Glycomic Characterization of Platelets from Patients with Immune Thrombocytopenia

Abstract Introduction: Platelet glycoproteins are key contributors to platelet function but their glycans structure is unclear. Alterations in glycan composition have been reported to impact platelet clearance under physiological conditions and in the disease mechanism of immune thrombocytopenia (ITP). Therefore, this study sought to characterize glycan structures in human platelets from healthy control individuals and ITP patients using mass spectrometry (MS)-based glycomics approach, andto compare their glycomic profiles to facilitate understanding of glycan alterations in ITP. Methods: Glycan residues on platelet surface were determined by flow cytometry. Platelet lysates (1×10 8 platelets) from 4 healthy controls and from 4 ITP patients with a clear anomalous glycosylation pattern were characterized by MALDI-MS based glycomic approaches. N-linked glycans were released from platelet glycoproteins by PNGase F digestion and subsequently purified with a Sep-Pak C18 reverse phase cartridge. O-linked glycans were released by reductive elimination. Both pools of glycans were permethylated prior to MALDI-TOF MS to obtain an initial carbohydrate profile. Selected glycan molecular ion species were analyzed by MALDI-TOF-TOF MS/MS before and after digesting with exoglycosidases. Results: Glycans present in platelets from healthy controls and from ITP patients were largely consistent. The MS spectra for N-glycans showed a mixture of high mannose glycans (m/z 1579.8, 1783.9, 1988.0, 2192.1 and 2396.2); complex glycans (m/z 2966.5, 3776.9 and 4587.4); and bisected or truncated glycans (m/z 3211.6 and 4022.1). The spectra showed the presence of bi-, tri- and tetra-antennary complex glycans, which varied in their level of sialylation.Figure 1 shows the relative abundance ratio within eight families of core glycan structures. Platelets from ITP patients showed a consistent increase in desialylated structures such as Hex 5HexNAc 4Fuc 1. In addition to different amounts of attached sialic acid, varying levels of fucosylation were observed; ranging from the addition of a single core Fuc (m/z 2244.1), to the addition of up to three Fuc residues (m/z 3402.7). Collision-activated decomposition (CAD) MALDI-TOF/TOF analysis was performed to generate fragment ions from molecular ions detected in MALDI-TOF profiling for detailed sequencing of platelet N-glycans. This analysis suggested the presence of three isoforms: (i) sialylated tetra-antennary structure with core fucosylation and one Lewis x/a antenna; (ii) sialylated tetra-antennary structure with one Lewis y/b antenna; (ii) sialylated, core-fucosylated tri-antennary structure with one LacNAc extension and one Lewis x/a antenna. Sialidase S digestion was used to highlight the extent of desialylation in the presence of sialidase. Noteworthy, the ratio of non-sialylated bi-and tri-antennary N-glycans in ITP patients were higher than that in controls. This enzymatic digestion confirms the presence of α2,3-linked Neu5Ac on platelet glycans. Remaining sialylated structures may possess α2,6-linked Neu5Ac. O-glycan profiles obtained showed the predominance of core 1 and core 2 structures (Figure 2). The two most dominant glycan structures in core 1 were sialyl T antigen (GalNAc 1Gal 1NeuAc 1; m/z 895.5) and disialyl T antigen (GalNAc 1Gal 1NeuAc 2; m/z 1256.7), being the latter less abundant in ITP patients. The core 2 structure was modified by the addition of fucose and/or sialic acid residues (m/z 1157.7, 1344.8, 1518.9 and 1706.0). Conclusion: N- and O-glycan structures in human platelets were characterized by MALDI-TOF MS profiling to reveal interesting structural features including the presence of sialylLewis x/a epitope, Lewis x/a epitope, Lewis y/b epitope and LacNAc extensions in complex type N-glycans. Presence of terminal sialic acid and sialylLewis x/a on platelet N-glycan antenna also suggest their potentialfunction as ligands for siglecs that are associated with cell signaling functions. Siglec-1 and -2 have been suggested to have potential roles in ethiopathogenesis of autoimmune diseases. Desialylation observed in glycans of platelets from ITP patients, might trigger immune system activation in these patients. This research was funded by ISCIII-Fondos FEDER PI19/00772 and Platelet Disorder Support Association Figure 1 Figure 1. Disclosures Butta: Roche: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; CSL-Behring: Research Funding; Novo-Nordisk: Speakers Bureau. Canales: F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Speakers Bureau; Karyopharm: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Speakers Bureau; iQone: Honoraria; Sandoz: Honoraria, Speakers Bureau; Incyte: Consultancy; Janssen: Consultancy, Honoraria, Speakers Bureau; Gilead/Kite: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Sanofi: Consultancy; Eusa Pharma: Consultancy, Honoraria; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria. Jiménez-Yuste: Pfizer: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; BioMarin: Consultancy; Sobi: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding. Alvarez Román: Octapharma: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding; Novo-Nordisk: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding.

