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Immunoglobulin light chain (LC) amyloidosis is a life-threatening disease whose understanding and treatment is complicated by vast numbers of patient-specific mutations. To address molecular origins of the disease, we explored 14 patient-derived and engineered proteins related to κ1-family germline genes IGKVLD-33*01 and IGKVLD-39*01. Hydrogen-deuterium exchange mass spectrometry analysis of local conformational dynamics in full-length recombinant LCs and their fragments was integrated with studies of thermal stability, proteolytic susceptibility, amyloid formation, and amyloidogenic sequence propensities using spectroscopic, electron microscopic and bioinformatics tools. The results were mapped on the atomic structures of native and fibrillary proteins. Proteins from two κ1 subfamilies showed unexpected differences. Compared to their germline counterparts, amyloid LC related to IGKVLD-33*01 was less stable and formed amyloid faster, whereas amyloid LC related to IGKVLD-39*01 had similar stability and formed amyloid slower. These and other differences suggest different major factors influencing amyloid formation. In 33*01-related amyloid LC, these factors involved mutation-induced destabilization of the native structure and probable stabilization of amyloid. The atypical behaviour of 39*01-related amyloid LC tracked back to increased dynamics/exposure of amyloidogenic segments in βC’Vand βEVthat could initiate aggregation, combined with decreased dynamics/exposure near the Cys23-Cys88 disulfide whose rearrangement is rate-limiting to amyloidogenesis. The results suggest distinct amyloidogenic pathways for closely related LCs and point to the antigen-binding, complementarity-determining regions CDR1 and CDR3, which are linked via the conserved internal disulfide, as key factors in amyloid formation by various LCs.

The amyloidogenic transthyretin (TTR) variant, V122I, occurs in 4% of the African American population and frequently presents as a restricted cardiomyopathy. While heterozygosity for TTR V122I predominates, several compound heterozygous cases have been previously described. Herein, we detail features of ATTRv amyloidosis associated with novel compound heterozygous TTR mutation, T60I/V122I and provide evidence supporting the amyloidogenecity of T60I.A 63-year-old African American female presented with atrial fibrillation, congestive heart failure, autonomic and peripheral neuropathy.Congophilic deposits in a rectal biopsy were immunohistochemically positive for TTR. Serum screening by isoelectric focussing revealed two TTR variants in the absence of wild-type protein. DNA sequencing identified compound heterozygousThis report provides clinical and experimental results supporting the amyloidogenic nature of a novel TTR T60I variant.

In ATTR amyloidosis, transthyretin (TTR) protein is secreted from the liver and deposited as toxic aggregates at downstream target tissues. Despite recent advancements in treatments for ATTR amyloidosis, the mechanisms underlying misfolded TTR-mediated cellular damage remain elusive. In an effort to define early events of TTR-associated stress, we exposed neuronal (SH-SY5Y) and cardiac (AC16) cells to wild-type and destabilized TTR variants (TTRV122I and TTRL55P) and performed transcriptional (RNAseq) and epigenetic (ATACseq) profiling. We subsequently compared TTR-responsive signatures to cells exposed to destabilized antibody light chain protein associated with AL amyloidosis as well as ER stressors (thapsigargin, heat shock). In doing so, we observed overlapping, yet distinct cell type- and amyloidogenic protein-specific signatures, suggesting unique responses to each amyloidogenic variant. Moreover, we identified chromatin level changes in AC16 cells exposed to mutant TTR which resolved upon pre-incubation with kinetic stabilizer tafamidis. Collectively, these data provide insight into the mechanisms underlying amyloid-mediated cellular damage and provide a robust resource representing cellular responses to aggregation-prone proteins and ER stress.

Background Transthyretin amyloidosis cardiomyopathy (ATTR-CM) is an underappreciated cause of heart failure that results from misfolded TTR (prealbumin) protein. Diflunisal is an approved non-steroidal anti-inflammatory drug that stabilizes TTR, with limited data available regarding effects on cardiac structure and function. Methods and Results ATTR-CM patients (n=81, 41% treated with 250 mg twice-daily diflunisal by clinical practice) were retrospectively identified with baseline and follow-up (median interval 1 year) serum biomarker and echocardiographic data compared, including global longitudinal strain (GLS). Chi-squared and Wilcoxon tests assessed differences between subjects, divided by treatment group, and univariable and multivariable linear regression was performed. At baseline, patients treated with diflunisal were younger (68 vs 77 years, P = .0001), with lower B-type natriuretic peptide (BNP; 249 vs 545 pg/mL, P = .009) and serum creatinine (1.1 vs 1.2 mg/dL, P = .04), but similar TTR concentration (P = .31), cardiac troponin I (P = .06), and GLS (P = .67). At follow-up, diflunisal untreated versus treated patients showed differences in TTR concentration (19 vs 33 mg/dL, P = .01) and favorable differences in left atrial volume index (+4.6 vs −1.4 mL/m2, P = .002) and cardiac troponin I (+0.03 vs −0.01 ng/mL, P = .01) for the entire cohort. Among the subset with wild-type ATTR (n=53), diflunisal treatment was associated with differences in GLS (+1.2% untreated vs +0.1% treated, P = .03). Changes in wall thickness (P = .2), left ventricular ejection fraction (P = .71), and BNP (P = .42) were similar between groups. Conclusions In ATTR-CM, diflunisal treatment resulted in measurable differences in some parameters of cardiac structure and function after only 1 year of administration. Further longer-term analysis is warranted.

Symptomology of AL amyloidosis can be vague, with a broad range of manifestations and potential etiologies. We sought to determine whether time from initial patient-reported symptom onset to diagnosis was associated with survival.The Boston University Amyloidosis Patient Database was queried for patients with AL amyloidosis who presented to the Center for initial evaluation from 2010 to 2015.A total of 324 patients with AL amyloidosis were evaluated for initial evaluation. The median time to diagnosis from initial symptom onset was 7.1 months (range, 0-61). At data cutoff, 60.2% (n = 195) of patients were alive; of those, the majority were diagnosed6 months from initial symptoms (52.3%, n = 102). In contrast, time to diagnosis from symptom onset was6 months in 63.6% (n = 82) of patients who did not survive at the time of data cutoff (P = .0005). Survival analysis of time from diagnosis to death or data cutoff stratified by time from patient-reported symptom onset to diagnosis (6, 6-12, and12 months) showed significant differences among groups (P = .001). Additionally, multivariable regression demonstrated that an increase in time from self-reported symptom onset to diagnosis was significantly associated with an increased risk of death (HR = 1.02, 95% CI = 1.01-1.04, P = .002).These results support the importance of early diagnosis for patients with AL amyloidosis.

