ATTR Amyloid Research Articles

Oxidative conversion of transthyretin in formalin-fixed clinical amyloid samples results in the formation of the His90Asp and His90Asn variants

Background Proteomics is routinely used to type clinical amyloid deposits, and offers additional benefit of identifying genetic variants, which can be diagnostically useful. Reviewing the proteomics data for ATTR patients attending our Centre revealed an unusually large number of samples containing a rare pathogenic H90D TTR variant alongside the more common H90N variant. Methods These findings raised questions to their source. Proteomics was used to monitor the generation of H90D/H90N variants in fresh, frozen, stored samples during extraction and digestion, and also following Cu2+-mediated oxidation. Results There was no evidence that the variants were present in the circulation, except in one patient with genetically confirmed H90D TTR, in fresh fat aspirates or tissues from an ATTR amyloid mouse model. The variant could be generated in vitro from both wild-type TTR and ex vivo ATTR fibrils by non-enzymic oxidation of histidine at position 90. These data suggest that the H90D variant can be generated artefactually from wild-type 90H TTR through a radical-mediated oxidation of histidine, followed by its conversion to asparagine and aspartic acid. This probably occurs during storage. Conclusions In the absence of genetic data, the identification of H90D TTR in stored tissue by proteomics should be treated with caution.

Histopathological and Immunohistochemical Characteristics of Different Types of Cardiac Amyloidosis.

Cardiac involvement is the most important factor determining prognosis in patients with systemic amyloidosis. This retrospective observational study of 98 patients with amyloidosis was undertaken to assess the amyloid types that are most likely to affect the heart, describe histopathological and clinical features of cardiac amyloidosis, and estimate the number of cases not diagnosed clinically prior to death. All cases were divided into two groups based on the method of examination. The first group included 46 patients with cardiac amyloidosis revealed via endomyocardial biopsies (EMBs), and the second group included 52 amyloidosis patients who did not undergo EMBs, in whom cardiac involvement was identified only at autopsy. The EMBs demonstrated that AL amyloidosis was detected in 21 (46%) specimens, ATTR amyloid in 24 cases (52%), and AA amyloid in 1 case (2%). The autopsy reports defined 15 (46%) cases of AL amyloidosis, 21 (40%) of ATTR and 16 (31%) of AA amyloidosis. It should be noted that a clinical diagnosis of ATTR amyloidosis was made only in 9.5% of patients from the autopsy group, suggesting that ATTR may be an underdiagnosed cause of heart failure in elderly patients. The most intense amyloid deposits were determined in biopsy and autopsy specimens of patients with AL kappa amyloidosis, underlying a poorer prognosis.

ELISA-4-amyloid: diagnostic accuracy of an ELISA panel for typing the four main types of systemic amyloidosis in subcutaneous abdominal fat tissue samples

Background Reliable typing of amyloid is essential. Amyloid extraction from tissue enables immunochemical typing of the precursor protein using an enzyme-linked immunosorbent assay (ELISA). Objective To assess the diagnostic accuracy of a panel of ELISAs for typing the four main types (AA, ATTR, AL-kappa and AL-lambda amyloid). Methods From 1996 to 2023 subcutaneous abdominal fat tissue aspirates were obtained from 1339 amyloidosis patients and 868 controls. Amyloid was visually graded 0–4+ in Congo red-stained smears. Amyloid extracted from tissue by Guanidine was typed using a panel comprising four ELISAs. Results All amyloid protein concentrations in extracts correlated with amyloid grade in smears. Typing sensitivity was low (23.3%) in samples with grade 1+/2+ amyloid. Overall typing sensitivity of the panel was 81.6% for all easily visible amyloid (grade 3+/4+): high for AA (98.8%) and ATTR (96.8%) and fair for AL-kappa (66.7%) and AL-lambda (75.9). Overall typing specificity was 98.0% and the overall positive predictive value was 98.0%. Conclusions We describe a highly specific ELISA panel for routine typing of the main amyloid types in fat tissue. Until more sensitive typing techniques will become generally available, typing easily visible amyloid in fat tissue using this ELISA panel is reliable, affordable and straightforward.

