To see the other types of publications on this topic, follow the link: Euroflow.
Journal articles on the topic 'Euroflow'
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 journal articles for your research on the topic 'Euroflow.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse journal articles on a wide variety of disciplines and organise your bibliography correctly.
1
Narita, Kentaro, Takeshi Yoroidaka, Momoko Fujisawa, Hiroyuki Takamatsu, Shinji Nakao, and Kosei Matsue. "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)." Blood 132, Supplement 1 (November 29, 2018): 3187. http://dx.doi.org/10.1182/blood-2018-99-116108.
Full textAbstract:
Abstract 【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. Disclosures Takamatsu: 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.
2
Okazuka, Kiyoshi, Tadao Ishida, Jun Sakamoto, Nobuhiro Tsukada, and Kenshi Suzuki. "MRD Detection in Myeloma Cells: Comparison between Inexpensive 1-Tube 10 Color Multiparameter Flow Cytometry and Euroflow Multiparameter Flow Cytometry." Blood 134, Supplement_1 (November 13, 2019): 1832. http://dx.doi.org/10.1182/blood-2019-123900.
Full textAbstract:
Background: Achievement of MRD-negative status in MM patients has been demonstrated to be associated with better outcome. EuroFlow MM-MRD has been a global standard for MRD detection by MFC. In Japan, EuroFlow is limited to research use only, because the dynamics of the population have raised concerns about increasing health care expenditure in Japan. Therefore, it is desirable to develop an inexpensive FCM method with equivalent sensitivity to EuroFlow for detecting MRD. Aims: In this study, we analyzed the number and percentage of total plasma cells (PC), abnormal PC with EuroFlow MM-MRD and 10-color-MFC in 50 MM patients. Patients and Methods: Bone marrow samples collected from 50 MM patients were subjected to MRD detection by EuroFlow MM-MRD and 10-color-MFC simultaneously. EuroFlow MM-MRD was performed according to EuroFlow standard operating procedure. 10-color MFC was performed at BML corporation. Briefly, CD38 multiepitope FITC, CD138 V450, CD45 V500-C, CD56 PE, CD19 APC-H7, CD27 APC R700, CD81 BV605, CD117 PE-Cy7, cIgk APC and cIgl PerCP-Cy5.5 of monoclonal antibody were used according to the ICCS(International Clinical Cytometry Society) guidelines. Data were analyzed with FACSuite ver 1.2.1(Becton Dickinson) using a FACSLylic(Becton Dickinson). Wilcoxon signed-rank test was used for the comparison of paired variables. Results: The representative example of 10-color-MFC were shown in figure 1. We first compared the percentage of normal plasma cells in bone marrow. The percentage of normal plasma cells analyzed with 10-color-MFC and EuroFlow were 0.287% (range 0.001-1.0322%) and 0.257%(range0.0002-1.37%), respectively. The good correlation between them was observed (r=0.509). The percentage of myeloma cells determined with 10-color-MFC and EuroFlow were 0.0705%(range 0-0.9086%) and 0.0940% (range 0-1.3000%), respectively. Quantification of abnormal plasma cells were highly similar between 2 tests (r=0.977, figure 2). To compare the percentage of abnormal plasma cells analyzed with 10-color-MFC and EuroFlow, p-value determined with Wilcoxon signed-rank test was 0.0069 in 50 sample. However, when total bone marrow cells were 3 million cells, limit of detection (LOD) and limit of quantification (LOQ) in 10-color MFC were defined as 0.001% and 0.0017%, respectively. The correlation in samples with less than 3 million of bone marrow cells was not good (p=0.424). Only when 3 million or higher number of bone marrow cells were analyzed, better correlation is thought to be observed (p=0.0001). High concordant analytical results were observed with an overall qualitative concordance of 95%. Conclusion: Our 10-color-MFC demonstrated high sensitivity to detect MRD with good correlation and also inexpensive (about 100 dollars as of July 2019). However, 3 million or higher number of bone marrow cells is inevitable to demonstrate good correlation. In the country where high medical expenditure is not acceptable, our inexpensive MFC is considered as good alternative methods to detect MRD in MM patients. Disclosures Tsukada: Takeda Pharmaceutical Co., Ltd.: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Celgene: Honoraria; Ono Pharmaceutical: Honoraria; Sanofi: Honoraria; Kyowa Kirin: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; Fujimoto Pharmaceutical: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Asahi Kasei Pharma Corporation: Honoraria; MOCHIDA PHARMACEUTICAL CO., LTD.: Honoraria. Suzuki:Ono: Research Funding; BMS: Honoraria, Research Funding; Takeda: Honoraria; Janssen: Honoraria; Celgene: Honoraria.
3
van Dongen, Jacques J. M., Maurice R. G. O'Gorman, and Alberto Orfao. "EuroFlow and its activities: Introduction to the special EuroFlow issue of The Journal of Immunological Methods." Journal of Immunological Methods 475 (December 2019): 112704. http://dx.doi.org/10.1016/j.jim.2019.112704.
Full text
4
Sanoja-Flores, *Luzalba, *Bruno Paiva, Juan A. Flores-Montero, Noemi Puig, Leire Burgos, Omar García, Felipe Prosper, et al. "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)." Blood 126, no. 23 (December 3, 2015): 4180. http://dx.doi.org/10.1182/blood.v126.23.4180.4180.
Full textAbstract:
Abstract 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<0.0001) in MGUS -0.0002% (<0.0001%-0.05450%) and 0.011 cPC/µL (range: <0.0001 cPC/µL -3.2 cPC/µL)- than in SMM -0.0026% (0.00020%-0.23%) and 0.14 cPC/µL (range: 0.022 cPC/µL - 14.58 cPC/µL) and MM -0.0033% (0.00064%-1.05%) and 2.01 cPC/µL (range: 0.043 cPC/µL -103.8 cPC/µL)-, respectively. Interestingly, a clear relationship was found between the presence of circulating cPC in PB of both MGUS, SMM and MM cases, and BM involvement by >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 (cont'd): 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.
5
Kalina, Tomas, Nadezda Brdickova, Hana Glier, Paula Fernandez, Marieke Bitter, Juan Flores-Montero, Jacques J. M. van Dongen, and Alberto Orfao. "Frequent issues and lessons learned from EuroFlow QA." Journal of Immunological Methods 475 (December 2019): 112520. http://dx.doi.org/10.1016/j.jim.2018.09.008.
Full text
6
Roshal, Mikhail, Juan A. Flores-Montero, Qi Gao, Maesa Koeber, Jessica Wardrope, Brian G. M. Durie, Ahmet Dogan, Alberto Orfao, and Ola Landgren. "MRD detection in multiple myeloma: comparison between MSKCC 10-color single-tube and EuroFlow 8-color 2-tube methods." Blood Advances 1, no. 12 (May 3, 2017): 728–32. http://dx.doi.org/10.1182/bloodadvances.2016003715.
Full text
7
Takamatsu, Hiroyuki, Takeshi Yoroidaka, Takeshi Yamashita, Ryoichi Murata, Mikio Ueda, Shinji Nakao, and Kosei Matsue. "Comparison of Minimal Residual Disease Detection between the Manual and Automated Gating Strategies of Euroflow Next-Generation Flow in Patients with Multiple Myeloma." Blood 134, Supplement_1 (November 13, 2019): 3083. http://dx.doi.org/10.1182/blood-2019-122398.
Full textAbstract:
Background: The rate of complete response (CR) in multiple myeloma (MM) has dramatically increased because of the development of novel agents. In addition, the development of methods for measuring minimal residual disease (MRD), such as multiparameter flow cytometry and next-generation sequencing, has made it possible to stratify CR patients according to the MRD extent. EuroFlow next-generation flow (EuroFlow-NGF) is considered one of the gold standard methods for evaluating the negative status of MRD in MM. The automated gating strategy of EuroFlow-NGF has been shown to detect MRD as accurately as the manual gating strategy by experts. Oberle et al. (Haematologica, 2017) have found that daratumumab persisted on the surface of myeloma cells treated with it and that the anti-CD38 multi-epitope antibody used in EuroFlow-NGF has partial cross-reactivity with daratumumab, leading to generally lower mean fluorescence intensities of CD38. Therefore, MRD levels may have been underestimated in patients who were treated with anti-CD38 monoclonal antibodies (mAbs) using the automated gating strategy, leading to inappropriate management of the patients. Because no studies have examined the correlation of MRD extent between the manual and automated gating strategies in patients with MM who have received anti-CD38 mAbs, we compared MRD detection between the two gating strategies of EuroFlow-NGF in patients with MM. Methods: The study included bone marrow samples from 51 patients with MM (27 male and 24 female patients), including 13 patients treated with anti-CD38 mAb (12 treated with daratumumab and 1 treated with isatuximab). The median patient age was 70 years (range, 32-92 years) at MRD assessment. The disease statuses at MRD assessment were stringent CR in 26 patients (51%), CR in 7 (14%), very good partial response in 13 (26%), partial response in 1 (2%), and progressive disease in 4 (8%). The sample preparation protocol, Ab panel, and automated gating strategy of EuroFlow-NGF have been reported previously (Flores-Montero et al. Leukemia 2017). Briefly, we performed the EuroFlow-NGF method, which involved ammonium chloride-based bulk lysis, followed by surface staining using antibodies against CD138-BV421, CD27-BV510, CD38 multiepitope (ME)-FITC, CD56-PE, CD45-PerCP Cy5.5, CD19-PECy7, CD117-APC, and CD81-APC C750 in tube 1 and surface/intracellular staining using antibodies against CD138-BV421, CD27-BV510, CD38 ME-FITC, CD56-PE, CD45-PerCP Cy5.5, CD19-PECy7, CD117-APC, CD81-APC C750, cytoplasmic (cy) Igκ-APC, and cyIgλ-APC C750 after permeabilization in tube 2. For data analysis, events from both eight-color tubes (tubes 1 and 2) were merged, and the values of all parameters per tube were mathematically calculated using the merge and calculation functions of Infinicyt software (Cytognos SL, Salamanca, Spain). Automatic identification and enumeration of total plasma cells (tPCs) and abnormal plasma cells (MRD) were performed using the automatic gating function of Infinicyt software as described previously (Flores-Montero et al. Leukemia 2017). We compared the total nucleated cell number, tPC ratio, and MRD ratio between the manual (by experts) and automated gating strategies of EuroFlow-NGF. Results: In patients with MM who did not receive any anti-CD38 mAb therapy, we observed high correlations for both the tPC (r = 0.959, P < 0.0001) (Figure A) and MRD (r = 0.974, P < 0.0001) (Figure B) ratios between the manual and automated gating strategies of EuroFlow-NGF. On the other hand, in patients with MM who received anti-CD38 mAb therapy, we did not observe good correlations for both the tPC (r = 0.349, P = 0.2) (Figure A) and MRD (r = 0.292, P = 0.3) (Figure B) ratios between the two strategies owing to a lower fluorescence intensity of CD38 on PCs. In addition, when the MRD threshold was set to 10-5, the discordance of MRD positivity/negativity between the two strategies was significantly higher in patients who received anti-CD38 mAb therapy than in those who did not receive anti-CD38 mAb therapy [4/13 (31%) vs. 1/38 (3%), P = 0.012]. Conclusion: Although the automated gating strategy of EuroFlow-NGF could be a viable alternative to the manual strategy for the assessment of MRD in MM, we may have to utilize the manual strategy to obtain precise MRD results for patients with MM who received anti-CD38 mAbs. Figure Disclosures Takamatsu: Celgene: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ono pharmaceutical: Honoraria, Research Funding; CSL Behring: Research Funding; SRL: Consultancy, Research Funding; Janssen Pharmaceutical: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Fujimoto Pharmaceutical: Honoraria; Becton, Dickinson and Company: Honoraria; Abbvie: Consultancy; Daiichi-Sankyo Company: Honoraria. Yoroidaka:Ono Pharmaceutical: Honoraria. Yamashita:Janssen Pharmaceutical K.K.: Honoraria; Daiichi-Sankyo Company: Honoraria; Kyowa Kirin: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; TEIJIN PHARMA LIMITED: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Bristol-Myers Squibb: Honoraria; Ono Pharmaceutical: Honoraria; Celgene: Honoraria. Murata:Celgene: Honoraria; Ono pharmaceutical: Honoraria. Nakao:Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; SynBio Pharmaceuticals: Consultancy; Ohtsuka Pharmaceutical: Honoraria; Celgene: Honoraria; Ono Pharmaceutical: Honoraria; Novartis Pharma K.K: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; Kyowa Kirin: Honoraria; Alaxion Pharmaceuticals: Honoraria. Matsue:Novartis Pharma K.K: Honoraria; Ono Pharmaceutical: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Celgene: Honoraria; Janssen Pharmaceutical K.K.: Honoraria.
8
Szczepański, T. "Nowoczesna diagnostyka i monitorowanie ostrych białaczek wg standardów EuroFlow." Acta Haematologica Polonica 46 (September 2015): 1. http://dx.doi.org/10.1016/j.achaem.2015.07.016.
Full text
9
Takamatsu, Hiroyuki, Naoki Takezako, Rachel K. Wee, Takeshi Yoroidaka, Takeshi Yamashita, Ryoichi Murata, Kosei Matsue, and Shinji Nakao. "Comparison of Minimal Residual Disease Detection in Autografts of Patients with Multiple Myeloma between 8-Color Multiparameter Flow Cytometry (EuroFlow) and Next-Generation Sequencing." Blood 132, Supplement 1 (November 29, 2018): 258. http://dx.doi.org/10.1182/blood-2018-99-111250.
Full textAbstract:
Abstract 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. Figure 1. Figure 1. Disclosures Takamatsu: 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.
10
Flores-Montero, Juan A., Bruno Paiva, Luzalba Sanoja-Flores, Noemi Puig, Omar García, Sebastian Böttcher, José J. Pérez-Morán, et al. "Next Generation Flow (NGF) for High Sensitive Detection of Minimal Residual Disease (MRD) in Multiple Myeloma (MM)." Blood 126, no. 23 (December 3, 2015): 367. http://dx.doi.org/10.1182/blood.v126.23.367.367.