β2-microglobulin amyloidosis was first described in the 1980s as a protein deposition disease associated with long-term haemodialysis. More recently, two inherited forms resulting from separate point mutations in the β2-microglobulin gene have been identified. In this report, we detail a novel β2M variant, P32L, caused by a unique dinucleotide mutation that is linked to systemic hereditary β2-microglobulin amyloidosis. Three family members from a Portuguese kinship featured cardiomyopathy, requiring organ transplantation in one case, along with soft tissue involvement; other involvements included gastrointestinal, neuropathic and sicca syndrome. In vitro studies with recombinant P32L, P32G, D76N and wild-type β2-microglobulin were undertaken to compare the biophysical properties of the proteins. The P32L variant was caused by the unique heterozygous dinucleotide mutation c.154_155delinsTT. Amyloid disease featured lowered serum β2-microglobulin levels with near equal amounts of circulating P32L and wild-type proteins; amyloid deposits were composed exclusively of P32L variant protein. In vitro studies of P32L demonstrated thermodynamic and chemical instability and enhanced susceptibility to proteolysis with rapid formation of pre-fibrillar oligomeric structures by N- and C-terminally truncated species under physiological conditions. This work provides both clinical and experimental evidence supporting the critical role of P32 residue replacement in β2M amyloid fibrillogenesis.

Objective: To characterize the changing spectrum of amyloidosis classes, as well as patient demographics, at a major US referral centre. Patients and methods: A retrospective analysis was conducted of all referrals to the Amyloidosis Centre at Boston University and Boston Medical Centre over the last 3 decades. Results: A total of 3987 new patients with amyloidosis were evaluated between 1990 and 2018 with the average number of new cases per year increasing 2.5-fold during this period. Systemic immunoglobulin light-chain (AL) amyloidosis decreased in proportion with each decade from 77% to 69% to 50% of new cases. Meanwhile, ATTR amyloidosis increased from 12% to 16% to 29%, predominately due to more diagnosis of ATTRwt and ATTRV122I amyloidosis. Gender and race profile differences, while changing over the observed time period, persisted among amyloidosis patients. Conclusion: Amyloid diseases are more widely recognized and classes of amyloidosis, including ATTRwt and ATTRV122I, once considered rare are now increasingly diagnosed. These data likely reflect a national trend of increased amyloidosis awareness facilitated by accessible diagnostic approaches, emerging treatments, and coordinated educational initiatives. ClinicalTrials.gov identifier: NCT00898235.

Cardiac involvement in systemic amyloidosis (AL) occurs in ~50% of all AL patients. However once symptomatic heart failure develops, therapeutic options are limited thereby conferring a poor overall prognosis. The median survival is treatment-naïve, AL cardiac amyloidosis with heart failure. Patients with biopsy-proven AL cardiac amyloid, who presented with heart failure and did not received prior AL treatment, were enrolled between 2004–2014, at the initial visit to the Amyloidosis Center at Boston University Medical Center. Routine laboratory tests, physical examination and echocardiography data were collected. There were 165 predominantly white (76.4%), and male (61%) patients, with a mean age of 61.6 ± 9.5 years. Median survival was 10.9 months (95% CI 6.2–14.7). By multivariate analysis increased relative wall thickness (RWT) [HR 6.70; 95% CI 2.45–18.30), older age (HR 1.04; 95% CI 1.01–1.06), higher New York Heart Association (NYHA) functional class (HR 1.50; 95% CI 1.02–2.2), log brain natriuretic peptide (BNP) levels (HR 1.45; 95% CI 1.15–1.81) and C-reactive protein (CRP) levels (HR 1.02; 95% CI 1.00–1.04) were significant predictors for increased mortality. In conclusion, in treatment-naïve, AL cardiac amyloidosis patients with heart failure symptoms who lack these high-risk features may have a better outcome. These findings might allow for better risk stratification although outcomes are still poor.

The recent decades have ushered in considerable advancements in the diagnosis and treatment of systemic light chain (AL) amyloidosis. As disease outcomes improve, AL amyloidosis-unrelated factors may impact mortality. In this study, we evaluated survival trends and primary causes of death among 2337 individuals with AL amyloidosis referred to the Boston University Amyloidosis Center. Outcomes were analyzed according to date of diagnosis: 1980-1989 (era 1), 1990-1999 (era 2), 2000-2009 (era 3), and 2010-2019 (era 4). Overall survival increased steadily with median values of 1.4, 2.6, 3.3, and 4.6 years for eras 1–4, respectively (P 10 years after diagnosis). Under changing standards of care, survival improved and early mortality declined over the last 40 years. These findings support a more optimistic outlook for patients with AL amyloidosis.

Introduction: Hereditary transthyretin amyloid cardiomyopathy (pV142I hATTR-CM) is an under-recognized cause of heart failure (HF) in elderly patients of African origin. While we have previously demonstrated that the endogenous TTR ligand retinol binding protein 4 (RBP4) identifies patients with hATTR-CM, its capacity to predict outcomes is unexplored. Further, comparative data are lacking describing clinical outcomes in hATTR-CM vs. matched patients with HF. Hypothesis: Reduced survival is associated with pV142I hATTR-CM when compared to an age, race, sex matched patients with HF. Methods: Retrospective, single center, cohort study of self-reported African American patients ≥ 60 years of age with normal TTR genotype, HF, and LV wall thickness ≥ 12mm (n=84) were compared to a cohort with biopsy-proven pV142I hATTR-CM (n=30). Patients did not receive ATTR-specific therapies, as study occurred prior to FDA approvals. Baseline laboratory values were measured. All-cause mortality was estimated and compared between groups based on a median followup of 45.4 months. Analyses were conducted with Fisher Exact, Kruskal Wallis, and Kaplan-Meier with log-rank testing. Results: hATTR-CM patients were slightly older but less likely to have concomitant HTN or DM (Table 1). Baseline RBP4 concentration was lower while troponin level higher in patients with hATTR-CM (Table 1). hATTR-CM patients had a higher hazard of all-cause mortality after adjusting for age (HR=3.5, 95% CI: 1.8 – 6.7, p Conclusions: Patients pV142I hATTR-CM demonstrate significantly reduced survival (a 3.5-fold increased risk of death, 43% 4-year survival) compared to control subjects with HF in the absence of amyloidosis. Baseline RBP4 concentration did not associate with survival. These data suggest that hATTR-CM recognition is essential to permit implementation of available therapies that can improve survival.

