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• W2003634626 abstract "Quantitative polymerase chain reaction (PCR) with patient-specific, allele-specific oligonucleotide (ASO) primers for individual immunoglobulin H VDJ region (ASO-PCR) amplification was performed using several sources of clinical material, including mRNA from peripheral blood cells (PBMNCs), whole bone marrow cells (BMMNCs), and the CD20+CD38− B-cell population in bone marrow, as well as cell-free DNA from the sera of patients with multiple myeloma (MM). We designed the ASO primers and produced sufficient PCR fragments to evaluate tumor burden in 20 of 30 bone marrow samples at diagnosis. Polymerase chain reaction amplification efficiency depended on primer sequences because the production of ASO-PCR fragments did not correlate with serum M-protein levels. However, the ASO-PCR levels in BMMNCs showed statistically significant correlations with those in PBMNCs and CD20+CD38− B-cells. The good association between the BMMNC and PBMNC data indicated that PBMNCs could be a suitable source for monitoring minimal residual disease (MRD). In the case of cell-free DNA, ASO-PCR levels showed a unique pattern and remained high even after treatment. Because the sequence information for each ASO-PCR product was identical to the original, the cell-free DNA might also be useful for evaluating MRD. Moreover, the ASO-PCR products were clearly detected in 17 of 22 mRNA samples from CD20+CD38− populations, suggesting that MM clones might exist in relatively earlier stages of B cells than in plasma cells. Thus, ASO-PCR analysis using various clinical materials is useful for detecting MRD in MM patients as well as for clarifying MM pathogenesis. Quantitative polymerase chain reaction (PCR) with patient-specific, allele-specific oligonucleotide (ASO) primers for individual immunoglobulin H VDJ region (ASO-PCR) amplification was performed using several sources of clinical material, including mRNA from peripheral blood cells (PBMNCs), whole bone marrow cells (BMMNCs), and the CD20+CD38− B-cell population in bone marrow, as well as cell-free DNA from the sera of patients with multiple myeloma (MM). We designed the ASO primers and produced sufficient PCR fragments to evaluate tumor burden in 20 of 30 bone marrow samples at diagnosis. Polymerase chain reaction amplification efficiency depended on primer sequences because the production of ASO-PCR fragments did not correlate with serum M-protein levels. However, the ASO-PCR levels in BMMNCs showed statistically significant correlations with those in PBMNCs and CD20+CD38− B-cells. The good association between the BMMNC and PBMNC data indicated that PBMNCs could be a suitable source for monitoring minimal residual disease (MRD). In the case of cell-free DNA, ASO-PCR levels showed a unique pattern and remained high even after treatment. Because the sequence information for each ASO-PCR product was identical to the original, the cell-free DNA might also be useful for evaluating MRD. Moreover, the ASO-PCR products were clearly detected in 17 of 22 mRNA samples from CD20+CD38− populations, suggesting that MM clones might exist in relatively earlier stages of B cells than in plasma cells. Thus, ASO-PCR analysis using various clinical materials is useful for detecting MRD in MM patients as well as for clarifying MM pathogenesis. Multiple myeloma (MM) is a B-cell malignancy characterized by the monoclonal expansion of plasma cells in bone marrow (BM) and the secretion of paraprotein in the serum and/or urine. Because high-dose chemotherapy with auto–hematopoietic stem cell transplantation, along with novel therapeutic agents, such as proteasome inhibitors and immunomodulatory drugs, increases response rates and extent of responses, the prognosis for MM patients has improved dramatically [1Gay F. Larocca A. Wijermans P. et al.Complete response correlates with long-term progression-free and overall survival in elderly myeloma treated with novel agents: analysis of 1175 patients.Blood. 2011; 117: 3025-3031Crossref PubMed Scopus (210) Google Scholar, 2Chanan-Khan A.A. Giralt S. Importance of achieving a complete response in multiple myeloma, and the impact of novel agents.J Clin Oncol. 