Full textAbstract:
Abstract Introduction: The clinical and prognostic utility of MRD monitoring in MM bone marrow (BM) by first generation (4-6-color) flow cytometry (flow-MRD), has been now demostrated for more than a decade. Thereby, flow-MRD is considered to be a well-suited technique for MRD monitoring in MM, due to its high applicability and specificity, and its broad availability in diagnostic laboratories. However, recent results have confirmed that 1st generation flow-MRD has a lower sensitivity than molecular techniques such as allele-specific oligonucleotyde (ASO)-PCR and next generation sequencing (NGS); in addition, the lack of standardization of conventional flow-MRD approaches, also has a negative impact on its reproducibility. Here we report on the validation of the next generation flow (NGF)-MRD approach developed by the EuroFlow Consortium and the International Myeloma Foundation (IMF) for ultrasensitive and standardized detection of MRD in MM. Methods: A total of 275 BM samples were included in the study: 1) a group of 31 normal/reactive and 63 diagnostic MM BM samples were evaluated to identify the most efficient candidate markers for the NGF panel, using a multivariate analysis-based approach; 2) next, a total of 181 BM samples from 15 healthy donors (HD), 36 MGUS, 15 MM and 3 solitary plasmacytoma (SP) patients studied at diagnosis, plus 112 MM follow-up samples, most of which (n=71) corresponded to BM samples from MM patients in very good partial response, complete remission (CR) or stringent CR were analyzed. These samples were evaluated by the EuroFlow-IMF NGF-MRD method. The method was based on a (standardized) lyse-wash-and-stain sample preparation protocol, the measurement of high numbers of BM cells (≥5x106 cells/tube) and an optimized 8-color, 2-tubes, antibody panel, for accurate identification of BM plasma cells (PCs) and discrimination between phenotypically aberrant (aPC) and normal PC (nPC): tube 1: CD138BV421 CD27BV510 CD38FITC CD56PE CD45PerCP Cy5.5 CD19PE Cy7 CD117APC CD81APC C750, and; tube 2: identical to tube 1 except for cytoplasmic (Cy) Immunoglobulin (Ig) kAPC/CyIglAPC C750). Results obtained with the NGF-MRD MM method in the 71 VGPR, CR and sCR samples, were then compared with a 2nd generation (routine) 8-color flow-MRD approach which involved a single 8-color combination staining for the same markers described above for tube 1, but in the absence of full optimization of the positions for the antibody-fluorochrome conjugates and no selection for treatment-independent antibody CD38 clones. In a subset of 16 of these 71 MRD samples in which enough material was available, comparison with NGS was also performed in parallel. Results: In all MGUS, MM and SP cases analyzed at diagnosis, aPC showed aberrant phenotypes vs. nPC from HD BM, based on multivariate analysis of individual cells from each of the patients against a reference data base of normal/reactive PCs (100% applicability). For the MM MRD BM samples, a median of 9.8x106 (range: 2.4-15.3) events were acquired (tube 1 plus 2) vs. 1x106 (range: 0.03-5) events for the 2nd generation flow-MRD approach with an (estimated) 10 times lower limit of detection and 10 times lower limit of quantitation of 3x10-6 and 5x10-6 vs. 3x10-5 and 5x10-5 for the NGF-MRD vs. the 2nd generation flow-MRD approaches, respectively. This led to a higher rate of MRD+ samples with the NGF-MRD method: 14/48 (29%) cases that were flow-MRD-negative with the 2nd generation 8-color flow-MRD method became MRD+ (median percentage of residual aPC of 0.0007%; range: 0.0002 to 0.02%) (Figure 1A). In contrast, 4/38 (11%) samples were negative by NGF, while positive by 2nd generation flow-MRD; one of these four proved to be MRD-negative by cytoplasmic immunoglobulin light chain restriction analysis. Further comparison of NGF vs NGS showed 9 of the 16 samples evaluable were MRD-negative by NGS; from them, one third (3/9) were positive by NGF with a median number of residual aPCs of 0.005% (range: 0.0002-0.006%) (Figure 1B). Conclusions: The EuroFlow-IMF NGF-MRD approach provides a fast, highly applicable, ultrasensitive, standardized and accurate approach for the assessment of MRD in BM samples from MM patients and overcomes the current limitations of both 1st and 2nd generation flow-MRD approaches; preliminary results showed higher sensitivity than NGS. Figure 1. Figure 1. Disclosures Paiva: Sanofi: Consultancy; Binding Site: Consultancy; EngMab AG: Research Funding; BD Bioscience: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Celgene: Consultancy; Janssen: Consultancy. Puig:Janssen: Consultancy; The Binding Site: Consultancy. Mateos:Janssen-Cilag: Consultancy, Honoraria; Takeda: Consultancy; Celgene: Consultancy, Honoraria; Onyx: Consultancy. San Miguel:Sanofi-Aventis: Honoraria; Novartis: Honoraria; Millennium: Honoraria; Janssen-Cilag: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Onyx: Honoraria. Durie:Celgene: Consultancy; Onyx: Consultancy; Takeda: Consultancy; Johnson & Johnson: Consultancy. van Dongen: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 (cont'd): 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; 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..
11
Kalina, T., J. Flores-Montero, V. H. J. van der Velden, M. Martin-Ayuso, S. Böttcher, M. Ritgen, J. Almeida, et al. "EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols." Leukemia 26, no. 9 (September 2012): 1986–2010. http://dx.doi.org/10.1038/leu.2012.122.
Full text
12
Arana, Paula, Bruno Paiva, Maria Belen Vidriales, Juan A. Flores-Montero, Noemi Puig, Teresa Cedena, Lourdes Cordon, et al. "Comparison Between First-Generation 4-Color Vs. Second-Generation 8-Color Multiparameter Flow Cytometry (MFC) to Monitor Minimal Residual Disease (MRD) in Multiple Myeloma (MM)." Blood 126, no. 23 (December 3, 2015): 2963. http://dx.doi.org/10.1182/blood.v126.23.2963.2963.
Full textAbstract:
Abstract Introduction: Several cooperative MM groups have shown that MRD monitoring may be relevant as biomarker to evaluate the efficacy of different treatment strategies, to support treatment decisions, and to act as surrogate for overall survival (OS) in MM. Because of its wider applicability, a significant fraction of available MRD data has been obtained using MFC that originally, was limited to 4- or 6-colors and measured a limited number of cells. It is assumed that the sensitivity of MFC can increase by usage of ≥8 markers and acquisition of greater cell numbers, but the degree of improved specificity and sensitivity remains unknown. Methods: We aimed at determining the increment in specificity and sensitivity upon transition from first-generation 4-color into a second-generation 8-color MFC assay, by applying new computational tools developed by the EuroFlow consortium in elderly MM patients, enrolled in the GEM2010MAS65 study, for which MRD monitoring was performed with an 8-color monoclonal antibody combination - CD45-PacB/CD138-OC515/CD38-FITC/CD56-PE/CD27-PerCPCy5.5/CD19-PECy7/CD117-APC/CD81-APCH7 - and acquisition of ≥2x106 leukocytes (detection limit: 10-5). Time-to-progression (TTP) and OS were measured from diagnosis. Results: First, we created a reference data file of normal (n=17) and clonal (n=71) plasma cells (PCs) derived from bone marrow samples of healthy individuals and MM patients (Figure 1A) in order to determine the individual contribution of each marker to the discrimination between normal vs. clonal PCs. Principal component analysis (PCA) showed that CD19 ranked as the most significant marker followed by CD56, CD81, CD27, CD117, CD45, forward scatter (FSC), CD38, CD138 and sideward scatter (SSC). Accordingly, the 8-color combination resulted in improved discrimination between normal vs. clonal PCs as compared to the former 4-color approach based only on CD38/CD56/CD19/CD45 (Figure 1B); in fact, CD81, CD27 and CD117 had higher independent value than CD45 in the PCA. Afterwards, we focused on 50 randomly selected MRD-positive patients enrolled in the GEM2010MAS65 study, to compare the performance of an 8- vs. 4-color software-guided classification of MRD cells. PCA based on 8-colors showed that all but two patients were accurately located in the clonal PC reference and outside 1 or 2 standard deviation (SD) curves of the normal PC reference (96% accuracy; Figure 1C); by contrast, using 4-color software-guided classification up to 9 patients became located in the overlapping area between 1 and 2 SD of the normal and clonal PCs references (82% accuracy; Figure 1D). Afterward, we investigated the increment in sensitivity due to the evaluation of 2x106 leukocytes with the second-generation 8-color flow assay instead of the standard 2x105 cells with the first-generation 4-color approach, by determining how many of the 50 MRD-positive patients would turn into MRD-negative if only 2x105 leukocytes had been analyzed (detection limit: 10-4). Interestingly, by reducing the number of visible events to 2x105, our results showed that up to 15 out of the 50 cases (30%) would become wrongly classified as MRD-negative. Then, we investigated the impact in TTP and OS of having MRD levels of 10-5 within a series of 163 patients enrolled in the GEM2010MAS65 and with MRD assessment. Accordingly, 88 cases had detectable MRD levels ≥10-4, 21 patients had persistent MRD at 10-5, and the remaining 54 cases were MRD-negative. Importantly, MRD-positive patients at 10-5 had similar outcome as compared to cases with MRD levels ≥10-4 (both had median TTP of 31 months; 3-year OS rates were 80% and 74%, respectively) and significantly inferior to that of MRD-negative patients [median TTP not reached (P <.001); 3-year OS rate of 93% (P =.05)]. Conclusions: We showed that the transition from a first-generation 4-color into a second-generation 8-color MFC assay that measured ten-times more cells resulted in increased specificity and sensitivity. MRD detection at the 10-5 level is clinically relevant, since it identifies a subset of patients with inferior survival than MRD-negative cases, similar to that of the overall MRD-positive patient population. Figure 1. Figure 1. Disclosures Paiva: Millenium: Consultancy; BD Bioscience: Consultancy; Celgene: Consultancy; Janssen: Consultancy; EngMab AG: Research Funding; Binding Site: Consultancy; Onyx: Consultancy; Sanofi: Consultancy. Puig:Janssen: Consultancy; The Binding Site: Consultancy. Gironella:Celgene Corporation: Consultancy, Honoraria. van Dongen:BD Biosciences (cont'd): 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.. Mateos:Takeda: Consultancy; Onyx: Consultancy; Celgene: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria. San Miguel:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees.
13
Pedreira, C. E., E. Sobral da Costa, Q. Lecrevise, G. Grigore, R. Fluxa, J. Verde, J. Hernandez, J. J. M. van Dongen, and A. Orfao. "From big flow cytometry datasets to smart diagnostic strategies: The EuroFlow approach." Journal of Immunological Methods 475 (December 2019): 112631. http://dx.doi.org/10.1016/j.jim.2019.07.003.
Full text
14
Beznos, OA, LYu Grivtsova, AV Popa, MA Shervashidze, IN Serebryakova, OYu Baranova, EA Osmanov, and NN Tupitsyn. "Evaluation of Minimal Residual Disease in B-Lineage Acute Lymphoblastic Leukemia Using EuroFlow Approaches." Clinical oncohematology 10, no. 2 (April 2017): 158–68. http://dx.doi.org/10.21320/2500-2139-2017-10-2-158-168.
Full text
15
Schilhabel, Anke, Henrik Knecht, Anton W. Langerak, Jacques J. M. van Dongen, Michael Kneba, Jill Ray, Elizabeth Punnoose, et al. "Analytical Validation of Patient-Specific PCR-Based MRD Assessment for Use As a Primary Endpoint in CLL Clinical Trials." Blood 126, no. 23 (December 3, 2015): 2924. http://dx.doi.org/10.1182/blood.v126.23.2924.2924.
Full textAbstract:
Abstract Introduction. Minimal residual disease (MRD) is an objective measure of disease status defined by the number of leukemic cells in the blood or bone marrow of leukemic patients. In recent clinical studies of chronic lymphocytic leukemia (CLL), undetectable MRD levels (< 1 tumor cell/10,000 leukocytes) have been shown to correlate with prolonged progression free survival (PFS) and overall survival, independent of treatment or known risk factors. MRD assessment has been proposed as an alternative to PFS as a primary endpoint in frontline CLL pivotal studies to evaluate the efficacy of novel therapies at an earlier time-point. Thorough standardization and validation are needed to use MRD as a primary surrogate endpoint. Allele specific oligonucleotide (ASO)-PCR of immunoglobulin (IG) gene rearrangements is a method for quantifying MRD using patient-specific PCR primers and has been standardized by the EuroMRD Consortium (www.EuroMRD.org). Given that each patient has individualized PCR primers designed for their leukemic clone, this posed a unique challenge for the analytical validation studies to demonstrate that the assays are uniform in their reproducibility and analytical sensitivity to measure MRD across patients with CLL. Here we report a comprehensive, IVD-guided analytical validation of the ASO-PCR technique according to the guidance of regulatory authorities. We provide evidence that the ASO-PCR methodology can reproducibly measure MRD to the required threshold of 10-4, across patients with CLL. Results. Performance of ASO-PCR was assessed using a combination of retrospective data from the CLL11 clinical trial and prospectively performed experiments. Patient assays from 60 CLL patients were tested in two EuroMRD laboratories to demonstrate linearity across the measurement range of 10-1 to 10-5, and a limit of detection of 6.3x10-5, which is below the cut-off of 10-4 used for defining MRD negativity. Concordance of the method to an orthogonal method was determined from the previously published comparison of flow cytometry with ASO-PCR (Boettcher et al., Leukemia 2009; 23: 2007) with 93.8 % overall agreement between both methods (n=452). Agreement of MRD status was >97% when comparing individually designed ASO primers for the same patient within the lab. The overall agreement between the two different laboratories using independently designed ASO-PCR assays was 93.5%. Precision was assessed above and below the threshold of 10-4 using ASO-PCR assays of 3 individual patient samples diluted to appropriate MRD levels listed in Table 1. Theexperiment was designed to mimic sources of variation by evaluating MRD samples over the course of the clinical study (3 days x 2 operators x 3 patients x 2 laboratories x 3 replicates). Overall variability was estimated using a mixed effects model including fixed patient effects and random effects for operator and day. Based on the known MRD distribution of frontline CLL patients, we estimate acceptable overall variability on the order of 80% CV at lower concentrations (≤ 3.2x10-4) and 40% CV at higher concentrations (> 3.2x10-4). This precision estimate provides reasonable misclassification rates (< 5%) due to the fact that the majority of patients had MRD levels either well above or below 10-4 level. Experiments also addressed stability of patient specimens and critical assay components. Table 1. Precision of ASO-PCR results obtained at MRD levels 10-2 to 10-5Table 1.Estimated Total CV (%) Averaged Across 3 patientsMRD levelKielErasmus1.00E-028.7034.791.00E-0310.0235.743.20E-0415.8233.271.00E-0431.7036.383.20E-0589.8978.801.00E-05258.06277.27 Conclusion. The analytical validation studies described here provide evidence that the ASO-PCR methodology, standardized by EuroMRD, performs well to reproducibly detect MRD status across CLL patients at the threshold of 10-4. These studies serve as an example for the validation of personalized, patient-specific quantitative clinical assays for use as a primary endpoint in clinical trials. The authors would like to acknowledge the valuable work of the following people who contributed to this work: M. Brüggemann (UKSH Kiel), R Raja, C. Cox, W. Darbonne, R. Desai, and K. Trunzer. Disclosures Langerak: 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.; Roche: Other: Lab services in the field of MRD diagnostics provided by Dept of Immunology, Erasmus MC (Rotterdam). van Dongen:BD Biosciences (cont'd): 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.. Ray:Genentech, Inc.: Employment. Punnoose:Genentech, Inc.: Employment. Kim:Genentech, Inc.: Employment. Haberberger:Genentech, Inc.: Employment. Bernaards:Roche: Employment. Zhu:Genentech, Inc.: Employment. Lewin-Koh:Genentech, Inc.: Employment. Ritgen:Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding.