Wild-type transthyretin-associated (ATTRwt) amyloidosis is an age-related disease that causes heart failure in older adults. This disease frequently features cardiac amyloid fibril deposits that originate from dissociation of the tetrameric protein, transthyretin (TTR). Unlike hereditary TTR (ATTRm) amyloidosis, where amino acid replacements destabilize the native protein, in ATTRwt amyloidosis, amyloid-forming TTR lacks protein sequence alterations. The initiating cause of fibril formation in ATTRwt amyloidosis is unclear, and thus, it seems plausible that other factors are involved in TTR misfolding and unregulated accumulation of wild-type TTR fibrils. We believe that clusterin (CLU, UniProtKB P10909), a plasma circulating glycoprotein, plays a role in the pathobiology of ATTRwt amyloidosis. Previously, we have suggested a role for CLU in ATTRwt amyloidosis based on our studies showing that (1) CLU codeposits with non-native TTR in amyloid fibrils from ATTRwt cardiac tissue, (2) CLU interacts only with non-native (monomeric and aggregated) forms of TTR, and (3) CLU serum levels in patients with ATTRwt are significantly lower compared to healthy controls. In the present study, we provide comprehensive detail of compositional findings from mass spectrometry analyses of amino acid and glycan content of CLU purified from ATTRwt and control sera. The characterization of oligosaccharide content in serum CLU derived from patients with ATTRwt amyloidosis is novel data. Moreover, results comparing CLU oligosaccharide variations between patient and healthy controls are original and provide further evidence for the role of CLU in ATTRwt pathobiology, possibly linked to disease-specific structural features that limit the chaperoning capacity of CLU.

In marked contrast to multiple myeloma, racial/ethnic minorities are underrepresented in publications of systemic light-chain (AL) amyloidosis. The impact of race/ethnicity is therefore lacking in the narrative of this disease. To address this gap, we compared disease characteristics, treatments, and outcomes across racial/ethnic groups in a referred cohort of patients with AL amyloidosis from 1990 to 2020. Among 2416 patients, 14% were minorities. Non-Hispanic Blacks (NHBs) comprised 8% and had higher-risk sociodemographic factors. Hispanics comprised 4% and presented with disproportionately more BU stage IIIb cardiac involvement (27% vs. 4–17%). At onset, minority groups were younger in age by 4–6 years. There was indication of more aggressive disease phenotype among NHBs with higher prevalence of difference between involved and uninvolved free light chains >180 mg/L (39% vs. 22–33%, P = 0.044). Receipt of stem cell transplantation was 30% lower in Hispanics compared to non-Hispanic White (NHWs) on account of sociodemographic and physiologic factors. Although the age/sex-adjusted hazard for death among NHBs was 24% higher relative to NHWs (P = 0.020), race/ethnicity itself did not impact survival after controlling for disease severity and treatment variables. These findings highlight the complexities of racial/ethnic disparities in AL amyloidosis. Directed efforts by providers and advocacy groups are needed to expand access to testing and effective treatments within underprivileged communities.

© 2020 American Chemical Society. Structural characterization of misfolded protein aggregates is essential to understanding the molecular mechanism of protein aggregation associated with various protein misfolding disorders. Here, we report structural analyses of ex vivo transthyretin aggregates extracted from human cardiac tissue. Comparative structural analyses of in vitro and ex vivo transthyretin aggregates using various biophysical techniques revealed that cardiac transthyretin amyloid has structural features similar to those of in vitro transthyretin amyloid. Our solid-state nuclear magnetic resonance studies showed that in vitro amyloid contains extensive nativelike β-sheet structures, while other loop regions including helical structures are disrupted in the amyloid state. These results suggest that transthyretin undergoes a common misfolding and aggregation transition to nativelike aggregation-prone monomers that self-assemble into amyloid precipitates in vitro and in vivo.

AIMS Wild-type transthyretin (ATTRwt) cardiac amyloidosis has emerged as an important cause of heart failure in the elderly. Atrial fibrillation (AF) commonly affects older adults with heart failure and is associated with reduced survival, but its role in ATTRwt is unclear. We sought to explore the clinical impact of AF in ATTRwt. METHODS AND RESULTS Patients with biopsy-proven ATTRwt cardiac amyloidosis (n = 146) were retrospectively identified, and clinical, echocardiographic, and biochemical data were collected. Patients were classified as AF or non-AF and followed for survival for a median of 41.4 ± 27.1 months. Means testing, univariable, and multivariable regression models were employed. A systematic review was performed. AF was observed in 70% (n = 102). Mean age was similar (AF, 75 ± 6 vs. non-AF, 74 ± 5 years, P = 0.22). Anticoagulant treatment of patients with AF was as follows: 78% warfarin, 17% novel anticoagulant, and 6% no anticoagulation. Amiodarone was prescribed to 24%. There were no differences in left ventricular ejection fraction (P = 0.09) or left atrial volume (P = 0.87); however, mean diastolic dysfunction grade was higher in AF (mean 2.7 ± 0.5 vs. 2.4 ± 0.5, P = 0.01). While creatinine (P = 0.52) and B-type natriuretic peptide (P = 0.48) were similar, patients with AF had lower serum transthyretin concentrations (221 ± 51 vs. 250 ± 52 μg/mL, P

Immunoglobulin light chain (LC) amyloidosis (AL) is a life-threatening human disease wherein free mono-clonal LCs deposit in vital organs. To determine what makes some LCs amyloidogenic, we explored patient-based amyloidogenic and non-amyloidogenic recombinant LCs from the λ6 subtype prevalent in AL. Hydrogen-deuterium exchange mass spectrometry, structural stability, proteolysis, and amyloid growth studies revealed that the antigen-binding CDR1 loop is the least protected part in the variable domain of λ6 LC, particularly in the AL variant. N32T substitution in CRD1 is identified as a driver of amyloid formation. Substitution N32T increased the amyloidogenic propensity of CDR1 loop, decreased its protection in the native structure, and accelerated amyloid growth in the context of other AL substitutions. The destabilizing effects of N32T propagated across the molecule increasing its dynamics in regions ∼30 A away from the substitution site. Such striking long-range effects of a conservative point substitution in a dynamic surface loop may be relevant to Ig function. Comparison of patient-derived and engineered proteins showed that N32T interactions with other substitution sites must contribute to amyloidosis. The results suggest that CDR1 is critical in amyloid formation by other λ6 LCs.

