Minimal Residual Disease In Multiple Myeloma Research Articles

Identification of glutathione S-transferase π 1 as a prognostic proteomic biomarker for multiple myeloma using proteomic profiling.

Multiple myeloma (MM) is a B-cell hematological malignancy with monoclonal plasma cell proliferation in the bone marrow. Early diagnosis of MM remains difficult due to the lack of specific symptoms and biomarkers. In the present study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the ClinProt system was used to detect potential biomarkers for MM from the bone marrow samples of 30 patients and 30 healthy controls. A total of 10 of the most significantly differentiated peaks between the patients and controls were identified. When patients with MM were compared with controls, 6 peaks with m/z values of 1,779.24, 1,866.32, 2,022.36, 2,878.9, 4,417.76 and 7,155.38 were upregulated, and 4 peaks with m/z values of 1,466.54, 1,520.02, 1,546.53 and 2,991.05 were downregulated. Of these 10 peaks, 4 peaks (pk 8, 1,866.32 Da; pk 15, 2,878.90 Da; pk 17, 2,991.05 Da; and pk 3, 1,520.02 Da) were further sequenced and identified using liquid chromatography/electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Furthermore, the expression of fibronectin 1 and glutathione S-transferase π 1 (GSTP1) were validated in patients with MM via ELISAs. Clinical data and statistical analysis indicated that GSTP1 expression was closely associated with the clinical stage of patients with MM. High GSTP1 levels were an independent risk factor for worse prognosis in patients with MM. These results demonstrate that GSTP1 may be a novel biomarker for early diagnosis, prognosis and monitoring of minimal residual disease in MM.

The Pathogenesis of Multiple Myeloma (MM) Is Preceded By Mutated Lymphopoiesis and B Cell Oligoclonality That Persist in Patients with Negative Minimal Residual Disease (MRD)

The Pathogenesis of Multiple Myeloma (MM) Is Preceded By Mutated Lymphopoiesis and B Cell Oligoclonality That Persist in Patients with Negative Minimal Residual Disease (MRD)

Flow Cytometry-Based Photodynamic Diagnosis with 5-Aminolevulinic Acid for the Detection of Minimal Residual Disease in Multiple Myeloma.

Multiple myeloma is the cancer of plasma cells. Along with the development of new and effective therapies, improved outcomes in patients with multiple myeloma have increased the interest in minimal residual disease (MRD) monitoring. However, the considerable heterogeneity of immunophenotypic and molecular markers of myeloma cells has limited its clinical application. 5-Aminolevulinic acid (ALA) is a natural compound in the heme biosynthesis pathway. Following ALA treatment, tumor cells preferentially accumulate porphyrins because of the differential activities of aerobic glycolysis, known as Warburg effect. Among various porphyrins, protoporphyrine IX is a strong photosensitizer; thus, ALA-based photodynamic diagnosis has been widely used in various solid cancers. Here, the feasibility of flow cytometry-based photodynamic detection of MRD was tested in multiple myeloma. Among various human cell lines of hematological malignancies, including K562 erythroleukemia, Jurkat T-cell leukemia, Nalm6 pre-B cell leukemia, KG1a myeloid leukemia, and U937 monocytic leukemia, human myeloma cell line, KMS18, and OPM2 abundantly expressed ALA transporters, such as SLC36A1 and SLC15A2, and 1 mM ALA treatment for 24 h resulted in nearly 100% porphyrin fluorescence expression, which could be competitively inhibited by ALA transport with gamma-aminobutyric acid. Titration studies revealed that the lowest ALA concentration required to achieve nearly 100% porphyrin fluorescence in KMS18 cells was 0.25 mM, with an incubation period of 2 h. Under these conditions, incubation of primary peripheral blood mononuclear cells resulted in only 1.8 % of the cells exhibiting porphyrin fluorescence. Therefore, flow cytometry-based photodynamic diagnosis is a promising approach for detecting MRD in multiple myeloma.

Validation of a Sensitive Flow Cytometry Assay to Assess Minimal Residual Disease of Multiple Myeloma Cells in Apheresis Product

For treatment of patients with multiple myeloma (MM), flow cytometry has become a widely used and valuable method for the evaluation of minimal residual disease (MRD) in bone marrow. Use of an optimized single-tube, 10-color flow cytometry panel for assessment of MM MRD has shown to be beneficial in patient monitoring and has shown correlation to the acknowledged EuroFlow 8-color, 2-tube method. In order to further correlate levels of MM and relapse in patients, validation of a sensitive MM MRD assay that can be applied to testing for residual disease in apheresis product prior to autologous stem cell transplant, a standard treatment for patients with multiple myeloma. This approach can exhibit great value in patient monitoring and treatment. As new combination therapies are developed for treatment of multiple myeloma in concert with autologous stem cell transplantation, evaluation of MRD in apheresis will likely continue to grow in importance. The purpose of this study is to show the validation and sensitivity of a flow cytometry assay designed to detect Multiple Myeloma (MM) cells in G-CSF and other mobilized apheresis samples from human Multiple Myeloma patients. Using GSM-mobilized apheresis product, spiked with different levels of patient de-identified MM plasma cells, validation of a 10-color MM MRD panel can be evaluated, intended to mimic patient apheresis at varying levels of residual disease post treatment. As with any minimal residual disease assessment, a high number of events are required to ensure sensitivity and precision. Typically, acquisition of 3-5 x 107 events is required to ensure precision of MM MRD levels to 0.001%. DisclosuresNo relevant conflicts of interest to declare.