16
van Dongen, J. J. M., and A. Orfao. "EuroFlow: Resetting leukemia and lymphoma immunophenotyping. Basis for companion diagnostics and personalized medicine." Leukemia 26, no. 9 (September 2012): 1899–907. http://dx.doi.org/10.1038/leu.2012.121.
Full text
17
Najidh, Safa, A. J. van der Sluijs-Gelling, W. H. Zoutman, C. P. Tensen, T. H. van Hall, J. Almeida, J. J. M. van Dongen, and M. H. Vermeer. "Standardized flow cytometry (EuroFlow) demonstrates heterogeneous T-cell origin of Sézary lymphoma cells." European Journal of Cancer 101 (September 2018): S8. http://dx.doi.org/10.1016/j.ejca.2018.07.163.
Full text
18
Edwards, Emily S. J., Julian J. Bosco, Pei M. Aui, Robert G. Stirling, Paul U. Cameron, Josh Chatelier, Fiona Hore-Lacy, Robyn E. O’Hehir, and Menno C. van Zelm. "Immunophenotyping of B-Cell AND T-CELL ABNORMALITIES IN PID USING EUROFLOW-BASED PANELS." Pathology 52 (February 2020): S46. http://dx.doi.org/10.1016/j.pathol.2020.01.166.
Full text
19
Flores-Montero, Juan, Georgiana Grigore, Rafael Fluxá, Juan Hernández, Paula Fernandez, Julia Almeida, Noemí Muñoz, et al. "EuroFlow Lymphoid Screening Tube (LST) data base for automated identification of blood lymphocyte subsets." Journal of Immunological Methods 475 (December 2019): 112662. http://dx.doi.org/10.1016/j.jim.2019.112662.
Full text
20
Takamatsu, Hiroyuki, Naoki Takezako, Takeshi Yoroidaka, Takeshi Yamashita, Ryoichi Murata, Atsuko Yamazaki, Masahiro Takeuchi, et al. "Minimal Residual Disease in Autografts and Bone Marrow of Patients with Multiple Myeloma: 8-Color Multiparameter Flow Cytometry (EuroFlow-NGF) Vs. Next-Generation Sequencing." Blood 136, Supplement 1 (November 5, 2020): 22–23. http://dx.doi.org/10.1182/blood-2020-137014.
Full textAbstract:
Background: Autologous stem cell transplantation (ASCT) in conjunction with novel therapeutic drugs can dramatically improve response rates and the prognoses of patients with multiple myeloma (MM). However, most patients with MM ultimately relapse due to minimal residual disease (MRD). Next-generation multiparameter flow cytometry (MFC) (EuroFlow-NGF) and next-generation sequencing (NGS) are currently the standard methods to assess MRD. Aims: To compare the prognostic value of MRD detection in autografts and bone marrow (BM) cells using 8-color MFC (EuroFlow-NGF) and NGS (Adaptive Biotechnologies), and also MRD levels between fresh and cryopreserved autografts using NGF. Methods: The study enrolled 52 newly-diagnosed MM patients who underwent ASCT. The median age ASCT was 61 (range 41-69) years and included 29 males and 23 females at ISS I (n = 17), II (n = 23), and III (n = 12). Of these, 18 patients harbored high-risk chromosomal abnormalities including t(4;14) (n = 15), del17p and t(4;14) (n = 2), and complex (n = 1). Bortezomib-based chemotherapy was used for induction together with melphalan at 140 mg/m2 (n = 1) and 200 mg/m2 (n = 51) for conditioning before ASCT. 39 of 52 (75%) patients received maintenance therapy until progressive disease. The best responses achieved post-ASCT included 30 sCR, 4 CR, 15 VGPR, and 3 PR. Forty autografts, one from each MM patient, were analyzed using NGF and NGS protocols, and BM cells at pre/post-ASCT and autografts derived from 16 patients were analyzed using NGS. The EuroFlow-NGF method uses standard sample preparation; large numbers of cells are evaluated using an optimized 8-color antibody panel that facilitates accurate identification of discrimination between phenotypically aberrant plasma cells (aPCs) and their normal counterparts (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). Eight additional autografts were used to assess MRD in both fresh and cryopreserved samples by NGF. Results: MRD was evaluated in 48 of 52 autografts (92%) using NGF and in 44 of 52 autografts (85%) using NGS. We identified aPCs in autografts based on multivariate analysis of individual cell populations (e.g., CD56+, CD19−, CyIgκ+, and CD117+). As the results of NGF revealed a strong correlation with respect to MRD in fresh vs. thawed autografts (r = 0.999, P < 0.0001), MRD was subsequently evaluated in thawed autografts. The sensitivity of NGF was 1 × 10−5-2 × 10−6; the sensitivity of NGS was 1 × 10−6. 28 of 48 (58%) of the autografts were MRD-positive by NGF; 30 of 44 (68%) of the autografts were MRD-positive by NGS. MRD levels in autografts using NGF and NGS correlated with one another (r = 0.69, P < 0.0001; Fig. 1A). MRD negative in autografts by NGF cases (MRDNGF (-)) and MRDNGS (-) tended to show better progression-free survival (PFS) than MRDNGF (+) (P = 0.195) and MRDNGS (+) (P = 0.156), respectively. Furthermore, MRDNGS (-) showed significantly better overall survival (OS) than MRDNGS (+) (P = 0.03) (Fig. 1C) while MRDNGF (-) showed better OS than MRDNGF (+) (P = 0.09) (Fig. 1B). Our data revealed only a minimal correlation between MRD in the autografts (median 1.1 × 10−5,range 0-7.29 × 10−4) and in the BM cells at pre-ASCT (median 5.05 × 10−3,range 6 × 10−6-2.64 × 10−1; r = 0.09, P = 0.7) or at post-ASCT (median 2.11 × 10−4,range 0-9.09 × 10−3; r = 0.14, P = 0.6); MRD detected in the autografts was > 27 times lower than that detected in pre-ASCT BM cells, and MRD detected in the post-ASCT BM cells was > 3 times lower than that detected in pre-ASCT BM cells except for one case in which the ratio was increased by two times. Interestingly, while MRD was detected in all BM cells at pre-ASCT (n = 16), 4 of 16 (25%) of these autografts were MRDNGS-negative. The median of MRD levels of the 4 cases in pre-ASCT and post-ASCT BM cells were 4.14 × 10−4 (range 6-583 × 10−6)and 1.8 × 10−5 (range 0-27 × 10−6), respectively. Conclusion: Although EuroFlow-NGF is a rapid and accurate method for detecting MRD, NGS was more sensitive and provided greater prognostic value than EuroFlow-NGF. Disclosures Takamatsu: Adaptive Biotechnologies: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Janssen Pharmaceutical: Consultancy, Honoraria, Research Funding; Ono pharmaceutical: Honoraria, Research Funding; SRL: Consultancy, Research Funding. Takezako:Bristol-Myers Squibb: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Janssen: Research Funding; Abbvie: Research Funding. Nakao:Symbio: Consultancy; Kyowa Kirin: Honoraria; Alexion: Research Funding; Novartis: Honoraria.
21
van der Velden, Vincent H. J., Juan Flores-Montero, Martin Perez-Andres, Marta Martin-Ayuso, Oliver Crespo, Elena Blanco, Tomas Kalina, et al. "Optimization and testing of dried antibody tube: The EuroFlow LST and PIDOT tubes as examples." Journal of Immunological Methods 475 (December 2019): 112287. http://dx.doi.org/10.1016/j.jim.2017.03.011.
Full text
22
Gaipa, Giuseppe, Cristina Bugarin, Sergio Matarraz, Chiara Buracchi, Lukasz Sedek, Vincent H. J. van der Velden, Tomasz Szczepanski, et al. "Euroflow-Based Immunophenotypic Characterization of CD34+ Cell Compartment in Juvenile Myelomonocytic Leukemia (JMML): A New Tool for Differential Diagnosis." Blood 128, no. 22 (December 2, 2016): 3127. http://dx.doi.org/10.1182/blood.v128.22.3127.3127.
Full textAbstract:
Abstract Background Juvenile myelomonocytic leukemia (JMML) is a lethal myeloproliferative disease (MPD) of young childhood characterized by an overproduction of myelomonocytic cells and an increased in vitro sensitivity of hematopoietic progenitors to granulocyte-macrophage colony-stimulating factor [GM-CSF] (Emanuel PD et al, Blood 1991). Diagnostic criteria for JMML are currently well-established and based on clinical features and laboratory findings. However, in some patients diagnosis of JMML vs other overlapping disease entities, still remains a challenge, immunophenotyping being not part of the diagnostic work-up of JMML. Here, we aimed at detailed characterization of the CD34+ cell compartment in JMML bone marrow (BM) using the standardized EuroFlow myeloid panel in combination with innovative EuroFlow software maturation tools. Our major goal was to determine the potential utility of immunophenotyping of CD34 cells in the diagnostic work-up of JMML. Methods Overall, we analyzed BM cells from 10 JMML patients at diagnosis (age range: 0-7 years), 17 control subjects (age range: 0-15 years) and 5 patients (age range: 0-5 years) with a suspected diagnosis of JMML that was subsequently not confirmed following standardized EuroFlow antibody combinations: 1) cyCD3/ CD45/ cyMPO/ cyCD79a/ CD34/ CD19 / CD7/smCD3 (for early lineage assignement); 2) HLADR/CD45/CD16/CD13/CD34/CD117/CD11b/CD10 (neutrophilic maturation); 3) HLADR/CD45/CD35/CD64/CD34/CD117/CD300e (IREM2)/CD14 (monocytic maturation); 4) HLADR/CD45/CD36/CD105/CD34/CD117/CD33/CD71 (erythroid vs plasmacytoid dendritic cell maturation). Samples were processed and analyzed according to the Euroflow standard operating protocols (van Dongen JJM et al, Leukemia 2012, Kalina T et al, Leukemia 2012). Data analysis was specifically focused on the immunophenotypic profile of CD34+ gated cells. Results Within the CD34+ BM cell compartment the proportion (mean % ± 1SD) of granulocytic and monocytic precursors were not significantly different in JMML as compared to controls: 33% ± 15% vs 25% ± 12% (p = 0.16) and 14% ± 6.3% vs 12% ± 7.1% (p = 0.68) respectively. Otherwise we observed a slightly decreased in erythroid CD34+ progenitors in JMML vs controls (1.0% ± 1.2% vs 2.8% ± 1.7%, p<0.05). Moreover, a significantly different distribution of lymphoid precursors was observed: B-cell precursors were strongly reduced in JMML vs controls (3.0% ± 3.5% vs 53% ± 16%, p<0.0001), while CD7+ lymphoid precursors resulted significantly enhanced (28% ± 18% vs 2.3% ± 1.2%, p<0.0001). We then investigated the presence of unusual immunophenotypes in JMML CD34+ BM cells, including CD7+/MPO+, CD79a+/CD7+, and CD79a+/MPO+ cells. Interestingly, we consistently found CD7+/MPO+ and/or CD79a+/CD7+ cells in 7/7 JMML patients analyzed (mean 7.9% ± 6.4%), while in control subjects these cells were virtually absent (0.02% ± 0.00%, p<0.0001). In contrast, no CD79a+/MPO+ cells were detected among CD34+ precursors. Those 5 patients suspected of having JMML showed a CD34+ BM cell immunophenotypic profile that was not significantly different from that of normal subjects. These patients were finally diagnosed as not having JMML (two had CMV infection, one a Leukocyte Adhesion Deficiency II, one a Noonan Syndrome, the final diagnosis is the other patient being still pending). Of note, JMML peripheral blood (PB) CD34+ cells from 6 JMML patients (3 paired BM-PB samples and 3 additional PB samples) fully confirmed the aberrant immunophenotypic signature seen in BM-derived samples. Conclusions CD34+ precursor cells from JMML patients display a unique immunophenotypic profile characterized by an inverted ratio of CD19+ B/CD7+ lymphoid precursors, associated with unusual marker coexpressions, which might contribute to fast and more precise diagnostic work-up of JMML. Further studies in larger patient series are required to confirm our observations. Disclosures Biondi: Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; BMS: Membership on an entity's Board of Directors or advisory committees; Cellgene: Other: Advisory Board.
23
Kalina, Tomas, Juan Flores-Montero, Quentin Lecrevisse, Carlos E. Pedreira, Vincent H. J. van der Velden, Michaela Novakova, Ester Mejstrikova, et al. "Quality assessment program for EuroFlow protocols: Summary results of four-year (2010-2013) quality assurance rounds." Cytometry Part A 87, no. 2 (October 23, 2014): 145–56. http://dx.doi.org/10.1002/cyto.a.22581.
Full text
24
van Dongen, J. J. M., L. Lhermitte, S. Böttcher, J. Almeida, V. H. J. van der Velden, J. Flores-Montero, A. Rawstron, et al. "EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes." Leukemia 26, no. 9 (May 3, 2012): 1908–75. http://dx.doi.org/10.1038/leu.2012.120.
Full text
25
Nováková, Michaela, Hana Glier, Naděžda Brdičková, Marcela Vlková, Ana Helena Santos, Margarida Lima, Mikael Roussel, et al. "How to make usage of the standardized EuroFlow 8-color protocols possible for instruments of different manufacturers." Journal of Immunological Methods 475 (December 2019): 112388. http://dx.doi.org/10.1016/j.jim.2017.11.007.
Full text
26
Bazinet, Alexandre, Ryan N. Rys, Claudia M. Wever, Amadou Barry, Celia Greenwood, Christian Young, Alma Mendoza, et al. "A 10-Color Flow Cytometry Panel for Both Diagnosis and Minimal Residual Disease Measurement in Chronic Lymphocytic Leukemia." Blood 134, Supplement_1 (November 13, 2019): 5451. http://dx.doi.org/10.1182/blood-2019-123904.
