Combination of Flow Cytometry and Functional Imaging for Monitoring of Residual Disease in Myeloma

Abstract Introduction The iliac crest is the usual sampling site for minimal residual disease (MRD) monitoring in Multiple Myeloma (MM). However, the disease distribution in the bone marrow (BM) is often heterogeneous. Functional imaging can be used to complement MRD detection at a single site, thereby accounting for asymmetrically distributed disease. Diffusion weighted MRI with background suppression (DWIBS) is a novel functional imaging method that can detect disease in a higher proportion of newly diagnosed MM (NDMM) patients than 18F-fluorodeoxyglucose positron emission tomography (PET), as it is independent of the tumor metabolism. Yet, its performance for monitoring of residual disease has not been described. The aims of this study were 1) to compare DWIBS to PET for the detection of residual disease in patients achieving complete remission (CR), and 2) to test whether DWIBS and PET could complement MRD flow cytometry with a sensitivity of 1x10-5. To address these aims, we investigated 168 NDMM and 33 relapsed patients for whom DWIBS, PET, and MRD were available at the onset of CR during first-line and salvage therapy, respectively. Methods All patients signed written consent in accordance with the Declaration of Helsinki. Residual focal lesions (FLs) were defined as well delineated focal intensities above the surrounding BM background. For DWIBS FLs were considered if restriction could be confirmed on ADC maps. 8-color MRD flow cytometry with a limit of detection of 1x10-5 was available for 83 NDMM and all 33 salvage therapy patients. The Kaplan-Meier method was used for survival analyses. PFS time was measured from onset of CR to relapse or death from any cause or censored at the date of last contact. Paired-end whole exome sequencing of CD138-enriched MM cells was performed on an Illumina HiSeq 2500. Mutations were called from BWA aligned sequencing reads using MuTect. Subclonal reconstruction was done using SciClone. Results Compared to PET, DWIBS detected more CR patients with residual FLs (21% vs. 6%), and the concordance between PET and DWIBS was low. Only 6 of the DWIBS-positive patients also presented with FLs in PET. Yet, 5 patients had PET+/DWIBS- FLs, suggesting that the two techniques are complementary. Both, DWIBS+ and PET+ FLs negatively impacted PFS (p<0.05). For 83 patients MRD data were available. Combining MRD and imaging, residual disease was detectable in 53 patients (64%). The best outcome was seen for 30 double negative (MRD-/Imaging-) patients (3 events with a median follow-up of 3.6 years), the worst outcome was seen for 10 double positive (MRD+/Imaging+) patients (median PFS: 2.1 years). Only 4 of 86 patients were MRD-/Imaging+, indicating that residual FLs are rare in MRD-negative NDMM patients at a sensitivity of 1x10-5. A heterogeneous disease distribution is a common feature of late-stage patients. To test if this increased heterogeneity confounded MRD, we investigated a set of 33 heavily pretreated patients who achieved CR during salvage therapy. Combining MRD and imaging data, we detected residual disease in 25 patients (76%). Of note, the proportion of patients, who were MRD-negative but had residual FLs on functional imaging was significantly higher compared to NDMM (8/16 vs 4/34 patients, p=0.01). At the same time, 10 patients (30%) were MRD+ but Imaging-, supporting the idea that a combined MRD/Imaging approach can improve detection of residual disease and should be used in late-stage patients. To obtain insights in the underlying biology, we performed longitudinal multi-region sequencing of a subset of these CR patients. Our findings support the concept of persistence and progression of multiple spatially separated clones in the BM irrespective of being in an MRD-negative CR. Thereby, focal residual disease could be shown to contribute to relapse. Conclusion DWIBS is a promising tool for detection of residual disease and complements PET. The combination of MRD diagnostics and functional imaging improves prediction of outcome, with double-negativity and double positivity defining groups with excellent and dismal PFS, respectively. Prospective trials using this information to tailor therapy are warranted. From a biological perspective, this study highlights the confounding effects of spatial heterogeneity and limited dissemination of clones within the BM on MRD diagnostics. This may especially be true for patients achieving deep responses during salvage therapies. Disclosures Roy Choudhury: University of Arkansas for Medical Sciences: Employment, Research Funding. Epstein:University of Arkansas for Medical Sciences: Employment. Barlogie:International Workshop on Waldenström's Macroglobulinemia: Other: travel stipend; Millenium: Consultancy, Research Funding; Multiple Myeloma Research Foundation: Other: travel stipend; ComtecMed- World Congress on Controversies in Hematology: Other: travel stipend; Myeloma Health, LLC: Patents & Royalties: : Co-inventor of patents and patent applications related to use of GEP in cancer medicine licensed to Myeloma Health, LLC; European School of Haematology- International Conference on Multiple Myeloma: Other: travel stipend; Celgene: Consultancy, Research Funding; Dana Farber Cancer Institute: Other: travel stipend. Davies:Takeda: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Morgan:Celgene: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Research Funding.