Whole Exome Sequencing of Residual Disease in Multiple Myeloma: Searching for Novel Therapeutic Targets

Introduction:Multiple myeloma (MM) is a plasma cell dyscrasia causing damage of multiple organs with fatal consequences for patients. Despite the success of modern therapies eliminating a vast bulk of the aberrant cells, surviving residual clones eventually lead to the relapse of the disease. Accumulation of genomic alterations during the stage of minimal residual disease (MRD) likely contributes to a selective grow advantage and survival under the drug pressure. Identification of specific mutations in MM patients with MRD can provide unique opportunities to target the residual plasma cell clones. Here we present the first whole exome sequencing (WES) analysis of 22 MM samples of patients with MRD that identified 814 mutated genes with 4% of genes previously implicated in the pathogenesis of MM.Methods:Aberrant plasma cells (A-PCs) and peripheral blood (PB) were collected from patients after signing informed consent form. Presence of MRD was assessed with EuroFlow protocol and A-PCs were sorted out from bone marrow according to their pathological immunophenotype based on the expression of antigens CD38, CD45, CD19 and CD56. DNA from A-PCs was isolated and amplified by Repli-g Single cell kit (QIAGEN). Sequencing libraries were prepared using SureSelect Human All Exon V6 Kit (Agilent Technologies) and sequenced by Macrogen Inc. on Illumina HiSeq 4000 platform with average coverage 50x and 2x 100bp read length. Sequencing data were processed using the Bcbio framework following the standard workflow for tumor-matched-normal studies. Specifically, reads were mapped to the human reference genome GRCh37, successively marking duplicates using Picard. Germline mutations were identified using GATK HaplotypeCaller, whereas somatic mutations were identified using MuTect2 reporting as significant variants observed in at least 5 reads and minimum allele frequency of 10%. Variants in homopolymer regions longer than 5 nucleotides were filtered out. The final set of calls were further characterised by assessing their functional impact using snpEff and by annotating each variant using data from 1000 Genomes Phase 3, ExAC, and ClinVar. We then used OncodriveCLUST to identify putative oncogenic genes, and later compared these results with a literature curated list of MM driver genes (Weaver & Tariman, 2017).Results:Our dataset comprises 22 samples from 21 patients (one patient was sampled in two time points) with MM MRD, who received bortezomib-based regimen (age 41-71, average 59 years, 11/22 males, 10/22 females). 8 patients reached complete response, 9 patients had very good partial response and 4 patients had partial response. In our analysis, we detected 1,014 tumour somatic variants (8-287 per sample, median 36), most of them being missense mutations (676/1014), splice site mutations (145/1014) and frameshift insertions (134/1014). The variants affected a total of 814 genes, 97 genes were shared in at least two samples. The most frequently mutated genes were KIAA1211 (11/22), the immunoglobulin gene IGLV3-1 (8/22), apoptotic chromatin condensation inducer ACIN1 (7/22) and CCR4-associated factor 3 CNOT3 (7/22). We also identified 32 genes known to be mutated in MM in 64% of our samples (14/22). We found mutations shared by at least 2 samples in KRAS (4/22), DIS3 (3/22), TRAF3 (3/22), NRAS (2/22), ANK2 (2/22), BRAF (2/22) and RBM15 (2/22). Further analysis with OncodriveCLUST identified 18 putative oncogenic genes (FDR < 0.1), including KRAS, DIS3, ACIN1.Conclusion:We presented the first whole exome study of MM MRD, providing a characterisation of the mutations observed in A-PCs. We overcame problem with low amount of A-PCs in this disease stage by using whole genome amplification and a highly customised bioinformatic analysis pipeline. Our study suggests that A-PCs are characterised by new MM MRD specific set of mutated genes, along with the presence of mutations in well-known multiple myeloma cancer driver genes. This offers a great potential for design of novel precise treatments targeting MRD after standard MM therapies.Supported by Ministry of Health of the Czech Republic (17-30089A, CZ-DRO-FNOs/2016) and Ministry of Education of the Czech Republic (SGS18/PřF/2017-2018) DisclosuresKryukov:JSC BIOCAD: Employment. Maisnar:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Hajek:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Novartis: Research Funding.

Next Generation Flow for Multiple Myeloma Minimal Residual Disease: Igh Rearrangement NGS Is Complement to the NGF

Background: Detection of leukemia-associated aberrant immuno-phenotype is used to assess minimal residual disease (MRD) by multi-parameter flow cytometry (MFC). However, detection of MRD by MFC remains to be a challenging due to the possible change in aberrant immunophenotype during disease progress. In our present study, we compared International Myeloma Working group (IMW) treatment response and NGF MRD, including BM PC% and cytogenetics. Thereon, we conducted IgH rearrangement study by NGS in cases showing discrepant results.Methods: A total of 35 BM (35 myeloma patients at follow-up) was enrolled. We performed NGF using 8-color antibody panel using Navios flow cytometer and Infinicyt. Linearity of NGF was validated with myeloma cell line (U266) and BM specimen at initial diagnosis in myeloma patient. IgH rearrangement NGS was performed using Immunoseq assay (Adaptive Biotechnologies, Seattle, WA, USA). Paired specimen at initial diagnosis BM and follow-up BM were subjected to NGS study.Results: Detection sensitivity of NGF was <0.001%. Patients who achieved CR or sCR showed MRD negativity in 63.6% (7/11). Twenty-three patients showed neoplastic PCs above LLOQ and their response criteria were 1 sCR, 3 CR, 2 VGPR, 3 PR, 1 MR, 5 SD, 3 progressive disease, 3 relapse, and 2 with unavailable response. Four patients who did not achieve CR (1 VGPR, 1 PR, 1 MR, and 1 SD) showed MRD negativity by NGF. In 4 patients with discrepancy between IMW treatment response and NGF, we compared the results of IgH NGS at initial BM with those after treatment. NGS revealed a persistence of residual clone in 1 patient and an acquisition of new clone after treatment. One patient had same dominant clone both initial diagnosis BM (95.2%; proportion of clone) and follow-up BM (45.8%). The other patient had newly appeared clones in follow-up BM (6.12%, 5.63%, 3.42%, 3.11%, 3.09%) which clones were absent in initial diagnosis BM. The other 2 patients showed heterogeneous clones without dominant clone at follow-up BM by NGS. Results of FISH and immunofixation are summarized in Table 1. This results show IgH rearrangement NGS can detect malignant clone that could not be identified by using NGF.Conclusions: Thirty-six percent of patients who did not achieve CR showed NGF MRD negativity and NGS revealed residual clones in half of them. Switching of immunophenotypes of neoplastic PC can escape monitoring of NGF, and complementary NGS test is needed to catch such drifting clones for monitoring of MRD in MM. DisclosuresNo relevant conflicts of interest to declare.

