Ig Gene Clonality Analysis Using Next-Generation Sequencing for Improved Minimal Residual Disease Detection with Significant Prognostic Value in Multiple Myeloma Patients

Next-generation sequencing (NGS) of rearranged Ig genes is an effective technology for identifying pathologic clonal cells in multiple myeloma (MM) and tracking minimal residual disease. The clinical effect of implementing NGS in Ig gene clonality analysis was evaluated via a retrospective chart review. A total of 312 patients diagnosed with MM were enrolled in the study. Ig gene clonality was determined by fragment analysis using BIOMED-2 multiplex PCR assays and by NGS using the LymphoTrack IGH FR1 Assay and LymphoTrack IGK Assay. The clonality detection rates in diagnostic samples obtained using fragment analysis and NGS were 96.7% and 95.4%, respectively (statistically nonsignificant difference; P = 0.772). Among samples of patients in complete remission, the clonality detection rates obtained using fragment analysis and NGS were 33.3% and 60.3%, respectively (statistically significant difference; P = 0.034). Progression-free survival was significantly longer in negative than positive patients by NGS analysis (P = 0.03). Clonality detection by NGS-based methods using IGH FR1 and IGK assays in routine clinical practice is feasible, provides good clonality detection rates in diagnostic samples, and allows monitoring of samples in MM patients with significant prognostic value. Next-generation sequencing (NGS) of rearranged Ig genes is an effective technology for identifying pathologic clonal cells in multiple myeloma (MM) and tracking minimal residual disease. The clinical effect of implementing NGS in Ig gene clonality analysis was evaluated via a retrospective chart review. A total of 312 patients diagnosed with MM were enrolled in the study. Ig gene clonality was determined by fragment analysis using BIOMED-2 multiplex PCR assays and by NGS using the LymphoTrack IGH FR1 Assay and LymphoTrack IGK Assay. The clonality detection rates in diagnostic samples obtained using fragment analysis and NGS were 96.7% and 95.4%, respectively (statistically nonsignificant difference; P = 0.772). Among samples of patients in complete remission, the clonality detection rates obtained using fragment analysis and NGS were 33.3% and 60.3%, respectively (statistically significant difference; P = 0.034). Progression-free survival was significantly longer in negative than positive patients by NGS analysis (P = 0.03). Clonality detection by NGS-based methods using IGH FR1 and IGK assays in routine clinical practice is feasible, provides good clonality detection rates in diagnostic samples, and allows monitoring of samples in MM patients with significant prognostic value. Multiple myeloma (MM) is characterized by clonal proliferation of neoplastic plasma cells in bone marrow.1Palumbo A. Anderson K. Multiple myeloma.N Engl J Med. 2011; 364: 1046-1060Crossref PubMed Scopus (1706) Google Scholar Traditionally, several factors have been known to be associated with the prognosis of MM patients, such as cytogenetic abnormalities and serum levels of β2-microglobulin, lactate dehydrogenase, and albumin. Recent myeloma therapies have achieved high response rates; however, most patients eventually relapsed due to the persistently low levels of malignant plasma cells after treatment. Such minimal residual disease (MRD) assessment is crucial for evaluating treatment response and risk stratification in MM patients. The attainment of MRD negativity is associated with prolonged progression-free and overall survival in MM patients.2Perrot A. Lauwers-Cances V. Corre J. Robillard N. Hulin C. Chretien M.L. et al.Minimal residual disease negativity using deep sequencing is a major prognostic factor in multiple myeloma.Blood. 