Quantitative 99mTc-pyrophosphate myocardial uptake: Changes on transthyretin stabilization therapy

BackgroundQuantitative technetium-99m-pyrophosphate cardiac single-photon emission computed tomography (99mTc-PYP SPECT/CT) is an emerging method for estimating myocardial burden of transthyretin cardiac amyloidosis (ATTR-CA), but its efficacy in monitoring longitudinal changes remains uncertain. We aimed to investigate longitudinal changes in cardiac ATTR amyloid burden following transthyretin stabilization therapy using visual and quantitative 99mTc-PYP SPECT/CT and to relate these with changes in cardiac biomarkers and function. MethodsThis prospective longitudinal cohort study investigated changes in 99mTc-PYP SPECT/CT in 23 participants with ATTR-CA on transthyretin stabilization therapy (median: 2.6 years). Quantitative analysis included left ventricular (LV) standardized uptake values (SUVs) (SUVmax, SUVmean), cardiac amyloid activity (CAA; SUVmean∗LV activity volume), and percent injected dose (%ID) (mean activity concentration∗LV activity volume/injected activity), calculated using a threshold of >1.5 times left atrial blood pool activity concentration on SPECT/CT. Longitudinal changes of paired continuous and ordinal variables were analyzed using Wilcoxon signed-rank test. ResultsFollowing therapy, visual grade decreased significantly (P = 0.003). Several quantitative 99mTc-PYP metrics also decreased significantly: SUVmax (median −0.75, P = 0.011), CAA (median: −406.6, P < 0.001), and %ID (median: −0.45, P < 0.001). Serum transthyretin levels improved (median: +6.5 mg/dL, P = 0.008). Echocardiographic parameters (global longitudinal strain, LV mass index, and LV wall thickness), N-terminal pro-B-type natriuretic peptide, and estimated glomerular filtration rate remained stable. ConclusionsFavorable changes in 99mTc-PYP myocardial uptake were observed in participants on transthyretin stabilization therapy, whereas echocardiographic parameters and biomarkers remained stable. These results likely signify myocardial ATTR amyloid stabilization rather than amyloid burden regression. Further investigation is needed to understand the implications of these findings.

Cutaneous biomarkers of therapeutic efficacy in early treatment of hereditary ATTR amyloid polyneuropathy with tafamidis.

ATTR (ATTRv) amyloidosis neuropathy is characterized by progressive sensorimotor and autonomic nerve degeneration secondary to amyloid deposition caused by a misfolded transthyretin protein (TTR). Small nerve fiber neuropathy is an early clinical manifestation of this disease resulting from the dysfunction of the Aδ and C small nerve fibers. Tafamidis, a selective TTR stabilizer, has proven its efficacy in the earlier stages of hATTR. To evaluate the clinical course and utility of cutaneous pathological biomarkers in patients with ATTR amyloidosis treated with tafamidis compared to control patients. Forty patients diagnosed with early stages of ATTRv amyloidosis (polyneuropathy disability [PND] scores 0-II) underwent small and large nerve fiber neurological evaluations, and annual skin biopsies for intraepidermal nerve fiber density (IENFD) and amyloid deposition index (ADI) estimation. Thirty patients were allocated to receive tafamidis, and 10 patients served as controls. Tafamidis pharmacokinetics analysis was performed in patients who received the treatment. At baseline, 12% of patients in stage PND 0 and 28% in PND I displayed small nerve fiber denervation in the distal thigh, whereas 23% and 38%, respectively, in the distal leg. Similarly, 72% and 84% had amyloid deposition in the distal thigh and 56% and 69% in the distal leg. Following 1 year of treatment, the tafamidis group showed significant clinical improvement compared to the control group, revealed by the following mean differences (1) -9.3 versus -4 points (p = <.00) in the patient's neuropathy total symptom score 6 (NTSS-6) questionnaire, (2) -2.5 versus +2.8 points (p = <.00) in the Utah Early Neuropathy Score (UENS), and (3) +1.2°C versus -0.6 (p = .01) in cold detection thresholds. Among the patients who received tafamidis, 65% had stable or increased IENFD in their distal thigh and 27% in the distal leg. In contrast, all patients in the control group underwent denervation. The ADI either decreased or remained constant in 31% of the biopsies in the distal thigh and in 24% of the biopsies in the distal leg of the tafamidis-treated patients, whereas it rose across all the biopsies in the control group. At the 4-year follow-up, the tafamidis group continued to display less denervation in the distal thigh (mean difference [MD] of -3.0 vs. -9.3 fibers/mm) and the distal leg (mean difference [MD] -4.9 vs. -8.6 fibers/mm). ADI in tafamidis-treated patients was also lower in the distal thigh (10 vs. 30 amyloid/mm2) and the distal leg (23 vs. 40 amyloid/mm2) compared to control patients. Plasma tafamidis concentrations were higher in patients with IENFD improvement and in patients with reduced amyloid deposition. Patients without amyloid deposition in the distal leg at baseline displayed delayed disease progression at 4 years. Cutaneous IENFD and amyloid deposition assessments in the skin of the distal thigh and distal leg are valuable biomarkers for early diagnosis of ATTR amyloidosis and for measuring the progression of small nerve fiber neuropathy. Early treatment with tafamidis slows the clinical progression of the disease, skin denervation, and amyloid deposition in the skin. Higher plasma concentrations of tafamidis are associated with better disease outcomes, suggesting that increasing the drug dose could achieve better plasma concentrations and response rates. This study describes the longest small nerve fiber neuropathy therapeutic trial with tafamidis and is the first to report small fiber symptoms, function, and structural assessments as outcomes.