Full textAbstract:
Background: Chronic lymphocytic leukemia (CLL) has a classic immunophenotype, consisting of light chain restriction, CD5+, CD19+, dim CD20, CD23+, CD43+, CD200+, CD10- and CD79b-. This distinguishes it from normal B cells and other lymphoproliferative disorders (LPDs). Antibodies targeting these antigens are included in two 8-color flow cytometry panels developed by the Euroflow consortium for the work up of B cell LPDs. Combining these antibodies into one 10-color panel would be more cost-effective. Furthermore, new CLL therapies can induce deep remissions, creating an increasing demand to measure minimal residual disease (MRD), defined as having over 1 residual leukemic cell per 10,000 leukocytes (10-4). The current international standardized approach for measuring MRD established by the European Research Initiative on CLL (ERIC) uses a panel of antibodies targeting CD3, CD5, CD19, CD20, CD22, CD43, CD79b and CD81. However, these antibody-fluorochrome combinations are different than those used by the Euroflow diagnostic panels. Thus laboratories considering implementing MRD testing would need to purchase antibodies for 3 different panels (2 diagnostic and 1 MRD). We expanded the Euroflow 8-color lymphocyte screening tube (LST) to include CD200 and CD23, such that CLL can be detected in one 10-color tube, at levels as low as 0.01%. The goal of this study was to determine the potential cost savings in implementing this new panel and to determine if it is sensitive enough to detect MRD. Methods: We calculated the number of samples analyzed with our modified 10-color LST tube (mLST1, obtained lyophilized) from April 2018 to March 2019 to rule out an LPD and the number of antibody aliquots saved using this approach compared to the standard 2-tube Euroflow method. We also created a version of the above-mentioned panel (mLST2) using liquid antibodies to increase the generalizability of our results, substituting CD38 with CD43 to see if this improved MRD detection (see panels below). For MRD testing, we used CLL samples from 24 different patients to produce 60 MRD samples at various concentrations of leukemic cells. Samples were prepared by spiking CLL cells into suspensions of normal leukocytes at approximate concentrations of 0.1%, 0.01% and 0.001%. Each sample was aliquoted and stained with the three panels: ERIC, mLST1 and mLST2. Data was acquired using a BD FACSCanto II or a BD FACSAria Fusion and analysed using BD FACSDiva software. CLL cells were identified based on differential expression of key markers and MRD was calculated as the number of CLL cells/total leukocytes. MRD positivity was defined as ≥ 0.01%. Agreement between the panels was assessed using the Bland-Altman plot method. We also calculated the percentage agreement between the panels in identifying MRD positivity. Results: In 1 year, mLST1 was performed on 474 samples, of these 220 had an LPD and 123 (56%) had a classic CLL phenotype, obviating the need for further testing. This resulted in the net savings of 476 antibody aliquots. For MRD assessment, differential expression of CD5 and CD20 were the most significant contributors in distinguishing CLL from normal B cells using the mLST1 and mLST2. We identified one CLL case with an atypical immunophenotype (dim CD5, bright CD20) which proved difficult to gate using a mLST panel. There was agreement in MRD results obtained with the mLST panels and the ERIC panel. For values above the limit of quantification, the 95% limit of agreement was ±0.3369 log for the ERIC vs mLST1 comparison and ±0.3485 log for the ERIC vs mLST2 comparison. Thus, variability in MRD levels between the panels was less than 2-fold the majority of the time, which we considered clinically acceptable as MRD is measured on an exponential scale. The ERIC panel and the mLST1 had 88.3% agreement in distinguishing MRD-positive versus MRD-negative samples. Agreement was 93.3% between the ERIC panel and the mLST2. Conclusions: Using a modified 10-color LST panel for both diagnosis and MRD measurement of CLL is feasible. The advantages are increased familiarity with the antibodies and potential cost savings, making MRD accessible to more cytometry laboratories. Atypical CLL cases without the usual CD5 positivity and dim CD20 are very difficult to gate using an LST panel. In these cases, the ERIC panel is clearly superior as CD22, CD79b, CD81 and CD43 can still provide separation between the malignant and normal lymphocytes. Disclosures Bazinet: BD Biosciences: Other: Provided a significant amount of the antibodies used in this project free of cost.. Wever:Teva Canada Innovation: Employment. Gimmig:BD Biosciences: Employment. Johnson:Lundbeck: Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding; Merck: Consultancy, Honoraria; Roche: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding; Abbvie: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BD Biosciences: Other: Provided a significant proportion of the antibodies used in this project free of cost.; BMS: Consultancy, Honoraria; Seattle Genetics: Honoraria.
27
Nørgaard, Jakob Nordberg, Niels Abildgaard, Anna Lysén, Mona-Elisabeth R. Revheim, James P. Connelly, and Fredrik H. Schjesvold. "Krd Consolidation in Myeloma Patients with a Positive PET-CT after Standard First Line Treatment: A Phase II Study (CONPET)." Blood 134, Supplement_1 (November 13, 2019): 3172. http://dx.doi.org/10.1182/blood-2019-125523.
Full textAbstract:
Background: [18F]-Fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET/CT) positivity after first line treatment with autologous stem cell transplantation (ASCT), is strongly correlated with reduced progression free survival and overall survival (Moreau et al., JCO, 2017). However, FDG PET/CT positive patients who obtain FDG PET/CT negativity after treatment can have comparable outcomes to patients who were FDG PET/CT negative at baseline (Davies et al., Haematologica 2018). Aiming for FDG PET/CT negativity may therefore be an important goal in myeloma treatment. The use of FDG PET/CT positivity as an indication for consolidation therapy after ASCT has not been studied before. Methods: This is an ongoing, multicenter phase II study. Patients with multiple myeloma who have received standard first line treatment including ASCT and achieved very good partial response (VGPR) or better, are eligible for the study and examined by FDG PET/CT. Patients who are FDG PET/CT positive defined by the Italian Myeloma criteria for PET USe (IMPETUS) (Nanni C et al., EJNMMI 2016 and 2018) are included in the treatment phase of the study and are assessed for minimal residual disease (MRD) by Euroflow (sensitivity: 10-5) before treatment. The treatment consists of four 28-day cycles of KRd (carfilzomib 36 mg/m2 day 1,2,8,19,15 and 16 (except 20 mg/m2 day 1 and 2 first cycle), lenalidomide 25 mg day 1-21 all cycles and dexamethasone 40 mg day 1,8,15 and 22 all cycles). After four cycles, FDG PET/CT and Euroflow for MRD are repeated for response evaluation. Both patients with FDG PET/CT negativity and patients with FDG PET/CT positivity at baseline are followed for progression free survival (PFS) and overall survival (OS). Results: As of 1st of July 2019, 43 patients have been screened in the study. Sixteen patients (37%) had a positive FDG PET/CT result. Eight of 13 (62%) patients with a FDG PET/CT positive result were MRD negative. Eight patients have completed four cycles of KRD consolidation; two patients were converted into FDG PET/CT negativity; one of the two remained MRD positive, the other remained MRD negative. Three patients had reduced FDG uptake but were still considered FDG PET/CT positive; one of these converted from MRD positive to negative. One had stable disease and two had progression on FDG PET/CT whereof one converted from MRD negative to positive. Conclusion: A significant proportion (37%) of patients treated with standard first line treatment including ASCT with very good partial response or better was considered FDG PET/CT positive. Sixty-two percent of these patients were MRD negative by Euroflow, confirming the complementary features of these two methods. Treatment with four cycles of KRd improved disease status based on FDG PET/CT in 5 of 8 patients (62,5%) and converted 2 out of 8 patients to FDG PET/CT negativity. One patient was converted from MRD positivity to MRD negativity. This study is ongoing and will enroll 50 FDG PET/CT positive patients. Before the ASH 2019 meeting, we plan to screen approximately fifteen additional patients with FDG PET/CT and about five more patients will have completed KRd consolidation therapy. Table Disclosures Nørgaard: Bayer, Astrazeneca: Honoraria. Abildgaard:Janssen: Research Funding; Celgene: Research Funding; Takeda: Research Funding; Amgen: Research Funding. Revheim:South-Eastern Norway Regional Healt Authority: Research Funding. Schjesvold:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; SkyliteDX: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
28
Takamatsu, Hiroyuki, Takeshi Yoroidaka, Takeshi Yamashita, Ryoichi Murata, Kyoko Yoshihara, Satoshi Yoshihara, Mikio Ueda, Shinji Nakao, and Kosei Matsue. "Minimal residual disease assessment using EuroFlow in patients with relapsed/refractory multiple myeloma who received carfilzomib+lenalidomide+dexamethasone (KRD) therapy." Clinical Lymphoma Myeloma and Leukemia 19, no. 10 (October 2019): e184. http://dx.doi.org/10.1016/j.clml.2019.09.308.
Full text
29
Yoroidaka, Takeshi, Hiroyuki Takamatsu, Takeshi Yamashita, Ryoichi Murata, Kyoko Yoshihara, Satoshi Yoshihara, Mikio Ueda, Shinji Nakao, and Kosei Matsue. "Minimal Residual Disease Assessment Using Euroflow-NGF in Patients with Multiple Myeloma Treated with a Combination of Carfilzomib, Lenalidomide, and Dexamethasone (KRD)." Blood 134, Supplement_1 (November 13, 2019): 3130. http://dx.doi.org/10.1182/blood-2019-126552.
Full textAbstract:
Background: Owing to the development of novel agents, the rate of complete response (CR) in multiple myeloma (MM) has increased. Additionally, the development of methods for measuring minimal residual disease (MRD) (e.g., multiparameter flow cytometry [MFC] and next-generation sequencing) has enabled us to stratify CR patients according to MRD levels. In this study, we hypothesized that deep response predicts better prognosis in MM. To investigate this hypothesis, we assessed the response of patients treated with carfilzomib + lenalidomide + dexamethasone (KRD) using MFC and compared survival outcomes between different groups defined by the MRD status. Methods: The response of patients with relapsed/refractory MM treated with KRD at four different centers between September 2016 and October 2018 was prospectively investigated using the EuroFlow next-generation flow (EuroFlow-NGF) method. In this method, ammonium chloride-based bulk lysis was used, followed by surface staining with antibodies against CD138-BV421, CD27-BV510, CD38 multiepitope (ME)-FITC, CD56-PE, CD45-PerCP Cy5.5, CD19-PECy7, CD117-APC, and CD81-APC C750 in tube 1 and surface/intracellular staining with antibodies against CD138-BV421, CD27-BV510, CD38 ME-FITC, CD56-PE, CD45-PerCP Cy5.5, CD19-PECy7, CD117-APC, CD81-APC C750, cytoplasmic (cy) Igκ-APC, and cyIgλ-APC C750 after permeabilization in tube 2. MRD levels were assessed using bone marrow (BM) cells after several KRD cycles, with the lower limit of detection set at 1 × 10−5. Presence of high-risk cytogenetics [del 17p, t(4;14) and/or t(14;16)] in BM cells was analyzed through FISH. Results: A total of 21 patients (12 males, 9 females) were treated with KRD and assessed for MRD levels. The median age of these patients was 66 years at KRD initiation (range 30-83 years), and 11 patients had ISS 1, 6 had ISS 2, and 4 had ISS 3. Four patients displayed high-risk chromosomal abnormalities, including del 17p (n = 3) and t(14;16) (n = 1). The median number of prior treatments was 3 (range 1-6); these included bortezomib (n=12), lenalidomide (n=19), and autologous stem-cell transplantation (n=12). The median number of KRD cycles was 4 (range 1-22). The proportion of patients achieving ≥CR and overall response (≥ partial response [PR]) was significantly higher after KRD treatment than the proportion that had been achieved by previous therapies (71% vs. 9.5%, p < 0.001; 100% vs. 71%, p = 0.008, respectively). Pre-KRD responses included 2 stringent CR (sCR), 7 very good PR (VGPR), 6 PR, 3 stable disease, and 3 progressive disease. Post-KRD responses included 13 sCR, 2 CR, 3 VGPR, and 3 PR. A total of 95% (20/21) of patients achieved sCR, and 5% (1/21) VGPR as best response. After KRD, response was upgraded in 19 (90%) patients and maintained in two PR (10%) patients. During and after KRD treatment, MRD negativity was achieved in 12 of 16 (75%) and in 15 of 21 (71%) patients, respectively. The median number of therapy lines after KRD was 1 (range 0-5). All 4 high-risk cytogenetic cases achieved MRD negativity. Among MRD-positive cases, both 2-year progression-free survival (PFS) and 2-year overall survival (OS) from KRD initiation were 100%. Among MRD-negative cases, 2-year PFS and OS from KRD initiation were 92% and 100%, respectively. The median follow-up was 1.8 years (range 0.5-2.5 years). One MRD-negative case showed extramedullary relapse 1.4 years after the last KRD cycle. This patient did not have high-risk cytogenetics and achieved "flow MRD negativity" after two KRD cycles, and the treatment was stopped after 7 KRD cycles due to peripheral neuropathy. Paiva et. al. also reported that only 6 of 225 (3%) MRD-negative patients relapsed. Strikingly, all 6 relapsing cases in the report had extramedullary plasmacytomas at diagnosis; all relapsed with extramedullary plasmacytomas and only 2 developed concomitant serological relapse (ASH 2017, abstract #905). Conclusions: KRD induced deep responses in relapsed/refractory MM patients who eventually displayed excellent PFS. All patients with high-risk cytogenetics achieved EuroFlow-NGF negativity. Post-remission imaging studies such as MRI/PET-CT may be necessary for patients who presented with extramedullary plasmacytomas even when they achieved flow MRD negativity. Figure Disclosures Yoroidaka: Ono Pharmaceutical: Honoraria. Takamatsu:Celgene: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ono pharmaceutical: Honoraria, Research Funding; CSL Behring: Research Funding; SRL: Consultancy, Research Funding; Janssen Pharmaceutical: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Fujimoto Pharmaceutical: Honoraria; Becton, Dickinson and Company: Honoraria; Abbvie: Consultancy; Daiichi-Sankyo Company: Honoraria. Yamashita:Celgene: Honoraria; Ono Pharmaceutical: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; Kyowa Kirin: Honoraria; Daiichi-Sankyo Company: Honoraria; TEIJIN PHARMA LIMITED: Honoraria. Murata:Celgene: Honoraria; Ono pharmaceutical: Honoraria. Yoshihara:Kyowa Kirin: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Eisai Co., Ltd.: Honoraria. Yoshihara:Chugai Pharmaceutical Co.,Ltd: Honoraria; Bristol-Myers Squibb: Honoraria; Novartis Pharma K.K.: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Sumitomo Dainippon Pharma: Honoraria; Kyowa Kirin: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Celgene: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria. Nakao:Bristol-Myers Squibb: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Celgene: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Novartis Pharma K.K: Honoraria; Kyowa Kirin: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Ono Pharmaceutical: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; SynBio Pharmaceuticals: Consultancy. Matsue:Takeda Pharmaceutical Company Limited: Honoraria; Novartis Pharma K.K: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Celgene: Honoraria; Ono Pharmaceutical: Honoraria.
30
Glier, Hana, Michaela Novakova, Jeroen te Marvelde, Andre Bijkerk, Daniela Morf, Daniel Thurner, Katerina Rejlova, et al. "Comments on EuroFlow standard operating procedures for instrument setup and compensation for BD FACS Canto II, Navios and BD FACS Lyric instruments." Journal of Immunological Methods 475 (December 2019): 112680. http://dx.doi.org/10.1016/j.jim.2019.112680.
Full text
31
Terpos, Evangelos, Ioannis V. Kostopoulos, Aristea-Maria Papanota, Konstantinos Papadimitriou, Panagiotis Malandrakis, Paraskevi Micheli, Ioannis Ntanasis-Stathopoulos, et al. "Next Generation Flow Cytometry Provides a Standardized, Highly Sensitive and Informative Method for the Analysis of Circulating Plasma Cells in Newly Diagnosed Multiple Myeloma: A Single Center Study in 182 Patients." Blood 134, Supplement_1 (November 13, 2019): 4338. http://dx.doi.org/10.1182/blood-2019-127989.
