4 results on '"Zac McDonald"'
Search Results
2. M-Protein Sequencing and Monitoring in Serum of LC-Only Multiple Myeloma Patients
- Author
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Elisabet E. Manasanch, Suzanne Trudel, Zac McDonald, Jesus Giovanni Piza, Robert Z. Orlowski, Liqiang Yang, Donna E. Reece, Gregory P. Kaufman, Mariya S. Liyasova, Bin Ma, and Natalia Migdal
- Subjects
Myeloma protein ,business.industry ,Immunology ,medicine ,Cell Biology ,Hematology ,medicine.disease ,business ,Biochemistry ,Molecular biology ,Multiple myeloma - Abstract
Background M-protein, a secreted antibody of malignant plasma cells, is a gold standard biomarker for monitoring the disease status in multiple myeloma (MM) patients (pts). The development of peripheral blood based ultrasensitive (MRD) methods of M protein detection is of high interest and importance. In 20% of MM pts, the M-protein consists of only the light chain (LC) of the immunoglobulin (Ig) molecule. For these LC-only MM pts, disease monitoring is challenging due to low levels of M-protein in serum. Urine protein electrophoresis (UPEP) and immunofixation electrophoresis (IFE), or serum free light chain (FLC) lack the sensitivity and/or specificity to track the M-protein in these pts, while bone marrow-based assays cannot be performed frequently due to their invasive nature. Thus, we evaluated the performance of EasyM, a mass spectrometry(MS)-based, non-invasive, sensitive assay for monitoring M-protein levels in LC-only patients in the MCRN-001 Canadian national and MD Anderson VRD-panobinostat frontline trials. Methods MCRN-001 trial is evaluating enhanced conditioning prior to ASCT for newly diagnosed MM (NDMM). After treatment with bortezomib (BTZ) based induction eligible MM pts received BuMel prior to ASCT. Busulfan was administered via IV at 3.2 mg/kg on days -5 to -3, or days -6 to -4 pre-ASCT (day 0) and melphalan was given at 140 mg/m 2 on day -2 or -3 pre-ASCT. Lenalidomide (LEN) administration began 100 days post-ASCT at 10 mg/d and continued until progressive disease (PD) onset. In the MDACC 2011-0192 frontline study in newly diagnosed MM, transplant-eligible pts received the novel combination of LEN, BTZ, dexamethasone (DEX) and panobinostat (RVD-panobinostat). The IMWG criteria were used to monitor clinical response in both trials. A total of 13 LC-only MM pts were selected for the study. Local IRB approval was obtained prior to the study. To derive the M-protein's full amino acid sequence, FLC was first enriched from the diagnostic serum sample. The FLC enrichment consisted of IgG depletion with protein A/G beads, followed by affinity purification of kappa or lambda LC containing Igs. Non-reducing PAGE was then used to separate FLC monomers, FLC dimers and full-length Igs. Finally, in-gel digestion of FLC monomers and dimers by multiple proteolytic enzymes were analyzed on a Q-Exactive mass spectrometer. Data analysis and sequence assembly were performed with the REmAb protein sequencing platform. To monitor M-protein levels in serum, unique, tryptic peptides from sequenced FLC were selected and quantified in diagnostic and follow-up samples with a PRM assay on a Q-Exactive instrument. Results M-protein sequencing The full FLC sequence was derived for 8 out of 13 (61.5%) LC-only pts. The M-protein was successfully sequenced even when the FLC concentration was as low as 147 mg/L. However, FLC concentration measured by FreeLite assay was not a reliable predictor of our ability to derive the full sequence. The appearance of a sharp FLC monomer and dimer bands on PAGE after FLC enrichment was a better predictor of the M-protein sequencing success. M-protein monitoring The M-protein of one pt did not contain any unique peptides. This pt was excluded for further analysis. In the remaining 7 pts, the M-protein contained at least one unique peptide and could thus be monitored by EasyM. For 6 LC-only pts, the M-protein monitored by EasyM correlated with the disease status measured by serum FLC, UPEP and urine IFE. A separate serial dilution test estimated that the limit of quantification can reach as low as 0.13 mg/L. Figure 1 shows representative data for two LC-only patients. One pt experienced relapse during the study; however, this relapse could not be detected