Renal Amyloidosis: A Narrative Literature Review

Amyloidosis is a disorder in which soluble proteins aggregate and are deposited extracellularly in tissues as insoluble fibrils, causing progressive organ dysfunction. Amyloid fibril formation begins with misfolding of amyloidogenic precursor proteins. The fibrils have a characteristic appearance by electron microscopy and produce double refraction under polarized light when stained with Congo red dye. Classification of amyloidosis is based on the precursor proteins that form amyloid fibrils and the distribution of amyloid deposition both systemically and locally. The main form of systemic amyloidosis; AL amyloid, AA amyloidosis, ATTR amyloid. The kidney is the organ most frequently involved in systemic amyloidosis. Systemic amyloidosis may originate from anomalous proteins, such as immunoglobulin light chains or serum amyloid protein in chronic inflammation or may arise from hereditary disorders. The clinical manifestations of renal amyloidosis vary with the type of amyloid protein and the location and extent of amyloid deposition. Treatment of amyloidosis should be a two-part process; managing symptoms and reducing or stabilizing amyloid protein. Treatment of amyloidosis is focused on reducing the production of amyloidogenic proteins and inhibiting their aggregation.

Patisiran Treatment in Patients with Transthyretin Cardiac Amyloidosis

BackgroundTransthyretin amyloidosis, also called ATTR amyloidosis, is associated with accumulation of ATTR amyloid deposits in the heart and commonly manifests as progressive cardiomyopathy. Patisiran, an RNA interference therapeutic agent, inhibits the production of hepatic transthyretin.MethodsIn this phase 3, double-blind, randomized trial, we assigned patients with hereditary, also known as variant, or wild-type ATTR cardiac amyloidosis, in a 1:1 ratio, to receive patisiran (0.3 mg per kilogram of body weight) or placebo once every 3 weeks for 12 months. A hierarchical procedure was used to test the primary and three secondary end points. The primary end point was the change from baseline in the distance covered on the 6-minute walk test at 12 months. The first secondary end point was the change from baseline to month 12 in the Kansas City Cardiomyopathy Questionnaire–Overall Summary (KCCQ-OS) score (with higher scores indicating better health status). The second secondary end point was a composite of death from any cause, cardiovascular events, and change from baseline in the 6-minute walk test distance over 12 months. The third secondary end point was a composite of death from any cause, hospitalizations for any cause, and urgent heart failure visits over 12 months.ResultsA total of 360 patients were randomly assigned to receive patisiran (181 patients) or placebo (179 patients). At month 12, the decline in the 6-minute walk distance was lower in the patisiran group than in the placebo group (Hodges–Lehmann estimate of median difference, 14.69 m; 95% confidence interval [CI], 0.69 to 28.69; P=0.02); the KCCQ-OS score increased in the patisiran group and declined in the placebo group (least-squares mean difference, 3.7 points; 95% CI, 0.2 to 7.2; P=0.04). Significant benefits were not observed for the second secondary end point. Infusion-related reactions, arthralgia, and muscle spasms occurred more often among patients in the patisiran group than among those in the placebo group.ConclusionsIn this trial, administration of patisiran over a period of 12 months resulted in preserved functional capacity in patients with ATTR cardiac amyloidosis. (Funded by Alnylam Pharmaceuticals; APOLLO-B ClinicalTrials.gov number, NCT03997383.)