The Prognostic Significance of Minimal Residual Disease and Possibility of Its Correction in Multiple Myeloma

Introduction.Most patients with multiple myeloma (MM) are considered to be incurable, and relapse owing to minimal residual disease (MRD) is the main cause of death among these patients, the optimal methodology to assess MRD is not clear. The results of previous studies demonstrated the potential of multiparameter flow cytometry (MFC) and (PET-CT) in evaluation of MRD in MM. MRD monitoring should be applied in prospective clinical trials to compare and evaluate the efficacy of different treatment strategies, particularly in the consolidation and maintenance settings. The impact of MRD negativity is important, but further studies are needed to quantify the pharmacoeconomic and quality-of-life differences between early and delayed transplant strategies. Therefore, with the currently available evidence, upfront autologous stem cell transplantation (ASCT) is standard of care regardless of MRD status.Aim.We are aiming to determine the role of MRD and role of autologous stem cells transplantation in MM.Materials and methods.We've recently started a prospective one-center pilot study in subjects with MM. We analyzed 18 transplant-eligible patients with MM (the median age is 57 years, a male/female ratio is 3.5:1).The induction therapy Bortezomib-based only regimens was used in 12/18 (67%) patients, combination of Bortezomib-Immunomodulator-based regimens - in 6/18 (33%). High dose therapy (Mel200) and ASCT is carried out on 100% patients. The standard risk was established on 15 patients, 1 patient has an intermediate risk and 2 patients have high risk according to mSMART 2.0 stratification. The MFC MRD status of bone marrow was evaluated after 4-6 cycles of induction therapy and after ASCT on 5-color flow cytometry with use anti- CD38, CD138, CD45, CD19, CD20, CD27, CD56 and CD117 antibodies. We were based on two levels: MFC MRD- (<10-4) and MFC MRD- (<10-5) for assessing the significance of factors that affect MRD and for identifying the prognostic potential of MRD-negative status. The evaluation of MRD was carried out by genetic (cytogenetic and FISH) analysis of bone marrow plasma cells and PET-CT with 18-FDG before ASCT and on 100 day post ASCT.The results.The MFC MRD- (<10-4) before carrying out an ASCT reached 22.2% (4/18), the MFC MRD- (<10-5) - 0% and was not depended on the variant of pre-transplantation regimen. After the ASCT had been carried out there was a tendency to decrease the tumor burden in bone marrow from 0.65% to 0.1% and to increase the frequency of MFC MRD- (<10-4) status to 44.4% (8/18), of which MFC MRD- (<10-5) was 16.7% (3/18). MRD status was determined before ASCT and after ASCT by MFC and FISH in patients with high risk. The use of maintenance therapy with bortezomib (n = 5) or lenalidomide (n = 13) did not increase the frequency of MRD status. The PFS median in MFC MRD+ (>10-4) group was 23 months, in the MFC MRD- (<10-4) was not achieved; 2-year PFS was 43% and 100%, respectively (p=.04) We compared PFC between MFC MRD+ (>10-4) before ASCT (n = 4) and MFC MRD- (<10-4) after ASCT (n = 6) to assess the effect of ASCT in MM. The median PFS was not reached in both groups; 2-year PFS was 67% and 100%, respectively. The reliable difference between PFS in MFC MRD- (10-4-10-5) group and MFC MRD- (<10-5) was absent: the median of PFS was not achieved in both groups. PET-CT has been tested on 15 patients, PET-CT- response was achieved in 53% (8/15) patients. The PFS median in PET-CT+ group and PET-CT- group was not achieved. The 2-year PFS was higher in PET-CT+ group then PET-CT- probably due to patients with MFC MRD-. The 2-year PFS in «MFC MRD-PET-CT-» group was 100% to 55% in other patients.Conclusion.Carrying out ASCT demonstrated a tendency to increase the percentage of MFC MRD negative responses and improvement of PFS. The use of MFC in evaluation of MRD should be complemented with PET-CT and genetic methods for further analysis of the MFC MRD role status on MM patients. DisclosuresNo relevant conflicts of interest to declare.

Quantification of Residual Bone Marrow Plasma Cells By Eight-Color Flow Cytometry in Patients with Multiple Myeloma: Comparison between the Dura Clone RE PC Panel (Beckman Coulter) and the Euro Flow MM-MRD Panel (Cytognos)