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Real-World Experience with Minimal Residual Disease Testing with Next Generation Flow Cytometry and Functional Imaging in Multiple Myeloma

Blood ◽

10.1182/blood-2020-140772 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 17-18

Author(s):

David Böckle ◽

Paula Tabares Gaviria ◽

Xiang Zhou ◽

Janin Messerschmidt ◽

Lukas Scheller ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

High Risk ◽

Functional Imaging ◽

Real World ◽

Research Funding ◽

Residual Disease ◽

Focal Lesions ◽

High Risk Disease ◽

Minimal Residual

Background: Minimal residual disease (MRD) diagnostics in multiple myeloma (MM) are gaining increasing importance to determine response depth beyond complete remission (CR) since novel agents have shown to induce high rates of deep clinical responses. Moreover, recent reports indicated combining functional imaging with next generation flow cytometry (NGF) could be beneficial in predicting clinical outcome. This applies in particular to the subset of patients suffering from relapsed/refractory multiple myeloma (RRMM) who tend to show a higher incidence of residual focal lesions despite serological response. Here, we report our institutions experience with implementing both functional imaging and NGF-guided MRD diagnostics in clinical practice. Methods: Our study included patients with newly diagnosed multiple myeloma (NDMM) and RRMM achieving VGPR, CR or sCR. Bone marrow aspirates were obtained for MRD-testing according to IMWG 2016 criteria. Samples were collected between July 2019 and July 2020 and analyzed with NGF (according to EuroFlowTM guidelines) at a sensitivity level of 10-5. Results were compared to functional imaging obtained with positron emission tomography (PET) and diffusion-weighted magnetic resonance imaging (DW-MRI). High-risk disease was defined as presence of deletion 17p, translocation (14;16) or (4;14). Results: We included 66 patients with NDMM (n=39) and RRMM (n=27) who achieved VGPR or better. In patients with RRMM the median number of treatment lines was 2 (range 2-11). Fifteen patients suffered from high-risk disease. Median age at NGF diagnostics was 64 years (range 31-83). Among patients achieving VGPR (n=27), CR (n=10) and sCR (n=29) seventeen (26%) were MRD-negative by NGF testing. CR or better was significantly associated NGF MRD-negativity (p=0.04). Notably, rates of NGF MRD-negativity were similar among patients with NDMM (28%) and RRMM (26%). Even some heavily pretreated patients who underwent ≥ 4 lines of therapy achieved MRD-negativity on NGF (2 of 9). Functional imaging was performed in 46 (70%) patients with DW-MRI (n=22) and PET (n=26). Median time between NGF and imaging assessment was 2 days (range 0-147). Combining results from imaging and NGF, 12 out of 46 (26%) patients were MRD-negative with both methods (neg/neg). Three patients displayed disease activity as measured with both, imaging and NGF (pos/pos). Twenty-nine of the remaining patients were MRD-positive only according to NGF (pos/neg), while two patients were positive on imaging only (neg/pos). More patients demonstrated combined MRD-negativity on NGF and imaging (neg/neg) in the NDMM setting than in RRMM (32% versus 19%). We also observed that 30% of the patients with high-risk genetics showed MRD-negativity on both imaging and NGF. Of note, none of the patients with very advanced disease (≥4 previous lines) was MRD-negative on both techniques. Conclusion In the clinical routine, MRD diagnostics could be used to tailor maintenance and consolidation approaches for patients achieving deep responses by traditional IMWG criteria. Our real-world experience highlights that MRD-negativity can be achieved in patients suffering from high-risk disease and also in late treatment lines, supporting its value as endpoint for clinical trials. However, our data also support MRD diagnostics to be combined with functional imaging at least in the RRMM setting to rule out residual focal lesions. Future studies using MRD for clinical decision-making are highly warranted. Disclosures Einsele: Takeda: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; GlaxoSmithKline: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau. Rasche:Celgene/BMS: Honoraria; GlaxoSmithKline: Honoraria; Oncopeptides: Honoraria; Skyline Dx: Research Funding; Janssen: Honoraria; Sanofi: Honoraria.