Full textAbstract:
Introduction: The apparent heterogeneity of multiple myeloma (MM) constitutes a key challenge in the clinical management and the design of effective therapeutic interventions, while it entails the identification of biomarkers with a strong prognostic value. In this context and taking into account patients' inconvenience to invasive bone marrow (BM) aspiration, the assessment of circulating plasma cells (CPCs) in liquid biopsies, at the time of diagnosis, has been proposed as a useful assay with prognostic value. Different methodologies have been applied for the detection of CPCs; the most common is the use of multicolor flow cytometry (MFC), mainly of 2-, 4-, or 6-color combination panels, which however yielded heterogeneous results due to variations in the detection efficacy of each approach. In the present study, we applied the standardized and highly sensitive Next Generation Flow Cytometry (NGF) approach, to detect CPCs in diagnostic MM peripheral blood (PB) samples, we compared their phenotypic characteristics with the aberrant clonal cells of BM matched samples and we correlated their presence with disease characteristics. Patients and Methods: PB and BM matched samples from 182 consecutive MM patients, at diagnosis, were evaluated for the presence of aberrant plasma cells (APCs) following the standard operating procedures (SOP) of NGF, according to EuroFlow guidelines. All these patients were diagnosed and treated in a single center (Department of Clinical Therapeutics, N.K. University of Athens, Greece). Samples were collected in EDTA-anticoagulated tubes and treated with the bulk-lysis procedure. Recovered cells were stained with antibodies against surface CD19-PEC7, CD27-BV510, CD38-FITC, CD45-PERCP, CD56-PE and CD138-BV421 and the intracellular CyIgκ-APC and CyIgλ-APCC750 to verify clonality. Six to ten million cells were acquired per sample, thus reaching a median Limit of Detection (LOD) of 3.5x10-6. Optimal PMT voltages were set according to the EuroFlow SOP for instrument set-up and daily performance status of FACSCANTOII was monitored with both CS&T (BD) and Rainbow beads (Spherotech Inc, Lake Forest, IL). Results: CPCs were detected in 158/182 (86.8%) MM diagnostic samples within a range of 0.0002% to 63.8% of total PB nucleated cells (PBNCs). The CPCs showed the same aberrant phenotype as the one detected in the BM for all cases, although with a significantly reduced intensity for the markers CD27, CD38, CD138 and CD56. When more than one phenotypically distinct subgroups were detectable in the BM, the same phenotypic subsets were present in the PB with the same relative frequency for >90% of bi/multi-phenotypic cases. The higher number of CPCs (>0.1% of all PBNCs) strongly correlated with an increased BM infiltration rate by myeloma cells (p<0.0001), with ISS-3 disease stage (p<0.0001) and with the presence of high-risk cytogenetics [t(4;14), t(14;16) and/or del(17p53); p<0.0001]. There was also weaker correlation between high number of CPCs and high serum creatinine levels (p=0.015). Inversely, the absence of CPCs or the presence of CPCs at numbers ≤0.001% correlated with lower serum β2-microglobulin (p<0.0001), with higher hemoglobin levels (p<0.0001) and with the presence of an elevated normal plasma cell compartment within the BM (i.e. ≥5% of all PCs; r2=0.84, p<0.0001). There was no association between the CPC number and the therapeutic response to induction treatment (IMWG criteria). Despite the short follow-up period (median of 16 months), there is a trend for inferior PFS in patients with high CPCs (p=0.16). Conclusions: The NGF approach using the EuroFlow protocol enables the detection of even rare CPCs in diagnostic MM PB samples, due to the high number of cells acquired and the elegantly elaborated 8-color marker combinations which allows for the detection of CPCs with even a non-typical phenotype. Our matched PB and BM analysis revealed that BM APCs and CPCs share very similar characteristics suggesting that liquid biopsy offers a representative alternative for the phenotypic characterization of BM APCs. The correlation of high CPCs with adverse disease characteristics suggests that the quantification of CPCs by standardized NGF may emerge as a valuable surrogate prognostic biomarker which could replace other invasive methods or other less informative assays. Disclosures Terpos: Janssen: Honoraria, Other: Travel expenses, Research Funding; Medison: Honoraria; Amgen: Honoraria, Research Funding; Takeda: Honoraria, Other: Travel expenses, Research Funding; Genesis: Honoraria, Other: Travel expenses, Research Funding; Celgene: Honoraria. Gavriatopoulou:Amgen: Honoraria; Janssen: Honoraria, Other: Travel expenses; Genesis: Honoraria, Other: Travel expenses; Takeda: Honoraria, Other: Travel expenses. Kastritis:Genesis: Honoraria; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Honoraria; Prothena: Honoraria. Dimopoulos:Sanofi Oncology: Research Funding.
32
Askeland, Frida Bugge, Anne-Marie Rasmussen, and Fredrik Schjesvold. "Relapse from MRD Negativity As Indication for Treatment in Multiple Myeloma - the Remnant Study." Blood 136, Supplement 1 (November 5, 2020): 21–22. http://dx.doi.org/10.1182/blood-2020-139230.
Full textAbstract:
Background: Early intervention can reduce the rate of progression and improve overall survival (OS) in smouldering multiple myeloma (SMM) patients (pts) (1, 2). The most powerful predictor of prognosis in active MM is whether pts achieve minimal residual disease (MRD) negativity in bone marrow with treatment (3), however treating MRD relapse has not been evaluated in a randomized fashion. The REMNANT study will evaluate whether treating MRD relapse after first line (1.L) treatment prolongs progression free survival (PFS) and OS for MM pts versus treating relapse after 1.L. treatment at progressive disease (PD) (4). To establish a homogenous group of MRD negative pts after 1.L treatment including autologous stem cell transplantation (ASCT), pts are enrolled at diagnosis and treated with Norwegian standard of care (SOC) 1.L treatment. MRD negative pts will move on to the randomized part. Methods: The REMNANT study is an academic, multicenter, open-label, randomized phase II/III study of NDMM pts eligible for ASCT (see Figure 1). 391 pts across Norway will be included in the phase II part of the study and receive SOC 1.L treatment according to Norwegian national guidelines; VRd (V: 1,3 mg/m2 SC Days 1, 4, 8, 11; R: 25 mg PO Days 1-14; d: 20 mg PO Days 1, 2, 4, 5, 8, 9, 11, 12) for 4 pre-transplant induction and 4 post-transplant consolidation cycles (all 21-d cycles). After induction pts will undergo tandem or single ASCT, depending on toxicity and response to first ASCT. The primary endpoint of the phase 2 part of the study is the number of pts who achieve MRD negative (Euroflow NGF 10 -5) complete response (CR) 30-45 days post consolidation. Safety evaluations and pts-reported outcome assessment will be measured. Pts (176) achieving MRD negative CR will be randomly assigned in a 1:1 ratio to receive 2.L treatment at MRD reappearance (arm A) or at progressive disease (PD) as defined by the IMWG criteria (4) (arm B). Randomization will be stratified by R-ISS stage at diagnosis and single vs tandem ASCT. Pts in arm A will be followed with MRD assessment every 4 month and start 2.L treatment at loss of MRD negative CR. Pts in arm B will be followed up by standard criteria and start 2.L treatment at PD. Both arms will receive the same 2.L treatment; KdD (all 28-d cycles) (K: 70mg/m2 iv Days 1,8,15 d: 40 mg Days 1, 8, 15, 22 D: 1800 mg SC Days 1, 8, 15 during C 1-2, Days 1, 8 during cycle 3-6, Day 1 from cycle 7,). 2.L treatment will continue until disease progression, unacceptable AEs or patient withdrawal. In arm A MRD Euroflow will be assessed after 6 and 18 months of 2L therapy. In arm B MRD Euroflow will be assessed if >CR is achieved but not before 6 months of 2 L therapy, and again after 12 consecutive months. The co-primary endpoint is progression and death by any cause (PFS) and death by any cause alone (OS). Secondary endpoints includes TTNT and the proportion pf pts who achieve MRD negative CR during 2.L treatment in arm A and arm B, safety evaluations and pts-reported outcome. The trial is approved and will start enrollment Q3 2020. 1. Mateos MV, Hernandez MT, Giraldo P, de la Rubia J, de Arriba F, Corral LL, et al. Lenalidomide plus dexamethasone versus observation in patients with high-risk smouldering multiple myeloma (QuiRedex): long-term follow-up of a randomised, controlled, phase 3 trial. Lancet Oncol. 2016;17(8):1127-36. 2. Lonial S, Jacobus S, Fonseca R, Weiss M, Kumar S, Orlowski RZ, et al. Randomized trial of lenalidomide versus observation in smoldering multiple myeloma. 2020;38(11):1126-37. 3. Munshi NC, Avet-Loiseau H, Rawstron AC, Owen RG, Child JA, Thakurta A, et al. Association of Minimal Residual Disease With Superior Survival Outcomes in Patients With Multiple Myeloma: A Meta-analysis. JAMA Oncol. 2017;3(1):28-35.5. 4. Kumar S, Paiva B, Anderson KC, 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(8):e328-e46. Disclosures Schjesvold: Amgen, Celgene, Janssen, MSD, Novartis, Oncopeptides, Sanofi, SkyliteDX, Takeda: Honoraria; Celgene, Amgen, Janssen, Oncopeptides: Research Funding; Amgen, Celgene, Janssen, MSD, Novartis, Oncopeptides, Sanofi, Takeda: Consultancy. OffLabel Disclosure: Carfilzomib-dexamethason-daratumumab (KdD) as second line treatment
33
Spencer, Andrew, Tiffany Khong, Flora Yuen, Hannah Victoria Giles, Malgorzata Gorniak, Hang Quach, Noemi Horvath, et al. "A Longitudinal Evaluation of Euroflow and Combined Quantitative Immunoprecipitation (QIP) and Free Light Chain (FLC) Mass Spectometry (MS) in Functional High Risk Multiple Myeloma." Blood 134, Supplement_1 (November 13, 2019): 3090. http://dx.doi.org/10.1182/blood-2019-129760.
Full textAbstract:
Introduction: The achievement of minimal residual disease (MRD) negativity is being increasingly recognised as the optimal measure of therapeutic response for both newly diagnosed and relapsed and/or refractory multiple myeloma (MM) patients. Bone marrow (BM) evaluation with either Next Generation Sequencing (NGS) or Next Generation Flow-cytometry (NGF) affords a high level of sensitivity and the attainment of MRD negativity (< 1 in 10-5 MM cells) with either approach is a powerful predictor of superior progression free survival (PFS). Both, however, are limited by the requirement for invasive bone marrow biopsy and the technical limitations imposed by variability in sample quality. Moreover, we and others have demonstrated the presence of significant spatial heterogeneity in MM that increases in the context of disease progression. Against this background we have evaluated a blood-based strategy for disease burden evaluation, Quantitative ImmunoPrecipitation (Mass Spectometry (QIP MS) and Free Light Chain Mass Spectometry (FLC MS) in a uniformly treated cohort of functional high-risk MM patients also undergoing sequential NGF (EuroFlow platform) MRD evaluation. Methods: Newly diagnosed MM patients failing (<partial remission [PR] as best response) front-line bortezomib-based induction therapy were enrolled onto the Australasian Leukaemia and Lymphoma Group (ALLG) MM17 trial (ACTRN12615000934549) evaluating an intensive salvage approach utilising a combination of carfilzomib, thalidomide and dexamethasone (KTd) as re-induction (KTd x 6 cycles) and as post autologous stem cell transplantation (ASCT) consolidation (KTd x 2 cycles followed by Td x10 cycles). NGF MRD status was determined pre-ASCT, post-ASCT and post-KTd consolidation utilising the standardised 8-colour EuroFlow platform. Matched serum samples from the 3 time-points were evaluated in parallel with QIP and FLC MS. Briefly, polyclonal antibodies (anti-IgG, -IgA, -IgM, -total κ, -total λ, free κ and free λ) covalently attached to paramagnetic microparticles were incubated with serum, washed and treated to simultaneously elute and reduce patient immunoglobulins. Light chain mass spectra were generated on a MALDI-TOF-MS system. Concordance between NGF and MS was assessed via the derivation of Cohen's kappa values. Results: Fifty patients were enrolled onto the ALLG MM17 trial. QIP and/or FLC MS identified the serum monoclonal paraprotein (PP) at baseline in all cases (100% sensitivity). Serum samples for MS with matched BM for NGF were available on 33 patients pre-ASCT, 32 post-ASCT and 26 post-KTd consolidation (91 matched samples in total). Sequential MS demonstrated serological complete remission (disappearance of MS baseline detectable monoclonal intact immunoglobulin [PP] and/or FLC) (CRMS) in 11%, 47% and 53% of patients pre-ASCT, post-ASCT and post-KTd, respectively. NGF MRD negativity at the same time points was 39%, 52% and 71% (the latter equivalent to a 50% MRD negativity rate within the original n=50 intention-to-treat population). The Cohen's kappa values for the 3 time-points were 0.21, 0.18 and 0.35 indicating fair to moderate concordance with the best concordance at the post-KTd consolidation time-point and with a Cohen's kappa value for the entire cohort (n=91) of 0.30. The sequential MS demonstrated that 12 patients had discordant disappearance of baseline PP and free light chains (FLC) prior to achieving CRMS. In 11 the FLC disappeared before the PP and in 1 the PP prior to the FLC. The former though to be due to either the FLC falling below the sensitivity of the technique following successful therapy or the presence of 2 sub-clones with differential drug sensitivity, whereas the latter was likely secondary to the persistence of a FLC expressing sub-clone. Post-KTd MS demonstrated good concordance with serological response (Cohen's kappa value = 0.61) but with 18% of patients demonstrating sCR/CR despite persisting MS detectable PP and/or FLC. Conclusion: These preliminary data confirm the utility of QIP MS and FLC MS for the sequential monitoring of tumour burden in HR MM. Concordance with standard monitoring was good with MS detectable disease in some patients with serological sCR/CR consistent with the higher sensitivity of MS. Concordance with NGF was only fair to moderate mandating the future comparison of larger sample sets to better understand the relationship between the 2 methodologies. Disclosures Spencer: Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau; Secura Bio: Consultancy, Honoraria; Servier: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria; Specialised Therapeutics Australia: Consultancy, Honoraria. Khong:Novartis Oncology: Research Funding. Quach:Janssen: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Sanofi: Research Funding; GSK: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees. Kalff:Amgen: Honoraria; Celgene: Honoraria; pfizer: Honoraria. Reynolds:Novartis Australia: Honoraria; Alfred Health: Employment, Other: Biostatistician for trials funded by the Australian government and Abbvie, Amgen, Celgene, GSK, Janssen-Cilag, Merck, Novartis, Takeda, but sponsored by Alfred Health.; AUSTRALASIAN LEUKAEMIA & LYMPHOMA GROUP (ALLG): Consultancy; Novartis AG: Equity Ownership.