2018; 132: 2456-2464Crossref PubMed Scopus (181) Google Scholar,3Rustad E.H. Hultcrantz M. Yellapantula V.D. Akhlaghi T. Ho C. Arcila M.E. Roshal M. Patel A. Chen D. Devlin S.M. Jacobsen A. Huang Y. Miller J.E. Papaemmanuil E. Landgren O. Baseline identification of clonal V(D)J sequences for DNA-based minimal residual disease detection in multiple myeloma.PLoS One. 2019; 14: e0211600Crossref PubMed Scopus (16) Google Scholar Conventional methods of MRD assessment include allele-specific oligonucleotide PCR and multiparametric flow cytometry (MFC).4Flores-Montero J. Sanoja-Flores L. Paiva B. Puig N. Garcia-Sanchez O. Bottcher S. van der Velden V.H.J. Perez-Moran J.J. Vidriales M.B. Garcia-Sanz R. Jimenez C. Gonzalez M. Martinez-Lopez J. Corral-Mateos A. Grigore G.E. Fluxa R. Pontes R. Caetano J. Sedek L. Del Canizo M.C. Blade J. Lahuerta J.J. Aguilar C. Barez A. Garcia-Mateo A. Labrador J. Leoz P. Aguilera-Sanz C. San-Miguel J. Mateos M.V. Durie B. van Dongen J.J.M. Orfao A. Next generation flow for highly sensitive and standardized detection of minimal residual disease in multiple myeloma.Leukemia. 2017; 31: 2094-2103Crossref PubMed Scopus (295) Google Scholar,5Martinelli G. Terragna C. Zamagni E. Ronconi S. Tosi P. Lemoli R.M. Bandini G. Motta M.R. Testoni N. Amabile M. Ottaviani E. Vianelli N. de Vivo A. Gozzetti A. Tura S. Cavo M. Molecular remission after allogeneic or autologous transplantation of hematopoietic stem cells for multiple myeloma.J Clin Oncol. 2000; 18: 2273-2281Crossref PubMed Scopus (146) Google Scholar Although allele-specific oligonucleotide PCR provides high sensitivity for the detection of residual clonotype sequences, it is not widely used for routine clinical testing because it is a laborious and time-consuming process, owing to the design and validation of patient-specific primers and probes for quantitative PCR. The most commonly used method in clinical laboratories for detecting clonal Ig gene rearrangements is fragment analysis using multiplex PCR primers established by the EuroClonality/BIOMED-2 consortium.6van Dongen J.J. Langerak A.W. Bruggemann M. Evans P.A. Hummel M. Lavender F.L. Delabesse E. Davi F. Schuuring E. Garcia-Sanz R. van Krieken J.H. Droese J. Gonzalez D. Bastard C. White H.E. Spaargaren M. Gonzalez M. Parreira A. Smith J.L. Morgan G.J. Kneba M. Macintyre E.A. 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: 2257-2317Crossref PubMed Scopus (2387) Google Scholar However, the detection sensitivity of fragment analysis is approximately 5%, which makes it unsuitable for MRD assessment. MFC, another MRD assessment technique, is widely available in hematology laboratories, and has broad applicability in patients. However, obtaining consistent sensitivity between laboratories requires additional standardization efforts, such as those dedicated toward selecting antibody panels and gating strategies.7Flanders A. Stetler-Stevenson M. Landgren O. Minimal residual disease testing in multiple myeloma by flow cytometry: major heterogeneity.Blood. 2013; 122: 1088-1089Crossref PubMed Scopus (77) Google Scholar Recently, the next-generation sequencing (NGS) technology has been applied to clonality detection and quantification of Ig gene rearrangements in MM.3Rustad E.H. Hultcrantz M. Yellapantula V.D. Akhlaghi T. Ho C. Arcila M.E. Roshal M. Patel A. Chen D. Devlin S.M. Jacobsen A. Huang Y. Miller J.E. Papaemmanuil E. Landgren O. Baseline identification of clonal V(D)J sequences for DNA-based minimal residual disease detection in multiple myeloma.PLoS One. 2019; 14: e0211600Crossref PubMed Scopus (16) Google Scholar,8Martinez-Lopez J. Lahuerta J.J. Pepin F. Gonzalez M. Barrio S. Ayala R. Puig N. Montalban M.A. Paiva B. Weng L. Jimenez C. Sopena M. Moorhead M. Cedena T. Rapado I. Mateos M.V. Rosinol L. Oriol A. Blanchard M.J. Martinez R. Blade J. San Miguel J. Faham M. Garcia-Sanz R. Prognostic value of deep sequencing method for minimal residual disease detection in multiple myeloma.Blood. 2014; 123: 3073-3079Crossref PubMed Scopus (305) Google Scholar NGS has the advantage of universal applicability, with the use of off-the-shelf consensus primers. In addition, high sensitivity and specificity can be expected because the initial clonotype sequence is tracked in massively parallel sequencing data from follow-up samples. The purpose of this study was to explore the clinical usefulness of NGS-based clonality tests in MM patients. A total of 312 patients diagnosed with MM from January 2013 to July 2019 were included in this study. The medical records of the patients were retrospectively reviewed. This study protocol was approved by the institutional review board of the Severance Hospital, Yonsei University College of Medicine, Seoul, Republic of Korea (institutional review board number 4-2019-0815). Clinical response and disease progression were assessed according to the International Myeloma Working Group criteria for MM.9Kumar S. Paiva B. Anderson K.C. Durie B. Landgren O. Moreau P. et al.International Myeloma Working Group consensus criteria for response and minimal residual disease assessment in multiple myeloma.Lancet Oncol. 