2010; 28: 2612-2624Crossref PubMed Scopus (165) Google Scholar]. Nonetheless, most patients eventually relapse or develop progressive phases, which suggests the survival of malignant cells with proliferative capacity even after administration of those powerful regimens [3Watanabe R. Tokuhira M. Kizaki M. Current approaches for the treatment of multiple myeloma.Int J Hematol. 2013; 97: 333-344Crossref PubMed Scopus (25) Google Scholar]. Improving treatment response and survival for MM patients requires evaluating and monitoring minimal residual disease (MRD) during treatment [4Takamatsu H. Ogawa Y. Kobayashi N. et al.Detection of minimal residual disease in patients with multiple myeloma using clonotype-specific PCR primers designed from DNA extracted from archival bone marrow slides.Exp Hematol. 2013; 41: 894-902Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 5Ferrero S. Drandi D. Mantoan B. Ghione P. Omedè P. Ladetto M. Minimal residual disease detection in lymphoma and multiple myeloma: impact on therapeutic paradigms.Hematol Oncol. 2011; 29: 167-176Crossref PubMed Scopus (39) Google Scholar]. Among methods for MRD evaluation in MM patients, polymerase chain reaction (PCR) with patient-specific allele-specific oligonucleotide (ASO) primers for individual immunoglobulin H (IgH) VDJ regions (ASO-PCR) is considered the most sensitive [6Harousseau J.L. Attal M. Avet-Loiseau H. The role of complete response in multiple myeloma.Blood. 2009; 114: 3139-3146Crossref PubMed Scopus (195) Google Scholar, 7Novella E. Giaretta I. Elice F. et al.Fluorescent polymerase chain reaction and capillary electrophoresis for IgH rearrangement and minimal residual disease evaluation in multiple myeloma.Haematologica. 2002; 87: 1157-1164PubMed Google Scholar]. Although previous reports have described measuring MRD with ASO-PCR, they usually involved analysis of BM cells by qualitative or semiquantitative methods [8Sarasquete M.E. García-Sanz R. González D. 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: 1365-1372PubMed Google Scholar, 9Ladetto M. Donovan J.W. Harig S. 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: 241-253Abstract Full Text PDF PubMed Scopus (89) Google Scholar].In this study, we quantified ASO-PCR products using peripheral blood mononuclear cells (PBMNCs) as well as BM mononuclear cells (BMMNCs). In addition, we quantified ASO-PCR products for mRNA from CD20+38− B-cells in BM to examine whether clonogenic cells are present in a relatively earlier B-cell fraction; we also quantified cell-free DNA from the sera to examine whether DNA fragments from MM cells are present in peripheral blood.Materials and methodsPatients and samplesThis study was performed according to the guidelines of the ethical committee of Osaka University Hospital and was approved by the Institutional Review Board of Osaka University Hospital and 19 related hospitals (HANDAI Clinical Blood Club). We analyzed 30 MM cases registered from December, 2011, until November, 2012. All patients gave written, informed consent for the molecular analysis. Remission status and disease progression were defined according to the International Myeloma Working Group criteria [10Durie B.G.M. Harousseau J.L. Miguel J.S. et al.International uniform response criteria for multiple myeloma.Leukemia. 2006; 20: 1467-1473Crossref PubMed Scopus (2193) Google Scholar], with the only exception being that complete remission (CR) was not confirmed by BM biopsy. Fluorescence in situ hybridization analysis of t(4;14), t(11;14), t(14;16), and del(17p13) for BM-derived interphase cells was performed.Design of a sense primer for detection of a specific immunoglobulin H VDJ regionThe mononuclear cells were separated from the BM of MM patients at diagnosis. The CD38highCD20− BMMNCs were sorted by using a FACS Aria cell sorter (Becton Dickinson, Heidelberg, Germany). Genomic DNA was extracted from the sorted CD38highCD20− BMMNCs using the QIAGEN DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany). The IgH VDJ regions were amplified by PCR with primers from the VH family of primers for sense and the JH consensus primer (5′-CTTACCTGAGGAGACGGTGACC) for antisense [11van Dongen J.J.M. Langerak A.W. Brüggemann M. 