Background: Natural history studies, which describe changing disease outcomes under real-world clinical practice, can help support drug development for rare diseases by defining appropriate endpoints for clinical trials. The current study provides natural history information on survival and mortality in light chain (AL) amyloidosis, a rare disease that was historically considered inevitably fatal. With the recent expansion of effective therapeutics, patients with this disease are living longer, which in turn necessitates a better understanding of the factors leading to death in later disease course. Aims: To evaluate (1) trends in overall survival (OS) over time and (2) primary causes of death across the course of AL amyloidosis disease. Methods: We identified 2,337 patients diagnosed with systemic AL amyloidosis between 1980 and 2019 from the prospectively maintained database at the Boston University Amyloidosis Center (ClinicalTrials.gov Identifier: NCT00898235). Disease outcomes were analyzed according to date of tissue diagnosis: 1980-1989 (era 1, n = 185), 1990-1999 (era 2, n = 575), 2000-2009 (era 3, n = 865) and 2010-2019 (era 4, n =712). Survival information was verified through yearly clinical evaluations, contact by phone/letter for patients who did not return for follow-up, or the U.S. Social Security Death Index if contact was not established. We defined deaths as AL amyloidosis-related when clinical data indicated organ progression or complications from plasma cell-directed therapy. Deaths occurring while in remission, off treatment and without evidence of organ progression were considered disease-unrelated. Results: OS increased steadily across eras 1-4 with median values of 1.4, 2.6, 3.3 and 4.6 years, respectively (P < .001). Six-month mortality decreased over time from 23% to 13%. Notably, median age at diagnosis increased from 59 to 63 years (P < .001), and time interval to diagnosis from patient-reported symptom onset shortened from 10 months to 6 months (P = .065). At data cutoff (October 2020), 1,660 (71%) patients had died. Primary cause of death was ascertainable for 1,160 (70%) cases. Organ failure was most common, accounting for 564 (49%) deaths, amongst which cardiac failure predominated. Sudden unexpected death was the next most frequent cause, contributing to 266 (23%) deaths. Together, cardiac failure and sudden death decreased in proportion with longer survival from diagnosis, representing 67% (236/354) of deaths occurring within ≤6 months; 56% (322/575) within >6 months to ≤5 years; 36% (54/151) within >5 years to ≤10 years; and 36% (29/80) after >10 years (P < .001). Meanwhile, renal failure and infections emerged as important causes of later-occurring deaths. There was sufficient data on 1,243 (75%) deaths to assign relation to AL amyloidosis. The vast majority were disease-related. Yet, disease-unrelated deaths increased with longer survival from diagnosis, accounting for 2% (9/373) of deaths occurring within ≤6 months; 8% (48/616) within >6 months to ≤5 years; 16% (25/161) within >5 years to ≤10 years; and 29% (27/93) after >10 years (P < .001). Conclusions: Under changing standards of care, OS improved and early mortality declined over the last 40 years, supporting a much-improved outlook for current and future patients with AL amyloidosis. Cardiac failure and sudden deaths decreased across the course of disease. Even so, progression of amyloidosis remained a top cause of death even among long-term survivors. Figure 1 Figure 1. Disclosures Sanchorawala: Takeda: Research Funding; Celgene: Research Funding; Prothena: Membership on an entity's Board of Directors or advisory committees, Research Funding; Caelum: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Regeneron: Membership on an entity's Board of Directors or advisory committees; Proclara: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; Karyopharm: Research Funding.

Transthyretin (TTR), normally a plasma circulating protein, can become misfolded and aggregated, ultimately leading to extracellular deposition of amyloid fibrils usually targeted to heart or nerve tissues. Referred to as TTR-associated amyloidoses (ATTR), this group of diseases is frequently life threatening and fatal if untreated. ATTR, caused by amyloid-forming variant TTR proteins (ATTRm) that arise from point mutations in the TTR gene, were classically referred to as familial amyloid cardiomyopathy (FAC) or familial amyloid polyneuropathy (FAP), reflecting the clinical phenotype. FAC and FAP are pathologies that can be challenging to diagnose as there are no definitive biomarkers of disease; moreover, disease-specific measures of progression are lacking, and treatment options are limited. Thus, the discovery of sensitive and specific indicators of disease has the potential to improve recognition, enable accurate measurement of amyloid progression and response to treatment, and reveal key information regarding FAC and FAP pathobiological mechanisms. In this study, the goal was to investigate serum proteomic features unique to FAC and FAP types of ATTRm. Multiple-reaction monitoring mass spectrometry (MRM-MS), a powerful technique in profiling proteomes, was used to measure the serum concentrations of 160 proteins in samples from FAC and FAP patients. Results were compared to data from healthy control sera obtained from individuals matched to age (≥60 years), gender (male), and race (Caucasian). Proteomic analyses of ATTRm (FAC and FAP) and control samples showed significant concentration differences in 107 of 192 (56%) of the serum proteins that were studied. In comparing FAC to FAP, differences in concentrations as well as interactions and functions of several proteins were identified as unique to each disease; significantly lower levels of TTR were specific to FAC, but not to FAP. Annotated functional clustering identified extracellular region, signal, and signal peptide as terms common to FAC and FAP. Conversely, disulfide bond was unique to FAC; secreted, glycosylation site: N-linked, glycosylation, glycoprotein, polymorphism, and sequence variant were associated solely with FAP. Predicted protein-protein associations in FAC were seen for reaction, binding, and activation processes; no associations were found in FAP. This study demonstrates significant proteomic differences between ATTRm patient and control sera, as well as ATTRm phenotype-associated variations in the circulating levels of several proteins including TTR. The identification of serum proteins unique to FAC and FAP may have diagnostic and prognostic utility and could possibly provide important clues about disease mechanisms.

Immunoglobulin (Ig) light chain (LC) amyloidosis (AL) is characterized by the overproduction and tissue deposition of monoclonal LC in various organs and tissues. The plasma circulating monoclonal LC is believed to be the precursor of the deposited protein and in vitro studies aimed at understanding AL pathobiology have mainly focused on LC and its variable domain. While 33% of patients have free circulating monoclonal LC, ∼40% feature LC complexed to heavy chain (HC) forming a monoclonal intact Ig; the significance of free vs. bound LC in the amyloid forming pathway is unknown. To address this issue, we developed a cell-based model using stable mouse plasmacytoma Sp2/0 cells that co-express patient-derived amyloidogenic LC and HC proteins. The system was designed using amyloidogenic kappa and lambda LC, and gamma HC sequences; stable production and secretion of either free LC and/or intact Ig were accomplished by varying the LC to HC ratios. This novel cell-based system provides a relevant tool to systematically investigate LC and HC interactions, and the molecular events leading to the development of AL amyloidosis.

Summary The systemic amyloidoses are diverse disorders in which misfolded proteins are secreted by effector organs and deposited as proteotoxic aggregates at downstream tissues. Although well described clinically, the contribution of synthesizing organs to amyloid disease pathogenesis is unknown. Here, we utilize hereditary transthyretin amyloidosis (ATTR amyloidosis) induced pluripotent stem cells (iPSCs) to define the contribution of hepatocyte-like cells (HLCs) to the proteotoxicity of secreted transthyretin (TTR). To this end, we generated isogenic, patient-specific iPSCs expressing either amyloidogenic or wild-type TTR. We combined this tool with single-cell RNA sequencing to identify hepatic proteostasis factors correlating with destabilized TTR production in iPSC-derived HLCs. By generating an ATF6 inducible patient-specific iPSC line, we demonstrated that enhancing hepatic ER proteostasis preferentially reduces the secretion of amyloidogenic TTR. These data highlight the liver's capacity to chaperone misfolded TTR prior to deposition, and moreover suggest the potential for unfolded protein response modulating therapeutics in the treatment of diverse systemic amyloidoses., Graphical Abstract, Highlights • A singular gene-editing strategy can correct all mutations in ATTR amyloidosis • Mutant TTR iPSC-derived hepatic cells employ ER stress and proteostasis machinery • Hepatic cells of ATTR amyloidosis patients exhibit cellular and molecular changes • ATF6 branch-specific activation of the UPR reduces secretion of proteotoxic TTR, In this article, Giadone and colleagues identify proteostasis factors, including the unfolded protein response (UPR), that drive response to systemic amyloid disease in patient-specific iPSCs. This work demonstrates that stress-independent, selective activation of the adaptive UPR is capable of selectively reducing the secretion of toxic, destabilized protein.