2016; 17: e328-e346Abstract Full Text Full Text PDF PubMed Scopus (1113) Google Scholar Disease staging and results of ancillary test results, including morphologic assessment of bone marrow aspiration and biopsy, multicolor flow cytometry, serum protein electrophoresis, serum immunofixation electrophoresis, and serum free-light chain assay, were reviewed. The overall patient characteristics and disease types are summarized in Table 1.Table 1Patient and Disease Characteristics at the Time of Initial DiagnosisCharacteristicsFragment analysis (n = 182)NGS (n = 130)P valueSex Male89 (48.9)78 (60.0)0.06834 Female93 (51.1)52 (40.0)0.06834Age, median (range), years64.5 (37–92)66 (31–91)0.1355Ig isotype∗Ig heavy chain was not detected in light chain disease (n = 72; 24.0%) and nonsecretory type (n = 3; 1.0%). IgG97 (53.3)68 (52.3)0.9541 IgA35 (19.2)23 (17.7)0.844 IgD8 (4.4)5 (3.8)1.000 IgM0 (0.0)1 (0.8)0.8656Light chain only41 (22.5)31 (23.8)0.8916Nonsecretory1 (0.5)2 (1.5)0.7686Creatinine, mg/dL, median (range)0.9 (0.46–11.55)1.0 (0.46–19.57)0.05799Hemoglobin, g/dL, median (range)11.9 (4.2–18.0)11.2 (6–17.6)0.4347Calcium, mg/dL, median (range)9.4 (7.4–17.1)9.2 (7.3–15.1)0.9259β2-Microglobulin, mg/L, median (range)4.2 (1.05–36.92)3.9 (1.24–54.03)0.3553Bone marrow plasma cell %, median (range)35.5 (4.9–97.9)37.5 (3.3–97.8)0.8456ISS I33 (25.8)26 (29.2)0.6863 II40 (31.2)29 (32.6)0.9526 III55 (43.0)34 (38.2)0.5742 Missing5441Data are expressed as n (%) of patients, unless otherwise indicated.ISS, International Staging System; NGS, next-generation sequencing.∗ Ig heavy chain was not detected in light chain disease (n = 72; 24.0%) and nonsecretory type (n = 3; 1.0%). Open table in a new tab Data are expressed as n (%) of patients, unless otherwise indicated. ISS, International Staging System; NGS, next-generation sequencing. Bone marrow aspirate samples at initial diagnosis and follow-up were obtained in K2 EDTA tubes. Genomic DNA was extracted using the QIAamp DNA Blood Mini Kit (Qiagen, Venlo, the Netherlands) for clonality testing. clonality the IGH Clonality Assay and the IGK Clonality Assay San were used for PCR The assays use BIOMED-2 multiplex in the IGH and and the IGK and The PCR were by fragment analysis DNA and Clonality in samples was the of the the in diagnostic samples the be at than the of the in the In follow-up samples, with in diagnostic samples and with a of an were as NGS analysis was with the LymphoTrack IGH FR1 Assay and LymphoTrack IGK Assay according to the Genomic DNA was using the to of DNA was using a multiplex for FR1 and PCR the were using the were the using Kit San the positive and negative in the were analysis was with available LymphoTrack Data The for clonality and clonotype sequence was by The of MRD in follow-up samples was using the LymphoTrack MRD Data In the sequencing data from the follow-up of the characterized clonotype sequences as an and to sequence were detected and The median sequencing for monitoring samples were to The a sensitivity of with sequencing the of DNA with flow was using and the for and were included in were a flow was In this are criteria of multicolor flow cytometry for MRD assessment were as the of a of at cells with an test was to by the test for the of was as were β2-microglobulin, and bone marrow plasma cells were The was in and data the with test was applied were using analysis was used to the between plasma cell and was used to NGS and Time to progression was evaluated using and with P were analysis was using for The clonality detection rates in diagnostic samples obtained using fragment analysis and NGS were 96.7% and with significant (P = Among the monitoring samples, the clonality detection rates obtained using fragment analysis and NGS were and with a significant (P Among the samples of patients in complete remission, the