Development and characterization of a prototypic pan-amyloid clearing agent - a novel murine peptide-immunoglobulin fusion.

Systemic amyloidosis is a progressive disorder characterized by the extracellular deposition of amyloid fibrils and accessory proteins in visceral organs and tissues. Amyloid accumulation causes organ dysfunction and is not generally cleared by the immune system. Current treatment focuses on reducing amyloid precursor protein synthesis and slowing amyloid deposition. However, curative interventions will likely also require removal of preexisting amyloid deposits to restore organ function. Here we describe a prototypic pan-amyloid binding peptide-antibody fusion molecule (mIgp5) that enhances macrophage uptake of amyloid. The murine IgG1-IgG2a hybrid immunoglobulin with a pan amyloid-reactive peptide, p5, fused genetically to the N-terminal of the immunoglobulin light chain was synthesized in HEK293T/17 cells. The binding of the p5 peptide moiety was assayed using synthetic amyloid-like fibrils, human amyloid extracts and amyloid-laden tissues as substrates. Binding of radioiodinated mIgp5 with amyloid deposits in vivo was evaluated in a murine model of AA amyloidosis using small animal imaging and microautoradiography. The bioactivity of mIgp5 was assessed in complement fixation and in vitro phagocytosis assays in the presence of patient-derived amyloid extracts and synthetic amyloid fibrils as substrates and in the presence or absence of human serum. Murine Igp5 exhibited highly potent binding to AL and ATTR amyloid extracts and diverse types of amyloid in formalin-fixed tissue sections. In the murine model of systemic AA amyloidosis, 125I-mIgp5 bound rapidly and specifically to amyloid deposits in all organs, including the heart, with no evidence of non-specific uptake in healthy tissues. The bioactivity of the immunoglobulin Fc domain was uncompromised in the context of mIgp5 and served as an effective opsonin. Macrophage-mediated uptake of amyloid extract and purified amyloid fibrils was enhanced by the addition of mIgp5. This effect was exaggerated in the presence of human serum coincident with deposition of complement C5b9. Immunostimulatory, amyloid-clearing therapeutics can be developed by incorporating pan-amyloid-reactive peptides, such as p5, as a targeting moiety. The immunologic functionality of the IgG remains intact in the context of the fusion protein. These data highlight the potential use of peptide-antibody fusions as therapeutics for all types of systemic amyloidosis.

Amyloid myopathy: expanding the clinical spectrum of transthyretin amyloidosis—case report and literature review

We identified two patients with transthyretin (ATTR) amyloid myopathy (one ATTR variant amyloidosis, ATTRv; one wild-type ATTR amyloidosis, ATTRwt). Myopathy was the initial manifestation in ATTRwt, whereas it followed neuropathy and cardiomyopathy in ATTRv. The ATTRwt patient showed muscular tracer uptake on 99mTc-DPD planar scintigraphy at the time of initial diagnosis, consistent with ATTR amyloid myopathy. The ATTRv patient underwent heart transplantation because of progressive heart failure. Within the next two years, progressive myopathic symptoms and extracardiac tracer uptake on 99mTc-DPD planar scintigraphy were documented, attributable to ATTR amyloid myopathy. Interstitial amyloid deposits were confirmed by muscle biopsy in both patients, with a particularly high amyloid burden in the adipose tissue. This case report highlights the frequent concomitant presence of cardiac ATTR amyloidosis and ATTR amyloid myopathy. ATTR amyloid myopathy may precede cardiac manifestation in ATTRwt or occur after heart transplantation in ATTRv. Due to the high diagnostic accuracy of 99mTc-DPD scintigraphy for detecting ATTR amyloid myopathy and the emergence of novel therapeutics, it is important to increase the awareness of its presence.