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Anti-Glycoprotein V Autoantibodies in Patients with Immune Thrombocytopenia: Regularly Detectable and Functionally Relevant

Blood ◽

10.1182/blood-2018-99-118090 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1141-1141

Author(s):

Richard Vollenberg ◽

Rabie Jouni ◽

Peter A. A. Norris ◽

Monika Burg-Roderfeld ◽

Nina Cooper ◽

...

Keyword(s):

Monoclonal Antibody ◽

Board Of Directors ◽

Disease Severity ◽

Research Funding ◽

Immune Thrombocytopenia ◽

Human Platelets ◽

High Avidity ◽

Advisory Committees ◽

Tail Vein ◽

Positive Direct

Abstract Immune thrombocytopenia (ITP) results from autoimmunization against platelet antigens. Autoantibodies (aabs) are considered to represent a major mechanism of thrombocytopenia in ITP by inducing platelet clearance from the circulation. Several lines of evidence demonstrate that aabs against glycoproteins (GP) IIb/IIIa and Ib/IX are predominant in ITP patients. Both disease severity and treatment response rates to specific therapeutics have been associated with aabs patterns. GP V is a well characterized immune target in Varicella-associated and drug-induced thrombocytopenia, but has never been studied systematically in ITP. In this study, patients with a suspected diagnosis of primary ITP were included once they met pre-defined clinical inclusion criteria. The presence of GP IIb/IIIa-, GP Ib/IX, and GP V-specific aabs was investigated by monoclonal antibody immobilization of platelet antigens (MAIPA) assay, both on patients' autologous platelets (direct MAIPA) and in serum (indirect MAIPA). In addition, serum IgG fractions were prepared from all patients with a positive direct MAIPA. IgG fractions were tested by surface plasmon resonance (SPR) technology for the presence of anti-GP V aabs. Complete data sets were obtained from 1,140 qualified patients. Platelet-bound aabs were detected in 343/1,140 patients (30.1%). Of these, 222 (64.7%) had platelet-bound anti-GP V aabs, either alone (10/222), or together with other specificities (211/222). Free anti-GP V aabs were detected in 30/222 patients by indirect MAIPA, but in 88/222 by SPR. The avidity of aabs detected by both methods (n=29; R700/R350=0.73±0.14) was significantly higher than the avidity for aabs detected by SPR only (n=59; R700/R350=0.32±0.13, p < 0.001). In order to study the potential biological relevance of anti-GP V, a phagocytosis assay using CD14+ positively-selected macrophages from spleen specimens from splenectomized ITP patients was performed. Anti-GPV aabs induced a modest amount of platelet uptake above the normal human serum-incubated control. No difference was observed between high avidity and low avidity anti-GP V. The effect of anti-GPV on platelet clearance was further studied in a NOD/SCID mouse model. Freshly isolated human platelets were injected into the lateral mouse tail vein; after 30 min, IgG fractions isolated from human sera containing anti-GPV antibodies or control sera from healthy donors were injected into the other lateral tail vein, and the survival of human platelets in the mouse circulation was analyzed by taking murine blood 60, 120, 300 min and 24h after baseline. High avidity and low avidity anti-GP V aabs (n=3 per group) eliminated human platelets with no detectable difference between the groups (mean platelet survival at t=300 min: 40% [range 27-55] versus 35% [range, 16-46]). A comparable, dose-dependent platelet clearance was also obtained with monoclonal antibody SW16 against GP V. In summary, we have demonstrated that anti-GP V autoantibodies are regularly detectable in ITP patients; and that they are able to induce phagocytosis and platelet clearance. Our findings have implications for both, further development of laboratory testing, and guidance for clinical decision making. First, comparison between MAIPA and SPR reveals that free aabs may be more frequent than reported, since aabs appear to escape detection by standard laboratory methods because of low avidity. Better test methods are required. Second, predicting disease severity and/or tailoring ITP therapy can possibly not be restricted to the postulated difference between anti-GP IIb/IIIa and anti-GP Ib/IX. Prospective studies are required to understand the impact of different GP-specific platelet aabs on the clinical course of ITP including, anti-GP V. Disclosures Rummel: Celgene: Honoraria; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria; Mundipharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Symbio: Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Eisai: Honoraria. Bakchoul:Aspen Germany gGmbH, CLS Behring, Stago gGmbH: Honoraria; German Research Society (DFG): Research Funding; Robert Bosch gGmbH: Research Funding.