Wild-type transthyretin amyloidosis (ATTRwt) results in an infiltrative cardiomyopathy often culminating in symptomatic heart failure. The use of cardiopulmonary exercise testing (CPET) in determining outcomes in ATTRwt cardiac amyloidosis is unknown. Given the emergence of novel therapies to treat transthyretin amyloidosis, we sought to investigate the utility of CPET on outcomes in patients with ATTRwt cardiomyopathy. Fifty-six patients, with biopsy and immunohistochemically proved ATTRwt, were enrolled between 2005 and 2015, as part of an NIH ATTRwt substudy at the Boston University Amyloidosis Center. Patients were prospectively studied, which included laboratory tests, electrocardiogram, echocardiography, in addition to CPET. In this cohort of ATTRwt patients who performed CPET were elderly, all were male, and predominantly white (69.9%). The overall median survival was 59.01 months (95% confidence interval [CI] 49.29 to 88.69). By multivariate analysis, C-reactive protein (CRP; hazard ratio [HR] 1.10 [1.03 to 1.18]), decreased sodium (HR 0.75 [0.58 to 0.97]), creatinine (HR 7.48 [2.44 to 22.98]) and VE/VCO2 (HR 1.10 [1.05 to 1.16]) were significant risk factors for mortality (p 40 had a worse median survival of 38.54 months (95% CI 32.63 to 51.47) versus 88.69 months (95% CI 56.26 to 89.49) than patients with VE/VCO2 slope ≤40. Receiver-operating characteristic curve showed that the combination of VE/VCO2, CRP, sodium, and creatinine (Area under the ROC Curve [AUC], 0.89) predicted 1-year mortality in ATTRwt cardiac amyloidosis. In conclusion, increased VE/VCO2, in combination with CRP, sodium, and creatinine, may identify patients at increased risk of death in ATTRwt cardiomyopathy. VE/VCO2 might have a role in objectively assessing therapeutic response in ATTRwt cardiac amyloidosis.

Transthyretin-associated forms of cardiac amyloidosis are fatal protein misfolding diseases that can be inherited (ATTRm) or acquired (ATTRwt). An accurate diagnosis of ATTR amyloidosis can be challenging as biopsy evidence, usually from the affected organ, is required. Precise biomarkers for ATTR disease identification and monitoring are undiscovered, disease-specific therapeutic options are needed, and the current understanding of ATTR molecular pathogenesis is limited. The aim of this study was to investigate and compare the serum proteomes in ATTRm and ATTRwt cardiac amyloidosis to identify differentially expressed blood proteins that were disease-specific. Using multiple-reaction monitoring mass spectrometry (MRM-MS), the concentrations of 160 proteins were analyzed in serum samples from ATTRm and ATTRwt patients, and a healthy control group. Patient and control sera were matched to age (≥60 years), gender (male), and race (Caucasian). The circulating concentrations of 123/160 proteins were significantly different in patient vs control sera; TTR and retinol-binding protein (RBP4) levels were significantly decreased (p0.03) in ATTRm compared to controls. In ATTRm, 14/123 proteins were identified as unique to that group and found generally to be lower than controls; moreover, the concentrations of RBP4 and 6 other proteins in this group were significantly different (p0.04) compared to ATTRwt. Predicted interactions among the 14 proteins unique to ATTRm were categorized as reaction and binding associations. Alternatively, 27 proteins were found to be unique to ATTRwt with associated interactions defined as activation, catalysis, and inhibition, in addition to reaction and binding. This study demonstrates significant proteomic differences between ATTR patient and control sera, and disease-associated variations in circulating levels of several proteins including TTR and RBP4. The identification of serum proteins unique to ATTRm and ATTRwt cardiac amyloidosis may have diagnostic and prognostic utility, and may provide important clues about disease mechanisms.

Serum and urine immunofixation electrophoreses (SIFE/UIFE) are used for clonal detection in plasma cell dyscrasias, while serum free light chain (sFLC) testing provides quantitation of clonal disease. Up to 20% of patients with light chain (AL) amyloidosis may present with normal FLC ratio (FLCr).We assessed the diagnostic, quantitative and prognostic potential of serum heavy light chain ratio (HLCr) in 199 untreated patients at initial evaluation.An abnormal HLCr was found in 37.2%, abnormal FLCr in 81.9% and positivity by SIFE/UIFE in 94% of patients. HLCr together with SIFE/UIFE identified clonality in 94% patients; the combination with FLCr yielded 100% sensitivity. An HLCr abnormality was significantly over-represented in normal compared to abnormal FLCr group (63.9% versus 31.3%). HLCr did not predict overall survival (OS) (log rank, p = 0.09), while an abnormal FLCr was associated with decreased OS (log rank, p = 0.03). The combined use of both ratios trended toward increased OS in the abnormal HLCr/normal FLCr group (log rank, p = 0.11; Wilcoxon, p = 0.04). On multivariate analysis, HLCr was not predictive of OS, whereas an abnormal FLCr was associated with shorter OS (HR = 1.7, p = 0.04).The HLC assay has potential as a supplemental test to quantify monoclonal protein in patients with normal FLCr results.

A valine to isoleucine amino acid substitution at position 122 (V122I or p.V142I) of the transthyretin (TTR) gene induces misfolding of this hepatic transport protein, conferring a clinical phenoty...