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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 ◽

10.1182/blood-2020-137014 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 22-23

Author(s):

Hiroyuki Takamatsu ◽

Naoki Takezako ◽

Takeshi Yoroidaka ◽

Takeshi Yamashita ◽

Ryoichi Murata ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Research Funding ◽

Prognostic Value ◽

Residual Disease ◽

Multiparameter Flow Cytometry ◽

Next Generation ◽

Generation Sequencing ◽

Minimal Residual

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.

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Flow Cytometry Based Detection of MRD in Bone Marrow of Patients with Multiple Myeloma: A Comparison Between Fluorescent-Based Cytometry Versus Cytof

Blood ◽

10.1182/blood.v126.23.4195.4195 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4195-4195 ◽

Cited By ~ 1

Author(s):

Arnab Ghosh ◽

Nicole Carreau ◽

Alessandra Moscatello ◽

Adeeb Rahman ◽

Jingjing Qi ◽

...

Keyword(s):

Heavy Metal ◽

Multiple Myeloma ◽

Flow Cytometry ◽

Metal Ions ◽

Research Funding ◽

Heavy Metal Ions ◽

Residual Disease ◽

Response To Therapy ◽

Institutional Research ◽

Highly Sensitive

Abstract The introduction of novel agents for the treatment of multiple myeloma (MM) has shifted the emphasis towards achieving a molecular complete remission (CR). Traditional methods to detect malignant plasma cells (PC) in minimal residual disease (MRD) utilize multicolor flow cytometry (MFC) to detect aberrant phenotypes. However fluorescent-based MFC assays are limited by the number of markers, difficulty in standardization of assays and overlap of signal between the fluorescent channels. These limitations can be overcome by cyTOF, a flow cytometry assay based on time-of-flight mass spectrometry using antibodies labeled with heavy-metal ions, permitting simultaneous assessment of large panels of markers. We have developed a novel highly sensitive mass cytometry protocol for the detection of MRD. BM from 5 MM patients and 1 non-myeloma control were RBC-lysed and labeled with MM markers for MFC and cyTOF. cyTOF is a flow cytometry based on mass spectometry where antibodies are labeled with heavy-metal ions, permitting utilization of more markers without concern for spillover. The MFC panel included CD38, CD138, CD45, CD56, CD19, CD117. Labeled cells for MFC were acquired by BD LSR Fortessa.. The panel for cyTOF comprised markers for cells across the hematopoietic spectrum and those specific for MM. Labeled cells acquired with cyTOF were analyzed using SPADE. Limits of detection (LOD) and quantification (LOQ) for MFC are based on prior reports. The results were compared with an independent commercial MFC-based assay (Genoptix). Malignant PC could be detected by MFC and cyTOF based assasys (See Figure). We were able to detect MRD in three subjects that had no detectable disease by an independent commercial MFC based assay (Genoptix). This can be attributed to acquiring at least 1x106 for our assays leading to a lower LOD and LOQ (greater sensitivity). Using cyTOF and SPADE, we could detect MRD in 4/5 patients. Overall our panel with cyTOF has a lower LOD than MFC for detection of MRD. Early detection of MRD in MM patients using a highly sensitive flow cytometry like cyTOF will help in risk stratification, predicting relapse and studying response to therapy. Disclosures Chari: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium/Takeda: Consultancy, Research Funding; Onyx: Consultancy, Research Funding; Array Biopharma: Consultancy, Other: Institutional Research Funding, Research Funding; Biotest: Other: Institutional Research Funding; Novartis: Consultancy, Research Funding. Jagannath:Bristol Myers Squibb: Honoraria; Janssen: Honoraria; Merck: Honoraria; Novartis: Honoraria; Celgene: Honoraria.