【Background】The use of novel agents with different mechanisms of action has resulted in high complete response (CR) rates and improved the survival of patients with multiple myeloma (MM). Obtaining minimal residual disease (MRD) negativity following treatment is an important determinant for longer survival in patients with MM. The EuroFlow MM-MRD panel (EuroFlow panel) using the Infinicyt software (Cytognos, Salamanca, Spain) is a two-tube six-color antibody panel. By the addition of two drop-in antibodies (CD138, CD27; vendor of user's choice) into both tubes, an eight-color configuration, which has been validated by the EuroFlow consortium is obtained. The DuraClone RE PC kit (DuraClone panel) is a recently developed one-tube eight-color dry antibody panel using the Kaluza software (Beckman Coulter, Miami, USA) for manual identification of abnormal plasma cells (PCs) to detect MRD in MM. Although both methods are available, a comparison between them has yet to be performed. This study aimed to compare the EuroFlow panel and the DuraClone panel analysis of residual abnormal plasma cells by eight-color flow cytometry.【Methods】Patients with International Myeloma Working Group defined symptomatic MM treated at Kanazawa University Hospital and Kameda Medical Center in Japan from January 2017 to July 2018 were included. Bone marrow samples were obtained as part of routine clinical practice for evaluating treatment response when appropriate. Bone marrow aspirates (4 mL; first pull of bone marrow aspiration) anti-coagulated with ethylenediamine tetraacetic acid were evenly split (2 mL each) for the EuroFlow panel and the DuraClone panel. PC quantification by the EuroFlow panel was performed at Kanazawa University Hospital and the DuraClone panel was performed at Kameda Medical Center. The monoclonal antibodies used in the EuroFlow and the DuraClone panels are detailed in Table 1. Identification of neoplastic PCs required ≥ 20 cells. Sample processing and measurement of PCs were performed within 48 hours of collection.The correlation of total leukocyte acquisition, total plasma cells and MRD were analyzed for both methods. Qualitative comparison of MRD negativity was also performed. Spearman's correlation coefficient was used for evaluating the correlation of paired data. The Bland-Altman plot was used for detection of fixed bias and proportional bias.【Results】A total of 79 samples were analyzed. We first assessed the number of total leukocytes using the EuroFlow panel and the DuraClone panel. The median number of total leukocytes acquired with the EuroFlow panel was significantly higher than with the DuraClone panel (median: 8,505,172 cells, interquartile range [IQR]: 6,164,094 - 9,105,097 cells and median: 2,858,026 cells, IQR: 1,466,004 - 4,284,926 cells, respectively; p < 0.01), but the percentage of total plasma cells showed good concordance between both methods (regression coefficient = 0.91, p < 0.01). The median percentage of abnormal plasma cells was 0.0035% (IQR: 0.0005 - 0.05) and 0.0046% (IQR: 0.0005 - 0.05) using the EuroFlow panel and the DuraClone panel, respectively. Figure 1A shows scatter plots comparing the percentage of abnormal cells analyzed using the EuroFlow panel and the DuraClone panel. Both methods showed a high degree of concordance (regression coefficient = 0.90, p < 0.01). Bland-Altman plots also showed good agreement between both methods. There was no statistically significant fixed bias with a mean difference of 10% (95% confidence interval [CI]: -12~14%). In addition, no significant proportional bias was detected between the two methods (regression coefficient: -0.01, p = 0.85; Figure 1B).Qualitative analysis of MRD negativity also showed substantial agreement between the two methods (kappa = 0.7); 11 of 79 (13.9 %) samples showed discrepancy for determination of MRD negativity (< 1 × 10-5). Nine of these samples were MRD negative by the EuroFlow panel, but MRD positive by the DuraClone panel, and two samples were MRD positive by the EuroFlow panel, but MRD negative by the DuraClone panel. However, all discrepancies occurred near the limit of detection of both methods (1.0 × 10-5).【Conclusion】Our findings indicate that the DuraClone panel has good overall concordance with the EuroFlow panel for the detection of abnormal PCs in MM samples. The one-tube DuraClone panel enables to reduce processing time and test cost compared with the two-tube EuroFlow panel. [Display omitted] DisclosuresTakamatsu:Celgene: Honoraria, Research Funding; Janssen: Honoraria; Bristol-Myers Squibb: Research Funding; Ono: Research Funding. Nakao:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria; Novartis: Honoraria.

Comparison of Minimal Residual Disease Detection in Autografts of Patients with Multiple Myeloma between 8-Color Multiparameter Flow Cytometry (EuroFlow) and Next-Generation Sequencing