Background: Racial/ethnic minorities have been underrepresented in most population-based studies of AL amyloidosis published to date. This observation stands in marked contrast to multiple myeloma, a closely related disorder with a twofold higher incidence among blacks vs. whites. Given the scarcity of information about health disparities in AL amyloidosis, we aimed to characterize the clinical presentation and outcomes of this unique plasma cell disorder according to race/ethnicity. Methods: Data on consented patients with systemic AL amyloidosis seen at the Boston University Amyloidosis Center between 1990 and 2020 were obtained from a prospectively maintained database (ClinicalTrials.gov Identifier: NCT00898235). Sociodemographic factors, hematologic and organ disease markers, along with the use of high-dose melphalan and stem cell transplantation (HDM/SCT) were stratified by self-reported race/ethnicity. Groups included non-Hispanic white (NHW), non-Hispanic black (NHB), Hispanic, and non-Hispanic other (NHO). Differences were assessed by χ2 test for categorical variables and one-way ANOVA test for continuous variables. Multivariable logistic regression was used to determine the influence of variables on HDM/SCT utilization. The effect of race/ethnicity on mortality was estimated by multivariate Cox proportional hazards regression with adjustment for relevant confounders. Results: In a cohort of 2,416 patients, only 14% were underrepresented minorities (Figure 1A). At diagnosis, racial/ethnic minority groups were younger than NHWs by a median of 4-6 years. NHBs comprised 8% of the cohort and had higher-risk sociodemographic factors (e.g. educational attainment of high school level or less among 39% vs. 25% for NHWs; P < .001). Furthermore, there was an indication of more aggressive disease among NHBs with more patients having a difference between involved and uninvolved free light chains (dFLC) of > 180 mg/L (39% vs. 22-33%; P = .044). Hispanics comprised 4% of the cohort and presented with more advanced cardiac disease (i.e. median BNP of 1,041 pg/mL vs. 221-480 pg/mL; P = .001). Among a subcohort of 1,668 patients with available treatment data, 33% of Hispanics and 39% of NHBs were treated with HDM/SCT as compared to 47% of NHWs (P = .071). This treatment disparity was accounted for by sociodemographic (i.e. lower educational level) and physiologic risk factors (i.e. stage III cardiac involvement), rather than race/ethnicity itself. At data cutoff in March 2020, 1,622 (67%) patients were deceased. Compared with NHWs, the median survival for NHBs was shorter by nearly one year (Figure 1B) with an age/sex-adjusted HR for death of 1.24 (95% CI 1.03-1.49; P = .020). After further adjusting for disease and treatment variables, this survival difference was eliminated altogether (HR 0.82; 95% CI 0.50-1.34; P = .427). Conclusions: For the first time, this study shines a spotlight on racial/ethnic disparities in AL amyloidosis. Systematic underdetection, along with delayed diagnosis and referral, may explain why minority patients bear a disproportionate burden of late-stage cardiac involvement and high dFLC at the time of initial presentation. In turn, these factors contribute to underutilization of effective treatments such as HDM/SCT (particularly among Hispanics) and poorer survival (among NHBs) as observed in our study. The possibility of predisposing genetic and/or biological factors will need to be considered in future studies. Ultimately, greater awareness of AL amyloidosis within underrepresented communities, together with more widespread and accessible testing, are necessary to mitigate these health disparities. Figure 1: (A) The distribution of race/ethnicity in a large cohort of patients with AL amyloidosis and (B) Kaplan-Meier survival curves are shown. NHW indicates non-Hispanic white; NHB, non-Hispanic black; and NHO, non-Hispanic other. Figure 1 Disclosures Sanchorawala: Caleum: Other: advisory board; Proclara: Other: advisory board; Abbvie: Other: advisory board; UpToDate: Patents & Royalties; Regeneron: Other: advisory board; Janssen: Research Funding; Takeda: Research Funding; Celgene: Research Funding; Prothena: Research Funding; Caelum: Research Funding; Oncopeptide: Research Funding.

Hereditary transthyretin amyloidosis (ATTR amyloidosis) is an autosomal dominant protein-folding disorder caused by over 100 distinct mutations in the transthyretin (TTR) gene. In ATTR amyloidosis, protein secreted from the liver aggregates and forms amyloid fibrils in downstream target organs, chiefly the heart and peripheral nervous system. Few animal models of ATTR amyloidosis exist and none recapitulate the multisystem complexity and clinical variability associated with disease pathogenesis in patients. Induced pluripotent stem cells (iPSCs) stand to revolutionize the way we study human development, model disease, and perhaps treat patients afflicted with highly variable multisystem diseases such as ATTR amyloidosis. Here, we fully characterize six representative iPSC lines from a library of previously reprogrammed iPSC lines and reprogrammable blood samples derived from ATTR amyloidosis patients. This unique resource, described herein, can be harnessed to study diverse disorder.

Systemic amyloidosis represents a class of disorders in which misfolded proteins are secreted by effector organs and deposited as proteotoxic aggregates at downstream target tissues. Despite being well-described clinically, the contribution of effector organs such as the liver to the pathogenesis of these diseases is poorly understood. Here, we utilize a patient-specific induced pluripotent stem cell (iPSC)-based model of hereditary transthyretin (TTR) amyloid disease (ATTR amyloidosis) in order to define the contributions of hepatic cells to the distal proteotoxicity of secreted TTR. To this end, we employ a gene correction strategy to generate isogenic, ATTR amyloidosis patient-specific iPSCs expressing either amyloidogenic or wild-type TTR. We further utilize this gene editing strategy in combination with single cell RNAseq to identify multiple hepatic proteostasis factors, including many components of adaptive unfolded protein response (UPR) signaling pathways, whose expression correlates with the production of destabilized TTR variants in iPSC-derived hepatic cells. We further demonstrate that enhancing ER proteostasis within ATTR amyloidosis iPSC-derived hepatic lineages via stress-independent activation of aforementioned adaptive UPR signaling preferentially reduces the secretion of destabilized amyloidogenic TTR. Together, these results suggest the potential of the liver to chaperone-at-a-distance and impact pathogenesis at downstream target cells in the context of systemic amyloid disease, and further highlight the promise of UPR modulating therapeutics for the treatment of TTR-mediated and other amyloid diseases.

Transthyretin (TTR) is a globular tetrameric transport protein in plasma. Nearly 140 single amino acid substitutions in TTR cause life-threatening amyloid disease. We report a one-of-a-kind pathological variant featuring a Glu51, Ser52 duplication mutation (Glu51_Ser52dup). The proband, heterozygous for the mutation, exhibited an unusually aggressive amyloidosis that was refractory to treatment with the small-molecule drug diflunisal. To understand the poor treatment response and expand therapeutic options, we explored the structure and stability of recombinant Glu51_Ser52dup. The duplication did not alter the protein secondary or tertiary structure but decreased the stability of the TTR monomer and tetramer. Diflunisal, which bound with near-micromolar affinity, partially restored tetramer stability. The duplication had no significant effect on the free energy and enthalpy of diflunisal binding, and hence on the drug-protein interactions. However, the duplication induced tryptic digestion of TTR at near-physiological conditions, releasing a C-terminal fragment 49-129 that formed amyloid fibrils under conditions in which the full-length protein did not. Such C-terminal fragments, along with the full-length TTR, comprise amyloid deposits in vivo. Bioinformatics and structural analyses suggested that increased disorder in the surface loop, which contains the Glu51_Ser52dup duplication, not only helped generate amyloid-forming fragments but also decreased structural protection in the amyloidogenic residue segment 25-34, promoting misfolding of the full-length protein. Our studies of a unique duplication mutation explain its diflunisal-resistant nature, identify misfolding pathways for amyloidogenic TTR variants, and provide therapeutic targets to inhibit amyloid fibril formation by variant TTR.

Wild-type transthyretin amyloidosis (ATTRwt) is a sporadic protein misfolding disease with no specific biomarkers or treatment [1]. The disease most often features cardiomyopathy and heart failure ...