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Retrospective Analysis of Minimal Residual Disease Testing By High Throughput Immunosequencing Versus High Sensitivity Flow Cytometry in Multiple Myeloma

Blood ◽

10.1182/blood-2021-154418 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1625-1625

Author(s):

Anwar Khan ◽

Nagehan Pakasticali ◽

Omar Fathalla ◽

Taiga Nishihori ◽

Mohammad O Hussaini

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Minimal Residual Disease ◽

Correlation Coefficient ◽

Research Funding ◽

Residual Disease ◽

High Sensitivity ◽

Spearman Correlation ◽

Color Flow ◽

Minimal Residual

Abstract Introduction: Detection of minimal residual disease (MRD) is one of the strongest predictors of outcome in multiple myeloma (MM). Until recently, the most commonly available method to detect MRD in clinical practice has been high sensitivity flow cytometry (FC) which can detect MRD with at 10 -5 sensitivity. In recent years, next-generation sequencing (NGS) has become a viable method to assess the MRD in MM patients with a 10 -6 sensitivity. NGS appears to have some advantages over HC-FC by circumventing subjectivity of analysis. However, real-world comparison between these two methodologies in the literature is limited and is important to inform daily hematopathology and oncology ordering practices. Methods: We retrospectively identified all cases of MM with NGS MRD data from bone marrow specimens at the Moffitt Cancer Center and collated corresponding flow MRD data and clinical data (OS, patient demographics) electronically and via chart review. 10-color flow cytometry was performed on a Gallios System and analyzed on Kaluza (Beckman Coulter, IN). Two million events were collected on all cells. Validated lower limit of detection was at least 0.01%. Antibodies included CD28, CD81, CD56, CD138, CD319, CD20, CD19, CD117, CD38, CD45, CD27, CD200 (BD, Biolegend, Beckman Coulter). clonoSEQ ® (Adaptive Biotechnologies, Seattle, WA) testing was performed which uses multiplex polymerase chain reaction (PCR) and NGS to identify, characterize, and monitor clonotypes of immunoglobulin (Ig) IgH (V-J), IgH (D-J), IgK, and IgL receptor gene sequences, and translocated BCL1/IgH (J) and BCL2/IgH (J) sequences Statistical analysis was performed by Spearman correlation coefficient and Kaplan-Meier analysis. Results: 192 samples from 122 unique patients were identified that had both NGS and FC data performed on the same sample. FC+ values ranged from 1x10 -7 to 0.39. NGS+ values ranged from 2.3 x 10 -7 to 0.15. Spearman correlation coefficient showed moderate concordance between NGS and FC at r=0.67 (p<0.001). Six samples were positive by FC (mean tumor burden (MTB)= 0.0007) but missed by NGS; whereas 59 samples were positive by NGS (MTB= 0.002) but missed by flow cytometry. Two cases by FC were equivocal and these were both definitively designated as MRD+ by NGS. Overall survival was worse for MRD+ (by NGS or FC) vs MRD(-) (Figure 1). Conclusion: Our study confirms the importance of MRD detection in MM and shows the robust utility of NGS for MRD detection in routine hematopathology practice. While both FC and NGS are complementary given that each can potentially detect MRD missed by another method, the data supports the increased sensitivity of NGS over FC. Figure 1 Figure 1. Disclosures Nishihori: Novartis: Research Funding; Karyopharm: Research Funding. Hussaini: Stemeline Therapeutics: Honoraria.

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Measurable Residual Disease in Extracellular Vesicles from Bone Marrow and Peripheral Blood of Patients with Multiple Myeloma: A Proof-of-Concept Study

Blood ◽

10.1182/blood-2021-149398 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4712-4712

Author(s):

Rui Bergantim ◽

Mélanie A.G. Barbosa ◽

Sara Peixoto da Silva ◽

Bárbara Polónia ◽

Hugo R. Caires ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Complete Remission ◽