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Comparison of MALDI-TOF Mass Spectrometry Analysis of Peripheral Blood and Bone Marrow Based Flow Cytometry for Tracking Measurable Residual Disease (MRD) in Patients with Multiple Myeloma

Blood ◽

10.1182/blood-2019-123838 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3060-3060

Author(s):

Marion Eveillard ◽

Even H Rustad ◽

Mikhail Roshal ◽

Yanming Zhang ◽

Amanda Kathryn Ciardiello ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Research Funding ◽

M Protein ◽

Advisory Committees ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Active Disease ◽

Tof Ms

Introduction In multiple myeloma (MM), the absence of measurable residual disease (MRD) after completed therapy is associated with longer progression free survival. Different techniques are available to detect low levels of plasma cells in bone marrow (BM) either by flow cytometry or by next-generation sequencing as a gold standard of molecular methods. But these techniques are limited because they require a representative bone marrow sample obtained by an invasive procedure. Therefore, detecting low levels of disease in blood would be ideal, because serial sampling is much easier and fully representative, and it would allow for the detection of extramedullary disease. Mass spectrometry-based methods have been shown to be more sensitive for detecting monoclonal proteins (M-protein) in serum. In this study, we were motivated to evaluate MALDI-TOF mass spectrometry (MALDI-TOF MS) head-to-head with an established BM-based MRD assays. Patients and Methods This cohort included 71 patients treated at Memorial Sloan Kettering Cancer Center (MSKCC) who had serum samples available at 2 timepoints including during active disease and within 60 days of MRD results as determined by flow cytometry of bone marrow aspirates (Flow-BM-MRD). The cohort enrolled 26 females and 45 males with a median age of 61 years (range 37-78 years). Twenty-seven patients had high-risk cytogenetics at baseline. The median time between diagnosis and the MRD timepoint was 13.4 months (3.4-91 months). MALDI-TOF MS analysis was performed according to the method published by Mills et al. Immunoglobulins were purified from serum samples using CaptureSelect beads specific of each isotype and were then eluted from the beads. Light chains and heavy chains were separated by the addition of a reducing agent. Purified samples were mixed in matrix and spotted onto a stainless steel MALDI plate and were analyzed using a Microflex LT MALDI-TOF mass spectrometer (Bruker). Samples taken during active disease were used to identify the mass to charge ratio (m/z) of the M-protein and served as a surrogate marker in the analysis of subsequent samples. MALDI-TOF MS results were compared to the Flow-BM-MRD assay, performed using the MSKCC's ten-color, single-tube method. Results MALDI-TOF MS detected an M-protein in all 71 active disease samples and in 25 MRD samples. MALDI-TOF-MS results at the MRD timepoint were concordant with Flow-BM-MRD for 44/71 patients (p=0.342, chi-square test). Eight patients were positive and 36 negative by both techniques. Twenty-seven patients were discordant, including 10 patients detectable only by Flow-BM-MRD and 17 detectable only by MALDI-TOF MS. Among the 10 patients detectable by flow cytometry but not by MALDI, the median MRD level was 0.00092% (+<0.0001% - 0.011%). The M-protein could have been present but below the polyclonal background. Regarding the 17 patients positive only by MALDI-TOF-MS, the BM sample for flow analysis was not suitable for 3 patients due to hemodilution. The others 14 samples reached the target of sensitivity with a limit of detection of 0.0001%. Alternatively, the MALDI-TOF result could be a false positive in terms of disease detection. MS is likely not falsely detecting M-proteins and indeed, immunofixation was also positive in 11/17 of these samples. However, low levels of M-protein may not indicate the presence of active disease. Indeed, a confounding factor is that immunoglobulins have a long half-life in serum. To determine the clinical utility of more sensitive M-protein detection, we evaluated the clinical outcome for the 48 newly diagnosed MM patients in CR at the MRD timepoint with a median follow-up of 11 months. Of these 48 patients, 2 of the 3 that were positive by both techniques relapsed during follow-up. One out of 27 patients that were negative by both techniques relapsed. None of the 10 patients who were positive only by MALDI-TOF relapsed and 1 of the 8 patients who were positive only by Flow-BM-MRD relapsed. Conclusions This study is important because it is a first step in understanding how to use a more sensitive blood test for the follow-up of MM patients. MALDI-TOF MS analysis may provide complementary results to Flow-BM-MRD especially for the follow-up of patients in CR and during maintenance therapy to detect poor responders that would be positive by both techniques. In summary, our results suggest that MALDI-TOF may be quite useful for early detection of relapse. Disclosures Roshal: Physicians' Education Resource: Other: Provision of services; Celgene: Other: Provision of Services; Auron Therapeutics: Equity Ownership, Other: Provision of services. Hassoun:Celgene: Research Funding; Janssen: Research Funding; Novartis: Consultancy. Smith:Celgene: Consultancy, Patents & Royalties, Research Funding; Fate Therapeutics and Precision Biosciences: Consultancy. Lesokhin:Takeda: Consultancy, Honoraria; Serametrix Inc.: Patents & Royalties; Genentech: Research Funding; GenMab: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding; Janssen: Research Funding; Juno: Consultancy, Honoraria. Mailankody:Juno: Research Funding; Celgene: Research Funding; Janssen: Research Funding; Takeda Oncology: Research Funding; CME activity by Physician Education Resource: Honoraria. Landgren:Abbvie: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Theradex: Other: IDMC; Adaptive: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Other: IDMC; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Mass Spectrometry to Measure Response in Immunoglobulin Light Chain Amyloidosis (AL)