clonality detection rates obtained using fragment analysis and NGS were 33.3% and with a significant (P = 0.034). the which the methods were from January 2013 to 2017; NGS, from to was in the results obtained from tests with to such as serum light chain and multicolor flow of Ig and in and NGS of fragment analysis clonality testing was with the Clonality Assay use BIOMED-2 multiplex in the IGH and and the IGK and of NGS analysis was with the LymphoTrack IGH FR1 Assay and LymphoTrack IGK Assay of clonality of Ig gene (n = (n = (n = (n = of serum light chain of serum light chain was as or (n = (n = (n = (n = of multicolor flow cytometry, of multicolor flow cytometry was as the of a of at cells with an (n = (n = (n = (n = next-generation sequencing.∗ clonality testing was with the Clonality Assay use BIOMED-2 multiplex in the IGH and and the IGK and NGS analysis was with the LymphoTrack IGH FR1 Assay and LymphoTrack IGK Assay of serum light chain was as or of multicolor flow cytometry was as the of a of at cells with an Open table in a new tab NGS, next-generation sequencing. rates for IGH clonality assessment using fragment analysis and NGS assays in the diagnostic samples were and respectively rates for IGK clonality assessment using fragment analysis and NGS assays in the diagnostic samples were and In the monitoring samples, positive rates for IGH clonality assessment using fragment analysis and NGS were and rates for IGK clonality assessment using fragment analysis and NGS in the monitoring samples were and with Ig in diagnostic samples, positive rates of IGH clonality tests in light chain myeloma were than in the of myeloma (P = and P by fragment analysis and NGS In IGH clonality using NGS, the positive rates of in light chain in light chain and in light chain disease. IGK clonality assessment for light chain myeloma and significantly positive rates and than those obtained for type myeloma and (P and P = using fragment analysis and NGS of Ig to Ig in clonality testing was with the Clonality Assay use BIOMED-2 multiplex in the IGH and and the IGK and assay, analysis was with the LymphoTrack IGH FR1 Assay and LymphoTrack IGK Assay clonality testing was with the Clonality Assay use BIOMED-2 multiplex in the IGH and and the IGK and assay, analysis was with the LymphoTrack IGH FR1 Assay and LymphoTrack IGK Assay chain to myeloma with Ig with heavy and light chain next-generation sequencing.∗ P clonality testing was with the Clonality Assay use BIOMED-2 multiplex in the IGH and and the IGK and NGS analysis was with the LymphoTrack IGH FR1 Assay and LymphoTrack IGK Assay Conventional to myeloma with Ig with heavy and light chain Open table in a new tab NGS, next-generation sequencing. the between plasma cell and clonality an analysis was using the plasma cell from the aspiration and the median were to and to for fragment analysis and NGS In diagnostic samples, the plasma cell aspirate of patients with negative clonality results were to and to which were significantly than those of the patients with positive clonality to and to using fragment analysis and NGS respectively (P = and P = In the monitoring samples, positive rates were associated with plasma cell using aspirate in the analysis (P = In bone marrow samples with plasma cell the positive rates of clonality obtained using fragment analysis and NGS assays were and respectively the quantitative results as clonal from NGS NGS and assays were with clonal plasma cells detected by and for which data the were available were A total diagnostic samples were was the of determined by flow cytometry: = of total from the sequence by of as and plasma cells as and positive determined by was an between the NGS and assays for levels = of clonal plasma cells the and plasma cell were the of as determined by flow cytometry and with the clonal obtained using was an between the NGS and assays The median follow-up was to analysis was to the multiple myeloma patients gene testing using monitoring from the The follow-up gene test was a median of to after the diagnosis and follow-up to obtained negative results of gene test were the negative and those not have negative results were to the positive the for progression-free survival from the of was significantly longer in