Research Funding ◽

Residual Disease ◽

Disease Diagnosis ◽

Invasive Monitoring ◽

Protein Markers ◽

Measurable Residual Disease

Abstract BACKGROUND: Multiple myeloma (MM) treatment improved substantially in the last years, with unprecedented survival outcomes. However, even when achieving complete remission, patients ultimately relapse. Therefore, monitoring measurable residual disease (MRD) is crucial to assess treatment response and define the depth of patients' remission status. However, this currently still requires invasive bone marrow (BM) aspirates, which severely hinders real-time monitoring of the disease. Therefore, the identification of biomarkers of MRD in the peripheral blood (PB) of patients would allow a more frequent and minimally invasive monitoring of MRD. Extracellular Vesicles (EVs) are small particles (30-1000nm) shed by all cells, which are found in all biofluids including the BM and PB. These particles carry a specific cargo from their cell of origin, including proteins, enclosed by a lipidic layer. Therefore, they have been described as a possible source of cancer biomarkers, with potential to monitor MRD. AIMS: This study aimed to implement a protocol for the isolation of EVs from the BM and PB of MM patients at distinct stages of the disease (diagnosis and remission), in order to detect and compare the levels of known MRD biomarkers in their cargo. METHODS: The study was previously approved by the Ethical Committee of CHSJ and patient's consent was obtained. EVs from BM and PB Platelet-Poor Plasma (PPP) were isolated by size-exclusion chromatography (SEC), and further concentrated by ultrafiltration (UF). Then, the EVs were characterized according to their size and concentration (by Nanoparticle Tracking Analysis), morphology (by Transmission Electron Microscopy), protein concentration (Lowry protein assay) and presence of EV-associated protein markers (Western Blot - WB). In addition, 16 known MRD and MM biomarkers were analyzed by WB in the isolated EVs from PB and BM of seven patients, at two main stages of the disease - diagnosis versus response after autologous stem cell transplant (ASCT). Clinical features regarding cytogenetics and immunophenotypic markers using multi-parameter flow cytometry (MFC) were analyzed and compared. RESULTS: The two-step protocol described allowed the isolation of size-resolved EVs from both PB and BM of MM patients. The EVs isolated (both from PB and BM) presented a size-range from 50 to 500nm and presented EV-associated protein markers, such as CD81 and CD63. Moreover, several MM MRD biomarkers (e.g. CD56, CD45, CD38 and light chain) were detected in the cargo of the EVs from BM and PB at diagnosis and complete remission. The biomarkers of MM and MRD detected in the cargo of PB EVs were mainly the same as the ones detected in the cargo of BM EVs. The complete remission after ASCT was mostly associated with a decrease in the expression of EV-associated MM markers in both the BM and the PB; however, in some patients a few of the markers persisted at this stage when compared to diagnosis. In fact, the expression of CD45 and HLA-DR persisted at the remission stage in 3 and 2, respectively, out of 5 patients presenting these markers at diagnosis. Moreover, an increased expression of CD56 was also detected at remission in 3 out of 7 patients. By correlating these data with patient's routine work-up it was found that patients with persistent CD45 didn't reach 10^-5 MRD negative by flow cytometry. CONCLUSIONS: Taken together, this work suggests that it is possible to detect MM markers in EVs from either BM or PB of MM patients and compare their expression at different stages of the disease (diagnosis and remission after ASCT). Importantly, our results demonstrate the importance and potential of analyzing EVs cargo from PB, suggesting the possibility of using them for minimally invasive monitoring of MRD in MM patients. ACKNOWLEDGEMENTS: The authors acknowledge Celgene/BMS for providing funding to this work (Project Looker - Grant_138800). The authors acknowledge Cytogenetics Laboratory, Department of Clinical Hematology, Centro Hospitalar e Universitário São João and Flow Cytometry Laboratory, Department of Clinical Pathology, Centro Hospitalar e Universitário São João. Disclosures Bergantim: Amgen: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; BMS: Consultancy, Research Funding, Speakers Bureau; Takeda: Consultancy, Speakers Bureau. Barbosa: BMS: Research Funding. Silva: BMS: Research Funding. Polónia: BMS: Research Funding. Caires: BMS: Research Funding. Guimarães: BMS: Research Funding; Amgen: Research Funding. Vasconcelos: BMS: Research Funding; Amgen: Research Funding.

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Evolution of Multiparametric Flow Cytometry Testing for Minimal Residual Disease Assessment in Multiple Myeloma and Its Impact on Clinical Outcomes: A Single Institution Experience

Blood ◽

10.1182/blood.v128.22.2274.2274 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2274-2274 ◽

Cited By ~ 1

Author(s):

Nischala Ammannagari ◽

Paul K. Wallace ◽

Theresa Hahn ◽

Yali Zhang ◽

Christine M. Ho ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Board Of Directors ◽