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: 2257-2317Crossref PubMed Scopus (2470) Google Scholar]. The amplified PCR fragments were sequenced. Based on each sequence, each patient's ASO primers were individually designed according to the methods described by van der Velden et al. [11van Dongen J.J.M. Langerak A.W. Brüggemann M. 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: 2257-2317Crossref PubMed Scopus (2470) Google Scholar].Preparation of standard samples for real-time polymerase chain reactionDNA from CD38highCD20− BMMNCs of each patient was amplified using each specific IgH VDJ region primer and a consensus probe corresponding to the JH region. Subsequently, the PCR products were incorporated into the PCR 2.1-TOPO vector (TOPO TA Cloning Kit, Life Technologies, Grand Island, NY), and sequence analysis was carried out to confirm whether the insert carried the target sequence. These plasmids were used as templates to draw each standard curve.Extraction of mRNA from bone marrow and peripheral blood mononuclear cells and CD20+CD38− B-cells and of cell-free DNA from patient seraThe mRNA was extracted from MM patient BMMNCs, PBMNCs, and CD20+38− B-cells using the QIAGEN RNeasy Mini Kit (QIAGEN) at diagnosis, as well as at the follow-up time points (6 months [6mo] and 12 months [12mo] after the start of the treatment). DNA was extracted from the sera of the patients at the same time using the WAKO DNA Extractor SP kit (Wako Pure Chemical Industries, Osaka, Japan).Real-time quantitative polymerase chain reaction of the IgH geneAfter mRNA was reversed transcribed into cDNA using M-MLV Reverse Transcriptase (Life Technologies), real-time reverse transcription PCR (RT-PCR) was performed on a Takara PCR Thermal Cycler Dice (Takara, Shiga, Japan) according to the protocol described below. The pairs of primers used for the quantification of IgH mRNA were MM-MRD-5′ (0.3 μmol/L; sequences in Supplementary Table E1, online only, available at www.exphem.org) and JH-3′ (0.3 μmol/L; 5′-CTTACCTGAGGAGACGGTGACC-3′). The primer pairs for the quantification of β-actin mRNA as an internal control were ACF (0.3 μmol/L; 5′-TGGACATCCGCAAAGACCTG-3′) and ACR (0.3 μmol/L; 5′- AAGTACTCCGTGTGGATCGG-3′). The reaction mix for real-time RT-PCR was the SYBR Premix Dimer Eraser (Takara). The conditions for real-time RT-PCR were as follows: 95°C for 30 sec, 40 cycles of 95°C for 5 sec, 60°C for 30 sec, and 72°C for 30 sec. Values for IgH mRNA/β-actin mRNA (IgH/β-actin) were considered to indicate the real-time RT-PCR product levels (ASO-PCR levels). We confirmed that the sequences of the ASO-PCR products of interest from BMMNCs, PBMNCs, CD20+CD38− B-cells, and cell-free DNA were identical to the originally designed versions.Intra-assay precision of quantitative real-time reverse transcription polymerase chain reactionIntra-assay precision of real-time RT-PCR was evaluated with 10 replicates of the plasmid. Plasmid was subjected to seven phases of dilution, from 9.81 × 106 copies to 9.81 × 10° copies, then measured 10 times each. For example, in the case of patient #8, the variation coefficients for the Ct values were 1.06% for 9.81 × 106 copies, 1.01% for 9.81 × 105 copies, 0.66% for 9.81 × 104 copies, 0.89% for 9.81 × 103 copies, 0.86% for 9.81 × 102 copies, 1.12% for 9.81 × 101 copies, and 2.09% for 9.81 × 10° copies.Limit of detection of quantitative real-time reverse transcription polymerase chain reactionThe smallest plasmid copy number was measured 10 times, and the minimal copy number detected by real-time RT-PCR was calculated by Ct applied to a standard curve. The resulting sensitivity was 9.81 copies.Statistical analysisTo examine the correlations of the IgH/β-actin levels in BMMNCs with the ASO-PCR products in various clinical materials, as well as M-protein and percent of BM plasma cells, we performed univariate regression analysis and calculated the corresponding Spearman's correlation. An effect was always considered to be statistically significant if the p value of its corresponding statistical test was smaller than 0.05. For the statistical analysis, R version 3.0.2 (R Foundation for Statistical Computing, Vienna, Austria) was used.ResultsPatient characteristicsA summary of patient characteristics is given in Table 1, and all patient data are shown in Supplementary Table E2 (online only, available at www.exphem.org). Thirty patients with MM (7 with stage I, 9 with stage II, and 14 with stage III, according to the International Staging System [12Greipp P.R. San Miguel J. Durie B.G.M. et al.International staging system for multiple myeloma.J Clin Oncol. 