Blood ◽

10.1182/blood-2018-99-119969 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4502-4502

Author(s):

Angela Dispenzieri ◽

Surendra Dasari ◽

Bonnie Kaye Arendt ◽

Mindy Kohlhagen Kohlhagen ◽

Taxiarchis Kourelis ◽

...

Keyword(s):

Board Of Directors ◽

Light Chain ◽

Research Funding ◽

Complete Response ◽

Serum Samples ◽

Advisory Committees ◽

Monoclonal Protein ◽

Maldi Tof ◽

Monoclonal Proteins ◽

Tof Ms

Abstract INTRODUCTION: Our group has demonstrated that MASS-FIX is a quick, inexpensive, and accurate means to diagnose and monitor the serum and urine of patients with plasma cell disorders. Screening can be done with a MALDI-TOF MS and samples reflexed to microflow liquid chromatography coupled with electrospray ionization (ESI) and Q-TOF MS (microLC-ESI-Q-TOF MS). Because this technique provides a mass/charge (m/z) for a given patient's monoclonal protein, this method can provide greater sensitivity and specificity to monitor for complete response (CR), especially in patients receiving therapeutic monoclonal antibodies. Our goal was to assess the performance of miRAMM in patients with AL who have been classified as complete response using conventional means. METHODS: We identified 77 patients with AL who had both documented CR by immunofixation (Seibia) of the serum (SIFE), urine IFE (UIFE), and serum free light chain (FLC; The Binding Site) and paired serum samples to test by MALDI-TOF and ESI-TOF. No urine samples were available to test. Paired serum samples from baseline and approximately one year post-therapy were immunoaffinity purified using nanobodies targeting kappa, lambda, alpha, gamma and mu as previously described. For the MALDI-TOF (Bruker Microflex, LT), a range of 9,000 to 32,000 m/z was acquired. The m/z distribution was then visually inspected for the presence of a peak that was distinct from the polyclonal background in both the M+1 and M+2 light chain mass ranges. For the ESI-TOF, spectra were also collected on an TripleTOF 5600 quadrupole time-of-flight mass spectrometer (ABSciex, Vaughan ON, CA) in ESI positive mode with a Turbo V dual ion source with an automated calibrant delivery system. TOF MS scans were acquired from m/z 600−2500 with an acquisition time of 100 ms. RESULTS: Median age of the cohort was 58 (range 42, 81). Fifty-eight percent were male. No test was 100% sensitive at baseline with positive results as follows: abnormal FLC ratio, 82%; positive SIFE, 70%; 73% positive UIFE; positive serum MASS-FIX, 71%; and ESI-TOF, 79% . There was light chain isotype agreement in all cases, except for 2 patients for whom the SIFE and the FLCr disagreed. Of the 56 patients with baseline positive MASS-FIX, there was evidence of the original monoclonal protein in 5 patients (see figure for example of spectra illustrating same m/z before and after therapy). Of the 63 patients who had baseline positive ESI-TOF, there was evidence of the original monoclonal protein in 8 patients. Small unrelated monoclonal proteins were seen both by SIFE and by mass spectrometry techniques. With the spectrometry techniques, however, these