Clinical Outcomes ◽

Research Funding ◽

Plasma Cells ◽

Residual Disease ◽

Advisory Committees ◽

Multiparametric Flow Cytometry ◽

Minimal Residual

Abstract Minimal residual disease (MRD) after autologous hematopoietic cell transplant (AHCT) in multiple myeloma (MM) has been shown to be an important predictor of clinical outcomes, suggesting that MRD negativity may be a new goal of therapy. Multiparametric flow cytometry (MFC) is a commonly used method for MRD assessment, however this technique is still evolving and efforts are underway to standardize this testing. The key factors which enable detection of residual malignant plasma cells by MFC remain an area of active investigation. We performed a retrospective review of 172 consecutive MM patients who received AHCT between 10/1/2007 and 5/31/2015 at our institution and had undergone MRD assessment by MFC at day +100 post-AHCT. Day +100 post-AHCT response was determined using the International Myeloma Working Group (IMWG) Uniform Response Criteria (URC) and was correlated with MRD assessment as well as progression free survival (PFS) and overall survival (OS). Data were collected on the specific MFC panel utilized, including the epitopes analyzed and the total plasma cell number (PCN) counted (normal and malignant PC). These variables were correlated with clinical outcomes including day +100 MM response, PFS and OS. Of 172 patients, 30 were MRD-positive, 133 MRD-negative, and 9 were equivocal at day +100 post-AHCT, the latter of which were excluded from further analyses. Day+100 MRD-negative status by MM response was: 31/37(84%) for VGPR, 35/41 (85%) for CR, and 42/42 (100%) for sCR. Patients who achieved a CR or sCR had improved PFS and OS rates compared with patients who achieved ≤VGPR: 3-year PFS: 61% (95% CI 49-74%) vs 46% (95% CI 32-59%), P=0.03; 3-year OS: 96% (95% CI 91-100%) vs 69% (95% CI 56-81%), P=0.005)). Patients with MRD-negative disease at day +100 post-AHCT had significantly superior PFS and OS compared to those with MRD-positive disease: 3-yr PFS 62% (95% CI 52-72%) vs 33% (95% CI 12-53%), P <0.0001) (Figure 1); 3-year OS 85% (95% CI 78-93%) vs 64% (95% CI 44-85%), P=0.004). There was no association between MRD status and age (<60 vs ≥60 years), sex, race (white vs other), performance status (KPS ≤80 vs ≥90), or subsequent transplant (P>0.1). The details of the four different MRD MFC panels are shown in Table 1. Panels C and D were compared, at a similar PCN level, but different epitopes tested, and found no significant difference in PFS or OS. Further analysis of PCN within the MRD-negative cohort revealed a trend towards improved 3-yr PFS rates with increasing numbers of PCN analyzed: 42% (95% CI 20-63%) for PCN<250,000, 68% (95% CI 52-83%) for PCN=250,000-500,000, 59% (95% CI 42-76%) for PCN >500,000-1,000,000 and 89% (78-100%) for PCN>1,000,000 (P=0.099) (Figure 2). The 3-yr OS rates for MRD-negative patients were higher for increasing PCNs analyzed, but the PCN categories were not statistically significantly different: 74% (95% CI 54-94%) for PCN<250,000, 88% (95% CI 77-99%) for PCN=250,000-500,000, 85% (95% CI 73-98%) for PCN >500,000-1,000,000 and 100% for PCN>1,000,000 (P=0.2). Sensitivity analysis revealed similar trends when a cut-off of above or below 500,000 or 1,000,000 was used. Our results confirm that achievement of MFC MRD negativity at day +100 post-AHCT is associated with improved PFS and OS. Factors such as the long-half lives of immunoglobulins, the quality of the bone marrow aspirate obtained, and the presence of occult extramedullary disease may account for the patients who were MRD negative but did not achieve a CR at day +100 post AHCT by IMWG URC. MRD assessment by MFC at our institution has evolved over time to include higher numbers of acquired and analyzed events. Notably, there was a trend towards improved outcomes with greater numbers of plasma cells analyzed, suggesting that continued development of MRD assessment by MFC should focus on increasing PCN analyzed in order to improve detection of residual MM clones. Disclosures Hahn: Novartis: Equity Ownership; NIH: Research Funding. McCarthy:The Binding Site: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gamida Cell: Honoraria, Membership on an entity's Board of Directors or advisory committees. Holstein:Millennium: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

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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 ◽

10.1182/blood-2018-99-111250 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 258-258 ◽

Cited By ~ 1

Author(s):

Hiroyuki Takamatsu ◽

Naoki Takezako ◽

Rachel K Wee ◽

Takeshi Yoroidaka ◽

Takeshi Yamashita ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Research Funding ◽

Prognostic Value ◽

Plasma Cells ◽

Residual Disease ◽

Multiparameter Flow Cytometry ◽

Next Generation ◽

Generation Sequencing ◽

Minimal Residual

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.