Background: Autologous stem cell transplantation (ASCT) in conjunction with novel therapeutic drugs can dramatically improve response rates and the prognosis of patients with multiple myeloma (MM). However, most patients with MM are considered to be incurable, and relapse owing to minimal residual disease (MRD) is the main cause of death among these patients. Therefore, new technologies to assess deeper responses are required. Next-generation sequencing (NGS) and multiparameter flow cytometry (MFC) methods have been used to assess MRD. However, the lack of standardization of conventional MFC approaches has had a negative impact on its reproducibility. Recently, a next-generation MFC method (EuroFlow, NGF) has been developed by the EuroFlow Consortium and the International Myeloma Foundation (IMF) for a highly sensitive and standardized detection of MRD in MM.Aims: To compare the prognostic value of MRD detection in autografts in MM between NGS (Adaptive) and 8-color MFC method (EuroFlow, NGF), and also MRD levels between fresh and cryopreserved autografts.Methods: A total of 39 newly-diagnosed MM patients who underwent ASCT were enrolled in this study. Median age 60 at ASCT (range 41-69); males 22, females 17; ISS 1 (n=10), 2 (n=19), 3 (n=10). 10 patients showed high-risk chromosomal abnormalities (t(4;14) (n=9), del17p & t(4;14) (n=1)). The induction regimen was bortezomib-based chemotherapy. All patients received melphalan 200 mg/sqm as conditioning regimen before ASCT. 34 of 39 (87%) patients received maintenance therapy until progressive disease. The best response post-ASCT was as follows: 23sCR, 2CR, 12VGPR, 2PR. 39 autografts, one from each MM patient, were analyzed using NGF and NGS methods. The NGF method was based on a standardized lyse-wash-and-stain sample preparation protocol, the measurement of high numbers of cells and an optimized 8-color, 2-tubes, antibody panel, for accurate identification of plasma cells (PCs) and discrimination between phenotypically aberrant (aPC) and normal PC (nPC) (J Flores-Montero et al., Leukemia 2017). NGS-based MRD assessment was performed using Adaptive's standardized NGS-MRD Assay (Seattle, WA) (Martinez-Lopez et al., Blood 2014). To assess the correlation of MRD levels between fresh and cryopreserved autografts using NGF, 6 additional MM patients' autografts were used.Results: MRD levels in all 39 autografts were assessed using EuroFlow, while those in 32 of 39 (82%) were assessed with NGS due to limited availability of material for calibration. We identified abnormal plasma cells (aPC) in autografts based on multivariate analysis of individual cells from each patient (e.g. CD56+, CD19-, CyIgκ+, CD117+). Since there was a good correlation in MRD levels between fresh and thawed frozen autografts detected by EuroFlow (R=0.943, P=0.02), we assessed the MRD levels in thawed frozen autografts. For the MM MRD in autografts, the events from tube 1 and tube 2 were combined and a median of 7.3×106 (range: 2.2×106-37.6×106) events was acquired. The sensitivity of EuroFlow was 1×10-5-2×10-6 while that of NGS was 10-7 due to the high number of DNA derived from autografts (Takamatsu et al., Ann Oncol 2017). 21 of 39 (54%) cases were MRD positive by 8-color MFC while 22 of 32 (69%) cases were MRD positive by NGS. The correlation of MRD levels between 8-color MFC and NGS was relatively high (Fig. 1A). MRD negative by NGF (MRDMFC (-)) cases tended to show better PFS than MRDMFC (+) cases (P=0.145) (Fig. 1B) while MRD negative by NGS (MRDNGS (-)) cases showed significantly better PFS than MRDNGS (+) cases (P=0.03) (Fig. 1C). Furthermore, MRDMFC (-) MRDNGS (-) cases showed significantly better PFS than MRDMFC (-) MRDNGS (+) cases (P=0.01), but the PFS of MRDMFC (-) MRDNGS (+) cases was not different from that of MRDMFC (+) MRDNGS (+) cases (P=0.70). MRDMFC (-) and MRDNGS (-) cases showed better OS than MRDMFC (+) (P=0.14) and MRDNGS (+) (P=0.08) cases, respectively.Conclusions: Although EuroFlow is a fast and accurate method for detecting MRD of MM in autografts, in this study the NGS platform had a higher sensitivity and prognostic value than EuroFlow. The homogenous nature of the mobilized autograft relative to the focal nature of myeloma in bone marrow might provide a better sample to assess MRD. [Display omitted] DisclosuresTakamatsu:Celgene: Honoraria, Research Funding; Ono: Research Funding; Bristol-Myers Squibb: Research Funding; Janssen: Honoraria. Nakao:Novartis: Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria.

Minimal residual disease detection in multiple myeloma: current status, challenges and strategies

Long time clinical results have showed that multiple myeloma (MM) patients who achieved complete remission (CR) had heterogeneous prognosis. Some patients who achieved CR relapsed in a short time, even died within 2 years after initial therapy, while other patients acquired long-term survival. With the emergence of minimal residual disease (MRD) assessment, rigorous personal therapy monitoring and accurate prognostic evaluation could be realized. MRD can monitor inside the bone marrow by multi-parameter flow cytometry, allele-specific oligonucleotide-based quantitative polymerase chain reaction and next-generation sequencing, as well as the external conditions of the bone marrow by sensitive imaging techniques. This article focuses on clinical benefit, characteristics of different monitoring methods and future direction of MRD in MM. Key words: Multiple myeloma; Neoplasm, residual; Prognosis; Multi-parameter flow cytometry

Ocena minimalnej choroby resztkowej w szpiczaku plazmocytowym

Minimalną chorobę resztkową (MRD) u pacjenta z rozpoznaniem szpiczaka plazmocytowego defi-niuje się jako populację nowotworowych komórek plazmatycznych, która pozostała w organizmie chorego po osiągnięciu odpowiedzi całkowitej (CR). Ostatecznym celem badań MRD jest dążenie do identyfikacji chorych o odmiennym rokowaniu i indywidualizacji leczenia na tej podstawie. Ocenę MRD u chorych na szpiczaka plazmocytowego zaleca się u młodszych pacjentów, którzy osiągną CR po przeszczepieniu autologicznych krwiotwórczych komórek macierzystych oraz u chorych starszych osiągających CR po chemioterapii opartej na inhibitorach proteasomu. Powinno się oznaczać MRD wyłącznie w szpiku kostnym. Do rekomendowanych metod oceny MRD w szpiczaku plazmocytowym zalicza się sekwencjonowanie następnej generacji genów immunoglobulinowych oraz wieloparametryczną cytometrię przepływową, przy czym wybór jednej z tych metod powinien zależeć od doświadczenia danego ośrodka oraz możliwości wykonania badania cytometrycznego w czasie 24 godzin od pobrania próbki szpiku. Eradykację MRD powinna się obecnie uważać za istotny cel terapii szpiczaka plazmocytowego. Jednak, ze względu na brak wystarczających danych do podejmowania decyzji klinicznych wyłącznie na podstawie wyniku badania MRD, istnieje po¬trzeba dalszych, dobrze zaprojektowanych badań klinicznych w tym zakresie.

Imagerie des lésions osseuses du myélome

Multiple myeloma (MM) differs from other lymphoid B malignancy by its characteristic bone disease. During the course of their MM, up to 80% of patients will suffer from this osteolytic disease or its sometimes-revealing complications. Assessment of MM-related bone disease is important: it can conduct to decide the initiation of antitumoral therapy. Conventional skeletal X-rays (but more and more often whole-body low-dose computed tomography (CT)) and spine and pelvis or preferential whole-body magnetic resonance imaging (MRI) are the gold-standard for the assessment at diagnosis and relapse. If necessary, CT is a complementary imaging method. The role of 18F-fluorodeoxyglucose positron emission tomography (PET)/CT increases in assessment of response to therapy and detection of MM minimal residual disease. Future will clarify the role of diffusion-weighted and dynamic contrast-enhanced MRI or of new imaging technologies such as PET/MRI.