than patients (P = 0.03). Although the median was in median was not in The from MRD assessment in patients was in patients. this was a retrospective study positive rates and of fragment analysis was samples with available samples of patients were characterized by NGS to the of this study. In diagnostic samples, IGH and IGK clonality assessment using fragment analysis a positive with NGS In monitoring samples, IGH and IGK clonality assessment using NGS positive rates with fragment analysis In of and of samples were negative in fragment analysis for IGH and IGK clonality was in the of NGS NGS NGS analysis the was not because of the number of samples (P = between and NGS of analysis was with the LymphoTrack IGH FR1 Assay and LymphoTrack IGK Assay clonality testing was with the Clonality Assay use BIOMED-2 multiplex in the IGH and and the IGK and next-generation sequencing.∗ NGS analysis was with the LymphoTrack IGH FR1 Assay and LymphoTrack IGK Assay clonality testing was with the Clonality Assay use BIOMED-2 multiplex in the IGH and and the IGK and Open table in a new tab NGS, next-generation sequencing. Ig genes a to antibody A. F. Molecular and of Full Text PDF PubMed Scopus Google Scholar of malignant cells in of rearrangements of analysis of IGH and IGK is widely used as for residual disease assessment of MM A. J. A. rearrangements as clonal in Engl J Med. PubMed Scopus Google Scholar of the widely used methods for rearrangements is fragment assay, which clonality PCR by or S. N. N. Stetler-Stevenson M. Landgren O. Minimal residual disease in multiple the to the Clin Oncol. PubMed Scopus Google Scholar fragment assays between clonal have the PCR sequences, MRD detection is However, method using NGS a sequence at low MRD Y. Orfao A. minimal residual disease detection by next-generation sequencing in multiple Oncol. 2019; PubMed Scopus Google Scholar have been to NGS-based methods for clonality assessment of MM patients. detection rates of diagnostic samples were using IGH and IGK assays in MM.3Rustad E.H. Hultcrantz M. Yellapantula V.D. Akhlaghi T. Ho C. Arcila M.E. Roshal M. Patel A. Chen D. Devlin S.M. Jacobsen A. Huang Y. Miller J.E. Papaemmanuil E. Landgren O. Baseline identification of clonal V(D)J sequences for DNA-based minimal residual disease detection in multiple myeloma.PLoS One. 2019; 14: e0211600Crossref PubMed Scopus (16) Google M.E. M. L. J. Ho C. K. C. P. I. T. A. Landgren O. J. Roshal M. A. K. of immunoglobulin heavy chain clonality testing by next-generation sequencing for routine of and plasma cell 2019; Full Text Full Text PDF PubMed Scopus Google Scholar In this the clonality detection rates of diagnostic and monitoring samples of MM patients in routine clinical practice using an NGS-based method in to the widely used fragment analysis IGH gene using NGS were by FR1 assays and the assays in study. The IGH was in diagnostic samples, which was than obtained using the fragment with primers of in the for the PCR to the the use of multiple primers. In low clonal detection rates for the IGH gene were with for using the FR1 M.E. M. L. J. Ho C. K. C. P. I. T. A. Landgren O. J. Roshal M. A. K. of immunoglobulin heavy chain clonality testing by next-generation sequencing for routine of and plasma cell 2019; Full Text Full Text PDF PubMed Scopus Google Scholar However, in this the of the IGH FR1 and IGK assays a high clonality detection of 95.4%, which was not significantly than of the fragment The IGK the clonality detection by with the IGH FR1 the IGH FR1 and IGK assays be an to and and the positive detection in In this the positive of IGH gene rearrangements by NGS analysis was low in light chain disease This be by the in rearranged genes than in rearranged T. R. of the and of rearranged heavy chain Google Scholar In addition, clonal IGK rearrangements were in of which was significantly than for MM obtained using the NGS assays (P = The several such as rearrangements the are and is minimal in MM to a high positive of E.H. Hultcrantz M. Yellapantula V.D. Akhlaghi T. Ho C. Arcila M.E. Roshal M. Patel A. Chen D. Devlin S.M. Jacobsen A. Huang Y. Miller J.E. Papaemmanuil E. Landgren O. Baseline identification of clonal V(D)J sequences for DNA-based minimal residual disease detection in