transient oligoclonal bands could be distinguished from the original monoclonal protein when they shared the same isotype based on the difference in m/z. DISCUSSION: At baseline, the MALDI was able to identify the baseline monoclonal protein in a comparable number of patients as SIFE, UIFE, and FLC. Approximately 10% of patients thought to be in CR using routine screening tests were found to have persistence of their original clone by MALDI and by ESI-TOF. Small post-therapy unrelated monoclonal proteins were also seen both by IFE and by MALDI, but either the presence of a different isotype or in the case of MALDI, a different m/z, made it clear that the monoclonal protein was not related to the original clone. The sensitivity of the assay will improve significantly when we formalize the introduction of free light chain magnetic beads to capture the serum FLCs. Routine use of MASS-FIX of the urine will also increase performance characteristics. Figure. Figure. Disclosures Dispenzieri: Celgene, Takeda, Prothena, Jannsen, Pfizer, Alnylam, GSK: Research Funding. Gertz:Prothena: Honoraria; celgene: Consultancy; spectrum: Consultancy, Honoraria; Teva: Consultancy; Physicians Education Resource: Consultancy; annexon: Consultancy; Apellis: Consultancy; Alnylam: Honoraria; Medscape: Consultancy; Amgen: Consultancy; janssen: Consultancy; Research to Practice: Consultancy; Abbvie: Consultancy; Ionis: Honoraria. Kumar:Novartis: Research Funding; KITE: 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; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: 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; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Dingli:Alexion Pharmaceuticals, Inc.: Other: Participates in the International PNH Registry (for Mayo Clinic, Rochester) for Alexion Pharmaceuticals, Inc.; Alexion Pharmaceuticals, Inc.: Other: Participates in the International PNH Registry (for Mayo Clinic, Rochester) for Alexion Pharmaceuticals, Inc.; Millennium Takeda: Research Funding; Millennium Takeda: Research Funding. Kapoor:Takeda: Research Funding; Celgene: Research Funding. Russell:Vyriad: Equity Ownership.

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Sialic Acid Derivatization for the Rapid Subclass- and Sialic Acid Linkage-Specific MALDI-TOF-MS Analysis of IgG Fc-Glycopeptides

Methods in Molecular Biology - High-Throughput Glycomics and Glycoproteomics ◽

10.1007/978-1-4939-6493-2_5 ◽

2016 ◽

pp. 49-62 ◽

Cited By ~ 1

Author(s):

Noortje de Haan ◽

Karli R. Reiding ◽

Manfred Wuhrer

Keyword(s):

Sialic Acid ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Ms Analysis ◽

Sialic Acid Linkage ◽

Tof Ms

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MALDI-TOF Mass Spectrometry in Serum for the Follow-up of Newly Diagnosed Multiple Myeloma Patients Treated with Daratumumab-Based Combination Therapy

Blood ◽

10.1182/blood-2019-128938 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4377-4377

Author(s):

Marion Eveillard ◽

Malin Hultcrantz ◽

Alexander M. Lesokhin ◽

Sham Mailankody ◽

Eric L Smith ◽

...