by EasyM. This result could indicate a possible clonal switch at time of disease progression, but further investigation is needed to verify this. Conclusions Due to the rapid turnover and clearance of light chains conventional blood/urine tests have lacked the higher sensitivity needed to monitor disease state in LC only MM patients. To overcome the lower FLC concentration the current study successfully applied LC enrichment strategies to enable the sequencing and high sensitivity monitoring of FLC M-protein by MS in blood. The EasyM LC assay is non-invasive, sensitive, capable of assessing disease status, and has potential to be further investigated as a peripheral blood myeloma response and MRD monitoring biomarker. Figure 1 Figure 1. Disclosures Ma: Rapid Novor Inc.: Current holder of individual stocks in a privately-held company. Reece: Millennium: Research Funding; Amgen: Consultancy, Honoraria; GSK: Honoraria; Karyopharm: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Sanofi: Honoraria; BMS: Honoraria, Research Funding. Manasanch: GSK, Secura Bio,Takeda, Celgene, Sanofi, Janssen and Adaptive Biotechnologies: Consultancy; Sanofi, Quest Diagnostics, Novartis, JW Pharma, Merck: Research Funding. Orlowski: Amgen, Inc., BioTheryX, Inc., Bristol-Myers Squibb, Celgene, EcoR1 Capital LLC, Genzyme, GSK Biologicals, Janssen Biotech, Karyopharm Therapeutics, Inc., Neoleukin Corporation, Oncopeptides AB, Regeneron Pharmaceuticals, Inc., Sanofi-Aventis, and Takeda P: Consultancy, Honoraria; CARsgen Therapeutics, Celgene, Exelixis, Janssen Biotech, Sanofi-Aventis, Takeda Pharmaceuticals North America, Inc.: Other: Clinical research funding; Asylia Therapeutics, Inc., BioTheryX, Inc., and Heidelberg Pharma, AG.: Other: Laboratory research funding; Asylia Therapeutics, Inc.: Current holder of individual stocks in a privately-held company, Patents & Royalties; Amgen, Inc., BioTheryX, Inc., Bristol-Myers Squibb, Celgene, Forma Therapeutics, Genzyme, GSK Biologicals, Janssen Biotech, Juno Therapeutics, Karyopharm Therapeutics, Inc., Kite Pharma, Neoleukin Corporation, Oncopeptides AB, Regeneron Pharmaceuticals, I: Membership on an entity's Board of Directors or advisory committees. Trudel: GlaxoSmithKline: Consultancy, Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Roche: Consultancy; Genentech: Research Funding; Pfizer: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Sanofi: Honoraria; BMS/Celgene: Consultancy, Honoraria, Research Funding.
- Published
- 2021
3. Targeted Mass Spectrometry-Based Serum M-Protein Monitoring for Early Relapse Detection
- Author
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Suzanne Trudel, Kathleen Gorospe, Jesus Giovanni Piza, Mariya Liyasova, Chenyu Yao, Qixin Liu, Xin Xu, Liqiang Yang, Bin Ma, Paul J. Taylor, Zac McDonald, and Donna E. Reece
- Subjects
Immunofixation ,biology ,Myeloma protein ,business.industry ,Protein Sequence Determination ,Immunology ,Disease progression ,Complete remission ,Early Relapse ,Cell Biology ,Hematology ,Human serum albumin ,Biochemistry ,Targeted mass spectrometry ,medicine ,Cancer research ,biology.protein ,business ,medicine.drug - Abstract
Background For most Multiple Myeloma (MM) patients, M-protein is the gold standard biomarker for disease diagnosis and monitoring. Traditional methods such as serum protein electrophoresis (SPEP) and serum immunofixation electrophoresis (IFE) lack the sensitivity to detect M-protein in patients with minimal residual disease. At ASH 2018, we reported a Mass Spectrometry (MS)-based highly sensitive assay (REmAb) for detecting and quantifying M-protein. This assay exploits the uniqueness of the M-protein sequence and can detect 1 M-protein in 10,000 polyclonal IgG. The current study utilized this established assay to first sequence then monitor M-protein in sera from diagnosis through treatment for patients from the first MCRN-001 Canadian national trial with bortezomib (BTZ)-based pre-induction (PI), augmented high-dose chemotherapy with busulfan + melphalan (BuMel) before ASCT and lenalidomide (Len) maintenance post-ASCT. Methods MCRN-001 trial patients were first induced with BTZ before harvesting stem cells. Eligible MM patients received BuMel prior to ASCT. Busulfan was administered IV at 3.2 mg/kg on days -5 to -3, or days -6 to -4 days pre-ASCT (Day 0) and melphalan was given at 140 mg/m2 on day -2 or -3 pre-ASCT. Len administration began 100 days post-ASCT at 10 mg/d (increased when appropriate to 15 mg/d) and continued until progressive disease (PD) onset. IMWG criteria was used to monitor clinical response and PD. 78 patients enrolled in this trial; only 58 offered consent for M-protein monitoring with MS. Serum samples were acquired at time of pre-induction (PI), before ASCT (screening sample), post-ASCT on day 100, every 3 months for the first year and then every 6 months until PD. The M-protein sequence was determined with the REmAb protein sequencing platform from either PI or screening samples when the PI sample was unavailable. For M-protein quantification, each sample was digested with trypsin prior to analysis by liquid chromatography tandem MS with an Orbitrap Fusion Tribrid or Q-Exactive instrument. Patient-specific, unique tryptic peptides, with at least one peptide from each Heavy and Light chain, were targeted by the MS assay. To monitor changes in M-protein levels per patient post-diagnosis and through treatment to complete remission (CR) and/or PD, this study used MS peak areas of patient-specific unique peptides normalized against peak areas of human serum albumin or spiked-in standard peptides. Results M-protein sequencing In this study, the lowest M-protein serum concentration required for sequencing was 0.2 g/dL. 48 out of 58 (83%) patient-specific M-proteins were sequenced from serum. 5 out of 48 patients sequenced discontinued the study early and were excluded from further analysis. M-protein monitoring 24 out of the 43 achieved CR. The M-proteins of these patients were monitored by MS to study early relapse detection. M-protein levels could be monitored by MS in all these patients from diagnosis through CR to PD onset, even when M-protein was undetectable by SPEP and IFE. A separate serial dilution test estimated that the limit of quantification can reach as low as 0.03 mg/dL. 3 patients who had achieved CR eventually relapsed (PD). In all 3 PD patients, a 2 to 200 fold increase in M-protein in 2 consecutive tests half a year apart was detected by MS 6 months earlier than clinical confirmation of PD by conventional testing. The other 21 had not progressed at time of analysis. In the non-progressor group, the M-protein levels detected by MS either continued to decrease during CR or remained relatively stable. Only 3 out of 21 patients demonstrated a 2-fold increase in M-protein level in any 2 consecutive tests. Figure 1 shows representative data for two patients. Conclusions This study demonstrates that M-protein sequencing and targeted MS assay can be used to monitor serum M-protein levels sensitively even when SPEP and IFE fail to detect M-protein. Based on this assay, a 2-fold increase in serum M-protein levels in the past 6 months can reliably predict disease progression in the next 6 months for patients achieving CR. In the 24 studied patients, the method has 100% sensitivity and 86% specificity at predicting disease progression from CR prior to detection by standard methods. Future work will analyze more patient samples to confirm the findings and investigation into criteria surrounding the 2-fold M-protein increase observed in CR patients and relapse post CR. Disclosures McDonald: Rapid Novor Inc Kitchener: Employment, Equity Ownership, Research Funding. Liyasova:Rapid Novor Inc Kitchener: Employment. Taylor:Rapid Novor Inc Kitchener: Employment. Xu:Rapid Novor Inc Kitchener: Employment. Gorospe:Rapid Novor Inc Kitchener: Employment. Yao:Rapid Novor Inc Kitchener: Employment. Liu:Rapid Novor Inc Kitchener: Employment, Equity Ownership. Yang:Rapid Novor Inc Kitchener: Employment. Ma:Rapid Novor Inc Kitchener: Equity Ownership. Reece:Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Otsuka: Research Funding; Merck: Research Funding; BMS: Research Funding. Trudel:GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Research Funding; Genentech: Research Funding; Sanofi: Honoraria; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria; Pfizer: Honoraria; Janssen: Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: The use of IV busulfan and melphalan as conditioning for myeloma transplants is off-label.
