Detection of M-Protein in Acetonitrile Precipitates of Serum using MALDI-TOF Mass Spectrometry

AbstractPurpose of the researchMultiple myeloma and plasmacytomas belong to a group of disorders, namely plasma cell dyscrasias and are identified by the presence of a monoclonal protein (M-protein). MALDI-TOF-mass spectrometry (MS) has demonstrated superior analytical sensitivity for the detection of M-protein and is now used for screening of M-protein at some centres. We present the results of an alternative methodology for M-protein analysis by MALDI-TOF MS.MethodsSerum samples from patients with newly diagnosed multiple myeloma or plasmacytoma with positive M-protein detected by serum protein electrophoresis, immunofixation electrophoresis and serum free light chain analysis, underwent direct reagent-based extraction process using Acetonitrile (ACN) precipitation. Serum κ and λ light chains were validated using immunoenrichment by anti- κ and anti- λ biotin-labelled antibodies immobilised on streptavidin magnetic beads. MALDI-TOF MS measurements were obtained for intact proteins using alpha-cyano-4-hydroxycinnamic acid as matrix. The images obtained were overlaid on apparently healthy donor serum samples to confirm the presence of M-protein.Principle resultsCharacteristic M-protein peaks were observed in the ACN precipitates of serum in the predicted mass ranges for κ and λ. The κ and λ peaks were confirmed by immunoenrichment analysis. Twenty-seven patient samples with either newly diagnosed multiple myeloma or plasmacytoma with monoclonal gammopathy detected by the standard methods were chosen for Acetonitrile precipitation and analysed by MALDI-TOF MS. All 27 patient samples demonstrated a peak suggestive of M-protein with mass/charge (m/z) falling within the κ and λ range. The concordance rate with serum immunofixation electrophoresis and free light chain analysis was above 90%.Major conclusionsWe report the results of a low-cost reagent-based extraction process using Acetonitrile precipitation to enrich for κ and λ light chains, which can be used for the screening and qualitative analysis of M-protein.

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Detection of M-Protein in Acetonitrile Precipitates of Serum Using MALDI-TOF Mass Spectrometry: A Novel Methodology

Blood ◽

10.1182/blood-2020-141301 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 36-37

Author(s):

Nikita Mehra ◽

Gopal Gopisetty ◽

Jayavelu S ◽

Arivazhagan Rajamanickam ◽

Shirley Sundersingh ◽

...

Keyword(s):

