınez-Sanchez et al [51] have demonstrated that a set of BIOMED-2 primer pairs alone was required to detect clones in 91% of patients with myeloma using unsorted BM aspirates and that molecular minimal residual disease has prognostic value for PFS after auto-PBSCT, albeit with lower sensitivity than ASO-PCR and MFC detection and quantification of the abnormal clone Third, modern MFC is sufficiently sensitive to detect as few as 0.01% atypical plasma cells in a normal BM [52,53] The European Myeloma Network has recommended that CD38, CD45, and CD138 be tested simultaneously in at least tube and included in all tests if possible CD19 and CD56 are the minimum recommended markers for identifying abnormal PCs (along with assessing cytoplasmic k/l expression), with CD20, CD27, CD28, and CD117 preferably included to broaden applicability [54] Most of the reported studies use 4-color MFC, an older, less sensitive technique; however, 6-color and even 8-color MFC have been described [52,55,56] In 1999, Almeida et al [57] described that MFC DNA ploidy using 13 different 3-color combinations can detect residual MM cells with sensitivity up to 1025 at months after auto-PBSCT Rawstron et al [53], using CD19 and CD56 as markers of neoplastic PCs, demonstrated that patients with neoplastic PCs at months after auto-PBSCT had shorter PFS than those with only normal PCs MFC detected neoplastic PCs in 27% of patients in IFX-negative CR, which predicted a shorter PFS OS was significantly higher in a low-risk group (normal PCs present 2Â after auto-PBSCT) compared with high-risk patients (100% versus 54%; P \ 0001) Liu et al [58] retrospectively demonstrated a significant PFS difference in patients who, before auto-PBSCT, had #1.8% abnormal PCs compared with patients with 1.8% abnormal PCs as measured by CD38/CD19/ CD45/CD56 expression (P 017) No OS difference was demonstrated based on percent of PCs or achievement of CR/very good partial response (VGPR) Paiva et al [59] in a prospective analysis demonstrated by multivariate analysis that minimal residual disease status at day 100 after auto-PBSCT was the most important prognostic factor for PFS (median 71 versus 37 months; P \ 001) and OS (median not reached versus 89 months; P 002) in patients treated on the GEM2000 protocol When minimal residual disease was examined by IFX in this study by univariate analysis, there was a significant improvement in both Minimal residual disease detection by flow cytometry MFC is a technique increasingly described in the literature as a viable method for minimal residual disease monitoring in MM, with several examples summarized in Table MFC can distinguish between normal and malignant PCs by the aberrant expression of cell surface markers in approximately 90% of patients [27] MFC has become a useful tool in MM for a variety of reasons First, malignant PCs consistently have aberrant immunophenotypes to distinguish them from benign PCs Second, these immunophenotypes are relatively stable over time, allowing for consistent Table Studies Examining MFC for Minimal Residual Disease Monitoring in MM Authors No of Patients Method Sensitivity Technical Success Rate Treatment Regimen CR San Miguel et al [67] Paiva et al [59] Liu et al [58] 87 295 47 4-color MFC 4-color MFC 4-color MFC 1024 1024 NR 100% 100% 97% Auto-SCT GEM2000 (auto-PBSCT) Auto-SCT Mateo et al [60] Kumar et al [68] 685 132 4-color MFC MFC NR NR 100% NR GEM2000 (auto-PBSCT) Chemotherapy 45% 50% 66% CR/VGPR 36% 22% to 47% Minimal Residual Disease Negativity Rate 36% 42% 8% NR 46% MFC indicates multiparameter flow cytometry; MM, multiple myeloma; CR, complete response; Auto-SCT, autologous stem cell transplant; auto-PBSCT, autologous peripheral blood stem cell transplant; NR, not reported; VGPR, very good partial response; GEM2000, alternating cycles of vincristine, carmustine, melphalan, cyclophosphamide, prednisone (VBMCP) and vincristine, carmustine, doxorubicin, dexamethasone (VBAD) followed by high-dose melphalan and auto-PBSCT Minimal Residual Disease in Multiple Myeloma Biol Blood Marrow Transplant 18:1790-1799, 2012 1795 Table Studies Examining Multiple Methods for Minimal Residual Disease Monitoring in MM Authors No of Patients Rawstron et al [53] 45 Sarasquete et al [27] 32 Martınez-Sanchez et al [51] 53 Methods Sensitivity Technical Success Rate 3-color MFC PCR (not ASO) Real-time ASO PCR 4-color MFC Fluorescent PCR (not ASO) 4-color MFC 1024 1023-1025 1025 1024 1023-1024 1024 100% 64% 75% 90% 91% 94.5% Treatment Regimen CR Auto-SCT 73% GEM2000 (auto-PBSCT) 58% GEM2000 (auto-PBSCT) 51% Minimal Residual Disease Negativity Rate NR 56% (25/45) 29% (7/24) 54% (13/24) 53% (28/53) 33% (17/51) MM indicates multiple myeloma; CR, complete response; MFC, multiparameter flow cytometry; ASO, allele-specific oligonucleotide; Auto-SCT, autologous stem cell transplant; NR, not reported; GEM2000, alternating cycles of vincristine, carmustine, melphalan, cyclophosphamide, prednisone (VBMCP) and vincristine, carmustine, doxorubicin, dexamethasone (VBAD) followed by high-dose melphalan and auto-PBSCT; auto-PBSCT, autologous peripheral blood stem cell transplant PFS and OS in patients with negative IFX compared with