Keyword(s):

Mass Spectrometry ◽

Board Of Directors ◽

Research Funding ◽

M Protein ◽

Light Chains ◽

Advisory Committees ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Tof Ms

Introduction Mass spectrometry-based methods have been shown to be more sensitive for detecting monoclonal proteins (M-protein) in serum compared to current electrophoretic techniques, serum protein electrophoresis (SPEP) and immunofixation (IFE). In particular, MALDI-TOF mass spectrometry (MALDI-TOF MS) may soon replace these techniques for the routine monitoring of multiple myeloma (MM) patients due to its relatively low cost and high throughput. In this study, we evaluate the performance of MALDI-TOF MS in the follow up of newly diagnosed multiple myeloma (NDMM) patients treated with a daratumumab-based combination therapy. We report our findings compared to SPEP and IFE results and discuss the advantages and disadvantages of the technique in the serial analysis of patients. Patients and Methods Twenty-seven NDMM patients treated with daratumumab-based combination therapy were included in this study; median age 57 years (range 33-79 years) and 52% were males. Each patient had 10 time points of follow-up: baseline, day 15 of cycle 1, the first day of each cycle from cycle 2 to cycle 8, and at the end of treatment (EOT). All samples were analyzed in a blinded fashion by MALDI-TOF MS. First, immunoglobulins were purified from serum using magnetic beads specific for IgG and IgA heavy chains or kappa and lambda light chains. Immunoglobulins were eluted from the beads and the light chains and heavy chains were separated by adding a reducing agent. Purified samples were analyzed using a Microflex LT MALDI-TOF mass spectrometer (Bruker). Samples taken at baseline were used to identify the mass to charge ratio (m/z) of the M-protein which served as a surrogate marker in the analysis of subsequent samples. MALDI-TOF MS results were compared to SPEP, IFE and the kappa/lambda free light chain (κ/λ) ratio. Results At baseline, IFE and MALDI-TOF MS were positive for all 27 patients while SPEP was negative for M-protein in 2 patients. Different M-protein isotypes were observed including 3 free kappa, 1 free lambda, 15 IgG kappa, 3 IgG Lambda, 3 IgA kappa and 2 IgA lambda. The κ/λ ratio was abnormal for 26/27 patients. Twenty-three patients completed the 8 cycles of treatment. During the follow-up, 14 of the 23 patients remained positive until the EOT by MALDI-TOF MS. Regarding these patients, 3 were negative by SPEP and IFE at the EOT. Nine of the 23 patients became negative by MALDI-TOF MS in a median time of 5 cycles (range 2- 8). Among these 9 patients, 1 reached a complete response (CR) and 6 reached stringent CR in a median time of 3 cycles (range cycle 2 - EOT). The 2 patients that did not reach CR but were negative by MALDI are suspected to have a false positive IFE result. These patients' IgG kappa M-protein overlaps with daratumumab on IFE and the Hydrashift assay (Sebia) was unavailable at the time of analysis. In these cases, MALDI provided better specificity compared to IFE as the M-protein could be distinguished from daratumumab based on m/z. However, daratumumab could not always be distinguished from the M-protein at some timepoints for some patients. The patient that still had an abnormal κ/λ ratio but was negative by MALDI had κ light chain MM. MALDI-TOF MS may be less sensitive for the detection of free light chains in serum. We observed differences between the M-spike intensity of the heavy- and light-chain specific purifications especially when the M-protein was at low levels. This may be due to differences in the polyclonal background for each purification reaction and will affect the sensitivity of M-protein detection. Conclusions This study is important because it helps to understand the performance of MALDI-TOF MS in the follow-up of MM patients under therapy. The use of serial samples allowed us to characterize patterns of immune markers longitudinally in relation to given therapy. The m/z ratio at baseline is a key for the interpretation during the follow-up and to avoid interference with other monoclonal immunoglobulins, like daratumumab, for example. When more than one monoclonal immunoglobulin is present, their relative concentration, not just their m/z values, is important for distinguishing two different peaks. MALDI-TOF MS is useful for monitoring patients under therapy because it provides higher specificity and sensitivity than electrophoretic methods. This may be especially important in clinical trials and in accurately defining CR and sCR. Disclosures Lesokhin: BMS: Consultancy, Honoraria, Research Funding; GenMab: Consultancy, Honoraria; Juno: Consultancy, Honoraria; Genentech: Research Funding; Janssen: Research Funding; Serametrix Inc.: Patents & Royalties; Takeda: Consultancy, Honoraria. Mailankody:Takeda Oncology: Research Funding; CME activity by Physician Education Resource: Honoraria; Juno: Research Funding; Celgene: Research Funding; Janssen: Research Funding. Smith:Celgene: Consultancy, Patents & Royalties, Research Funding; Fate Therapeutics and Precision Biosciences: Consultancy. Hassoun:Janssen: Research Funding; Novartis: Consultancy; Celgene: Research Funding. Landgren:Abbvie: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Adaptive: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: 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; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Theradex: Other: IDMC; Merck: Other: IDMC; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Autoantibody-mediated desialylation impairs human thrombopoiesis and platelet lifespan