Multiple Myeloma ◽

Mass Spectra ◽

The Other ◽

M Protein ◽

Light Chains ◽

Serum Samples ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Tof Ms ◽

Apparently Healthy

Background: Multiple myeloma (MM) and plasmacytoma(s) belong to a group of clonal plasma cell dyscrasias. In 97-98% of all cases, they are characterised by the detection of a monoclonal protein (M-protein) in the blood, and sometimes in the urine. MALDI-TOF-mass spectrometry (MS) has demonstrated excellent analytical sensitivity for the screening and detection of M-protein. We present the results of a novel methodology for M-protein analysis by MALDI-TOF MS. Patients and Methods: Blood samples from patients and controls were collected after obtaining Institutional Ethics Committee approval. The work was carried out in accordance with the Declaration of Helsinki after obtaining written informed consent. Patients with confirmed multiple myeloma or plasmacytoma and M-protein detected by serum protein electrophoresis (SPEP), serum immunofixation electrophoresis (IFE), and serum free light chains (FLC) were included for MALDI-TOF MS analysis. IFE and FLC analysis were sent to independent laboratories for external validation of the MALDI-TOF MS results. Reagent-based extraction The serum fraction was separated from whole blood by centrifugation at 5000 rpm for 15 minutes and stored at -80oC until further analysis. Twenty-five μL of the serum sample was mixed with 50% acetonitrile (ACN) to form a precipitate. After precipitation and incubation, the mixture was centrifuged. The protein precipitate was washed with 20% ACN. After centrifugation, the supernatant was discarded, and the precipitate was reconstituted in a buffer comprising 10% formic acid (FA) and 50 mmol/L tris(2-carboxyethyl)phosphine hydrochloride (TCEP). The MALDI-TOF MS results were validated using immunoenrichment by anti-kappa (κ) and anti-lambda (λ) biotin-labelled antibodies immobilised on streptavidin magnetic beads. MALDI-TOF MS measurements were obtained for intact proteins using alpha-cyano-4-hydroxycinnamic acid as a matrix. The images obtained were overlaid on apparently healthy serum samples to confirm the presence of M-protein. The samples were then analysed using UltraflexTM LT, Bruker MALDI/TOF-TOF mass spectrometer. The mass spectra for each sample was exported to FlexAnalysis 3.3 (Bruker Daltonics) and background subtracted. A sample was considered positive for M-protein if there was a sharp or broad peak within the κ or λ mass/charge (m/z) range- κ m/z- [M+2H]2+: 11550-12300 Da; [M+H]+: 23100-24600 Da), and λ m/z- [M+2H]2+: 11100-11500 Da; [M+H]+: 22200-23100 Da. All the images were acquired at a m/z range of 10000-29000 Da. Mass measurement was analysed with a summation of 500-5000 shots depending on the intensity of the M-peak. Results: Twenty-seven patient samples: 24- multiple myeloma, and 3- plasmacytoma with an M-protein identified by other biochemical tests, were chosen for ACN precipitation and analysed by MALDI-TOF MS. The median age was 62 years (range:44-72); males-12 (44%). A mass spectrometrist, S.J was blinded to the IFE and FLC results- blinded analyst. N.M was the unblinded analyst. Neat sample (without dilution) was spotted on the MALDI plate for all the control and patient samples. The Gaussian distribution of κ and λ light chains were obtained by analysing 20 serum samples of apparently healthy blood donors. All the 27 samples (100%) with M-protein confirmed by the other biochemical techniques, demonstrated a peak suggestive of M-protein with mass/charge (m/z) falling within the κ or λ range on MALDI-TOF MS: 24 patients with κ peak, and 3 with λ peak. (Fig. 1a and 1b) Immunoenrichment was performed on two samples- 1 with κ peak, and the other with λ peak, analysed by MALDI-TOF-MS by ACN precipitation. The mass spectra by immunoenrichment and ACN precipitation were found to be identical with the light chain m/z falling within their respective range. (Fig. 2 and 3) Three samples were labelled as confounders due to low peak intensity. However, their peaks matched their corresponding IFE and FLC reports. Concordance between MALDI-TOF MS and IFE was observed in 21/23 patients (91%); concordance between MALDI-TOF MS and FLC was observed in 23/24 patients (96%). Conclusions: We report the results of a low-cost, reagent-based extraction process using ACN precipitation to enrich for κ and λ light chains, which can be used for screening and for qualitative analysis of M-protein. Further studies are required to identify the immunoglobulin isotype, and to quantify the M-protein by this methodology. Disclosures No relevant conflicts of interest to declare.

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Automation and validation of a MALDI-TOF MS (Mass-Fix) replacement of immunofixation electrophoresis in the clinical lab

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2020-0581 ◽

2021 ◽

Vol 59 (1) ◽

pp. 155-163

Author(s):

Mindy Kohlhagen ◽

Surendra Dasari ◽

Maria Willrich ◽

MeLea Hetrick ◽

Brian Netzel ◽

...

Keyword(s):

Limit Of Detection ◽

Turnaround Time ◽

Automated System ◽

Serum Samples ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Automated Method ◽