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MASS-FIX versus standard methods to predict for PFS and OS among multiple myeloma patients participating on the STAMINA trial.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.8009 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. 8009-8009

Author(s):

Angela Dispenzieri ◽

Amrita Y. Krishnan ◽

Bonnie Arendt ◽

Surendra Dasari ◽

Yvonne Adeduni Efebera ◽

...

Keyword(s):

Mass Spectrometry ◽

Multiple Myeloma ◽

Flow Cytometry ◽

Multiple Testing ◽

Residual Disease ◽

Progression Free Survival ◽

Complete Response ◽

Independent Effect ◽

Next Generation ◽

Different Response

8009 Background: Measuring response among patients with multiple myeloma is essential for the care of patients. Deeper responses have been associated with better progression free survival (PFS) and overall survival (OS). Serum (SIFE) and urine immunofixation are the currently used markers for biochemical documentation of CR after which marrow is tested for plasma cell clearance. Next generation flow cytometry and sequencing are used to document the presence of minimal residual disease (MRD). Mass spectrometry of blood by MALDI (Mass-Fix) is a new simple, inexpensive, sensitive, and specific means of detecting monoclonal immunoglobulins. To better test the hypothesis that Mass-Fix is superior to existing methodologies to predict for survival outcomes—especially SIFE-- samples from the STAMINA trial (NCT01109004), a trial comparing 3 transplant approaches among patients who have already received induction, were employed. Methods: Five-hundred and seventy-five patients were included. Samples from enrollment post-induction (post-I) and 1-year post enrollment (1YR) were tested when available. Four response parameters were assessed univariately: Mass-Fix, SIFE, complete response, and MRD by next generation flow cytometry. Mass spectrometry spectra were evaluated in a blinded fashion. Complete response was according to the 2006 International Myeloma Working Group criteria. Multivariate Cox proportional hazard models using stepwise regression were developed to explore the independent effect of the different response parameters on PFS and OS and interactions with other risk factors. Results: Of the 4 response measures, only MRD and Mass-Fix predicted for PFS and OS at multiple testing points on multivariate analyses (Table). Of the 4 post-I measurements, only MRD predicted for PFS; however, Mass-Fix was the only post-I measurement to predict for OS. Of all the 1-year measures, both 1YR Mass-Fix and 1YR MRD positivity predicted for inferior PFS and OS. In models including MRD and Mass-Fix, SIFE and CR were not prognostic for PFS or OS. Conclusions: Mass-Fix is a powerful means to track monoclonal proteins. The full utility of Mass-Fix was not exploited given the absence of a diagnostic sample and the fact that only serum (and not urine) was tested. Despite these limitations, it performed well at pre-induction and at 1 year. Mass-Fix provides a convenient and non-invasive means of predicting for myeloma outcomes. Clinical trial information: NCT01109004. [Table: see text]

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The role of CD44 in the assessment of minimal residual disease of multiple myeloma by flow cytometry

Journal of Hematopathology ◽

10.1007/s12308-021-00468-2 ◽

2021 ◽

Author(s):

Xiaojia Zuo ◽

Jinyi Feng ◽

Lilin Ye ◽

Miao Xing ◽

Zhikui Deng ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Minimal Residual Disease ◽

Residual Disease ◽

Minimal Residual

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Comparison of Minimal Residual Disease Detection between the Manual and Automated Gating Strategies of Euroflow Next-Generation Flow in Patients with Multiple Myeloma

Blood ◽

10.1182/blood-2019-122398 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3083-3083

Author(s):

Hiroyuki Takamatsu ◽

Takeshi Yoroidaka ◽

Takeshi Yamashita ◽

Ryoichi Murata ◽

Mikio Ueda ◽

...

Keyword(s):

Multiple Myeloma ◽

Partial Response ◽

Pharmaceutical Company ◽

Research Funding ◽

Plasma Cells ◽

Residual Disease ◽

Next Generation ◽

Chugai Pharmaceutical ◽

Company Limited ◽

Minimal Residual

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.

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