34
Takamatsu, Hiroyuki, Takeshi Yoroidaka, Momoko Fujisawa, Kazuya Kobori, Masako Hanawa, Takeshi Yamashita, Ryoichi Murata, Mikio Ueda, Shinji Nakao, and Kosei Matsue. "Comparison of minimal residual disease detection in multiple myeloma by SRL 8-color single-tube and EuroFlow 8-color 2-tube multiparameter flow cytometry." International Journal of Hematology 109, no. 4 (February 18, 2019): 377–81. http://dx.doi.org/10.1007/s12185-019-02615-z.
Full text
35
Takamatsu, Hiroyuki, Takeshi Yoroidaka, Momoko Fujisawa, Kazuya Kobori, Masako Hanawa, Takeshi Yamashita, Ryoichi Murata, Mikio Ueda, Shinji Nakao, and Kosei Matsue. "Comparison of minimal residual disease detection in multiple myeloma between SRL 8-color single-tube and EuroFlow 8-color 2-tube multiparameter flow cytometry methods." Clinical Lymphoma Myeloma and Leukemia 19, no. 10 (October 2019): e183-e184. http://dx.doi.org/10.1016/j.clml.2019.09.307.
Full text
36
Hofste op Bruinink, Davine, Stefania Oliva, Lucie Rihova, Bronno van der Holt, Milena Gilestro, Jeroen G. te Marvelde, Pavla Vsianska, et al. "Flowcytometric Minimal Residual Disease Assessment in the EMN-02/HOVON-95 MM Trial: Used Methods and a Comparison of Their Sensitivity." Blood 128, no. 22 (December 2, 2016): 2072. http://dx.doi.org/10.1182/blood.v128.22.2072.2072.
Full textAbstract:
Abstract Background The introduction of novel treatment strategies against multiple myeloma (MM) has resulted in a major improvement in the overall outcome, which has led to an increased need for highly sensitive methods to detect minimal residual disease (MRD) in each patient. MRD assessment by multicolor flowcytometry (MFC) has been shown to be of prognostic value in many treatment protocols over the last decade, making it an attractive method to assess response in clinical trials. However, it is currently not known (1) what the best timing is to perform MFC MRD analysis in the context of a treatment protocol including induction, intensification, consolidation and maintenance treatment, (2) which patients should be selected for this analysis, and (3) what its feasibility is in a large international trial. The ongoing EMN-02 MRD Study aims to answer these questions within the framework of the EMN-02/HOVON-95 MM trial. Here, we describe our methods and the results of our first quality assessment round to compare the sensitivity of the used protocols. Methods The EMN-02/HOVON-95 MM trial is a randomized, multicenter, phase 3 trial in which newly diagnosed MM patients 18-65 years received 4 cycles of bortezomib, cyclophosphamide and dexamethasone (VCD) as induction treatment, followed by a first randomization between either 4 cycles of bortezomib, melphalan and prednisone (VMP), or high dose melphalan (HDM) and 1 or 2 ASCT as intensification treatment. Subsequently, patients were randomized between 2 cycles of bortezomib, lenalidomide and dexamethasone (VRD) or no consolidation treatment, followed by lenalidomide maintenance treatment for all until progression or toxicity occurred. Patients undergoing a bone marrow (BM) aspiration for complete response (CR) confirmation according to the International Myeloma Working Group (IMWG) criteria (Rajkumar et al. - Blood 2011) anytime during the trial were eligible for the EMN-02 MRD Study. BM samples from patients from 13 European countries were sent to 4 central MFC MRD laboratories in the Netherlands (A), Czech Republic (B), Denmark (C) and Italy (D), either using the strict Euroflow protocol (A) (Van Dongen et al. - Leukemia 2012) or Euroflow-based methods (B, C & D). In order to check compatibility between protocols, 5 bone marrow samples from MM patients with a clinical response ranging from progressive disease (PD) to CR were each divided in equal volumes and sent to the respective laboratories on 3 different days. MFC MRD analysis was performed on a FACS Canto II (BD) (A-C) or Coulter Navios flowcytometer (D). Protocols A, B & C used the Euroflow Plasma Cell Disorder (PCD) tube 1 and 2 combination of antibodies, containing the backbone markers CD138-PO, CD38-FITC, CD45-PB and CD19-PE-Cy7, with CD56-PE, B2micro-PerCP-Cy5.5, cyIgK-APC and cyIgL-APC-C750 in tube 1, and CD28-PE, CD27-PerCP-Cy5.5, CD117-APC, CD81-APC-H7 in tube 2. Protocol D had the same backbone markers (CD138-PerCP-Cy5.5, CD38-PB, CD45-KO and CD19-PE-Cy7), together with CD27-PE, CD81-FITC and CD20-APC in tube 1 and cyIgK-FITC, cyIgL-PE, CD56-APC and CD117-APC-AF 750 in tube 2. Bulk lysis was performed in protocols A, B and D. Every laboratory acquired at least 2x10e6 leukocytes (or at least 1x10e4 plasma cells) and performed data-analysis in Infinicyt version 1.6 or higher (A, B & C) or Navios Kaluza (D), using a threshold ranging from 10-25 aberrant plasma cell events as cutoff for MFC MRD positivity. Results Acquisition of events occurred the day after BM aspiration for all samples. The total number of acquired events per sample was dependent on the level of MRD, ranging from 3x10e5 to 2x10e7 leukocytes. MFC MRD results were very comparable between labs with a 1:1 correlation between results at every level of residual disease, being 1x10e-2, 1x10e-4, 1x10-4, 1x10e-5 and 1 MRD negative sample at the level of <1x10e-5. Based on these findings, protocols have been further harmonized and a second quality assessment round will be organized in Fall 2016 to validate the suggested improvements. Conclusions This is the first time that a European framework has been set up between laboratories to test MFC MRD analysis in the context of an international trial. The sensitivity of the protocols has been compared in a quality assessment round, which showed a high correlation of results. Disclosures Oliva: Amgen: Honoraria; Takeda: Honoraria; Celgene: Honoraria. Boccadoro:CELGENE: Honoraria, Research Funding; Mundipharma: Research Funding; Janssen: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Abbivie: Honoraria; SANOFI: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Hajek:Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Sonneveld:Celgene: Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria, Research Funding. Palumbo:Takeda: Employment, Honoraria; Janssen Cilag: Honoraria.
37
Takamatsu, Hiroyuki, Takeshi Yoroidaka, Momoko Fujisawa, Kazuya Kobori, Masako Hanawa, Takeshi Yamashita, Ryoichi Murata, Mikio Ueda, Shinji Nakao, and Kosei Matsue. "Correction to: Comparison of minimal residual disease detection in multiple myeloma by SRL 8-color single-tube and EuroFlow 8-color 2-tube multiparameter flow cytometry." International Journal of Hematology 109, no. 4 (March 21, 2019): 505. http://dx.doi.org/10.1007/s12185-019-02621-1.
Full text
38
Villa Cárdenas, Esteban Homero, Priscila Belén Cárdenas Valdez, Mónica Mercedes Valdez Cárdenas, and EDITOR: Dr Felipe Xavier Campoverde Merchán. "Linfomas de estirpe B: bases para su entendimiento." Oncología (Ecuador) 30, no. 2 (August 31, 2020): 167–77. http://dx.doi.org/10.33821/487.
Full textAbstract:
Propósito de la revisión: el objetivo de la revisión es delinear la fisiopatología de los linfomas de estirpe B. Buscamos reportes en donde se incluye la descripción del origen de los Linfomas B para una mejor comprensión de esta patología, a la luz de los avances en las diferentes áreas.
Recientes hallazgos: El Grupo Euroflow ha publicado una lista de paneles de Expresión de Antígenos de Superficie en Linfoma no Hodgkin, cuya lista se presenta en este artículo.
Extracto: Las neoplasias hematológicas han tenido grandes avances en los últimos años en varios campos, evolucionando desde la identificación citológica, pasando por su caracterización inmunofenotípica por medio de la Citometría de Flujo e Inmunohistoquímica y llegando a la caracterización molecular, iniciando por Técnicas de Cariotipo Convencional, continuando por técnicas de Inmunohibridación in situ y actualmente con la identificación molecular por medio de la Secuenciación de Nueva Generación. Esta es la razón por la que los sistemas de estadificación han ido evolucionando también, siendo el que está al momento en vigencia el propuesto por la Organización Mundial de la Salud en el año 2016. Los linfomas constituyen un grupo heterogéneo de neoplasias hematológicas con un amplio espectro de presentación clínica, cuyo origen se encuentra en los precursores de linfoides y que afectan a los diversos órganos linfoides. De estos, los linfomas de la línea B son los más comunes, motivo de esta revisión.
39
Mestrum, Stefan G. C., Anton H. N. Hopman, Frans C. S. Ramaekers, and Math P. G. Leers. "The potential of proliferative and apoptotic parameters in clinical flow cytometry of myeloid malignancies." Blood Advances 5, no. 7 (April 13, 2021): 2040–52. http://dx.doi.org/10.1182/bloodadvances.2020004094.
Full textAbstract:
Abstract Standardization of the detection and quantification of leukocyte differentiation markers by the EuroFlow Consortium has led to a major step forward in the integration of flow cytometry into classification of leukemia and lymphoma. In our opinion, this now enables introduction of markers for more dynamic parameters, such as proliferative and (anti)apoptotic markers, which have proven their value in the field of histopathology in the diagnostic process of solid tumors and lymphoma. Although use of proliferative and (anti)apoptotic markers as objective parameters in the diagnostic process of myeloid malignancies was studied in the past decades, this did not result in the incorporation of these biomarkers into clinical diagnosis. This review addresses the potential of these markers for implementation in the current, state-of-the-art multiparameter analysis of myeloid malignancies. The reviewed studies clearly recognize the importance of proliferation and apoptotic mechanisms in the pathogenesis of bone marrow (BM) malignancies. The literature is, however, contradictory on the role of these processes in myelodysplastic syndrome (MDS), MDS/myeloproliferative neoplasms, and acute myeloid leukemia. Furthermore, several studies underline the need for the analysis of the proliferative and apoptotic rates in subsets of hematopoietic BM cell lineages and argue that these results can have diagnostic and prognostic value in patients with myeloid malignancies. Recent developments in multiparameter flow cytometry now allow quantification of proliferative and (anti)apoptotic indicators in myeloid cells during their different maturation stages of separate hematopoietic cell lineages. This will lead to a better understanding of the biology and pathogenesis of these malignancies.
40
Dimier, Natalie, Paul Delmar, Carol Ward, Rodica Morariu-Zamfir, Gunter Fingerle-Rowson, Jasmin Bahlo, Kirsten Fischer, et al. "A Model for Predicting Effect of Treatment on Progression-Free Survival Using Minimal Residual Disease As a Surrogate Endpoint in Chronic Lymphocytic Leukemia." Blood 126, no. 23 (December 3, 2015): 720. http://dx.doi.org/10.1182/blood.v126.23.720.720.
Full textAbstract:
Abstract Introduction The standard primary endpoint in clinical trials of chronic lymphocytic leukemia (CLL) is progression-free survival (PFS). Given the increasingly long follow up required to detect differences in PFS between treatment arms in the era of more efficient therapeutics, valid surrogate endpoints are urgently needed to reduce clinical trial duration, thereby accelerating drug development, reducing costs and allowing patients (pts) earlier access to novel treatment options. Pts with CLL who achieve levels of minimal residual disease (MRD) of <1 clonal cell/10.000 leukocytes in peripheral blood (PB) as determined by multicolor flow cytometry or allele-specific oligonucleotide real-time quantitative polymerase chain reaction (ASO-RQ-PCR) at the end of initial treatment are considered MRD negative, and have been shown to experience significantly improved PFS. This analysis aimed to support the evaluation of MRD response at the end of treatment as a surrogate endpoint for PFS in CLL, based on a retrospective analysis of 3 multicenter, randomized, Phase 3 clinical trials. Methods MRD was prospectively assessed in PB of treatment-naive pts with CLL that participated in the German CLL Study Group CLL8, CLL10 and CLL11 multicenter, randomized, open-label, Phase 3 clinical trials, which included induction treatment followed by observation. The primary endpoint of each study was investigator-assessed PFS. MRD was quantified by 4-color flow cytometry in CLL8 and CLL10, and ASO RQ-PCR in CLL11. Both methods had a detection threshold of 1 CLL cell/10,000 leukocytes. Pts who had no MRD result but had disease progression or died shortly after 6 treatment cycles (within 90 [CLL8 and CLL10] or 56 [CLL11] days of last dose) were included and classed as MRD positive. A meta-regression model was developed to predict treatment effect on PFS using treatment effect on MRD. Log ratio was selected as the most reliable representation of MRD response based on the best model fit. To ensure no systemic bias with pt selection, demographic characteristics and efficacy results for the pt population used in the MRD analysis were compared with the respective intent-to-treat populations for each study. Results PB MRD levels at the end of treatment (CLL8 and CLL10, 75-195 days after last dose; CLL11, 56-190 days after last dose) were assessed in 393, 336, and 474 pts from CLL8, CLL10 and CLL11, respectively. PFS events occurred in 24% to 51% of pts assessed for MRD assigned to the experimental arm and in 34% to 67% assigned to active control treatment. Key efficacy data for the 3 trials are shown in Table 1. To fit a meta-regression model, each study was split into subgroups according to region (CLL8), country (CLL11) or randomly (CLL10). For each subgroup, the PFS hazard ratio (HR) was plotted against the ratio of MRD response rates (MRD negative rate in experimental arm to MRD negative rate in control arm, on a log-scale), and a regression line fitted to reflect the relationship between the two parameters (Figure 1). Circle size represents the weighting of each subgroup to the overall model; least variability in PFS HR have the largest circles. Clustering of circles by study reflects the overall treatment effect (for both MRD and PFS) in the studies. A statistically significant relationship between improved MRD response rates and reduction in the risk of disease progression or death was observed (for each unit increase in log of the ratio of MRD response rates, the log of PFS HR decreases by -0.299; 95% CI, -0.441 to -0.157; p=0.0004). Predictions based on this meta-regression model suggest that risk of progression or death decreases as the ratio of MRD response rates (MRD relative risk: MRD-negative rate in experimental arm/MRD-negative rate in control arm) increases (Table 2); i.e. a larger difference in MRD-response rates leads to lower PFS HR. Conclusion A surrogate endpoint (MRD) should not only provide prognostic value for the specific clinical outcome (PFS), but also evidence that treatment effect on the surrogate endpoint (MRD) reliably predicts treatment effect on the clinical outcome (PFS). Results of the meta-regression model show a significant association between treatment effect on MRD and treatment effect on PFS with regard to chemoimmunotherapy. The findings also suggest that treatment effect on PFS can be predicted based on treatment effect on MRD response. This model supports the use of MRD as a surrogate for PFS in pts with CLL. Disclosures Dimier: Roche: Employment. Delmar:F. Hoffmann-La Roche, Ltd.: Employment, Equity Ownership. Ward:F. Hoffmann-La Roche Ltd: Employment. Morariu-Zamfir:F. Hoffmann-La Roche Ltd: Employment. Fingerle-Rowson:Roche: Employment, Equity Ownership. Fischer:Roche: Other: Travel Grants. Eichhorst:Mundipharma: Consultancy, Research Funding, Speakers Bureau; AbbVie: Consultancy; Roche: Consultancy, Research Funding, Speakers Bureau. Goede:Mundipharma: Honoraria; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; GSK: Honoraria; Bristol-Myers Squibb: Honoraria. van Dongen: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.; DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; Cytognos: Patents & Royalties: Licensing of Patent on detection of IgE+ B-cells in allergic diseases. Royalties for 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. Royalty income for EuroFlow Consortium.; BD Biosciences: Other: Educational Lectures and Educational Workshops (+ related travelling costs). Laboratory Services in the field of technical validation of EuroFlow-OneFlow antibody tubes in dried format. Provided by the Laboratory of Medical Immunology, Erasums MC, Patents & Royalties; 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; 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. Ritgen:Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding. Böttcher:Celgene: Research Funding; AbbVie: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Beckton Dickinson: Honoraria. Langerak:InVivoScribe: Patents & Royalties: Licensing of IP and Patent on BIOMED-2-based methods for PCR-based Clonality Diagnostics. ; DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; Roche: Other: Lab services in the field of MRD diagnostics provided by Dept of Immunology, Erasmus MC (Rotterdam). Hallek:Janssen: Honoraria, Other: Speakers Bureau and/or Advisory Board, Research Funding; Gilead: Honoraria, Other: Speakers Bureau and/or Advisory Board, Research Funding; Roche: Honoraria, Other: Speakers Bureau and/or Advisory Board, Research Funding; Celgene: Honoraria, Other: Speakers Bureau and/or Advisory Board, Research Funding; Pharmacyclics: Honoraria, Other: Speakers Bureau and/or Advisory Board, Research Funding; Mundipharma: Honoraria, Other: Speakers Bureau and/or Advisory Board, Research Funding; Boehringher Ingelheim: Honoraria, Other: Speakers Bureau and/or Advisory Board; AbbVie: Honoraria, Other: Speakers Bureau and/or Advisory Board, Research Funding.