Positive Specimen ◽

Specimen Identification ◽

Tof Ms

AbstractObjectivesA matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) method (Mass-Fix) as a replacement for gel-based immunofixation (IFE) has been recently described. To utilize Mass-Fix clinically, a validated automated method was required. Our aim was to automate the pre-analytical processing, improve positive specimen identification and ergonomics, reduce paper data storage and increase resource utilization without increasing turnaround time.MethodsSerum samples were batched and loaded onto a liquid handler along with reagents and a barcoded sample plate. The pre-analytical steps included: (1) Plating immunopurification beads. (2) Adding 10 μl of serum. (3) Bead washing. (4) Eluting the immunoglobulins (Igs), and reducing to separate the heavy and light Ig chains. The resulting plate was transferred to a second low-volume liquid handler for MALDI plate spotting. MALDI-TOF mass spectra were collected. Integrated in-house developed software was utilized for sample tracking, driving data acquisition, data analysis, history tracking, and result reporting. A total of 1,029 residual serum samples were run using the automated system and results were compared to prior electrophoretic results.ResultsThe automated Mass-Fix method was capable of meeting the validation requirements of concordance with IFE, limit of detection (LOD), sample stability and reproducibility with a low repeat rate. Automation and integrated software allowed a single user to process 320 samples in an 8 h shift. Software display facilitated identification of monoclonal proteins. Additionally, the process maintains positive specimen identification, reduces manual pipetting, allows for paper free tracking, and does not significantly impact turnaround time (TAT).ConclusionsMass-Fix is ready for implementation in a high-throughput clinical laboratory.

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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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Nanoporous silica coupled MALDI-TOF MS detection of Bence-Jones proteins in human urine for diagnosis of multiple myeloma

Talanta ◽

10.1016/j.talanta.2019.03.067 ◽

2019 ◽

Vol 200 ◽

pp. 288-292 ◽

Cited By ~ 6

Author(s):

Shuping Long ◽

Qin Qin ◽

Yuning Wang ◽

Yi Yang ◽

Yan Wang ◽

...

Keyword(s):

Multiple Myeloma ◽

Human Urine ◽

Nanoporous Silica ◽

Maldi Tof Ms ◽

Ms Detection ◽

Maldi Tof ◽

Tof Ms ◽

Bence Jones Proteins

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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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Direct Detection of Monoclonal Free Light Chains in Serum by Use of Immunoenrichment-Coupled MALDI-TOF Mass Spectrometry

Clinical Chemistry ◽

10.1373/clinchem.2018.299461 ◽

2019 ◽

Vol 65 (8) ◽

pp. 1015-1022 ◽

Cited By ~ 10

Author(s):

Lusia Sepiashvili ◽

Mindy C Kohlhagen ◽

Melissa R Snyder ◽

Maria A V Willrich ◽

John R Mills ◽

...

Keyword(s):

Direct Detection ◽

Direct Method ◽

Light Chains ◽

Analytical Sensitivity ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Plasma Cell Disorders ◽

Immunofixation Electrophoresis ◽

Monoclonal Proteins ◽

Tof Ms

Abstract BACKGROUND Free light chain (FLC) quantification is the most analytically sensitive blood-based method commercially available to diagnose and monitor patients with plasma cell disorders (PCDs). However, instead of directly detecting monoclonal FLCs (mFLCs), FLC assays indirectly assess clonality based on quantifying κ and λ FLCs and determination of the к/λ FLC ratio. Often an abnormal FLC ratio is the only indication of a PCD, and confirmation by a direct method increases diagnostic confidence. The aim of this study was to develop an analytically sensitive method for direct detection of mFLCs. METHODS Patient sera (n = 167) previously assessed by nephelometric FLC quantification and immunofixation electrophoresis (IFE) were affinity enriched for IgG, IgA, and total and free κ and λ light chains and subjected to MALDI-TOF MS. Relative analytical sensitivity of these methods was determined using serially diluted sera containing mFLCs. RESULTS In sera with abnormal FLC ratios (n = 127), 43% of monoclonal proteins were confirmed by IFE, 57% by MALDI-TOF MS without FLC enrichment, and 87% with FLC enrichment MALDI-TOF MS. In sera with normal FLC ratios (n = 40), the FLC MALDI-TOF MS method identified 1 patient with an mFLC. Serial dilution and analysis of mFLC containing sera by IFE, nephelometry, and FLC MALDI-TOF MS demonstrated that FLC MALDI-TOF MS analysis had the highest analytical sensitivity. CONCLUSIONS FLC immunoenrichment coupled to MALDI-TOF MS enables direct detection of mFLCs and significantly increases the confirmation of abnormal serum FLC ratios over IFE and MALDI-TOF MS without FLC enrichment, thereby providing added confidence for diagnosing FLC PCDs.

