Barnidge, David R.

Abstract AL is a rare plasma cell disorder, resulting from clonal bone marrow plasma cells secreting immunoglobulin light chains (LC), which aggregate abnormally into fibrils, and leads to amyloid deposits in different organs. Previous studies using LC immunopurification and MALDI-TOF/MS (MASS-FIX) on patients with known plasma cell disorders (PCDs), demonstrated that monoclonal LC from AL patients display unusually complex mass to charge spectra (m/z) for their LCs at much higher frequencies than other PCDs such as multiple myeloma. We hypothesized that these were post-translational modification, presumably glycoforms. Here, we report investigations into LC glycosylation using MASS- FIX. Serum from 212 lambda and 72 kappa AL patients were collected following Mayo Clinic IRB guidelines. LC was purified from serum using nanobody enrichment method, followed by MASS-FIX. The LCs spectra acquired on MASS-FIX were visually analyzed and were classified into different patterns. The kappa and lambda LC mass distributions with abnormal complex patterns were interrogated for the presence of glycosylation by exposure to the LC to a deglycoslylating enzyme (PNGase F) and analysis of LC mass distribution using MASS-FIX and by LC-ESI-Orbitrap. Monoclonal LCs demonstrating significantly mass shifted peak outside the normal kappa and lambda mass distribution were observed in 31% of kappa and 6% of lambda patients with AL. In order to determine if this characteristic mass shift was due to glycosylation, 16 kappa and 6 lambda LCs were treated with a PNGase F and LC spectrum were re-examined by MASS-FIX and LC-ESI-Orbitrap. The monoclonal LCs whose masses were significantly shifted beyond normal kappa and lambda mass distributions were found to shift to lower molecular weight after PNGase F treatment, confirming the presence of N-glycosylation. Moreover, an additional 5 kappa and 23 lambda samples had other complex patterns, typically with multiple additional peaks at increments of 162 Da. These samples did not shift with PNGase F suggesting either glycation or O-glycosylation. This work is significant as it confirms that N-glycosylated LCs are over represented in patients with AL in comparison to other PCDs. In patients with AL, the frequency of N-glycosylation of kappa LC clones was 5 times higher than that of lambda LC clones. More work is needed to explore the mechanism for this finding, the role of glycosylation in fibril formation, and the other complex patterns observed. In addition, MASS-FIX method used in this work is currently being validated in the clinical lab such that rapid detection of N-glycosylation in patients with monoclonal gammopathies will be possible routinely, potentially leading to earlier clinical suspicion for AL among patients with monoclonal gammopathies and hence to earlier detection of AL. Citation Format: Sanjay Kumar, Angela Dispenzieri, Surendra Dasari, Paolo Milani, Giampaolo Merlini, MeLea Hetrick, David Barnidge, Benjamin Madden, David Murray. Mass spectrometric approach to identify N-glycosylation of light chain in patients with immunoglobulin light chain amyloidosis (AL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2693.

The detection and quantification of monoclonal‐proteins (M‐proteins) are necessary for the diagnosis and evaluation of response in plasma cell dyscrasias. Immunoglobulin enrichment‐coupled with matrix‐assisted laser desorption ionization time‐of‐flight mass‐spectrometry (MASS‐FIX) is a simple and inexpensive method to identify M‐proteins, but its clinical generalizability has not yet been elucidated. We compared MASS‐FIX to protein electrophoresis (PEL), serum/urine immunofixation‐electrophoresis (IFE), and quantitative serum free‐light chain (FLC) for the identification of M‐proteins in different clinical diagnoses. Paired serum and urine samples from 257 patients were tested. There were six patients for whom s‐IFE and FLC ratio were positive and serum MASS‐FIX was negative, but when serum and urine MASS‐FIX results were combined, only one patient with light chain‐MGUS was missed. Serum/urine‐MASS‐FIX detected M‐proteins in 18 patients with negative serum/urine‐PEL/IFE and serum‐FLC, 10 of whom had multiple myeloma or AL amyloidosis, who were mistakenly thought to have complete hematologic response by serum/urine‐PEL/IFE and serum‐FLC. Nearly half of the AL amyloidosis patients had atypical spectra, which may prove to be a clue to the diagnosis and pathogenesis of the disease. In conclusion, MASS‐FIX has a comparable sensitivity with PEL/IFE/FLC methods and can help inform the clinical diagnosis.