Peripheral neuropathy secondary to a ‘domino’ liver transplant: a case report

BackgroundPeripheral neuropathy caused by amyloidosis is one of the well-recognised sequelae of mutations in the transthyretin gene (TTR).Case presentationWe describe a case of peripheral neuropathy in a White British 74 year old man with wild-type TTR, 8 years following receipt of a ‘domino’ liver transplant (from a donor with a TTR mutation). The clinical phenotype and neurophysiology, coupled with presence of ATTR amyloid deposits on fat biopsy, established the diagnosis of ATTR amyloid neuropathy, as a consequence of receipt of a variant-TTR secreting liver. A nerve biopsy was not clinically appropriate for this patient. Such cases are rare since recipients of such livers are typically restricted to people whose natural lifespan is unlikely to stretch into the anticipated symptomatic period of ATTR amyloidosis. However, novel “gene silencing” therapeutics are now available which can dramatically alter the course of this disorder, by reducing the proportion of abnormal proteins.ConclusionsThis represents a rare but predictable iatrogenic side effect, and doctors should be aware of this eventuality occurring in a shorter time span than previously anticipated.

ATTR- and AFib amyloid - two different types of amyloid in the annular ligament of trigger finger

Introduction Histological examination of tissue specimens obtained during surgical treatment of trigger finger frequently encountered unclassifiable amyloid deposits in the annular ligament. We systematically explored this unknown type by a comprehensive analysis using histology, immunohistochemistry, and quantitative mass spectrometry-based proteomics. Methods 205 tissue specimens of annular ligaments were obtained from 172 patients. Each specimen was studied by histology and immunohistochemistry. Tissue specimens obtained from ten patients with histology proven amyloid in annular ligament were analysed by label-free quantitative proteomics. Histological and immunohistochemical findings were correlated with patient demographics. Results Amyloid was present as band like deposits along the surface of annular ligament, dot like or patchy deposits within the matrix. Immunohistochemistry identified ATTR amyloid in 92 specimens (mostly patchy in the matrix), while the band like deposits of 100 specimens remained unclassifiable. Proteomic profiles identified the unknown amyloid as most likely of fibrinogen origin. The complete cohort was re-examined by immunohistochemistry using a custom-made antibody and confirmed the presence of fibrinogen alpha-chain (FGA) in a hitherto unclassifiable type of amyloid in annular ligament. Conclusion Our study shows that two different types of amyloid affect the annular ligament, ATTR amyloid and AFib amyloid, with distinct demographic patient characteristics and histomorphological deposition patterns.

C66 PRIMARY RESULTS FROM APOLLO–B, A PHASE 3 STUDY OF PATISIRAN IN PATIENTS WITH TRANSTHYRETIN–MEDIATED AMYLOIDOSIS WITH CARDIOMYOPATHY

Abstract Introduction Transthyretin–mediated (ATTR) amyloidosis is a progressive, multisystem, fatal disease. Patisiran, an IV RNAi therapeutic, is approved for the treatment of hereditary ATTR amyloidosis with polyneuropathy. The safety and efficacy of patisiran in patients with wild–type or hereditary ATTR amyloidosis with cardiomyopathy (CM) is being investigated in the ongoing APOLLO–B study. Methods Patients were 18–85 yrs old with evidence of cardiac amyloidosis by echocardiography, and ATTR amyloid by tissue biopsy or fulfilling nonbiopsy diagnostic criteria for ATTR amyloidosis with CM. Medical history of heart failure (HF) due to ATTR amyloidosis with ≥1 prior hospitalization or current clinical evidence of HF was required. Patients were randomized 1:1 to patisiran IV 0.3 mg/kg or placebo Q3W for 12 months. The primary endpoint was change from baseline in 6–MWT at Month 12 (M12) with patisiran vs placebo. Results APOLLO–B enrolled 360 patients (patisiran, n=181; placebo, n=179): median age (range), 76.0 (41, 85) yrs; male, 89%; wtATTR, 80%. Baseline characteristics were consistent between patients on tafamidis at baseline (91/360 [25%]) and those who were not. Patisiran achieved a rapid and sustained reduction in serum TTR, irrespective of baseline tafamidis treatment. At M12, patisiran showed a significant benefit vs placebo in 6–MWT (median [95% CI] change from baseline: patisiran, –8.15 [–16.42, 1.50]; placebo, –21.35 [–34.05, –7.52]; HL estimate of median difference: 14.69 [0.69, 28.69]; p=0.0162) and KCCQ–OS (LS mean [SEM] change from baseline: patisiran, 0.300 [1.263]; placebo, –3.408 [1.277]; LS mean [SEM] difference: 3.709 [1.796]; p=0.0397). Consistent benefits with patisiran in 6–MWT and KCCQ–OS were observed across predefined patient subgroups. Time to first event of all–cause hospitalization, urgent HF visit, or death directionally favored patisiran vs placebo (HR [95% CI], 0.839 [0.557, 1.263]), as did all–cause mortality (HR [95% CI], 0.355 [0.110, 1.138]), but all composite outcomes endpoints did not achieve statistical significance over 12 months. Patisiran was well tolerated; most AEs were mild or moderate in severity, and there were no cardiac safety concerns. Conclusions Benefits at 12 months were observed with patisiran vs placebo in functional capacity, and health status and quality of life, in patients with ATTR amyloidosis with CM. The efficacy and safety of patisiran continues to be studied in the APOLLO–B open–label extension.