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Structure of pre-S2 N- and O-linked glycans in surface proteins from different genotypes of hepatitis B virus

Journal of General Virology ◽

10.1099/vir.0.79932-0 ◽

2004 ◽

Vol 85 (7) ◽

pp. 2045-2053 ◽

Cited By ~ 29

Author(s):

Sigrid Schmitt ◽

Dieter Glebe ◽

Tanja K. Tolle ◽

Günter Lochnit ◽

Dietmar Linder ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Surface Proteins ◽

M Protein ◽

Hbv Genotype ◽

Maldi Tof Ms ◽

Amino Terminal ◽

Maldi Tof ◽

B Virus ◽

Tof Ms

The middle-sized (M) surface proteins of hepatitis B virus (HBV) and other orthohepadnaviruses contain a conserved N-glycan in their pre-S2 domain, which is essential for the secretion of viral particles. Recently, we also found O-glycans in the pre-S2 domain of M protein from woodchuck hepatitis virus (WHV) and HBV genotype D. Since the O-glycosylation motif is not conserved in all genotypes of HBV, the glycosylation patterns of HBV genotypes A and C were analysed. Pre-S2 (glyco)peptides were released from HBV-carrier-derived HBV subviral particles by tryptic digestion, purified by reversed-phase HPLC and identified by amino acid and amino-terminal sequence analysis as well as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Pre-S2 N-glycans were characterized by anion-exchange chromatography, methylation analysis and on-target sequential exoglycosidase digestions in combination with MALDI-TOF-MS, demonstrating the presence of partially sialylated diantennary complex-type oligosaccharides in all genotypes examined. Pre-S2 O-glycans were characterized by on-target sequential exoglycosidase digestions in combination with MALDI-TOF-MS. The pre-S2 domain of M protein and, to a minor extent, of L (large) protein from HBV genotype C and D was partially O-glycosylated by Neu5Ac(α2–3)Gal(β1–3)GalNAcα- or Gal(β1–3)GalNAcα-units at Thr-37 within a conserved sequence context. Genotype A, containing no Thr at position 37 or 38, was not O-glycosylated. Analytical data further revealed that M protein is mostly amino-terminally acetylated in all examined genotypes and that the terminal methionine is partially oxidized. The findings may be relevant for the secretion and the immunogenicity of HBV.

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A Rapid MALDI-TOF Method for Isotyping and Quantitating M-Proteins in a Single Assay: Longitudinal Comparison to Serum Protein Electrophoresis and Hevylite for Monitoring Patients with Monoclonal Gammopathies

Blood ◽

10.1182/blood.v126.23.1780.1780 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1780-1780

Author(s):

David L Murray ◽

John R. Mills ◽

Maria Alice V Willrich ◽

David Barnidge ◽

Mindy C Kohlhagen ◽

...

Keyword(s):