2005; 23: 3412-3420Crossref PubMed Scopus (2076) Google Scholar]) were enrolled in this study. The median age in this cohort was 64.5 years (range = 36–83); the ratio of men to women was 16:14; and the numbers of IgG-, IgA-, and Bence Jones protein (BJP)-type patients were 18, 7, and 5, respectively (Table 1). The status of cytogenetic abnormality by fluorescent in situ hybridization was established in 4 for t(4;14), 4 for t(11;14), 1 for t(14;16), and 2 for del(17p13) (Table 1). Of 23 patients who were fully followed up, 4 reached CR, 9 achieved very good partial response (VGPR), 6 showed partial response (PR), and 4 remained in stable disease during the first year after the start of the treatment (Table 1).Table 1Patient characteristicsn = 30Median age (range)64.5 (36–83)Sex M16 (53.3%) F14 (46.7%)Type IgG18 (60%) IgA7 (23.3%) BJP5 (16.7%)ISS I7 (23.3%) II9 (30%) III14 (46.7%)Abnormal cytogenetics t(4;14)4 (13.3%) t(11;14)4 (13.3%) t(14;16)1 (3.3%) del(17p13)2 (6.7%)Best response (n = 23) CR4 (17.4%) VGPR9 (39.1%) PR6 (26.1%) SD4 (17.4%)ISS = International Staging System; SD = stable disease. Open table in a new tab Comparison of the IgH/β-actin values in between bone marrow and peripheral blood mononuclear cellsThe values for IgH/β-actin (ASO-PCR values) in BMMNCs varied from 10−9 to 102 at diagnosis (Fig. 1A); therefore, the efficiency of amplification by ASO-PCR seemed to depend on the designed primer sequences. This possibility was in part supported by the fact that the ASO-PCR levels in BMMNCs correlated with those in PBMNCs, but not to the percent of plasma cells in BM or the values for M-protein (Table 2).Table 2Correlation of the IgH/β-actin levels in BMMNCs with several biomarkers at diagnosis, 6mo, and 12moVariable (reference or unit)Coefficient95% CIp valueDiagnosis IgH/β-actin (PBMNCs)0.980.69–1.26<0.001 IgH/DNA (cell-free DNA)0.81−0.10–1.710.078 IgH/β-actin (CD20+CD38−)1.350.81–1.89<0.001 Serum IgG0.56−2.99–4.100.740 Serum IgA−4.37−19.15–10.410.458 % plasma cells−2.06−6.13–2.020.3046mo IgH/β-actin (PBMNCs)0.830.15–1.510.020 IgH/DNA (cell-free DNA)0.76−0.36–1.890.169 Serum IgG1.47−1.34–4.270.280 Serum IgA−1.03−16.43–14.370.845 % plasma cells1.32−0.28–2.910.10012mo IgH/β-actin (PBMNCs)1.900.96–2.840.001 IgH/DNA (cell-free DNA)−0.05−1.53–1.430.942 Serum IgG2.75−0.34–5.830.076 Serum IgA9.93−4.71–24.580.120 % plasma cells2.15−3.77–8.080.427CI = Confidence interval.All p values obtained by univariate regression analysis (R version 3.0.2). Open table in a new tab The ASO-PCR levels in PBMNCs from most of the patients rapidly decreased after treatment. At all time points, the ASO-PCR levels in PBMNCs were lower than those in BMMNCs (Fig. 1; Supplementary Table E2; online only, available at www.exphem.org).The IgH/β-actin levels in PBMNCs showed a statistically significant correlation with those in BMMNCs at diagnosis (Spearman's ρ = 0.98, p < 0.001; Table 2; Fig. 2), 6mo (Spearman's ρ = 0.83, p = 0.020; Table 2), and 12mo (Spearman's ρ = 1.90, p = 0.001; Table 2). Therefore, ASO-PCR using PBMNCs as well as BMMNCs is likely to be suitable for MRD evaluation. The ASO-PCR levels in BMMNCs did not correlate with either serum M protein (IgG and IgA) levels or the percent of plasma cells in BM (Fig. 2; Table 2).Figure 2Univariate regression analysis of IgH/β-actin (BMMNCs) with several biomarkers at diagnosis. Log values of the indicated parameters were used for univariate regression analysis (R version 3.0.2). Closed circles represent the correlation of IgH/β-actin (BMMNCs) with IgH/β-actin (PBMNCs), IgH/β-actin (CD20+CD38−), ASO-PCR (cell-free DNA), M-protein, and percent plasma cells in each patient. Dashed lines represent the linear regression line.