41
Beznos, O. A., L. Yu Grivtsova, A. V. Popa, M. A. Shervashidze, I. N. Serebryakova, and N. N. Tupitsyn. "PPROACHES TO THE ASSESSMENT OF MINIMAL RESIDUAL DISEASE IN B-CELL ACUTE LYMPHOBLASTIC LEUKEMIAS IN CONDITIONS OF TARGET THERAPY." Russian Journal of Biotherapy 16, no. 4 (December 30, 2017): 18–24. http://dx.doi.org/10.17650/1726-9784-2017-16-4-18-24.
Full textAbstract:
Background. Flow cytometry (FC) algorithms of detection of minimal residual disease (MRD) are well standardized, and approximate to molecular biologic methods. However, besides informative leukemia-associated aberrant immunophenotype, which are selected taking into account a tumor phenotype at diagnostics stage, it is necessary to consider specificity of the provided taget therapy and its influence on a cell. Objective: to offer stable combinations of antigens to identify B-cell precursors in patients on therapy of blinatumomab. Materials and methods. Clinical observation of patient G. 4 years old with B-cell precursors acute lymphoblastic leukemia (ALL) (pre-pre-B immunosubtype), whom after 3 bloks of reinduction therapy, taking into account MRD-positive status, blinatumomab was appointed as a monotherapy. Tumor immunophenotype was characterized in details by FC protocol according to EuroFlow in debute and relapse of the disease. MRD monitoring was provided by 8-color FC taking into account personalized leukemia-associated aberrant immunophenotypes. Results. In patient with B-cell precursors ALL received blinatumomab, the strategy of MRD monitoring was changed. Due to the lack of CD19 expression, identification of B-cell precursors was based on expression of cyCD22 in combination with nuclear TdT and CD10. Conclusion. In case of blinatumomab’s appointment during B-cell precursors ALL therapy, it is necessary to change the strategy of B-cell precursors identification, due to the lack of CD19 expression. Detection of B-cell precursors should be provided by assessment of other pan-B lineage antigens. First of all, it is cyCD22 or cyCD79a in combination with nuclear TdT and CD10, within the limits of nucleated cells of the sample.
42
Jones, Nicholas, Josette William Ragheb, Brian Ngo, David Uyeji, and Anselm L. Hii. "Validation of a Sensitive Flow Cytometry Assay to Assess Minimal Residual Disease of Multiple Myeloma Cells in Apheresis Product." Blood 132, Supplement 1 (November 29, 2018): 5646. http://dx.doi.org/10.1182/blood-2018-99-119858.
Full textAbstract:
Abstract 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%. Disclosures No relevant conflicts of interest to declare.
43
Fišer, Karel, Tomas Kalina, Martin Perez-Andres, Daniela Kužílková, Pablo Engel, Marta Cuenca, Elena Blanco Álvarez, Sophinus J. W. Bartol, and Menno C. van Zelm. "CD Maps - Dynamic Profiling of CD1 to CD100 Surface Expression on Human Leukocyte and Lymphocyte Subsets." Blood 134, Supplement_1 (November 13, 2019): 4878. http://dx.doi.org/10.1182/blood-2019-128123.
Full textAbstract:
CD molecules are surface molecules expressed on cells of the immune system that play key roles in immune cell-cell communication and sensing the microenvironment. These molecules are essential markers for the identification and isolation of leukocytes and lymphocyte subsets and their malignant counterparts. Here, we present the results of the first phase of the CD Maps study, mapping the expression of CD1-100 (n=110) on 47 immune cell subsets from blood, thymus and tonsil using an 8-color standardized EuroFlow approach and quantification of expression. The resulting dataset included median antibody binding capacities (ABC) and percentage of positivity for all markers on all subsets and was developed into an interactive CD Maps web resource. Using the resource, we examined differentially expressed proteins between granulocyte, monocyte and dendritic cell subsets, and profiled dynamic expression of markers during thymocyte differentiation, T-cell maturation, and between functionally distinct B-cell subset clusters. The CD Maps resource will serve as a benchmark of antibody reactivities ensuring improved reproducibility of flow cytometry-based research. Moreover, it will provide a full picture of the surfaceome of human immune cells and serves as a useful platform to increase our understanding of leukocyte biology, as well as, to facilitate the identification of new biomarkers and therapeutic targets of immunological and hematological diseases. CD Maps project is supported with reagents from BD Biosciences, BioLegend and Exbio. TK is supported by Ministry of Health Czech Republic grant 15-26588A and LO1604. KF is supported by Ministry of Health Czech Republic grant NV18-08-00385. Disclosures No relevant conflicts of interest to declare.
44
Pontes, Robéria, Juan Flores-Montero, Luzalba Sanoja-Flores, Noemi Puig, Roberto José Pessoa Magalhães, Alba Corral Mateos, Anna Beatriz Salgado, et al. "Impact of Treatment on B-Cell Regeneration By Next Generation Flow Cytometry in Patients with Multiple Myeloma." Blood 132, Supplement 1 (November 29, 2018): 4491. http://dx.doi.org/10.1182/blood-2018-99-117162.
Full textAbstract:
Abstract Introduction: Several studies have shown the role of the immune system in the development of MM, but there is no systematic description of normal B-cell regeneration during treatment points. Recently, EuroFlow consortium has developed NGF panel with high sensitivity to evaluated MRD and potentially, to assess of the normal B-cell compartment of patients with MM. Aims: Here we evaluated the B cell regeneration pattern of bone marrow (BM) from patients with MM by Next Generation Flow (NGF) cytometry at diagnosis and at minimal residual disease (MRD) time points of treatment. Material and Methods: Overall 190 samples of MM patients (45% female and 55% male, median age of 65 years - 37 to 87 years, of which: 16 at diagnosis, 30 post-induction, 76 D+100, 59 maintenance/consolidation and 9 out of treatment, and 8 samples of BM healthy donor - HD (≥ 50 years). At the moment treatment, D+100 were included samples from two research centers: HCS/USAL (n=64) and HUCFF/UFRJ (n=12). Induction regimens were composed of triple protocols (PI + IMIDs + Steroids); followed by high doses of melphalan with ASCT, while patients in maintenance/consolidation followed second line treatment regimens - IP+IMIDs and/or PIs+Steroids and/or monoclonal antibodies or INF or IPs. All samples of BM were subjected to bulk cell lysis with ammonium chloride solution for >107 cell acquisition and labeled with a combination of 10 antibodies - Panel EuroFlow MM MRD.Results: Of the 174 post treatment samples, 36% presented MRD- and 64% MRD+. At diagnosis, patients exhibit a significant reduction of precursors B cells and normal plasma cells (nPC) relative to HD, probably a reflection of the occupation of their binding sites by cPC. At post-induction, there was an increase in precursors B, compared to MM patients at diagnosis, associated with a reduction of mature B-cell (transitional/naïve and memory), regardless of MRD status. Concerning the nPC compartment, a reduction was observed, relative to HD. During treatment, reduction of the tumor burden leaves these sites free for precursors B, which rapidly recover while leads to a drastic decrease in mature B cells and regeneration of the precursors to mature B-cells is slower. On the other hand, in D+100, independent of the MRD status, there was a post treatment medullary recovery, with an increase in B precursors and transitional/naïve B-cells, in contrast, with a reduction of memory B-cells. Out of treatment, we observed a recovery of precursors B, mature B-cells and increase of nPC, but the immune recovery of these nPC is not sufficient to reach the levels of a healthy individual. Conclusion: NGF emerges as an optimal approach for simultaneous assessment of the BM regeneration profile of B-cells and the MRD status. After starting therapy, MM patients re-establish the compartments of B-cell precursors and transitional/naïve B-lymphocytes; however, memory B cells and nPC do not recovery until the end of treatment. This study is a starting point for exploring the importance of the B cells of the medullary microenvironment in the MM. Its potential impact on patient outcome deserving further investigations Disclosures Puig: Takeda: Consultancy, Honoraria; Celgene: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Mateos:Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees.
45
Du, Chenxing, An Gang, Yan Xu, Xuehan Mao, Yuting Yan, Jiahui Liu, Huishou Fan, et al. "Immunophenotype Drift of Residual Plasma Cells Indicates Therapeutic Response and Prognosis in Multiple Myeloma." Blood 134, Supplement_1 (November 13, 2019): 5491. http://dx.doi.org/10.1182/blood-2019-131730.
Full textAbstract:
Background Chemotherapy resistance remains a significant hurdle in the treatment of multiple myeloma (MM). However, it is difficult to discriminate the potential refractory patients from the very early stage. Flow cytometry is a convenient tool to detect the residual myeloma cell tiding, indicating therapeutic response sensitively. Methods From June, 2014 to December, 2016, 172 sequential patients with newly diagnosed multiple myeloma were enrolled in the BDH2008/02 clinical trial. Patient informed consent was obtained in accordance with the Declaration of Helsinki. 144 patients with at least two flow cytometry detections were analyzed. Bone marrow samples were detected by an eight-color EuroFlow panel. CD20 negative and CD81 positive is defined as normal phenotype. Results We conducted a median of 3-time (2-8) flow cytometry detection on each patient. When newly diagnosed and achieved best response, CD20, CD81 expression rates were 29.9%, 9.7% and 14.9%, 64.4% (P=0.0091, P<0.0001), respectively. According to the status variation of CD20 and CD81, all patients were divided into three groups: both markers were always normal (Group A), either CD20 or CD81 was abnormal at diagnosed and turned normal during therapy (Group B) and markers stayed abnormal (Group C). Patients with undetectable residual tumor cells were also classified as Group A. The overall response rate of the patients in Group C was inferior to Group B (>PR rate: 54.3% vs. 71.4%, P=0.021). And the OS of Group C was significantly worse than Group A and B (47.9 months vs. not reached vs. not reached, P=0.036). Conclusion CD20/CD81 switching to normal phenotype during therapy indicates therapeutic response and an improved outcome than that staying abnormal. The expression tiding of CD20 and CD81 may be a reasonable combination to dynamically stratify MM patients, directing the choice of maintenance therapy. Figure Disclosures No relevant conflicts of interest to declare.
46
Juárez-Avendaño, Gerardo, Nuria Citlalli Luna-Silva, Euler Chargoy-Vivaldo, Laura Alicia Juárez-Martínez, Mayra Noemí Martínez-Rangel, Noemí Zárate-Ortiz, Edith Martínez-Valencia, Briceida López-Martínez, Rosana Pelayo, and Juan Carlos Balandrán. "Poor Prognosis Biomolecular Factors Are Highly Frequent in Childhood Acute Leukemias From Oaxaca, Mexico." Technology in Cancer Research & Treatment 19 (January 1, 2020): 153303382092843. http://dx.doi.org/10.1177/1533033820928436.
Full textAbstract:
Objective: To investigate the cellular and molecular epidemiology of acute leukemias in vulnerable populations of children and adolescents in Oaxaca de Juarez, Mexico. Material and Methods: Descriptive, cross-sectional and retrospective study, conducted from 2014 to 2018 in which profiles of molecular and immunophenotypic aberrations were investigated in children and adolescents diagnosed with acute leukemia, by evaluating 28 molecular abnormalities by HemaVision-Q28 multiplex RT-PCR kit and standardized EuroFlow Immunophenotyping of bone marrow cells. Results: We included 218 patients, with 82.5% younger than 14 years and 17.5% adolescents. The median age was 9 years and a main peak of incidence was recorded at age of 4 to 5 years. B-cell acute lymphoblastic leukemia was diagnosed in 70.64% of all cases, acute myeloid leukemia was in 22.48%, T-cell acute lymphoblastic leukemia in 6.42%, and mixed lineage acute leukemia in 0.46% of cases. Overall, chromosomal translocations were positive in 29.82% of cases. While 65.31% of patients with acute myeloid leukemia reported aberrancies, only in 18.83% of B-cell acute lymphoblastic leukemia cases genetic abnormalities were obvious. Surprisingly, most prevalent translocations in B-cell acute lymphoblastic leukemia were t(9;22) in 20.7%, followed by t(4;11) in 17.2% and t(6;11) in 13.8%, whereas patients with acute myeloid leukemia showed t(15;17) in 40.6% and t(8;21) in 21.9%. In contrast, an homogeneous expression of t(3;21) and t(6;11) was recorded for T-cell acute lymphoblastic leukemia and mixed lineage acute leukemia cases, respectively. Except for t(1;19), expressed only by pre-B cells, there was no association of any of the studied translocations with differentiation stages of the B-leukemic developmental pathway. Conclusion: Our findings identify near 50% of patients with acute lymphoblastic leukemia at debut with high-risk translocations and poor prognosis in B-cell acute lymphoblastic leukemia as well as an unexpected increase of acute myeloid leukemia cases in young children, suggesting a molecular shift that support a higher incidence of poor prognosis cases in Oaxaca.