The clinical presentation of AL amyloidosis is highly variable. In this series, we describe five cases of AL amyloidosis with vertebral compression fractures as initial presentation. All five patients had evidence of bone marrow replacement on magnetic resonance imaging and bone marrow biopsies demonstrating diffuse interstitial amyloid deposition. Hepatomegaly and elevated liver enzymes, consistent with liver involvement with amyloidosis, were also seen in each case. All five patients responded well to anti-plasma cell chemotherapy, with normalization of serum free light chain levels, reduction in alkaline phosphatase and improvement in pain and functional status. Although rare, AL amyloidosis should be considered in the differential diagnosis of selected patients with spontaneous vertebral compression fractures. Moreover, there seems to be an association of vertebral compression fractures with liver involvement in AL amyloidosis.

To identify the prevalence of monoclonal gammopathy of undetermined significance (MGUS) in patients with transthyretin (ATTR) amyloidosis.We performed a retrospective analysis of patients with biopsy-proven ATTRwt (wild-type transthyretin amyloid protein) and genopositive ATTR V122I (valine-to-isoleucine substitution at position 122 of the TTR gene) amyloidosis evaluated at the Amyloidosis Center at Boston University and Boston Medical Center between 1 January 2003 and 31 December 2016.There were a total of 226 patients with ATTRwt and ATTR V122I amyloidosis evaluated during the specified time frame with 155 and 71 patients in each cohort, respectively. Those with complete medical records, 140 patients with ATTRwt and 57 V1221 ATTRm subjects, were included in the analyses. Fifty-five patients (39%) in the ATTRwt cohort and 28 patients (49%) in the ATTR V122I cohort had an MGUS, as indicated by an abnormality in the serum-free light-chain ratio and/or serum immunofixation electrophoresis.These data confirm the high prevalence of coexistent MGUS with ATTR amyloidosis in this patient population, with an MGUS rate that is higher than the general population. These findings also highlight the importance of a thorough diagnostic evaluation in patients with amyloidosis to determine the precursor protein, as the clinical course and treatment of AL (light-chain amyloid protein) and ATTR amyloidosis are distinct.

Aims Wild‐type transthyretin (ATTRwt) cardiac amyloidosis has emerged as an important cause of heart failure in the elderly. Atrial fibrillation (AF) commonly affects older adults with heart failure and is associated with reduced survival, but its role in ATTRwt is unclear. We sought to explore the clinical impact of AF in ATTRwt. Methods and results Patients with biopsy‐proven ATTRwt cardiac amyloidosis (n = 146) were retrospectively identified, and clinical, echocardiographic, and biochemical data were collected. Patients were classified as AF or non‐AF and followed for survival for a median of 41.4 ± 27.1 months. Means testing, univariable, and multivariable regression models were employed. A systematic review was performed. AF was observed in 70% (n = 102). Mean age was similar (AF, 75 ± 6 vs. non‐AF, 74 ± 5 years, P = 0.22). Anticoagulant treatment of patients with AF was as follows: 78% warfarin, 17% novel anticoagulant, and 6% no anticoagulation. Amiodarone was prescribed to 24%. There were no differences in left ventricular ejection fraction (P = 0.09) or left atrial volume (P = 0.87); however, mean diastolic dysfunction grade was higher in AF (mean 2.7 ± 0.5 vs. 2.4 ± 0.5, P = 0.01). While creatinine (P = 0.52) and B‐type natriuretic peptide (P = 0.48) were similar, patients with AF had lower serum transthyretin concentrations (221 ± 51 vs. 250 ± 52 μg/mL, P

AL amyloidosis is the consequence of clonal production of amyloidogenic immunoglobulin light chain (LC) proteins, often resulting in a rapidly progressive and fatal amyloid cardiomyopathy. Recent work has found that amyloidogenic LC directly initiate a cardio-toxic response underlying the pathogenesis of the cardiomyopathy; however, the mechanisms that contribute to this proteotoxicity remain unknown. Using human amyloidogenic LC isolated from patients with amyloid cardiomyopathy, we reveal that dysregulation of autophagic flux is critical for mediating amyloidogenic LC proteotoxicity. Restoration of autophagic flux by pharmacological intervention using rapamycin protected against amyloidogenic light chain protein-induced pathologies including contractile dysfunction and cell death at the cellular and organ level and also prolonged survival in an in vivo zebrafish model of amyloid cardiotoxicity. Mechanistically, we identify impaired lysosomal function to be the major cause of defective autophagy and amyloidogenic LC-induced proteotoxicity. Collectively, these findings detail the downstream molecular mechanisms underlying AL amyloid cardiomyopathy and highlight potential targeting of autophagy and lysosomal dysfunction in patients with amyloid cardiomyopathy.

Summary Familial transthyretin amyloidosis (ATTR) is an autosomal-dominant protein-folding disorder caused by over 100 distinct mutations in the transthyretin (TTR) gene. In ATTR, protein secreted from the liver aggregates and forms fibrils in target organs, chiefly the heart and peripheral nervous system, highlighting the need for a model capable of recapitulating the multisystem complexity of this clinically variable disease. Here, we describe the directed differentiation of ATTR patient-specific iPSCs into hepatocytes that produce mutant TTR, and the cardiomyocytes and neurons normally targeted in the disease. We demonstrate that iPSC-derived neuronal and cardiac cells display oxidative stress and an increased level of cell death when exposed to mutant TTR produced by the patient-matched iPSC-derived hepatocytes, recapitulating essential aspects of the disease in vitro. Furthermore, small molecule stabilizers of TTR show efficacy in this model, validating this iPSC-based, patient-specific in vitro system as a platform for testing therapeutic strategies., Highlights • Successful modeling of familial amyloidosis in vitro using iPSC technology • Proto-fibril formation leads to cellular damage in two target tissues of amyloidosis • iPSCs can be used in the testing of novel therapeutics for protein folding disorders, This work involves the modeling of hereditary transthyretin amyloidosis in vitro using iPSC technology. Murphy and colleagues demonstrate that it is possible to model a long-term, complex, multisystem disease using hepatic, cardiac, and neuronal lineages derived from patient-specific stem cells and validate this approach for the testing of therapeutic strategies.

Background: Wild-type transthyretin amyloidosis (ATTRwt), an underappreciated cause of heart failure in older adults, is challenging to diagnose and monitor in the absence of validated, disease-specific biomarkers. We examined the prognostic use and survival association of serum TTR (transthyretin) concentration in ATTRwt. Methods and Results: Patients with biopsy-proven ATTRwt were retrospectively identified. Serum TTR, cardiac biomarkers, and echocardiographic parameters were assessed at baseline and follow-up evaluations. Statistical analyses included Kaplan–Meier method, Cox proportional hazard survival models, and receiver-operating characteristic curve analysis. Median serum TTR concentration at presentation was 23 mg/dL (n=116). Multivariate predictors of shorter overall survival were decreased TTR, left ventricular ejection fraction and elevated cTn-I (cardiac troponin I); an inclusive model demonstrated superior accuracy in 4-year survival prediction by receiver-operating characteristic curve analysis (area under the curve, 0.77). TTR values lower than the normal limit, P =0.03). Further, TTR values at 1- and 2-year follow-ups were significantly lower ( P P =0.006) in untreated patients; conversely, the diflunisal-treated group showed increased TTR ( P =0.001) and stabilized cTn-I and left ventricular ejection fraction at 1 year. Conclusions: In this series of biopsy-proven ATTRwt, lower baseline serum TTR concentration was associated with shorter survival as an independent predictor of outcome. Longitudinal analysis demonstrated that decreasing TTR corresponded to worsening cardiac function. These data suggest that TTR may be a useful prognostic marker and predictor of outcome in ATTRwt.