P352 EXPLORATORY ANALYSES FROM APOLLO–B, A PHASE 3 STUDY OF PATISIRAN IN PATIENTS WITH ATTR AMYLOIDOSIS WITH CARDIOMYOPATHY

Abstract Introduction Transthyretin–mediated (ATTR) amyloidosis is a progressive and fatal disease. Patients with hereditary or wild–type ATTR amyloidosis frequently develop cardiomyopathy (CM). Patisiran, an IV RNAi therapeutic that inhibits synthesis of wt and variant TTR, is approved for the treatment of hATTR amyloidosis with polyneuropathy. Methods In APOLLO–B, patients were 18–85 yrs old with evidence of cardiac amyloidosis by echocardiography, ATTR amyloid deposition on tissue biopsy or fulfilling nonbiopsy diagnostic criteria for ATTR amyloidosis with CM, and had prior hospitalization for heart failure (HF) due to ATTR amyloidosis or clinical evidence of HF. Patients were randomized 1:1 to patisiran IV 0.3 mg/kg or placebo Q3W for 12 months. The primary endpoint was change from baseline in 6–MWT at Month 12 (M12) with patisiran vs placebo. Exploratory parameters at M12 included changes in cardiac biomarkers, echocardiography and Tc scintigraphy. Results APOLLO–B enrolled 360 patients (patisiran, n=181; placebo, n=179): median age, 76.0 yrs; male, 89%; wtATTR, 80%; on tafamidis at baseline, 25%. Patisiran showed a significant benefit compared with placebo in 6–MWT (median [95% CI] change from baseline: patisiran, –8.15 [–16.42, 1.50]; placebo, –21.35 [–34.05, –7.52]; HL estimate of median difference: 14.69 [0.69, 28.69]; p=0.0162). Patisiran demonstrated favorable trends in change from baseline of NT–proBNP (mean fold change ratio [95% CI]: 0.80 [0.73, 0.89]; p=1.825×10–5) and troponin I (mean fold change ratio [95% CI]: 0.87 [0.80, 0.95]; p=0.0011) at M12 vs placebo. Patisiran also demonstrated a trend towards benefit in change from baseline of most evaluated echocardiographic parameters at M12 vs placebo. Patisiran–treated patients evaluable for scintigraphy (n=37) experienced a reduction (37.8%) or no change (62.2%) in Perugini grade at M12 compared with baseline. Among placebo patients (n=28) at M12, none experienced a reduction from baseline in Perugini grade, and 1 (3.6%) increased in grade. Patisiran demonstrated an acceptable safety profile; AEs were mostly mild or moderate in severity, and there were no cardiac safety concerns. Conclusions Exploratory analyses after 12 months provide further evidence for the potential benefit of patisiran treatment on cardiac biomarkers and manifestations of cardiac amyloid involvement in patients with ATTR amyloidosis. The long–term impact of patisiran will be assessed in the ongoing APOLLO–B open–label extension.