Hedgehog and retinoid signaling alters multiple myeloma microenvironment and generates bortezomib resistance.

Interactions between multiple myeloma (MM) cells and the BM microenvironment play a critical role in bortezomib (BTZ) resistance. However, the mechanisms involved in these interactions are not completely understood. We previously showed that expression of CYP26 in BM stromal cells maintains a retinoic acid-low (RA-low) microenvironment that prevents the differentiation of normal and malignant hematopoietic cells. Since a low secretory B cell phenotype is associated with BTZ resistance in MM and retinoid signaling promotes plasma cell differentiation and Ig production, we investigated whether stromal expression of the cytochrome P450 monooxygenase CYP26 modulates BTZ sensitivity in the BM niche. CYP26-mediated inactivation of RA within the BM microenvironment prevented plasma cell differentiation and promoted a B cell-like, BTZ-resistant phenotype in human MM cells that were cocultured on BM stroma. Moreover, paracrine Hedgehog secretion by MM cells upregulated stromal CYP26 and further reinforced a protective microenvironment. These results suggest that crosstalk between Hedgehog and retinoid signaling modulates BTZ sensitivity in the BM niche. Targeting these pathological interactions holds promise for eliminating minimal residual disease in MM.

Research progress of minimal residual disease in multiple myeloma

In recent years, With the improvement of outcomes for patients with multiple myeloma(MM), the duration of progression-free survival(PFS) and overall survival (OS) were extended. Many patients achieved complete response(CR), but most patients will inevitably replased, and due to the presence of minimal residual disease (MRD). MRD is defined as the small number of cancer cells persisted in the patients who achieving CR after treatment. The detection of MRD in MM can provide the basis for diagnosis, treatment and prognosis of MM. In this review, we will introduce the techniques for MRD detection in patients with MM, and discuss the challenges and future opportunities for MRD testing. Key words: Multiple myeloma; Neoplasm, residual; Flow cytometry; Polymerase chain reaction

Phenotypic and genomic analysis of multiple myeloma minimal residual disease tumor cells: a new model to understand chemoresistance

AbstractPersistence of chemoresistant minimal residual disease (MRD) plasma cells (PCs) is associated with inferior survival in multiple myeloma (MM). Thus, characterization of the minor MRD subclone may represent a unique model to understand chemoresistance, but to our knowledge, the phenotypic and genetic features of the MRD subclone have never been investigated. Here, we compared the antigenic profile of MRD vs diagnostic clonal PCs in 40 elderly MM patients enrolled in the GEM2010MAS65 study and showed that the MRD subclone is enriched in cells overexpressing integrins (CD11a/CD11c/CD29/CD49d/CD49e), chemokine receptors (CXCR4), and adhesion molecules (CD44/CD54). Genetic profiling of MRD vs diagnostic PCs was performed in 12 patients; 3 of them showed identical copy number alterations (CNAs), in another 3 cases, MRD clonal PCs displayed all genetic alterations detected at diagnosis plus additional CNAs that emerged at the MRD stage, whereas in the remaining 6 patients, there were CNAs present at diagnosis that were undetectable in MRD clonal PCs, but also a selected number of genetic alterations that became apparent only at the MRD stage. The MRD subclone showed significant downregulation of genes related to protein processing in endoplasmic reticulum, as well as novel deregulated genes such as ALCAM that is prognostically relevant in MM and may identify chemoresistant PCs in vitro. Altogether, our results suggest that therapy-induced clonal selection could be already present at the MRD stage, where chemoresistant PCs show a singular phenotypic signature that may result from the persistence of clones with different genetic and gene expression profiles. This trial was registered at www.clinicaltrials.gov as #NCT01237249.

Multiple Myeloma Minimal Residual Disease.

Assessment of minimal residual disease (MRD) is becoming standard diagnostic care for potentially curable neoplasms such as some acute leukemias as well as chronic myeloid and lymphocytic leukemia. Although multiple myeloma (MM) remains as an incurable disease, around half of the patients achieve complete remission (CR), and recent data suggests increasing rates of curability with "total-therapy-like" programs. This landscape is likely to be improved with the advent of new antibodies and small molecules. Therefore, conventional serological and morphological techniques have become suboptimal for sensitive evaluation of highly effective treatment strategies. Although, existing data suggests that MRD could be used as a biomarker to evaluate treatment efficacy, help on therapeutic decisions, and act as surrogate for overall survival, the role of MRD in MM is still a matter of extensive debate. Here, we review the different levels of remission used to define depth of response in MM and their clinical significance, as well as the prognostic value and unique characteristics of MRD detection using immunophenotypic, molecular, and imaging techniques. Key facts The higher efficacy of new treatment strategies for MM demand the incorporation of highly sensitive techniques to monitor treatment efficacy MRD could be used as a more potent surrogate biomarker for survival than standard CR We need to understand the pros and cons of the different MRD techniques The time has come to incorporate highly sensitive, cost-effective, readily available, and standardized MRD techniques into clinical trials to assess its role in therapeutic decisions.

Better therapy requires better response evaluation: Paving the way for minimal residual disease testing for every myeloma patient.