47
Van Der Touw, William, Lin Kang, Joseph Dennis Tario, Bhavani Stout, Vanessa Voskinarian-Berse, Valentina Rousseva, Paul K. Wallace, Robert Hariri, and Xiaokui Zhang. "Immune Monitoring of CD34+ Placental Cell Derived Natural Killer Cell Therapy (PNK-007) in Phase I Study of Multiple Myeloma." Blood 134, Supplement_1 (November 13, 2019): 4457. http://dx.doi.org/10.1182/blood-2019-124509.
Full textAbstract:
Background: Natural Killer (NK) cells are innate immune lymphocytes with cytotoxic function and are critical for immune surveillance. Unlike T cells which rely on antigen-specific responses, NK cells recognize transformed cells through a variety of NK cell-specific receptor-ligand interactions. Clinical studies have highlighted the therapeutic potential of NK cell-based therapies and Celularity has developed a GMP procedure for generating PNK-007: an off-the-shelf and allogeneic NK cell product culture-expanded and differentiated from human placental CD34+ stem cells. PNK-007 was evaluated as a single-dose allogeneic cell therapy in a phase I dose-escalation study for multiple myeloma (MM) patients receiving autologous stem cell transplant (ASCT) (NCT02955550). 12 of 15 patients received subcutaneous IL-2 every 48 hours for 10 days following PNK-007 cell infusion while 3 patients in a separate cohort did not receive IL-2. Here, we report translational studies evaluating post-treatment immune reconstitution, minimal residual disease (MRD) detection at 10-5 threshold, and serum profiling of cytokines and chemokines. Methods: Patient bone marrow aspirate was collected for EuroFlow MRD assessment and immune phenotyping by flow cytometry at baseline, 90-100 days, 6 months and 1 year post-ASCT. Peripheral blood was collected at baseline, then weekly for six weeks following PNK-007 infusion, at 6 months and at 1 year post-ASCT. PNK-007 persistence, leukocyte populations, and HLA antibody panels were determined by flow cytometry. Serum was analyzed by Luminex for panel cytokines, chemokines and soluble cytokine receptors. All patients and PNK-007 drug product were typed for HLA and killer-cell immunoglobulin-like (KIR) genotype. Results: PNK-007 infusion did not interfere with immune reconstitution kinetics post-ASCT. Cohorts receiving PNK infusion 14 days post-ASCT had already recovered white blood cell counts to normal levels. One cohort receiving 3x107 PNK cells/kg 7 days post-ASCT showed an immune deficient environment (0.05x103 ± 0.004x103 leukocytes/ml, n=3), but recovered their white blood cell counts by day 21 (6.8x103 ± 1.8x103 leukocytes/ml, n=3). Using a validated Euro-flow MRD assay, 4/15 patients were MRD(-) at pre-ASCT baseline, and by day 90, 10/15 pts were MRD(-). PNK-007 persistence was not detected in patients by flow cytometry with the earliest timepoint tested being 7 days post-infusion. Panel HLA serum antibodies were not detected at any timepoint indicating the absence of alloantibodies. Withholding IL-2 administration in one cohort allowed us to evaluate its potential effects on NK cell recovery and immune reconstitution. We found that subcutaneous IL-2 q.o.d. for 10 days following PNK-007 infusion did not affect recovery kinetics and concentration of endogenous NK cells. However, these patients showed a 5-10 fold increase in serum soluble IL-2RA from baseline and elevated regulatory T cell (Treg) count in the blood versus baseline (167 ± 107 Treg/mL vs. 61 ± 33 Treg/mL, p=0.0075). Patients not receiving IL-2 saw no change in serum soluble IL-2RA or blood Treg level from baseline. CD4 and CD8 T cells from all patients retained their ability to become activated in response to ex vivo stimulation and favored release of IL-2 and IFNg. T cell effector function was maintained in all patients post-ASCT but was lost in a subset of patients at 1 year. Serum analysis showed low levels of free IL-15 at the time of PNK dosing despite the transient lymphodepleted state associated with ASCT. Cytokines associated with myeloid inflammation and T cell immunity in the month post dosing were within normal homeostatic level. Serum TGFβ was significantly lower at the day of PNK infusion compared to normal homeostatic levels. Conclusion: Translational data from a Phase I study of PNK-007 in post-ASCT MM established that dosing up to 3x107 cells/kg at 14 or 7 days post-transplant did not impair engraftment or immune reconstitution. EuroFlow MRD assessment of the bone marrow showed conversion of 6/11 patients from MRD(+) to MRD(-) by day 90 post transplant. The administration of IL-2 in this clinical study did not appear to benefit NK cell reconstitution but instead favored soluble IL2RA antagonism and increased systemic levels of Treg. These results demonstrate the feasibility of allogeneic NK cell therapy in the MM + ASCT setting and will help inform the design of further clinical studies. Disclosures Van Der Touw: Celularity, Inc.: Employment, Patents & Royalties. Kang:Celularity, Inc.: Employment, Patents & Royalties. Stout:Celularity, Inc.: Employment. Voskinarian-Berse:Celularity, Inc.: Employment. Rousseva:Celularity, Inc.: Employment. Hariri:Celularity Inc: Employment. Zhang:Celularity Inc: Employment.
48
Cheminant, Morgane, Stephanie Schmit, Aurore Touzart, Coralie Derrieux, Marie-Hélène Delfau-Larue, Catherine Thieblemont, Vincent Ribrag, et al. "Minimal Residual Disease Monitoring By 8-Color Flow Cytometry in Mantle Cell Lymphoma Is Complementary to Q-PCR Monitoring and Will Facilitate Pre-Emptive Treatment: An EU-MCL and Lysa Study." Blood 124, no. 21 (December 6, 2014): 1657. http://dx.doi.org/10.1182/blood.v124.21.1657.1657.
Full textAbstract:
Abstract Introduction: Mantle Cell Lymphoma (MCL) is characterized by frequent blood and bone marrow involvement. It has been demonstrated that use of Minimal Residual Disease (MRD) quantification in blood and/or bone marrow might be helpful in patient management. Gold standard MRD is based on Q-PCR clone specific amplification of IgH VDJ or IgH-BCL1 rearrangements, but these are relatively complex and time consuming and over half of the positive results are in a grey zone of borderline positivity. Flow cytometry (FCM) is more rapid and better adapted to individual patient management if quantitatively reproducible, but insufficiently sensitive when only 4 colors are used. We therefore developed a universal, 8-color, EuroFlow inspired, FCM strategy, which we compared with classical Q-PCR MRD in 61/97 patients included in (and 1 treated according to) the EU-MCL Younger and Elderly prospective trials who underwent Q-PCR MRD monitoring at Necker Hospital. Method: Q-PCR MRD from IgH VDJ (n=92) or BCL1-IgH (n=5) was performed prospectively from ficolled blood (PB) or bone marrow, from which residual material was cryopreserved in DMSO for FCM quantitation, using 10 antibodies labelled with 8 fluorochromes for positive and negative (CD45, CD19, CD5, LAIR1, CD11a, IGK, IGL, CD3, CD14 and CD56) gating, after diagnostic phenotyping of fresh material, using the same panel and a EuroFlow B lymphoid screening tube. Sensitivity of both techniques was at least 0.01% (1E-04). FCM was only considered positive if above 0.01%, whereas Q-PCR results were considered positive below quantifiable range (BQR) if borderline, above sensitivity, within Euro-MRD criteria for MRD positivity. BQR samples were separated based on the number of positive, triplicate samples. The objectives were to compare the two techniques and to determine their suitability for regular screening, with a view to pre-emptive treatment on molecular or phenotypic (MRD) relapse. Two patients were treated with Rituximab at MRD relapse, prior to clinical relapse, as proof of principle. Results: A total of 302 blood or bone marrow samples from 62 patients were quantified. Overall, 79% (42/53) of samples positive at or above 0.01% by PCR were also positive by FCM, compared to 29% (19/65) of those below 0.01%, but with at least 2 positive triplicates and virtually none of those with only 1 or no results above sensitivity (1%, 2/184). Quantification of the paired MRD results positive with PCR and/or FCM were significantly correlated (r2=0.74, P<0.0001). Amongst the 62 patients, 30 have relapsed and 19 have died. Nine relapsing patients (including one off protocol patient treated and monitored at initial and second MRD relapses) had sufficient MRD points to assess the capacity of PB Q-PCR or FCM to predict future clinical relapse sufficiently to justify pre-emptive treatment at MRD relapse. Clinical relapse was preceded by MRD relapse in 9/10 relapses by Q-PCR and 7/9 by FCM. Six of the 9 relapsing patients had achieved Q-PCR negativity in at least one PB sample. The mean latency for prediction by Q-PCR, when considering any increase in positivity to at least 2 positive triplicates as positive, was 11.3 months (range 1-24mths) and 5.4 months (0.5-11) when only results above 0.01% were considered positive. The equivalent latency by FCM was slightly shorter, at 6.5 months (0.5-21) Pre-emptive treatment of 2 patients at MRD relapse, prior to clinical relapse allowed re-establishment of molecular complete remission and a durable second remission in at least one with sufficient follow-up (Cf Fig.). Figure 1 Figure 1. Conclusion: Eight color flow cytometry is a promising alternative to classical clone-specific Q-PCR strategies in monitoring therapy in MCL, with an excellent correlation (29/31, 94%) for MRD levels of at least 0.1% and acceptable correlation at 0.01-0.1% (13/22, 59%). While less sensitive at very low levels on cryopreserved material, FCM may clarify the clinical relevance of low-level borderline positivity; however it remains to be determined prospectively which technique will have greater prognostic value in patient management. FCM sensitivity will be improved by prognostic testing of fresh whole blood or bone marrow, and this pilot data clearly justifies such studies. Finally, MRD relapse precedes clinical relapse by several months, justifying pre-emptive treatment, monitored by prospective FCM and IgH Q-PCR within clinical trials. Disclosures Dreyling: Roche: Honoraria, Research Funding.
49
Oliva, Stefania, Davine Hofste op Bruinink, Lucie ŘÍhová, Stefano Spada, Bronno van der Holt, Rossella Troia, Manuela Gambella, et al. "Minimal residual disease (MRD) monitoring by multiparameter flow cytometry (MFC) in newly diagnosed transplant eligible multiple myeloma (MM) patients: Results from the EMN02/HO95 phase 3 trial." Journal of Clinical Oncology 35, no. 15_suppl (May 20, 2017): 8011. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.8011.
Full textAbstract:
8011 Background: MRD detection is a sensitive tool to measure response in MM. We assessed MRD by MFC in newly diagnosed MM patients (pts) enrolled in the EMN02/HO95 phase 3 trial. Methods: Pts were ≤65 years old and received Bortezomib-Cyclophosphamide-Dexamethasone (VCD) induction, intensification with Bortezomib-Melphalan-Prednisone (VMP) vs High-Dose-Melphalan (HDM) followed by stem cell transplant, consolidation with Bortezomib-Lenalidomide-Dexamethasone (VRD) vs no consolidation, and Lenalidomide maintenance. MRD analysis was performed in pts achieving at least a very good partial response (VGPR) before starting maintenance (after HDM, VMP or VRD) and during maintenance every 6-12 months; samples were centralized to 3 European labs. MFC was performed on bone marrow according to Euroflow-based methods (8 colors, 2 tubes) with a sensitivity of 10-5. Quality checks were performed to compare sensitivity and to show correlation between protocols (Hofste op Bruinink D ASH 2016 abstract 2072). Results: 316 pts were evaluable before maintenance: median age was 57 years, 18% (57/316) pts had ISS III and 22% (70/316) had high risk cytogenetic (HR-C) defined as having at least one among del17, t(4;14) or t(14;16); 63% (199/316) had received HDM and 37% (117/316) VMP; thereafter 51% (160/316) had received VRD. 76% (239/316) were MRD negative (MRD-) of whom 64% (153/239) received HDM vs 36% (86/239) VMP, with a median follow-up time of 30 months from MRD enrolment. 3-year PFS was 50% in MRD positive (MRD+) vs 77% in MRD- pts (HR: 2.87, p < 0.001). Subgroup analyses were performed to evaluate the risk factors for MRD+ according to baseline characteristics and therapies: HR-C was the most important risk factor (HR 9.87, interaction-p = 0.001). Finally, 48% of MRD+ pts at pre-maintenance who had a second MRD evaluation after at least 1 year of lenalidomide became MRD-. Conclusions: MRD by MFC is a strong prognostic factor in MM pts receiving intensification with novel agents or transplant; lenalidomide maintenance further improved depth of response; HR-C is the most important prognostic factor in MRD+ pts. Clinical trial information: NCT01208766.
50
Tschautscher, Marcella, Dragan Jevremovic, Francis Buadi, Martha Lacy, Morie A. Gertz, Angela Dispenzieri, Prashant Kapoor, et al. "Implications and outcomes of MRD-negative multiple myeloma patients with immunofixation positivity." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): 8034. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.8034.
Full textAbstract:
8034 Background: Minimal residual disease (MRD) assessment in multiple myeloma (MM) has improved our ability to assess disease activity, resulting in more advanced prognostication. While MRD assessment remains confined to the bone marrow (BM) plasma cell population, serum studies including immunofixation (IFE) are required to complete response evaluation. The significance of those who are MRDneg yet have detectable monoclonal protein through IFE remains unclear. Methods: We retrospectively studied 256 MM patients who had MRD assessment via the Euroflow multiparametric flow cytometry on the BM with concomitant serum IFE testing. Patients who were MRDneg were included in the study. Outcomes included probability of disease progression (PD) at 1 year. The Cox-proportional hazards model was used to compare probability of PD among different groups. Time to progression (TTP) was calculated as the difference from date of MFC analysis to PD in months. Results: Among the entire cohort, 178 (70%) patients were MRDneg and median follow-up from MRD assessment was 6.3 months. Among these patients, 74 (42%) had a positive IFE at the time of MRD analysis. Within the MRDneg/IFEpos group, 31 (42%) patients remained IFEpos after a median follow up of 5.5 mo from initial MRD/IFE testing while 34 patients eventually became IFEneg after a median of 2.8 mo with no subsequent IFE available in 9 patients. The 1 year probability of PD in the MRDneg/IFEneg group was 20% compared to 41% in the MRDneg/IFEpos group (P < 0.01, Wilcoxon test). When comparing subsequent IFE status in those who were MRDneg/IFEpos, those who remained IFEpos had a trend towards shorter TTP compared to patients who later became IFEneg. Conclusions: Persistent monoclonal protein in the face of MRD negativity predicts for a shorter TTP. This likely reflects persistent disease that was not sampled on the BM aspirate in many of these patients compared to those who eventually become IFEneg with a trend towards longer TTP owing to the prolonged half-life and therefore clearance of M protein. This supports the current strategy of assessing for MRD at the time of suspected complete response to reduce the chance of positive MRD tests and thus avoidance of multiple BM exams.