- Published
- 2019
4. New Blood Based M-Protein Quantification Method 3,000 Times More Sensitive Than Standard SPEP
- Author
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Qixin Liu, Bin Ma, Paul Taylor, Liqiang Yang, and Zac McDonald
- Subjects
Immunofixation ,medicine.medical_specialty ,Hematology ,biology ,Serial dilution ,medicine.drug_class ,Myeloma protein ,business.industry ,Immunology ,Cell Biology ,Monoclonal antibody ,medicine.disease ,Biochemistry ,Molecular biology ,Polyclonal antibodies ,Internal medicine ,medicine ,biology.protein ,Progression-free survival ,business ,Multiple myeloma - Abstract
Summary of Work The amino acid sequence of the M-protein for multiple myeloma (MM) is unique compared to the polyclonal antibodies in patients' blood. In this study we utilize this uniqueness to develop an ultra-sensitive M-protein detection method with mass spectrometry (MS). The method involves the de novo sequencing of the amino acid sequence of the M-protein from the baseline blood sample collected at the time of diagnosis, and a targeted MS assay to detect and quantify the unique M-protein sequence in the follow-up blood samples. This non-invasive method is purely blood based and is 3,000 and 300 times more sensitive than SPEP and IFE, respectively. De Novo Protein Sequencing The M-protein sequencing is carried out on an MS based platform (REmAb). The effectiveness of de novo protein sequencing has been proven with the sequencing of hundreds of monoclonal antibodies (McDonald et al., Poster 294737, ASMS, 2018). In the current study the M-protein from 4 MM patients' baseline blood samples were successfully sequenced, demonstrating robustness of the method in the presence of the polyclonal background. As little as 50ul of the serum was required for the de novo sequencing. Sensitivity of M-protein Quantification The lower limit of quantification (LLOQ) was studied with a serial dilution experiment. The baseline blood sample of one patient was sequentially diluted with a healthy donor's serum. The peptide sequence unique to the M-protein was monitored with mass spectrometry to detect and quantify the M-protein in the serial dilution. The M-protein could still be detected and quantified when the dilution ratio was 1:10,000 (the amount of M-protein relative to background polyclonal serum IgG). In a separate experiment, synthesized heavy labelled proteotypic peptides were used to estimate the LLOQ in IgG enriched serum at 60 ug/dL, over 3,000 times more sensitive than SPEP (0.2g/dL) (Bergen et al., Clin Chem 62: 1 243-251, 2016) and 300 times more sensitive than IFE (0.02g/dL) (IMWG, British Journal of Haematology, 121, 749-757, 2003). As little as 30ul of serum was required in these experiments for monitoring of M-protein levels. Case Study A targeted MS based assay was developed to monitor the M-protein levels of a serial patient sample set (73-year-old male, treatment: Elotuzumab/Lenalidomide/Dexamethasone, progression free survival 32 months). M-protein levels were quantified in a total of 10 serial samples from partial remission, through complete remission (CR), until relapse over a period of 2 years and 3 months (Figure 1). M-protein could be detected and quantified in all samples collected during the CR period with estimated M-protein levels never below 10mg/dL. Notably, a 2-fold increase in M-protein levels (in comparison to the lowest historic level) could be detected 320 days prior to the timepoint of relapse. The upward trend continued in the next 3 serial CR samples preceding relapse. Except for one sample (see Figure 1, 'CR Before Relapse 12-14-2016'), CR was based on the absence of M-protein in the IFE result in the relevant clinical data. Conclusion The work represents a first step in the application of de novo sequencing and MS based detection for the sensitive monitoring of M-protein levels in serum. It shows a much-improved sensitivity over current standard approaches and has the great potential to provide non-invasive assessment of MRD for multiple myeloma. The preliminary data warrant further development of this MS based non-invasive and highly sensitive M-protein quantification method. Disclosures McDonald: Rapid Novor Inc: Employment, Equity Ownership. Liu:Rapid Novor Inc: Employment, Equity Ownership. Taylor:Rapid Novor Inc: Employment, Equity Ownership. Yang:Rapid Novor Inc: Employment, Equity Ownership. Ma:Rapid Novor Inc: Equity Ownership.
- Published
- 2018
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