multiple myeloma.PLoS One. 2019; 14: e0211600Crossref PubMed Scopus (16) Google D. M. R. Langerak A.W. M. Dongen J.J.M. San Miguel Morgan G.J. gene rearrangements and the of multiple myeloma.Blood. PubMed Scopus Google V. M.C. G. V. C. G. the of immunoglobulin light chain gene rearrangements via analysis of the light chain in PubMed Scopus Google Scholar detection was to be the use of good and cell E.H. Hultcrantz M. Yellapantula V.D. Akhlaghi T. Ho C. Arcila M.E. Roshal M. Patel A. Chen D. Devlin S.M. Jacobsen A. Huang Y. Miller J.E. Papaemmanuil E. Landgren O. Baseline identification of clonal V(D)J sequences for DNA-based minimal residual disease detection in multiple myeloma.PLoS One. 2019; 14: e0211600Crossref PubMed Scopus (16) Google Scholar In this was a significant in the plasma cell in patients with positive and negative results for clonality assays in the diagnostic samples. plasma cell was significantly with clonal in the monitoring samples. However, in bone marrow samples with low plasma cell the positive of clonality obtained using NGS analysis was than for fragment analysis in samples with plasma cell for NGS and fragment NGS-based MRD assessment is in samples with low to negative results from MRD from low levels of MRD in MM patients. The of of total sequence by NGS is from cells because gene rearrangements in such a of the total number of with PCR primers at a gene or an are an the of total cells can be by the obtained using NGS assays by the of and plasma In this the using and clonal plasma cell detected by good Such a method using obtained using flow cytometry be used to in routine clinical MRD determined by NGS patient with highly and MRD negativity was the prognostic to the known factors disease or cytogenetic risk A. Lauwers-Cances V. Corre J. Robillard N. Hulin C. Chretien M.L. et al.Minimal residual disease negativity using deep sequencing is a major prognostic factor in multiple myeloma.Blood. 2018; 132: 2456-2464Crossref PubMed Scopus (181) Google Scholar,8Martinez-Lopez J. Lahuerta J.J. Pepin F. Gonzalez M. Barrio S. Ayala R. Puig N. Montalban M.A. Paiva B. Weng L. Jimenez C. Sopena M. Moorhead M. Cedena T. Rapado I. Mateos M.V. Rosinol L. Oriol A. Blanchard M.J. Martinez R. Blade J. San Miguel J. Faham M. Garcia-Sanz R. Prognostic value of deep sequencing method for minimal residual disease detection in multiple myeloma.Blood. 2014; 123: 3073-3079Crossref PubMed Scopus (305) Google Scholar survival analysis of patients gene testing for MRD assessment in from with the MRD as assessed by NGS was a significant prognostic factor in multiple was significantly longer in patients than in patients by NGS NGS-based MRD assessment the risk stratification of multiple myeloma patients in clinical of this study be retrospective and Although it was evaluated using were significant in treatment the fragment analysis and NGS were it be such were which have the flow cytometry was using the method and were this method was sensitive than the next-generation flow cytometry, which NGS and next-generation flow cytometry not be cytometry was used to the of the J. Sanoja-Flores L. Paiva B. Puig N. Garcia-Sanchez O. Bottcher S. van der Velden V.H.J. Perez-Moran J.J. Vidriales M.B. Garcia-Sanz R. Jimenez C. Gonzalez M. Martinez-Lopez J. Corral-Mateos A. Grigore G.E. Fluxa R. Pontes R. Caetano J. Sedek L. Del Canizo M.C. Blade J. Lahuerta J.J. Aguilar C. Barez A. Garcia-Mateo A. Labrador J. Leoz P. Aguilera-Sanz C. San-Miguel J. Mateos M.V. Durie B. van Dongen J.J.M. Orfao A. Next generation flow for highly sensitive and standardized detection of minimal residual disease in multiple myeloma.Leukemia. 2017; 31: 2094-2103Crossref PubMed Scopus (295) Google Scholar In addition, the was using the the from NGS is a cells gene However, this method is a because plasma cells are by flow it is to cells gene In addition, as a number of patients were included in the survival analysis and the median follow-up was be to clonality detection by NGS-based method using IGH FR1 and IGK assays in routine clinical practice is feasible, provides good clonality detection rates in diagnostic samples, and MRD detection in MM patients with significant prognostic value.

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