Haematologica ◽

10.3324/haematol.2019.236117 ◽

2019 ◽

Vol 106 (1) ◽

pp. 196-207 ◽

Cited By ~ 5

Author(s):

Irene Marini ◽

Jan Zlamal ◽

Christoph Faul ◽

Ursula Holzer ◽

Stefanie Hammer ◽

...

Keyword(s):

Sialic Acid ◽

Immune Thrombocytopenia ◽

Platelet Adhesion ◽

Ex Vivo ◽

Human Platelets ◽

Matrix Proteins ◽

Ex Vivo Model ◽

Effector Functions ◽

Clinical Cohort ◽

Significant Impairment

Immune thrombocytopenia is a common bleeding disease caused by autoantibody-mediated accelerated platelet clearance and impaired thrombopoiesis. Accumulating evidence suggests that desialylation affects platelet life span in immune thrombocytopenia. Herein, we report on novel effector functions of autoantibodies from immune thrombocytopenic patients which might interfere with the clinical picture of the disease. Data from our study show that a subgroup of autoantibodies is able to induce cleave of sialic acid residues from the surface of human platelets and megakaryocytes. Moreover, autoantibody-mediated desialylation interferes with the interaction between cells and extracellular matrix proteins leading to impaired platelet adhesion and megakaryocyte differentiation. Using a combination of ex vivo model of thrombopoiesis, a humanized animal model, and a clinical cohort study, we demonstrate that cleavage of sialic acid induces significant impairment in production, survival as well as function of human platelets. These data may indicate that prevention of desialylation should be investigated in the future in clinical studies as a potential therapeutic approach to treat bleeding in immune thrombocytopenia.

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Linkage-Specific Sialic Acid Derivatization for MALDI-TOF-MS Profiling of IgG Glycopeptides

Analytical Chemistry ◽

10.1021/acs.analchem.5b02426 ◽

2015 ◽

Vol 87 (16) ◽

pp. 8284-8291 ◽

Cited By ~ 70

Author(s):

Noortje de Haan ◽

Karli R. Reiding ◽

Markus Haberger ◽

Dietmar Reusch ◽

David Falck ◽

...

Keyword(s):

Sialic Acid ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Tof Ms

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Evaluation of Platelet Function Defects in Patients with Immune Thrombocytopenia

Blood ◽

10.1182/blood-2021-153425 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1021-1021

Author(s):

Elena Monzón Manzano ◽

María Teresa Alvarez Román ◽

Andres Ramirez Lopez ◽

Elena G Arias-Salgado ◽

Paula Acuña ◽

...

Keyword(s):