Immunoglobulin light chain amyloidosis (AL) is a plasma cell disorder characterized by overproduction and deposition of monoclonal immunoglobulin (Ig) light chains (LC) or variable region fragments as amyloid fibrils in various organs and tissues. Much clinical evidence indicates that patients with AL amyloidosis sustain cardiomyocyte impairment and suffer from oxidative stress. We seek to understand the underlying biochemical pathways whose disruption or amplification during sporadic or sustained disease states leads to harmful physiological consequences and to determine the detailed structures of intermediates and products that serve as signposts for the biochemical changes and represent potential biomarkers. In this study, matrix-assisted laser desorption/ionization mass spectrometry provided extensive evidence for oxidative post-translational modifications (PTMs) of an amyloidogenic Ig LC protein from a patient with AL amyloidosis. Some of the tyrosine residues were heavily mono- or di-chlorinated. In addition, a novel oxidative conversion to a nitrile moiety was observed for many of the terminal aminomethyl groups on lysine side chains. In vitro experiments using model peptides, in-solution oxidation, and click chemistry demonstrated that hypochlorous acid produced by the myeloperoxidase - hydrogen peroxide - chloride system could be responsible for these and other, more commonly observed modifications.

Transthyretin (TTR) amyloidosis was originally described as an autosomal dominant inherited systemic disease with peripheral neuropathy being the paramount feature. It was obvious from the first de...

Transthyretin amyloid cardiomyopathy (ATTR cam) is likely an under-recognized cause of heart failure (HF) in the elderly [1], owing to misattribution of clinical features to other common co-morbidi...

Amyloid light chain (AL) amyloidosis is a rare hematologic disorder characterized by the accumulation of a misfolded monoclonal immunoglobulin (Ig) light chain (LC) as fibrillar protein deposits. Current treatments, including cytotoxic chemotherapy and immunomodulatory therapy, are directed at killing the plasma cells that produce the LCs, but have significant toxicity for other cell types. We have designed small interfering RNAs (siRNAs) targeting the amyloidogenic LC messenger RNA (mRNA) in order to reduce expression of the amyloid precursor protein. Using nanomolar concentrations of siRNAs, we have inhibited synthesis of LC in transfected cells in vitro in a dose-dependent fashion. Furthermore, in an in vivo plasmacytoma mouse model of AL amyloidosis, we have demonstrated that these siRNAs can significantly reduce local production and circulating levels of LC. This model system highlights the therapeutic potential of siRNA for AL amyloidosis.

Amyloidosis is an increasingly recognized group of disorders, characterized by the deposition of misfolded protein aggregates in various tissues and organs. For over 50 years, the Amyloidosis Center at Boston University Medical Center (BUMC) has taken lead in the comprehensive evaluation and treatment of patients with this disease. The center serves as a tertiary referral site both nationally and globally, with patients visiting from 18 countries in recent years. Historically, immunoglobulin light chain (AL) amyloidosis associated with a plasma cell dyscrasia has had the greatest prevalence at our center. Other forms of amyloidosis, such as the hereditary mutant transthyretin (ATTRm) and age-related wild type transthyretin (ATTRwt) amyloidoses, have been seen less commonly due in part to underdiagnosis and a perceived paucity of effective treatments. We report here the spectrum of referrals at the Amyloidosis Center at BUMC from 1990 to 2017. We used data from a prospectively maintained database of consented patients. Of the 3084 patients seen for an initial evaluation at our center during this period, patients with AL amyloidosis decreased from 86% to 79% to 65% in the first, second and third decade, respectively. In the same time intervals, ATTRm amyloidosis cases increased from 12% to 14% to 19% of the total patients; an even steeper increase, from 2% to 7% to 16%, was seen in patients with ATTRwt amyloidosis. There is a marked trend towards more referrals of ATTR amyloidosis at BUMC. We believe this reflects increasing awareness and advancements in the diagnostic and therapeutic landscape of ATTR amyloidosis. In the early 2000s, the potential for misdiagnosis of hereditary ATTR as AL amyloidosis was recognized (Lachmann, et al. N Engl J Med. 2002). Accordingly, biochemical analysis by immunogold electron microscopy or laser capture tandem mass spectrometry has become a standard for more accurate typing of amyloid protein. Availability and utilization of these specific pathological modalities may in part explain the growing proportion of ATTR amyloidosis seen at our center. Perhaps an even more significant factor is the development of nuclear cardiac imaging techniques, which accurately diagnose ATTR cardiac amyloidosis without the need for endomyocardial biopsy. In the last decade, technetium associated bone-avid tracers, such as pyrophosphate (PYP), have been found to be highly sensitive and specific in identifying ATTR cardiac amyloidosis among patients with heart failure (Gillmore, et al. Circulation. 2016). Ease of performance and interpretation, availability and standardization have spurred widespread adaptation of this imaging modality, thereby allowing for recognition of ATTR cases that were undiagnosed in prior decades. A recent surge in diagnosis of ATTR amyloidosis is coincident with an evolving therapeutic landscape. There have been a number of clinical trials for ATTR amyloidosis, investigating transthyretin protein suppression and stabilization, as well as fibril clearance. Three therapies have completed phase III trials and are pending US regulatory review and registration. Publicity surrounding these trials has led physicians and patients to seek referral to tertiary centers for evaluation for novel treatments. The rise in new ATTR referrals at BUMC is countered by a decline in AL amyloidosis referrals, from a former average of 102 new cases per year to 91 per year in the last decade. We believe this to be largely owed to increasing experience with and adoption by community hematologists of the therapeutic options for AL amyloidosis, which are derived from treatments for multiple myeloma. In conclusion, once thought to be exceedingly rare, ATTR is becoming an increasingly prevalent form of amyloidosis at tertiary centers, comprising nearly 1 of every 3 new referrals at BUMC in the past decade. We anticipate that this proportion will rise further as PYP nuclear imaging becomes incorporated into screening algorithms for heart failure and awareness spreads about the anticipated approval of new pharmaceuticals for ATTR amyloidosis. Disclosures Berk: Ionis: Honoraria, Other: Investigator; Alnylam: Honoraria, Other: Investigator; Pfizer: Other: Investigator.