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Comparing the kinetics of IgH/β-actin levels in BMMNCs and PBMNCs and M-protein levels, Figure 3 shows those in representative patients achieving VGPR or CR whose ASO-PCR values were more than 10−8 at each time point and more than 10−4 at diagnosis. It is noteworthy that the IgH/β-actin levels were decreased after treatment and reflected tumor burden well individually. In addition, the ASO-PCR products from BMMNC samples could be evaluated even when M-protein was not detected. The changes of ASO-PCR levels were more than those of M-protein levels in almost all cases, which means ASO-PCR kinetics are more sensitive to treatment effectiveness than M-protein kinetics. Interestingly, in one case (#3), ASO-PCR kinetics were almost the same as M-protein kinetics, which might indicate treatment refractoriness (Fig. 3B).Figure 3Representative cases, patients #1, #3, #8, #11, and #24. (A) Patient #1 was a 72-year-old male diagnosed with IgG κ–type MM without abnormal fluorescence in situ hybridization. (B) Patient #3 was a 56-year-old female diagnosed with IgG κ–type MM without abnormal fluorescence in situ hybridization. (C) Patient #8 was a 63-year-old female diagnosed with IgG κ–type MM with t(11;14). (D) Patient #11 was a 71-year-old male, with IgG κ–type MM without abnormal fluorescence in situ hybridization. (E) Patient #24 was a 49-year-old male diagnosed with IgA κ–type MM without abnormal fluorescence in situ hybridization. The IgH/β-actin values in the indicated clinical material, as well as serum IgG levels, are shown for diagnosis, 6mo, and 12mo.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The patient-specific ASO primers could be designed in 25 cases, but not in 3 BJP-type cases or 2 IgG-type cases. One BM sample (#6) at diagnosis was insufficient, and no PCR product was detected. Two sets (#28 and #29) of the designed ASO primers failed to amplify the PCR products, and the standard curves were not drawn. In addition, the IgH/β-actin values for two patients (#12 and #19) were somewhat too low to evaluate. Therefore, we could design the PCR primers and quantify the ASO-PCR products in 20 of 30 BMMNC samples at diagnosis (Supplementary Table E2, online only, available at www.exphem.org).Quantification of IgH gene fragments in cell-free DNA from the seraWe were able to detect patient-specific IgH DNA sequences in cell-free DNA extracted from the sera and quantify the ASO-PCR products. The ASO-PCR values in cell-free DNA were obtained in 18 cases at diagnosis, but not in 2 at diagnosis, 3 at 6mo, and 4 at 12mo. The changes in ASO-PCR levels in cell-free DNA over the clinical course differed from those of BMMNCs and PBMNCs (Table 2); myeloma cell-derived IgH DNA fragments in the sera stayed at similar levels and sometimes increased during treatment. Of importance, the sequences of the ASO-PCR products were identical to the originally designed sequence, suggesting that detection of the ASO-PCR products in cell-free DNA could reflect the persistence of myeloma cells somewhere in the body. Therefore, ASO-PCR using cell-free DNA is likely to have different significance from that using BMMNCs and PBMNCs.ASO-PCR products for mRNA in CD20+CD38− B-cells in bone marrowThe ASO-PCR products for CD20+CD38− B-cells in BM were relatively low, but were clearly detected, in 17 cases at diagnosis, whereas we could not detect them in 5 cases. The IgH/β-actin levels in CD20+CD38− B-cells in BM correlated with values for IgH/β-actin both in BMMNCs (Spearman's ρ = 1.35, p < 0.001; Table 2) and in PBMNCs (Spearman's ρ = 1.09, p < 0.001; data not shown). Thus, the evaluation of relatively earlier B-cell stages of myeloma cells seems to be of importance, including for the possible existence of MM clones in the CD20+CD38− B-cell population in BM.DiscussionWe quantified the PCR products of patient-specific IgH VDJ fragments in mRNA from PBMNCs, BMMNCs, and BM CD20+CD38− B-cells, as well as of cell-free DNA from the sera. All raw data are shown in a Supplementary Table E2 (online only, available at www.exphem.org). The ASO-PCR is powerful and sensitive for detecting MRD [4Takamatsu H. Ogawa Y. Kobayashi N. et al.Detection of minimal residual disease in patients with multiple myeloma using clonotype-specific PCR primers designed from DNA extracted from archival bone marrow slides.Exp Hematol. 