Flow Cytometry ◽

Sialic Acid ◽

Platelet Function ◽

Research Funding ◽

Immune Thrombocytopenia ◽

Healthy Controls ◽

Activation Markers ◽

Platelet Production ◽

Neuraminidase Activity ◽

Fibrinogen Receptor

Abstract Background: Primary immune thrombocytopenia (ITP) is a megakaryocytic (MK)/platelet-specific autoimmune disorder characterized by platelet count <100×10 9/L with or without bleeding manifestations, and diagnosed by exclusion of other causes of thrombocytopenia. It is widely accepted the involvement of platelet autoantibodies on deterioration of platelets from patients with ITP. Moreover, an enhanced activity of neuraminidase may also reduce sialic acid from glycoside residues on platelet surface, especially from the highly glycosylated von Willebrand factor (vWF) receptor. Because controversial results regarding the functionality of platelets from ITP patients can be found in literature, we aimed to determine platelet ability to be stimulated by agonists. Moreover, we aimed to determine the way anti-platelet auto- antibodies (abs) and neuraminidase activity may affect the function of platelets derived from MKs of healthy controls. Methods: This observational, prospective and transversal study included 42 patients with chronic primary ITP and 55 healthy controls. Platelet fibrinogen and vWF receptors and activation markers (PAC1 binding to activated fibrinogen receptor and exposure of P-selectin after agonists treatment), were evaluated by flow cytometry. Presence of Antibodies (abs) against platelet's glycoproteins in ITP serum was analysed with a Luminex based assay (LifecodesPak Lx). Neuraminidase (NEU) activity in serum was determined with the substrate 20-(4-methylumbelliferyl)-a-D-N-(MUNANA). Human CD34 + cell-enriched population was obtained with CliniMACS (MiltenyiBiotec) from G-CSF mobilized peripheral blood of a healthy donor. For MK differentiation, CD34 + cells were cultured 12 days in StemSpan™ Serum-Free Expansion Medium II (SFEM II) with 50ng/ml of recombinant human thrompoietin. Then, 10% of serum from healthy controls (4) or ITP patients (4) were added to the culture of mature MKs and incubated for 3 days. Phenotypic analysis of MKs and culture derived-platelets was carried out using abs against CD34, CD41, CD42a and CD42b.Platelet-like particles were considered as CD41-positive events with a size (FSC) and granularity (SSC) scatter properties similar to blood platelets. Culture-derived platelets were stimulated with 100 µM TRAP and 10 µM ADP and activation markers were analyzed by flow cytometry. Results: Expression of fibrinogen receptor on platelets from ITP patients were similar to those from healthy controls but showed a reduced capacity to be activated. Impairment in platelet degranulation measured as exposition of P-selectin after agonist's stimulation was also observed in platelets from these patients (Figure 1). Of note, surface content of CD42b subunit of vWF receptor was reduced (Figure 1). To determine whether diminished platelet function might be due to a plasma component, we induced platelet production from MK of healthy controls as referred in Methods. Abs against platelets and neuraminidase activity were determined in serum samples. Serum from 4 healthy controls or from 4 ITP patients (1 with anti-CD42b, 1 with anti-GPIa-IIa and 2 with undetectable abs) were added to MKs culture. No differences existed in MK differentiation and platelet production between MKs incubated with serum from healthy controls or from ITP patients, but similarly as observed in platelets from ITP patients, MK-derived platelets had an impaired ability to be activated (Table 1). Platelets derived from MKs incubated with ITP serum with anti-platelet abs had also a diminished exposure of CD42b (73±8% of controls). Moreover, neuraminidase content of these samples was slightly higher than that from ITP samples without abs (130 vs 100 % of controls). Conclusion: Platelets from ITP patients had a diminished ability to be stimulated. In vitro study showed that megakaryopoiesis was normal in presence of ITP serum, but released platelets had a lower ability to be activated. Involvement of abs in this effect cannot be ruled out despite we detected abs only in 2 of the tested sera because efficiency of method to detect these abs is ~ 50%. On the other hand, reduced levels of CD42b might be due to the increased activity of neuraminidase. Reduction of sialic acid from CD42b might initiate its metalloproteinase-mediated cleavage or change affinity of the ab used for its detection. Research funded by ISCIII-Fondos FEDER PI19/00772 and Platelet Disorder Support Association Figure 1 Figure 1. Disclosures Alvarez Román: Pfizer: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Novo-Nordisk: Consultancy, Honoraria, Research Funding. García Barcenilla: Roche: Speakers Bureau; Takeda: Speakers Bureau; Bayer: Speakers Bureau; SOBI: Speakers Bureau. Canales: Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria; Gilead/Kite: Consultancy, Honoraria; Eusa Pharma: Consultancy, Honoraria; Incyte: Consultancy; Karyopharm: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Sanofi: Consultancy; iQone: Honoraria; Sandoz: Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau. Jiménez-Yuste: Grifols: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; BioMarin: Consultancy; Sobi: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding. Butta: Novo-Nordisk: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Roche: Speakers Bureau; CSL-Behring: Research Funding.

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