Evaluation of methodologies for indirect comparison of eplontersen and vutrisiran for the treatment of hereditary transthyretin-mediated amyloidosis with polyneuropathy (P12-4.005)

<h3>Objective:</h3> To investigate comparative efficacy and safety of eplontersen and vutrisiran in patients with hereditary transthyretin-mediated amyloidosis with polyneuropathy (ATTRv-PN). <h3>Background:</h3> ATTRv-PN is a rare, autosomal dominant disease caused by mutations in the gene encoding transthyretin (TTR) protein, resulting in accumulation of ATTR amyloid in multiple tissues including the peripheral nervous system. TTR silencers developed and in development to treat ATTRv-PN include vutrisiran (recently approved by the FDA and EMA), and eplontersen, which met its co-primary endpoints and demonstrated an overall favourable safety profile in a planned Week 35 interim analysis of the phase III NEURO-TTRansform trial (NCT04136184). The eplontersen and vutrisiran trials both used an external placebo arm from earlier trials. No head-to-head studies have been conducted for eplontersen and vutrisiran to date. Therefore, the feasibility and implications of indirect treatment comparisons (ITCs) should be evaluated. ATTRv-PN exhibits substantial clinical heterogeneity, which challenges traditional ITC methods. Additional challenges relate to the trial designs, including the use of external placebo comparators. Different ITC approaches will be evaluated to assess the impact of these factors on the ability to compare efficacy and safety of eplontersen and vutrisiran. <h3>Design/Methods:</h3> In addition to a naïve comparison, population-adjusted methods that attempt to account for heterogeneity and differences in trial designs were evaluated, including matching-adjusted indirect comparison (MAIC) and simulated treatment comparison (STC). Whilst MAIC may fail to match characteristics demonstrating substantial heterogeneity, this is not an issue for STC. However, unlike MAIC, STC can falter when dealing with non-linear outcomes. Efficacy will be assessed based on the Modified Neuropathy Impairment +7 and Norfolk Quality of Life-Diabetic Neuropathy scores, and safety will also be assessed. <h3>Results:</h3> Results will become available ahead of AAN2023. <h3>Conclusions:</h3> This ITC feasibility assessment supports interpretation of comparative efficacy and safety analyses, which is important for decision-making by clinicians, payers and researchers. <b>Disclosure:</b> Dr. Karam has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for Alnylam. Dr. Karam has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Ionis. Dr. Karam has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for Sanofi. Dr. Karam has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for Argenx. Dr. Karam has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Alexion. Dr. Karam has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Neuroderm. Julian D. Gillmore has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for Alnylam Pharmaceuticals. Julian D. Gillmore has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Intellia. Julian D. Gillmore has received personal compensation in the range of $500-$4,999 for serving as a Consultant for AstraZenica. Julian D. Gillmore has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for BridgeBio. Julian D. Gillmore has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Pfizer. Mrs. Chen has received personal compensation for serving as an employee of AstraZeneca. Mrs. Chen has stock in AstraZeneca. Miss Jenkins has nothing to disclose. Dr. Hale has received research support from Cancer Research UK. Dr. Taylor has received personal compensation in the range of $0-$499 for serving on a Scientific Advisory or Data Safety Monitoring board for NIH France. Dr. Chen has received personal compensation for serving as an employee of AstraZeneca. Dr. Chen has stock in AstraZeneca. Nicholas Viney has received personal compensation for serving as an employee of Ionis Pharmaceuticals Inc.. Nicholas Viney has stock in Ionis Pharmaceuticals. Nicholas Viney has received intellectual property interests from a discovery or technology relating to health care. Dr. Schneider has received personal compensation for serving as an employee of ionis pharmaceuticals.

Tc-99m labelled bone scintigraphy in suspected cardiac amyloidosis.