In 2015, there is a large body of evidence demonstrating that minimal residual disease (MRD) negativity after therapy is a powerful predictor of progression-free survival and overall survival in multiple myeloma. On the basis of available data, we believe MRD provides a meaningful endpoint for regulatory purposes, academic studies, and a valuable prognostic evaluation of individual patients in the clinical setting. Similar to what has been shown in acute and chronic lymphocytic leukemia, based on emerging data, the prognostic impact of MRD in multiple myeloma appears to be independent of induction therapy received. This fact raises fundamental questions regarding best possible treatment strategies (e.g., fixed number of cycles versus response adapted number of cycles) as well as optimal treatment modalities (e.g., newer effective but less intense combination therapies versus high dose melphalan followed by autologous stem cell transplantation), in particular for patients newly diagnosed with multiple myeloma.

A Comparison of Minimal Residual Disease Detection Among ASO-PCR, Dd-PCR and Deep-Sequencing in Patients with Multiple Myeloma Who Underwent Autologous Stem Cell Transplantation

▪Background: Most patients with multiple myeloma (MM) are considered to be incurable, and relapse, due to minimal residual disease (MRD), is the main cause of death among these patients. Even though allele-specific oligonucleotide real-time quantitative PCR (ASO-qPCR) of immunoglobulin heavy chain gene rearrangement has been used to assess the MRD in MM due to its excellent sensitivity and specificity, ASO-qPCR has a major limitation in its relative quantification method because it needs a reference standard curve, which is usually made with dilutions of diagnostic myeloma DNA or from plasmids containing the target IgH rearrangement gene. Recently, droplet digital PCR (ddPCR) was developed to perform reliable absolute quantification of target genes. Based on the principle of single target gene detection, the sensitivity can be increased when the larger amount of DNA is analyzed. Here we assessed the prognostic value of MRD assessment in autografts from MM patients in the autologous stem cell transplantation (ASCT) setting using ASO-qPCR, ddPCR and next-generation sequencing (NGS) approaches.Methods: Twenty-three Japanese patients with newly diagnosed MM who received various induction regimens prior to ASCT without any post-ASCT therapy were retrospectively analyzed. Median age 57 (range 39-67); males 11, females 12; ISS 1 (n=7), 2 (n=12), 3 (n=3), not assessed (n=1). 11 patients were analyzed by G-banding and FISH (t(4;14), del17p, t(14;16)) and 4 patients showed high-risk chromosomal abnormalities (t(4;14) (n=2), t(14;16) (n=1), -13 by G-banding (n=1)). All patients had achieved a very good partial response (VGPR) or better after ASCT. Analyzed samples included: (1) BM slides from 20 MM patients at diagnosis, (2) fresh/frozen BM cells from 3 MM patients at diagnosis, and (3) obtained autografts. IGH-based ASO-qPCR was performed as described previously (Methods Mol Biol 2009). ddPCR was performed by the QX200 Droplet Digital PCR system (Bio-RAD Inc.) with a total 6000 ng of genomic DNA combined with the same ASO-primers and TaqMan-probes used in the ASO-qPCR. Droplets were generated by the QX200 droplet generator. End-point PCR (40 cycles) was performed on a C1000 Touch Thermal cycler (Bio-RAD Inc). The PCR product was loaded in the QX200 droplet reader and analyzed by QuantaSoft 1.7.4 (Bio-Rad Inc). NGS-based MRD assessment was performed using the immunosequencing platform (Adaptive Biotechnologies, South San Francisco, CA) (Martinez-Lopez et al Blood 2014).Results: Nineteen patients could be analyzed by ASO-qPCR and ddPCR, while all 23 patients could be analyzed by NGS. We compared MRD results in autografts between ddPCR and NGS. We observed a high correlation between ddPCR and NGS results of MRD (r=0.82, P<0.0001). Although 19 samples were MRD negative by ASO-qPCR (MRDASO (-)), 12 samples were MRD positive by NGS (0.6-46 x 10-6, median 3.5 x 10-6) (MRDNGS (+)) and 7 samples were MRD positive by ddPCR (1.2-102 x 10-6, median 7.5 x 10-6) (MRDddPCR (+)), demonstrating the higher sensitivity of NGS (10-6 or higher) and ddPCR compared to ASO-qPCR (10-4 -10-5). Two high-risk chromosomal abnormality cases (t(4;14) (n=1), -13 by G-banding (n=1)) could achieve MRDNGS (-) in autograft. We evaluated the association of clinical outcome with MRD in autografts using ddPCR and NGS. To investigate the value of sensitive MRD detection by ddPCR and NGS, we compared PFS in 7 MRDddPCR (+) cases (Group 1) with 12 MRDddPCR (-) cases (Group 2) and 12 MRDNGS (+) cases (Group 3) with 11 MRDNGS (-) (Group 4). Group 2 and Group 4 showed significantly better PFS than Group 1 (P = 0.028) and Group 3 (P=0.033), respectively (Figure 1A and 1B). We also compared 5 MRDddPCR (-) & MRDNGS (+) cases (Group 5) with 11 MRDNGS (-) cases (Group 4). Group 4 tended to show better PFS than Group 5 (P=0.063, Figure 1C).Conclusions: In this study, we showed the prognostic value of ddPCR and NGS-based MRD assessment in autografts of patients with MM. Although the ddPCR had improved sensitivity in detecting MRD in autografts and demonstrated higher prognostic value compared with ASO-qPCR, the NGS platform showed the highest sensitivity and prognostic value among these methods. [Display omitted] DisclosuresZheng:Adaptive Biotechnologies Corp: Employment, Equity Ownership. Moorhead:Adaptive Biotechnologies Corp: Employment, Equity Ownership. Faham:Adaptive Biotechnologies Corp.: Employment, Other: Stockholder.