AbstractImmunoglobulin light chain (AL) amyloidosis is a fatal complication of B‐cell proliferation secondary to deposition of amyloid fibrils in various organs. Urinary exosomes (UEX) are the smallest of the microvesicles excreted in the urine. Previously, we found UEX of patients with AL amyloidosis contained immunoglobulin light chain (LC) oligomers that patients with multiple myeloma did not have. To further explore the role of the LC oligomers, UEX was isolated from an AL amyloidosis patient with progressive renal disease despite achieving a complete response. LC oligomers were identified. Mass spectrometry (MS) of the UEX and serum identified two monoclonal lambda LCs. Proteomics of the trypsin digested amyloid fragments in the kidney by laser microdissection and MS analysis identified a λ6 LC. The cDNA from plasma cell clone was from the IGLV‐ 6‐57 family and it matched the amino acid sequences of the amyloid peptides. The predicted mass of the peptide product of the cDNA matched the mass of one of the two LCs identified in the UEX and serum. UEX combined with MS were able to identify 2 monoclonal lambda LCs that current clinical methods could not. It also identified the amyloidogenic LC which holds potential for response assessment in the future.

ABSTRACTTherapeutic monoclonal antibodies (MAbs) are an important class of drugs used to treat diseases ranging from autoimmune disorders to B cell lymphomas to other rare conditions thought to be untreatable in the past. Many advances have been made in the characterization of immunoglobulins as a result of pharmaceutical companies investing in technologies that allow them to better understand MAbs during the development phase. Mass spectrometry is one of the new advancements utilized extensively by pharma to analyze MAbs and is now beginning to be applied in the clinical laboratory setting. The rise in the use of therapeutic MAbs has opened up new challenges for the development of assays for monitoring this class of drugs. MAbs are larger and more complex than typical small-molecule therapeutic drugs routinely analyzed by mass spectrometry. In addition, they must be quantified in samples that contain endogenous immunoglobulins with nearly identical structures. In contrast to an enzyme-linked immunosorbent assay (ELISA) for quantifying MAbs, mass spectrometry-based assays do not rely on MAb-specific reagents such as recombinant antigens and/or anti-idiotypic antibodies, and time for development is usually shorter. Furthermore, using molecular mass as a measurement tool provides increased specificity since it is a first-order principle unique to each MAb. This enables rapid quantification of MAbs and multiplexing. This review describes how mass spectrometry can become an important tool for clinical chemists and especially immunologists, who are starting to develop assays for MAbs in the clinical laboratory and are considering mass spectrometry as a versatile platform for the task.

Abstract BACKGROUND Electrophoretic separation of serum and urine proteins has played a central role in diagnosing and monitoring plasma cell disorders. Despite limitations in resolution and analytical sensitivity, plus the necessity for adjunct methods, protein gel electrophoresis and immunofixation electrophoresis (IFE) remain front-line tests. METHODS We developed a MALDI mass spectrometry–based assay that was simple to perform, automatable, analytically sensitive, and applicable to analyzing the wide variety of monoclonal proteins (M-proteins) encountered clinically. This assay, called MASS-FIX, used the unique molecular mass signatures of the different Ig isotypes in combination with nanobody immunoenrichment to generate information-rich mass spectra from which M-proteins could be identified, isotyped, and quantified. The performance of MASS-FIX was compared to current gel-based electrophoresis assays. RESULTS MASS-FIX detected all M-proteins that were detectable by urine or serum protein electrophoresis. In serial dilution studies, MASS-FIX was more analytically sensitive than IFE. For patient samples, MASS-FIX provided the same primary isotype information for 98% of serum M-proteins (n = 152) and 95% of urine M-proteins (n = 55). MASS-FIX accurately quantified M-protein to <1 g/dL, with reduced bias as compared to protein electrophoresis. Intraassay and interassay CVs were <20% across all samples having M-protein concentrations >0.045 g/dL, with the ability to detect M-proteins <0.01 g/dL. In addition, MASS-FIX could simultaneously measure κ:λ light chain ratios for IgG, IgA, and IgM. Retrospective serial monitoring of patients with myeloma posttreatment demonstrated that MASS-FIX provided equivalent quantitative information to either protein electrophoresis or the Hevylite™ assay. CONCLUSIONS MASS-FIX can advance how plasma cell disorders are screened, diagnosed, and monitored.