positive IFX at day 100 after auto-PBSCT; however, this was not significant by multivariate analysis The following 4-color Ab combinations were used for MFC: CD38/CD56/CD19/CD45, CD138/ CD28/CD33/CD38, and CD20/CD117/CD138/ CD38 In more than 90% of cases, the CD38/CD56/ CD19/CD45 combination alone distinguished abnormal MM PCs from normal PCs Minimal residual disease negativity was defined as \1 MM PC in 104 normal PCs (sensitivity 1024) The same group further described the prognostic significance of the presence or absence of specific surface markers assessed by MFC and showed that high-risk genetic markers by FISH and persistent minimal residual disease by MFC at day 100 after auto-PBSCT predicted short CR, thus identifying patients with a poor prognosis and high risk of early progression [60,61] Thus, it has been prospectively demonstrated that minimal residual disease measurement by MFC is sensitive and predictive of survival outcomes However, MFC is a developing technology, with techniques still variable among institutions and laboratories Data is also dependent on the quality of the aspirate and can vary considerably depending on which aspirate sample is analyzed [40] Thus, further work needs to be done to standardize techniques and criteria for minimal residual disease assessment by MFC in MM Comparison Studies PCR and MFC have been directly compared in several small studies (Table 3) Sarasquete et al [27] retrospectively examined minimal residual disease by IFX, PCR, and MFC in 32 patients with MM in CR or near CR after HDT and auto-PBSCT The ASO RQ-PCR technique was only applicable in 75% of patients, whereas 90% of samples could be analyzed by MFC using Ab panels described in Paiva et al [59] In 25% of cases, PCR and MFC resulted in discordant results In all cases, PCR was positive with negative MFC; no cases were positive by MFC and negative by PCR The discordance rates for either PCR or MFC with IFX were 38%, with the more sensitive method varying from case to case PFS was significantly longer in patients with PCR #1024, but not significantly longer in patients with negative IFX or MFC \1024, although there was a trend toward significance with MFC Thus, in a small series of patients, minimal residual disease negativity by ASO RQ-PCR predicted longer PFS, whereas PFS was not significantly different for patients with negative IFX and negative MFC A second study by Lioznov et al [62] found that MFC and ASO-PCR correlated extremely well (r2 0.94; P \ 0001) in 69 samples, with only sample unevaluable by ASO-PCR (.98% evaluable) and none unevaluable by MFC Zhao et al [63] compared multiple methods of minimal residual disease monitoring in 121 BM samples Immunohistochemistry was the most effective method for detecting residual disease, with a 96% detection rate, followed by limited MFC (3-color analysis, CD38/CD45/k, CD38/CD45/l, CD138/CD45/ k, CD138/CD45/l) at 72% Cytogenetics (15%), FISH (50%), and detection of IgG and k light chain gene rearrangements by qualitative PCR with a limited set of non-ASO primers (60%) were less successful Although this study is an interesting comparison of multiple techniques, the sensitivity of both MFC and PCR are likely limited due to the use of older methodology Additionally, the majority of patients had residual disease at the time of BM examination, and no comparisons with serologic studies such as electrophoresis or IFX were made Paiva et al [64] compared IFX, sFLC, and immunophenotyping by MFC in 102 patients 65 years of age enrolled in the GEM05 65 year trial Three 4-color combinations (CD38/ CD56/CD19/CD45, CD38/CD27/CD45/CD28, and ß2-microglobulin/CD81/CD38/CD117) were used to detect phenotypic aberrancies in PCs Seven percent of patients with no minimal residual disease by MFC (sensitivity #1024 to 1025) after induction therapy (ie, immunophenotypic response) remained IFX positive initially, although all subsequently became IFX-negative Discrepant results were common among all methods tested Those patients with immunophenotypic response had significantly increased 1796 A J Hart et al PFS and time to progression compared to those with CR (IFX-negative and \5% PCs on BM biopsy) or sCR (CR and normalization of the sFLC ratio); however, no OS benefit was noted Measurement of Minimal Residual Disease in MM The challenges Measurement of minimal residual disease in MM has been a challenging endeavor Myeloma is a heterogeneous disease, making disease burden difficult to measure and follow in many circumstances Some patients present primarily with focal bone disease that is readily seen on imaging but not easily evaluated in the serum or by BM biopsy Others have patchy BM involvement with no evidence of focal bone involvement or have evidence of diffuse marrow enhancement with a paucity of serum and urinary paraprotein Thus, depending on their disease phenotype and molecular characteristics, certain methods of minimal residual disease measurement may not be applicable to some patients with myeloma, making it difficult to develop uniform guidelines for minimal residual disease monitoring in this disease The depth and prognostic role of minimal residual disease status after HDT/auto-PBSCT