Serum Protein ◽

Light Chain ◽

Patent Application ◽

Protein Electrophoresis ◽

M Protein ◽

Maldi Tof Ms ◽

Monoclonal Gammopathies ◽

Maldi Tof ◽

M Proteins ◽

Tof Ms

Abstract Background: Current laboratory methods for detection of monoclonal gammopathies include a panel of serum protein electrophoresis (SPEP), immunofixation electrophoresis (IFE) and serum free light chain quantitation (FLC). Our group has recently described a new assay based on matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) which is capable of detecting, isotyping and quantitating M-proteins in a single assay. The basic principle of the method involves leveraging the unique mass resulting from light chain (LC) Ig gene rearrangement in B-cells. In a similar fashion to SPEP, the mass distribution of LCs after dissociation from their heavy chains can be examined for the presence of an over expressed light chain. In this study we retrospectively compared the MALDI-TOF MS method to SPEP, IFE and Hevylite ratios for monitoring patients with monoclonal gammopathies. Methods: Serum from patients who had at least 1 diagnostic and 4 serial follow-up samples was enriched for IgG, IgA, IgM, kappa and lambda using nanobodies. After disassociating the heavy and light chains by reduction, the five purified samples were spotted onto a Bruker Microflex MALDI plate. Automated acquisition (~10 seconds/sample) was performed and the five LC mass spectra from each enrichment were overlaid. M-proteins were detected, quantitated and isotyped by the presence of distinct peaks (m-spike) within LC mass to charge regions. The serial patient samples were then measured by MALDI-TOF and a comparison of results was made to previous aquired data. The results were then analyze in light of clinical and lab data in order to evaluate the ability of the MALDI-TOF MS method to monitor patients with monoclonal gammopathies. Results: In M-protein positive patient samples, serial dilution revealed MALDI-TOF MS to have ~10-times lower limit of detection than IFE. M-protein isotype in the cohort by MALDI-TOF MS was 100 percent concordant with the isotype from IFE. The MALDI TOF M-protein quantitation was linear over a clinically acceptable range (0.1 to 6 g/dL) and had improved linearity over SPEP at lower M-protein levels. M-proteins quantitation by MALDI-TOF MS compared well with SPEP with a slope of 1.16 (R2 -0.93). Hevylite Ig ratios for each isotype were statistically similar to those from MALDI-TOF MS demonstrating the ability of the MALDI to measure isotype suppression. For some patients, the MALDI method was able to detect M-proteins in patients with normal Hevylite ratios. Finally, the results for each patient were then compared over the course of monitoring. The time evolution change in M-protein concentration was similar among the three methods with the exception of a few samples in which the MALDI-TOF detected residual M-proteins in treated patients not detected by the other methods. Conclusion: This preliminary study demonstrates that MALDI-TOF MS method can provide detection, quantitation and isotyping data suitable for monitoring patients. This is a significant finding since the MALDI-TOF method is amendable to automation, is rapid and in its current format is cost-competitive with current clinical assays. Disclosures Murray: Mayo Clinic: Patents & Royalties: Patent Application Filed. Mills:Mayo Clinci: Patents & Royalties: Patent Application Filed. Barnidge:Mayo Clinic: Patents & Royalties: Patent Application Filed.

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Bone Marrow-Based and Longitudinal Blood-Based MRD Tracking in Newly Diagnosed Multiple Myeloma Patients Treated with Daratumumab, Carfilzomib, Lenalidomide and Dexamethasone (DKRd): A Correlative and Clinical Phase II Study

Blood ◽

10.1182/blood-2018-99-114311 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3281-3281 ◽

Cited By ~ 3

Author(s):

Ola Landgren ◽

Katie Thoren ◽

Malin Hultcrantz ◽

Alexander M. Lesokhin ◽

Nikoletta Lendvai ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Peripheral Blood ◽

