Spencer, Brian

ABSTRACTBackgroundAdvances in fibril typing by mass spectrometry have improved the accuracy of amyloidosis diagnosis. Dual amyloidogenic proteins have been reported in deposits in sole and multiple different organs.MethodsFive patients with dual amyloidoses were diagnosed between 1995 and 2022 by Congo red staining and fibril typing using the best available methods at the time of evaluation. Sequencing of TTR and GSN genes was performed. Literature search identified 46 additional cases.ResultsThree patients exhibited Waldenström macroglobulinemia‐associated AL (n = 3) amyloidoses in conjunction with ATTRwt or AGel amyloidosis; two patients featured AL/ATTRwt and AA/ATTRwt amyloidoses. One patient demonstrated dual amyloidoses within one anatomical site; three patients featured two amyloidosis types at different anatomical sites; and one patient had dual amyloid deposits in a single anatomical site along with different sites. The time interval between diagnoses was 0–288 months, with the heart and kidneys being the most affected organs.ConclusionsOur findings underscore the complexity of clinical presentation in amyloidosis, as multiple amyloid types can co‐exist in a single individual and affect various anatomical sites. Accurate assessment of the clinical phenotype and thorough amyloid fibril typing from the target organs are essential for precise diagnosis and tailored treatment. Trial Registration: ClinicalTrials.gov Identifier: NCT00898235

Light chain amyloidosis (AL) is a rare, progressive, and often fatal protein misfolding disease. AL is caused by aberrant clonal plasma cell proliferation, where excess amounts of amyloidogenic monoclonal Free Light Chain (FLC) are produced, secreted into the circulation, misfolded and deposted as amyloid fibrils in various vital organs, such as the heart, kidney, liver, and nerves. The involed lambda (λ) FLC accounts for ~75% of all AL cases. Amyloidogenic FLCs (aFLCs) are believed to be the toxic species contributing to AL disease pathology. Specific quantification of aFLCs can provide critical guidance in clinical practice but has been hampered by the LCs sequence diversity as well as the lack of tests that are able to differentiate between circulating normal polyclonal FLCs and aFLCs. Contrary to non-amyloidogenic, aFLCs are kinetically and oftentimes thermodynamically unstable. aFLCs are prone to misfolding and formation of pathogenic conformations such as non-native structures, proteolytic fragments, soluble aggregates, and amyloid fibrils. We exploited such characteristics of aFLCs by applying limited proteolysis using Proteinase K, where less stable λ aFLC proteins readily yield a 23KDa fragment composed of the homodimeric LC constant domain (dLCCD) linked by a disulfide bond. Formation of the dLCCD fragment reveals a highly conserved, novel, cryptic epitope at the N-terminus of the fragment. We have generated monoclonal antibodies (mAbs) that explicitly bind to such novel epitope and developed immunoassays for the specific detection and sensitive quantification of λ aFLCs in biological samples. Western blot or Meso Scale Discovery (MSD) analyses were employed to detect and quantify dLCCD signal using the dLCCD mAbs on plasma samples from randomly selected, treatment naïve and λ-restricted cases with biopsy-proven AL amyloidosis before and after exposure to optimized proteolytic conditions. Tissue amyloid deposits of λ-type were confirmed in most AL cases. Informed consent for data and plasma sample collections were obtained from the Institutional Review Boards and in accordance with the Declaration of Helsinki. Multiple myeloma (MM) plasma samples of unknown LC restriction type, treatment or amyloid deposits status and normal volunteer control samples were purchased from BioIVT (USA). A set of representative results based on three runs are shown in Fig. 1. As little as 2 µL of biofluid was used for each assay. Upon limited proteolysis, dLCCD signal was detected in 32 of 35 AL plasmas, including two samples from patients with AL-associated IgM lymphoplasmacytic lymphoma (LPL). Of the three dLCCD negative AL samples, one presented with monoclonal IgAλ, and two cases were bi-clonal on serum immunofixation electrophoresis. dLCCD signal was not detected in 18 of 19 MM plasma samples. Similarly, dLCCD signal was undetectable in 23 normal plasmas. Overall, the dLCCD signals detected in AL plasmas were 10x-200x above that of normal control and most MM samples (Fig. 1). The low limit of quantification of the MSD assay was ~0.4 ng/dL (1.4 pM), with >500x dynamic range, enabling sensitive and robust quantification of λ aFLCs in biosamples. Analysis of Proteinase K recognition potential of the cleavage site of 389 λ LC sequences from the AL-Base, a database of amyloidogenic LC sequences (https://wwwapp.bumc.bu.edu/BEDAC_ALBase/) predicted that the dLCCD assay can detect λ aFLCs in up to 99% of λ AL patients, irrespective of the FLC sequences. This was confirmed with the 100% detection rate of the dLCCD signal in a validation cohort of 35 additional monoclonal λ-restricted AL samples. In summary, we have developed a quantitative blood-based immunoassay that is sensitive and highly specific for λ aFLC irrespective of the protein sequences. The preliminary data demonstrates the potential of our assay for AL diagnostics. Additional clinical applications for future exploration will include (i) monitoring and early detection of AL amyloidosis in plasma cell dyscrasias (monoclonal gammopathy of unknown significance, MM, and smoldering MM) and lymphoproliferative disorders, including LPL; and (ii) clinical management of patients with AL amyloidosis via the assessment and monitoring of minimal residual disease and therapeutic responses. The utility of the dLCCD assay in the quantification of λ aFLCs in bi-clonal AL cases warrants further investigation.

Significance We identified a one-of-a-kind duplication mutation in human transthyretin (TTR) that causes unusually aggressive systemic amyloidosis. To understand the poor response to treatment with a drug that stabilizes the TTR tetramer, we explored the structure, stability, and drug binding of recombinant proteins. The results suggested that amyloid formation could stem from global destabilization of the monomeric and tetrameric protein as well as the local disordering near the mutation site. This disordering induced proteolysis with release of aggregation-prone fragments. Alternatively, local disordering could trigger misfolding of the full-length protein by exposing an adhesive segment. Drug binding at a dimer interface distant from the mutation site did not significantly influence these pathological processes, indicating the need for alternative therapeutic targets.