and consolidation therapy in patients who have achieved CR or VGPR after initial induction therapy is not well defined Electrophoretic or sFLC ratio techniques used to detect PC products are valuable to detect disease when there are clear differences between monoclonal and polyclonal Igs, but these methods have clear limitations After treatments that affect production of all Igs, such as Total Therapy or auto-PBSCT, detection of residual disease by SPEP, UPEP, IFX, and sFLC becomes less precise and complicated by limited residual B cell repertoires, which lead to oligoclonality There have been no large randomized studies in MM comparing different treatment strategies that have evaluated minimal residual disease detected by MFC or PCR using either ASO or wider panels of consensus primers No studies prospectively examining minimal residual disease using MFC or PCR have been reported in patients treated with novel agents with or without HDT/auto-PBSCT Thus, more sensitive methods for detecting minimal residual disease are needed to compare the depth of remission using different treatment strategies MFC has been slightly less sensitive than PCR [27] These comparisons, however, are dependent upon the specific methodologies used as well as which combinations of primers (PCR) and antibodies (MFC) are used In both cases, the patchy nature of the disease makes false-negative results a concern New 6-color and 8color MFC techniques have not uniformly been shown to increase sensitivity beyond 1024 but can reduce costs Biol Blood Marrow Transplant 18:1790-1799, 2012 and time to perform the analysis [52,55,56] However, in the presence of background polyclonal plasma cells, MFC clearly has an advantage in the ability to identify the abnormal population without any decrement in analytical sensitivity, while sensitivity clearly decreases in PCR-based methods unless ASO primers are used Due to the wide availability, ease, and rapid turnaround time of MFC, this is likely to become the method of choice for minimal residual disease measurement in MM Timing of minimal residual disease assessment Multiple questions remain to be answered, including when is the best time to measure minimal residual disease in MM Korthals et al [44] showed that pre-auto-PBSCT minimal residual disease positivity by PCR was an independent prognostic factor for both EFS and OS, with patients with lowminimal residual disease having significantly improved EFS and OS However, this is a retrospective study of a small number of patients treated with idarubicin and interferon, agents that are not commonly used since the development of new nonchemotherapeutic agents Paiva et al [59] showed a significant increase in OS and PFS in patients with no minimal residual disease by MFC at day 100 after autoPBSCT However, obsolete induction chemotherapy regimens were also used in this study Ladetto et al [45] demonstrated the value of minimal residual disease measurement by PCR after auto-PBSCT and consolidation therapy with bortezomib and thalidomide This study also included induction chemotherapy (vincristine, doxorubicin, and dexamethasone) with agents that are now uncommonly used It thus remains unclear when is the best time to integrate minimal residual disease measurement into therapeutic decision making Several important questions remain unanswered based on the current literature Should only patients who are positive for minimal residual disease receive HDT/auto-PBSCT, and can minimal residual disease measurement be used to help determine whether this therapy should be administered early or later in a patient’s treatment course? Recent data suggests that OS is equivalent for early versus delayed autoPBSCT after induction therapy with immunomodulatory agents; however, minimal residual disease measurement could help to identify patients that could benefit more from of these strategies [65] Based on minimal residual disease measurement, should some patients receive consolidation therapy while others should not? Can minimal residual disease monitoring be used to determine the need for or duration of maintenance therapy? When is the best time after auto-PBSCT to measure minimal residual disease? Prospective clinical studies are needed to address these and other unanswered management dilemmas Biol Blood Marrow Transplant 18:1790-1799, 2012 To examine prospectively the prognostic significance of minimal residual disease monitoring by MFC, we are currently conducting a prospective study of newly diagnosed patients with MM Patients who meet criteria for MM and who are candidates for auto-PBSCT are treated with either bortezomib, lenalidomide, and low-dose dexamethasone (VRD) or bortezomib, liposomal doxorubicin, and low-dose dexamethasone (VDD) for cycles, followed by auto-PBSCT in those patients experiencing a partial response or greater MFC using three 7-color tubes is performed on the initial pretherapy marrow aspirate to determine the aberrant immunophenotypes of the MM cells In patients achieving $VGPR after cycles of induction therapy, minimal residual disease is being assessed before and at months after auto-PBSCT The aims of the study are to assess the frequency of minimal residual