Research Funding ◽

Newly Diagnosed ◽

Advisory Committees ◽

Single Tube ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Tof Ms

Abstract INTRODUCTION Using Carfilzomib, Lenalidomide and Dexamethasone (KRd) combination therapy in newly diagnosed multiple myeloma patients lead to ~40% minimal residual disease (MRD) negativity rate. Here, we use KRd in combination with daratumumab (DKRd); and treatment response is assessed with extensive correlative science including parallel bone-marrow-based and blood-based MRD tracking, together with targeted DNA sequencing of baseline bone marrow samples. Primary end-point is to rule out 60% and to target up to 80% MRD negativity rate. METHODS This is a single-arm, Phase II clinical trial based on Simon's optimal two-stage design. The first cohort (twice-a-week carfilzomib) (N=41) has the following treatment schedule: 8 cycles of treatment; 28-day cycles with carfilzomib 20/36 mg/m2 days 1, 2, 8, 9, 15 and 16; lenalidomide 25 mg days 1-21; dexamethasone 40 mg weekly cycles 1-4, 20 mg after cycle 4; and daratumumab 16 mg/kg days 1, 8, 15, and 22 cycles 1-2, days 1 and 15 cycles 3-6, and day 1 cycles 7-8. The second cohort (once-a-week carfilzomib) (N=41): 8 cycles of treatment; 28-day cycles with carfilzomib 20/56 mg/m2 days 1, 8, and 15; lenalidomide, dexamethasone, and daratumumab are given at the same doses/schedules as the first cohort. For fit patients, stemcell collection is recommended after 4 to 6 cycles of therapy; DKRd therapy is resumed after collection to a total of 8 cycles DKRd. Treatment response is being assessed with parallel bone-marrow-based (10-color single tube flowcytometry, invivoscribe V(D)J sequencing) as well as blood-based (MALDI-TOF and QTOF-mass spectrometry [MS]) for MRD tracking. Baseline bone marrow samples are evaluated with targeted DNA sequencing for FISH-Seq and somatic mutational characteristics (myTYPE). Here, we present the first stage (N=28) of the first cohort (twice-a-week carfilzomib). We are waiting for the results to mature before the second stage (N=13) of the first cohort can open. The second cohort (once-a-week carfilzomib) is opening for enrollment in August 2018 (N=41). RESULTS The first stage of the first cohort is fully enrolled; 28 patients meeting eligibility criteria were enrolled onto study (14 males, 14 females) between October 2017 and July 2018. Baseline characteristics include; median age 60 years (range 32-80 years); 12(43%) patients had high-risk FISH/SNP signature defined as one or more of the following: 1q+, t(4,14), t(14,16), t(14,20), and 17p-. At the submission of this abstract, 20 patients have completed one or more cycles DKRd; among these, 3 patients have completed all 8 cycles. The median number of cycles delivered is currently 4 (range 1-8). Full assessments with MRD assays have been completed in 3 patients: -Pt #1 obtained complete response (CR) after 3 cycles, and workup after the last cycle of therapy showed MRD-negativity (by 10-color single tube flowcytometry and V(D)J sequencing) in the bone marrow; and peripheral blood (serum) was negative by MALDI-TOF MS after completion of cycle 2. -Pt#2 obtained CR after 4 cycles, however, workup after cycle 5 showed MRD-positivity (by 10-color single tube flowcytometry and V(D)J sequencing) in the bone marrow; and peripheral blood (serum) was positive by MALDI-TOF MS throughout the end of the last cycle. -Pt#3 obtained CR after 4 cycles and after 6 cycles both 10-color single tube flowcytometry and V(D)J sequencing showed MRD-negativity in the bone marrow. However, MALDI-TOF MS detected small abnormal serum proteins in peripheral blood and remained positive throughout the end of cycle 8. Overall, the DKRd therapy is well tolerated and it has similar toxicity profile as KRd. Grade >3 adverse events were hypotension, musculoskeletal deformity, back pain, dyspnea, lung-infection, and febrile neutropenia. So far, 5 patients underwent dose reductions of lenalidomide. CONCLUSIONS In this pre-planned interim analysis of our phase II study, we show that DKRd is a highly effective and well tolerated combination therapy for newly diagnosed multiple myeloma patients. Based on small numbers of patients who have completed the planned DKRd cycles and been evaluated by bone marrow-based MRD and peripheral-blood based assays, we show that highly sensitive protein assays may allow longitudinal MRD tracking in peripheral-blood. At the meeting, we will present updated results using longitudinal testing with MALDI TOF-MS and QTOF-MS on the entire cohort. Disclosures Landgren: Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Pfizer: Consultancy; Karyopharm: Consultancy; Merck: Membership on an entity's Board of Directors or advisory committees. Lesokhin:Takeda: Consultancy, Honoraria; Janssen: Research Funding; Squibb: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Genentech: Research Funding; Serametrix, inc.: Patents & Royalties: Royalties. Mailankody:Juno: Research Funding; Physician Education Resource: Honoraria; Takeda: Research Funding; Janssen: Research Funding. Smith:Celgene: Consultancy, Patents & Royalties: CAR T cell therapies for MM, Research Funding. Hassoun:Oncopeptides AB: Research Funding. Shah:Amgen: Research Funding; Janssen: Research Funding. Arcila:Invivoscribe, Inc.: Consultancy, Honoraria. Ho:Invivoscribe, Inc.: Honoraria. Korde:Amgen: Research Funding.

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