2013; 41: 894-902Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 7Novella E. Giaretta I. Elice F. et al.Fluorescent polymerase chain reaction and capillary electrophoresis for IgH rearrangement and minimal residual disease evaluation in multiple myeloma.Haematologica. 2002; 87: 1157-1164PubMed Google Scholar, 8Sarasquete M.E. García-Sanz R. González D. 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: 1365-1372PubMed Google Scholar, 9Ladetto M. Donovan J.W. Harig S. 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: 241-253Abstract Full Text PDF PubMed Scopus (89) Google Scholar, 13Korthals M. Sehnke N. Kronenwett R. et al.Molecular monitoring of minimal residual disease in the peripheral blood of patients with multiple myeloma.Biol Blood Marrow Transplant. 2013; 19: 1109-1115Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 14Billadeau D. Prosper F. Verfaillie C. Weisdorf D. Van Ness B. Sequential analysis of bone marrow and peripheral blood after stem cell transplant for myeloma shows disparate tumor involvement.Leukemia. 1997; 11: 1565-1570Crossref PubMed Scopus (29) Google Scholar, 15Cremer F.W. Ehrbrecht E. Kiel K. et al.Evaluation of the kinetics of the bone marrow tumor load in the course of sequential high-dose therapy assessed by quantitative PCR as a predictive parameter in patients with multiple myeloma.Bone Marrow Transplant. 2000; 26: 851-858Crossref PubMed Scopus (21) Google Scholar, 16Cremer F.W. Kiel K. Wallmeier M. Goldschmidt H. Moos M.A. Quantitative PCR assay for the detection of low amounts of malignant cells in multiple myeloma.Ann Oncol. 1997; 8: 633-636Crossref PubMed Scopus (28) Google Scholar, 17Fenk R. Haas R. Kronenwett R. Molecular monitoring of minimal residual disease in patients with multiple myeloma.Hematology. 2004; 9: 17-33Crossref PubMed Scopus (13) Google Scholar], but it has some limitations. One problem is the cost of designing patient-specific ASO primers. The other is a failure to design primers because of the lack of clonal targets for amplification [8Sarasquete M.E. García-Sanz R. González D. 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: 1365-1372PubMed Google Scholar]. We applied this PCR method in 20 out of 30 cases, almost the same rate as previous reports [8Sarasquete M.E. García-Sanz R. González D. 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: 1365-1372PubMed Google Scholar]. In our cases, we often failed to design the patient's ASO primers and/or measure the PCR products in some BJP-type patients. We do not know the reason for this failure, which was not an issue in previous reports [4Takamatsu H. Ogawa Y. Kobayashi N. et al.Detection of minimal residual disease in patients with multiple myeloma using clonotype-specific PCR primers designed from DNA extracted from archival bone marrow slides.Exp Hematol. 2013; 41: 894-902Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 8Sarasquete M.E. García-Sanz R. González D. 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: 1365-1372PubMed Google Scholar, 14Billadeau D. Prosper F. Verfaillie C. Weisdorf D. Van Ness B. Sequential analysis of bone marrow and peripheral blood after stem cell transplant for myeloma shows disparate tumor involvement.Leukemia. 1997; 11: 1565-1570Crossref PubMed Scopus (29) Google Scholar, 15Cremer F.W. Ehrbrecht E. Kiel K. et al.Evaluation of the kinetics of the bone marrow tumor load in the course of sequential high-dose therapy assessed by quantitative PCR as a predictive parameter in patients with multiple myeloma.Bone Marrow Transplant. 