disease negativity at the end of cycles of VRD or VDD and to assess the frequency of minimal residual disease negativity at the end of auto-PBSCT compared to before auto-PBSCT If current novel regimens like VRD or VDD achieve a high incidence of minimal residual diseasenegativity, it may be reasonable to delay or not offer HDT/auto-PBSCT in first remission and to proceed with consolidation and/or maintenance therapy Minimal Residual Disease in Multiple Myeloma 1797 Minimal residual disease measurement by PCR and MFC seems to have prognostic value in MM; however, barriers to the uniform adoption of these techniques remain due to the heterogeneity of the disease, lack of availability of testing, and the relative novelty of agents that commonly lead to durable CRs in patients with MM A pragmatic approach to customizing disease management should include accurately defining disease phenotypes as well as proper risk stratification using validated pretreatment disease prognostic factors and posttherapy disease monitoring An individualized minimal residual disease monitoring algorithm based on individual disease genetic signature and pretreatment phenotype for each patient with MM, with a goal to help predict outcome and guide therapeutic decisions, will enable a riskadapted approach to disease management More clinical trials integrating these approaches are needed to determine how best to use improved disease monitoring methods and develop a truly personalized approach to MM therapy ACKNOWLEDGMENT Financial disclosure: The authors have nothing to disclose The goal: Risk adapted therapy Many techniques are available for the measurement of disease burden in MM Improving therapies leading to increased CR rates have made prolonged minimal residual disease-negative states achievable even without allo-SCT Radiographic studies, such as PET imaging, as well as examination of the serum and BM using highly sensitive techniques, such as PCR and MFC, are valuable tools for measurement of minimal residual disease in MM Investigation of minimal residual disease by PCR techniques in BM of patients with MM achieving CR after autoPBSCT provides relevant information on residual tumor load with a significant impact on the risk of relapse However, other minimal residual disease techniques, such as MFC, yield similar prognostic information with the advantage of being easier, faster, and more widely available Thus, it is reasonable to think that MFC will become the routine technique for assessing minimal residual disease in MM in clinical practice However, to reach this goal, additional studies including larger numbers of patients and longer follow-up are required At the moment, evidence indicates that RQ-PCR and MFC are complementary techniques in minimal residual disease evaluation for MM Both techniques show that decreases in the BM tumor load below malignant cell per 10,000 total BM cells could be used as a target for the definition of a molecular/immunophenotypic CR REFERENCES Durie BG, Salmon SE A clinical staging system for multiple myeloma Correlation of measured myeloma cell mass with presenting clinical features, response to treatment, and survival Cancer 1975;36:842-854 Greipp PR, San Miguel J, Durie BG, et al International staging system for multiple myeloma J Clin Oncol 2005;23:3412-3420 Fonseca R, Bergsagel PL, Drach J, et al International Myeloma Working Group molecular classification of multiple myeloma: spotlight review Leukemia 2009;23:2210-2221 Shaughnessy JD Jr, Zhan F, Burington BE, et al A validated gene expression model of high-risk multiple myeloma is defined by deregulated expression of genes mapping to chromosome Blood 2007;109:2276-2284 Decaux O, Lode L, Magrangeas F, et al Prediction of survival in multiple myeloma based on gene expression profiles reveals cell cycle and chromosomal instability signatures in high-risk patients and hyperdiploid signatures in low-risk patients: a study of the Intergroupe Francophone du Myelome J Clin Oncol 2008;26:4798-4805 Durie BG, Harousseau JL, Miguel JS, et al International uniform response criteria for multiple myeloma Leukemia 2006; 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myeloma: a tool for comparing the efficacy of different treatment strategies and predicting outcome Blood 2002;99:1853-1856 Kumar S, Flinn IW, Richardson PG, et al Novel three- and four-drug combination regimens of bortezomib, dexamethasone, cyclophosphamide, and lenalidomide, for previously untreated multiple myeloma: results from the Multi-Center, Randomized, Phase EVOLUTION Study ASH Annual Meeting 2010 Abstract 621 ... for minimal residual disease measurement in MM Timing of minimal residual disease assessment Multiple questions remain to be answered, including when is the best time to measure minimal residual. .. in minimal residual disease monitoring in MM Use of PCR allows the amplification of even single cells, providing an exquisitely sensitive method to detect minimal residual disease However, minimal. .. Measurement of Minimal Residual Disease in MM The challenges Measurement of minimal residual disease in MM has been a challenging endeavor Myeloma is a heterogeneous disease, making disease burden