Next Generation Flow (NGF): A High Sensitive Technique to Detect Circulating Peripheral Blood (PB) Clonal Plasma Cells (cPC) in Patients with Newly Diagnosed of Plasma Cell Neoplasms (PCN)

Introduction: Previous studies have shown that cPC can be detected in PB by conventional flow cytometry (FC) in around 70% of multiple myeloma (MM) and 37% of monoclonal gammopathy of undetermined significance (MGUS) patients at diagnosis. Its presence in MGUS has been associated with a higher risk of malignant transformation. We here investigated the utility and sensitivity of the EuroFlow-IMF NGF-MM minimal residual disease (MRD) approach for detecting circulating cPC in PB of patients with PCN. Methods: A total of 137 samples (including 71 PB and 66 bone marrow -BM- paired samples) from 71 newly-diagnosed PCN patients (37 MGUS; 21 MM; 5 SMM and 8 solitary plasmacytomas -SP-), plus 6 PB samples from healthy controls, were studied. Samples were processed following the EuroFlow Bulk Lysis Standard Operating Protocol (SOP) and stained with the EuroFlow-IMF MM MRD panel (Tube 1:CD138BV421/CD27BV510/CD38FITC/CD56PE/CD45PerCP-Cy5.5/CD19PE-Cy7/CD117APC/CD81APC-C750, and; Tube 2: identical to Tube 1 except for CyKappaAPC/CyLambdaAPC-C750). A median of 10.6 x106 events (range: 1.7 x106 - 15.7x106) were measured for PB samples using a FACSCanto II (BD Biosciences, San Jose, USA) instrument. Data were analyzed using the Infinicyt software (version 1.8.0RC6; Cytognos SL, Salamanca, Spain). Risk stratification of MGUS patients was established by the Mayo Clinic index. ROC analysis was used to define a cut-off to distinguish between MM and MGUS cases according to the percentage and absolute number of circulating PB cPC. Results: Overall, cPC were detected in the PB of all MM and SMM cases studied (100%) and more than half of MGUS patients (60%; p=0.005), while constantly absent in the eight patients with SP. Upon classifying MGUS patients according to the Mayo Clinic Index (n=32), positive PB samples were found in 25%, 62% and 73% of cases with scores of 0, 1 and 2, respectively. Median (range) percentage and absolute cPC numbers (per µL) were of 13 to 16 and 10 to 200 times lower (p 60% of cPCs within the PC BM compartment (R2 = 0.75; n=66). The cut-off obtained to distinguish between MM and MGUS cases according to the percentage and absolute number of cPCs circulating in PB was of 0.0009% and 0.055 cPC/µL with a sensitivity of 93% and 86%, and a specificity of 75% and 75% for relative and absolute numbers, respectively. Conclusions: The EuroFlow-IMF NGF-MM MRD panel and approach are well-suited for high sensitive detection of circulating cPC in the PB of virtually every newly-diagnosed MM and SMM patient and the majority of MGUS cases, particularly among MGUS at higher risk of malignant progression; interestingly in both patients groups, the presence of PB involvement and its levels were closely associated with the degree of involvement of the BM PC compartment by cPC. * Both authors have contributed similarly to this work and they should both be considered as first author. Disclosures Paiva:EngMab AG: Research Funding; Celgene: Consultancy; Janssen: Consultancy; Millenium: Consultancy; Binding Site: Consultancy; Sanofi: Consultancy; BD Bioscience: Consultancy; Onyx: Consultancy. Puig:Janssen: Consultancy; The Binding Site: Consultancy. Mateos:Celgene: Consultancy, Honoraria; Takeda: Consultancy; Onyx: Consultancy; Janssen-Cilag: Consultancy, Honoraria. Durie:Celgene: Consultancy; Onyx: Consultancy; Takeda: Consultancy; Johnson and Johnson: Consultancy. van Dongen:BD Biosciences (cont9d): Other: Laboratory Services in the field of technical validation of EuroFlow-OneFlow antibody tubes in dried format. The Laboratory Services are provided by the Laboratory of Medical Immunology, Dept. of Immunology, Erasmus MC, Rotterdam, NL; Cytognos: Patents & Royalties: Licensing of IP on Infinicyt software, Patents on EuroFlow-based flowcytometric Diagnosis and Classification of hematological malignancies, Patents on MRD diagnostics, and Patents on PID diagnostics. ; Cytognos (continued): Patents & Royalties: Royalty income for EuroFlow Consortium. The Infinicyt software is provided to all EuroFlow members free-of-charge.Licensing of Patent on detection of IgE+ B-cells in allergic diseases. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; InVivoScribe: Patents & Royalties: Licensing of IP and Patent on BIOMED-2-based methods for PCR-based Clonality Diagnostics.. Royalty income for EuroClonality-BIOMED-2 Consortium ; Immunostep: Patents & Royalties: Licensing of IP and Patents on immunobead-based dection of fusion proteins in acute leukemias and other tumors. Royalties for Dept. of Immunology, Erasmus MC and for EuroFlow Consortium ; BD Biosciences: Other: Educational Services: Educational Lectures and Educational Workshops (+ related travelling costs). The lectures and workshops fully focus on the scientific achievements of the EuroFlow Consortium (No advertisement of products of BD Biosciences). , Patents & Royalties: Licensing of IP and Patent on EuroFlow-based flowcytometric Diagnosis and Classification of hematological malignancies; Royalty income for EuroFlow Consortium.; Roche: Consultancy, Other: Laboratory Services in the field of MRD diagnostics, provided by the Laboratory of Medical Immunology, Dept. of Immunology, Erasmus MC, Rotterdam, NL.. San Miguel:Janssen-Cilag: Honoraria; Onyx: Honoraria; Millennium: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Sanofi-Aventis: Honoraria.

Next Generation Flow (NGF) for High Sensitive Detection of Minimal Residual Disease (MRD) in Multiple Myeloma (MM)

Next Generation Flow (NGF) for High Sensitive Detection of Minimal Residual Disease (MRD) in Multiple Myeloma (MM)