2000; 26: 851-858Crossref PubMed Scopus (21) Google Scholar]. Although the sensitivity of ASO-PCR has been reported to be 10−5 to 10−6, our results showed that gene amplification by ASO-PCR was highly dependent on the sequences of the designed primers. Thus, this restriction on designing primers is another problem. However, we could apply quantification of the ASO-PCR products to ample sources of clinical materials, such as PBMNCs, BM CD20+CD38− B-cells, cell-free DNA, and BMMNCs.There were statistically significant correlations in values for the ASO-PCR products between BMMNCs and PBMNCs, suggesting the possibility that clonogenic plasma cells or myeloma precursor cells may circulate in peripheral blood. In the previous reports [18Paiva B. Perez-Andres M. Vidrials M.B. Almeida J. de las Heras N. Mateos M.V. Competition between clonal plasma cells and normal cells for potentially overlapping bone marrow niches is associated with a progressively altered cellular distribution in MGUS vs myeloma.Leukemia. 2011; 25: 697-706Crossref PubMed Scopus (69) Google Scholar, 19Kiel K. Cremer F.W. Rottenburger C. et al.Analysis of circulating tumor cells in patients with multiple myeloma during the course of high-dose therapy with peripheral blood stem cell transplantation.Bone Marrow Transplant. 1999; 23: 1019-1027Crossref PubMed Scopus (29) Google Scholar], MM cells and clonogenic MM precursor B cells were detected in the peripheral blood. Thus, the ASO-PCR values in PBMNCs might reflect the sum of the circulating MM cells and clonogenic MM precursor B cells. Therefore, PBMNCs, rather than BMMNCs, have the potential to be evaluated for MRD in MM patients during and after treatment. Availability of PBMNCs for evaluation of MRD will relieve the pain of BM aspiration. Recently, next-generation DNA sequencing was shown to be more sensitive than ASO-PCR for detecting MRD in MM [20Martínez-López J. Lahuerta J.J. Pepin F. et al.Prognostic value of deep sequencing method for minimal residual disease detection in multiple myeloma.Blood. 2014; 123: 3073-3079Crossref PubMed Scopus (323) Google Scholar]. From our data, we anticipate that PBMNCs might be a good source for monitoring MRD even when the next-generation sequencing method is applied. Next-generation DNA sequencing could resolve some problems with ASO-PCR analysis, such as the high percentage of failure to design primers and the dependency of efficacy of amplification on primer sequences. However, the new technology is complicated and costs much more than the ASO-PCR method. Therefore, the ASO-PCR method could still be useful for MRD evaluation, even in the era of next-generation DNA sequencing, and the method of analyzing MRD should be determined on a case-by-case basis.We detected and quantified the ASO-PCR products of cell-free DNA from the sera. Thus, the presence of small amounts of circulating nucleic acids in plasma and serum might be important [21González-Masiá J.A. García-Olmo D. García-Olmo D.C. Circulating nucleic acids in plasma and serum (CNAPS): applications in oncology.Onco Targets Ther. 2013; 6: 819-832Crossref PubMed Scopus (58) Google Scholar]. Reports that the amount of circulating nucleic acids in plasma and serum is significantly increased in cancer patients have aroused great interest [21González-M" @default.
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• W2003634626 title "Quantitative polymerase chain reaction analysis with allele-specific oligonucleotide primers for individual IgH VDJ regions to evaluate tumor burden in myeloma patients" @default.
• W2003634626 cites W1502322024 @default.
• W2003634626 cites W1963853602 @default.
• W2003634626 cites W1991660588 @default.
• W2003634626 cites W2012271436 @default.
• W2003634626 cites W2019612632 @default.
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• W2003634626 cites W2045011755 @default.
• W2003634626 cites W2045343779 @default.
• W2003634626 cites W2047308944 @default.
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• W2003634626 cites W2086595383 @default.
• W2003634626 cites W2086653135 @default.
• W2003634626 cites W2110953306 @default.
• W2003634626 cites W2120474651 @default.
• W2003634626 cites W2121411022 @default.
• W2003634626 cites W2129573188 @default.
• W2003634626 cites W2136898525 @default.
• W2003634626 cites W2163130746 @default.
• W2003634626 cites W2323821297 @default.
• W2003634626 cites W2326331137 @default.
• W2003634626 cites W4235448637 @default.
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