Citations

Abstract Abstract 1909 AL amyloidosis is the most common type of systemic amyloidosis. It is caused by extracellular deposition of circulating immunoglobulin light chains (LC) thought to be secreted by a primarily bone marrow based clonal plasma cell proliferative disorder. Although, invariably, the type of LC secreted by the plasma cell clone is identical to the LC deposited in the amyloid plaque, a comprehensive analysis of the re-arranged clonal LC gene coding secreted LC protein, the protein sequence of the serum and urine monoclonal band and the LC protein deposited in the amyloid plaques have not been previously performed. In this study, we compared the protein sequence predicted by the clonal LC gene sequence with the protein sequence identified in the serum and urine monoclonal bands and protein plaques by mass spectrometry (MS) based proteomics. 10 cases with systemic AL amyloidosis were studied. Bone marrow samples containing the clonal plasma cells were obtained and clonal LC variable gene sequences were determined by PCR amplification, cloning and sequencing as described previously (Abraham et al, Blood 2003, 101: 3801). For protein analysis, monoclonal protein bands identified in serum and/or urine protein electrophoresis and immunofixation were isolated from archival gels. For amyloid plaque protein analysis, the plaques were microdissected from paraffin embedded tissue. To identify the protein amino acid sequence of serum and urine bands and amyloid plaques, the samples were digested into peptides by trypsin and analyzed by MS based proteomic analysis. (see Vrana et al. and Blood 2009; 114: 4957 for methods). The results for amino acid sequence predicted from the clonal LC gene sequence, determined directly by MS from the serum and/or urine monoclonal band and amyloid plaques were then aligned and compared. In all 10 cases, the LC amino acid sequences predicted by sequencing of the clonal LC gene from bone marrow plasma cells, the amino acid sequence obtained directly from the monoclonal serum and/or urine protein and the amino acid sequence obtained from the amyloid plaque were identical. In most cases, both the complete variable region and constant regions were deposited as amyloid plaques. Some amyloid plaques showed cleavage of first 16 amino acids of the variable region suggesting that this part was not required for amyloid deposition. All LC variable region gene sequences were affected by somatic hypermutation, and identical mutations were present in both the serum and urine bands and in the amyloid plaques. In conclusion, these results, for the first time, comprehensively show that the monoclonal protein secreted by the neoplastic plasma cells is the pathogenetic protein causing the amyloid deposits in AL amyloidosis. Amyloid deposits contain virtually all of the LC protein identified in the serum or urine including both variable and constant regions, suggesting that the presence of the constant region may also play a role in amyloidogenesis. The technological capability to determine the complete protein sequence of the amyloidogenic monoclonal serum and urine LC protein isolated from archived gels will create new opportunities to develop diagnostic, predictive and prognostic serum or urine based tests for amyloidosis. Disclosures: Theis: Mayo Clinic: Patents & Royalties. Vrana:Mayo Clinic: Patents & Royalties. Dogan:Mayo Clinic: Patents & Royalties.

Abstract BACKGROUND: Complement-mediated thrombotic microangiopathies (TMAs) are a subset of TMAs in which the thrombocytopenia, microangiopathic hemolytic anemia, and end organ damage are caused by mutations in genes encoding the alternative complement pathway. Numerous complement genes have been implicated in complement-mediated TMAs including complement factor H (CFH), CD46 (MCP), complement factor I (CFI), complement component 3 (C3), complement factor B (CFB), factor H related 5 (CFHR5), and thrombomodulin (THBD) among others. Genetic analysis typically yields a mutation in 60% of patients whose TMA is presumed to be mediated by complement dysregulation. However, the description of novel disease-associated variants may increase this proportion. METHODS: A retrospective study of patients with TMAs diagnosed between 2000 and 2014 was performed. TMA diagnosis was made based on thrombocytopenia and evidence of microangiopathic hemolytic anemia. Analysis was performed with Alamut¨ software with additional in silico prediction tools (SIFT, MutationTaster, and Polyphen) for classification of gene variants. Variants of unknown significance (VUS) and likely pathogenic variants were further assessed using several mutation databases, including HGMD, ClinVar, and Factor H database RESULTS: Of patients diagnosed with a TMA, genetic analysis was performed in only a 10% of patients. Of the 29 patients with genetic studies performed, mutations were identified in 18 patients (62%). The majority of the mutations had been described previously in the literature, but four novel variants were identified: three missense and one splice-site. The table below summarizes these variants as well as laboratory findings on presentation. These were two variants of CFH, one variant of CFHR5, and one variant of CFI. In silico modeling of these variants revealed two polymorphisms likely to be pathogenic, one polymorphism likely benign given the lack of predicted splicing changes, and one VUS. Table 1. Protein Mutation Classification Age Sex Hemoglobin (g/dL) Platelets (thousands) Creatinine (mg/dL) CFH c.245-10_245-9dup Likely benign 61 F 11.6 51 6.7 CFH c.476G>A, p.Ser159Asn Likely pathogenic 43 F 9.1 101 9.1 CFHR5 c.1412G>A p.Gly471Glu Likely pathogenic 30 F 9.8 129 4.9 CFI c.1190T>A p.Val397Glu Unknown significance 51 M 9.4 125 6.1 DISCUSSION: With therapy available to target the alternative complement pathway, genetic analysis to identify genetic variants capable of causing complement mediated TMAs is an essential part of the evaluation. This genetic data must be interpreted and correlated with functional analysis and clinical phenotype. The reporting of novel variants in clinical databases, with inclusion of relevant clinical findings, is necessary to accurately classify and verify variants as pathologic mutations or benign polymorphisms. The full understanding of this diverse disease requires a more complete understanding of its genetics. While complement pathway-directed therapies are available, their rational use requires thorough interpretation of laboratory data, including genetic analysis. Disclosures Murray: Mayo Clinic: Patents & Royalties: Patent Application Filed.

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.

SummaryThis study evaluated the differences in clinical features of 1077 newly diagnosed AL amyloidosis patients with renal involvement (n = 229, 21%), both cardiac and renal involvement (n = 443, 41%) and cardiac involvement (n = 405, 38%). Significant differences in dFLC (difference in involved and uninvolved light chains) were noted (renal, both, cardiac median: 83, 234 and 349 mg/l, P < 0.001). The proportion of patients with ≥ 10% bone marrow plasma cells (BMPCs) was lowest in renal only patients: 44%, 57%, 64%, respectively, P < 0.001. In a multivariate linear regression model incorporating organ involvement type and BMPCs ≥10%, organ involvement was a significant predictor of dFLC (P < 0.001). Median overall survival (OS) across the three groups was 83 vs. 19 vs. 16 months (P < 0.001) in patients not undergoing transplant and 5‐year OS in patients undergoing transplant was 90% vs. 75% vs. 64% (P = 0.007), respectively. In conclusion, renal involvement alone or renal + cardiac involvement in AL amyloidosis is associated with lower circulating light chain burden, which cannot be fully explained by BMPC burden alone. Increased sensitivity of the kidney to light chains, given significant interactions with the renal tubular system and secretion of modified light chain products may play a role in pathogenesis of renal AL amyloidosis and warrants further investigation.

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.