Murray, David

e19534 Background: Our group has developed a mass spectrometry (MS) method that can identify circulating monoclonal proteins, based on their unique molecular mass signatures. Given the propensity for patients with AL amyloidosis to have MS patterns consistent with post-translational modifications (PTMs), we posed the question whether these changes might be present long before the diagnosis of AL and potentially serve as a predictor for AL in patients with an asymptomatic plasma cell dyscrasia (aPCD). Methods: We included 64 patients with AL who had a preceding aPCD and who had stored serum samples at both time-points. The mass/charge (m/z) of monoclonal LC peaks (P) was noted for each sample. Presence of a PTM was defined as the presence of ≥1 “heavy” Ps at a particular charge state. A P was considered heavy if its m/z exceeded the right sided 95% C.I. of the m/z distribution of the respective normal LC. We used 9 patients with relapsed multiple myeloma (MM) without AL as controls. Results: Prior aPCDs were MGUS (N = 33), smoldering myeloma (n = 9) and MM (N = 1). In 21 patients aPCD was not further classified with a bone marrow biopsy. The median interval between aPCD and AL diagnosis was 77 months. At aPCD diagnosis, 42 patients had a single, typical P and 21 patients (33%) had PTMS: 13 patients had a single heavy P; 5 patients had 2 P (only one of which was heavy); and 3 patients had 2 heavy peaks. No PTMs were noted in our MM controls. The m/z of all typical and heavy P observed at aPCD remained unchanged for 63 patients as they transitioned to AL, which suggests no new PTMs occurred with the exception of one patient, who had a single PTM at aPCD diagnosis, but developed 2 additional heavy P appearing at +1232 and +1392 Dalton at AL diagnosis, suggestive of new PTMs. In all but 3 aPCD, the typical P was more abundant relative to heavy P. Heavy P abundance increased in 6 patients as they transitioned from the aPCD to the AL state and exceeded that of the typical P in 3 patients, suggestive of increasing abundance of existing PTMs in these cases. Conclusions: Pre-existing PTMs are present in 1/3 of AL patients and not in MM and can be used to identify patients at risk for AL. New PTMs are uncommon, but the relative abundance can change over time.

AbstractSymptomatic hyperviscosity is a well‐established phenomenon in Waldenström macroglobulinemia (WM). Monoclonal IgM can variably impact intrinsic serum viscosity, leading to widely disparate symptomatic thresholds for development of hyperviscosity‐related symptoms. Data regarding the predictors of symptomatic hyperviscosity and outcomes related to this complication remain scarce and a recent study proposed that IgM >6000 mg/dL be considered a new criterion for initiating therapy in otherwise asymptomatic (smoldering) WM to pre‐empt hyperviscosity‐related injury. Herein, we attempt to identify predictors of the development of symptomatic hyperviscosity and its impact in patients with WM. Of 997 WM patients evaluated from January, 1996 through June, 2017, symptomatic hyperviscosity was observed in 130 (13%) patients. Overall survival (OS) of these 130 patients was similar to that of patients without symptomatic hyperviscosity (median: 11.5 vs 11.6 years; P = 0.63). On multivariate‐analysis, only viscosity >1.8 cp (risk ratio: 4.0, P = 0.02) assessed at the time of WM diagnosis was an independent predictor for the development of subsequent symptomatic hyperviscosity. Among patients with smoldering WM and IgM >6000 mg/dL at diagnosis (n = 13) who were managed expectantly, the median time‐to‐initial therapy was 6.9 years and only 15% developed hyperviscosity‐related symptoms subsequently. In summary, the occurrence of symptomatic hyperviscosity does not impact OS. Serum viscosity at diagnosis of WM, and not IgM concentration, represents the single most important independent predictor for development of subsequent hyperviscosity‐related symptoms. Patients with smoldering WM and high serum IgM can be safely observed in the absence of any indications per the Consensus recommendations to initiate WM‐directed therapy.

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.

AbstractBackgroundSince the first monoclonal antibody (mAb) therapy hit the market in 1996, the number of disorders treated with this class of therapeutics has seen tremendous growth, with over 50 antibody-based therapeutics currently approved for use in the US and Europe. This class of therapeutics recently made profound progress in the treatment of refractory multiple myeloma (MM). Treating MM with the mAbs will challenge the laboratory's ability to differentiate exogenous mAbs being used to treat patients from endogenous mAbs associated with disease.ContentAn overview of the therapeutic mAbs (t-mAbs) developed for the treatment of MM is provided. The anticipated impact of these therapies on patient care, laboratory testing, and clinical research is discussed. Efforts underway to develop strategies and technologies to help laboratories address the growing challenge of mAb interferences are reviewed.SummaryLaboratories can implement risk mitigation strategies at the preanalytical and postanalytical phase of testing to reduce the likelihood of reporting false-positive M-protein results in patients receiving t-mAbs. However, at the analytical phase of testing, current laboratory methods are ill-suited to differentiate between residual disease and residual drugs. Mass spectrometry–based methods might be best positioned to aid laboratories with the rapidly evolving landscape of MM treatment.