To perform evaluation of widely embraced bone scintigraphy-based non-biopsy diagnostic criteria (NBDC) for ATTR amyloid cardiomyopathy (ATTR-CM) in clinical practice, and to refine serum free light chain (sFLC) ratio cut-offs that reliably exclude monoclonal gammopathy (MG) in chronic kidney disease. A multi-national retrospective study of 3354 patients with suspected or histologically proven cardiac amyloidosis (CA) referred to specialist centres from 2015 to 2021; evaluations included radionuclide bone scintigraphy, serum and urine immunofixation, sFLC assay, eGFR measurement and echocardiography. Seventy-nine percent (1636/2080) of patients with Perugini grade 2 or 3 radionuclide scans fulfilled NBDC for ATTR-CM through absence of a serum or urine monoclonal protein on immunofixation together with a sFLC ratio falling within revised cut-offs incorporating eGFR; 403 of these patients had amyloid on biopsy, all of which were ATTR type, and their survival was comparable to non-biopsied ATTR-CM patients (p = 0.10). Grade 0 radionuclide scans were present in 1091 patients, of whom 284 (26%) had CA, confirmed as AL type (AL-CA) in 276 (97%) and as ATTR-CM in only one case with an extremely rare TTR variant. Among 183 patients with grade 1 radionuclide scans, 122 had MG of whom 106 (87%) had AL-CA; 60/61 (98%) without MG had ATTR-CM. The NBDC for ATTR-CM are highly specific [97% (95% CI 0.91-0.99)] in clinical setting, and diagnostic performance was further refined here using new cut-offs for sFLC ratio in patients with CKD. A grade 0 radionuclide scan all but excludes ATTR-CM but occurs in most patients with AL-CA. Grade 1 scans in patients with CA and no MG are strongly suggestive of early ATTR-type, but require urgent histologic corroboration.

RNA Targeting and Gene Editing Strategies for Transthyretin Amyloidosis.

Transthyretin (TTR) is a tetrameric protein synthesized primarily by the liver. TTR can misfold into pathogenic ATTR amyloid fibrils that deposit in the nerves and heart, causing a progressive and debilitating polyneuropathy (PN) and life-threatening cardiomyopathy (CM). Therapeutic strategies, which are aimed at reducing ongoing ATTR amyloid fibrillogenesis, include stabilization of the circulating TTR tetramer or reduction of TTR synthesis. Small interfering RNA (siRNA) or antisense oligonucleotide (ASO) drugs are highly effective at disrupting the complementary mRNA and inhibiting TTR synthesis. Since their development, patisiran (siRNA), vutrisiran (siRNA) and inotersen (ASO) have all been licensed for treatment of ATTR-PN, and early data suggest these drugs may have efficacy in treating ATTR-CM. An ongoing phase 3 clinical trial will evaluate the efficacy of eplontersen (ASO) in the treatment of both ATTR-PN and ATTR-CM, and a recent phase 1 trial demonstrated the safety of novel in vivo CRISPR-Cas9 gene-editing therapy in patients with ATTR amyloidosis. Recent results from trials of gene silencer and gene-editing therapies suggest these novel therapeutic agents have the potential to substantially alter the landscape of treatment for ATTR amyloidosis. Their success has already changed the perception of ATTR amyloidosis from a universally progressive and fatal disease to one that is treatable through availability of highly specific and effective disease-modifying therapies. However, important questions remain including long-term safety of these drugs, potential for off-target gene editing, and how best to monitor the cardiac response to treatment.Kindly check and confirm the processed running title.This is correct.

Identification of a novel transthyretin mutation D39Y in a cardiac amyloidosis patient and its biochemical characterizations.

Hereditary transthyretin cardiac amyloidosis (hATTR-CA) is a rare autosomal dominantly inherited disease caused by mutations in the transthyretin (TTR) gene. TTR mutations often cause the instability of transthyretin, production of misfolded proteins, and ultimately excessive deposition of insoluble amyloid fibrils in the myocardium, thereby leading to cardiac dysfunction. Herein, we report a novel transthyretin D39Y mutation in a Chinese family. We characterized the kinetic and thermodynamic stabilities of D39Y mutant TTR, revealing that TTR D39Y mutant was less stable than WT TTR and more stable than amyloidogenic mutation TTR L55P. Meanwhile, the only FDA approved drug Tafamidis showed satisfactory inhibitory effect toward ATTR amyloid formation and strong binding affinity in test tube revealed by isothermal titration calorimetry. Finally, we measured the well-folded tetrameric TTR concentration in patient's and his descents' blood serum using a previously reported UPLC-based assay. Notably, the tetramer concentrations gradually increased from symptomatic D39Y gene carrier father, to asymptomatic D39Y gene carrier daughter, and further to wild type daughter, suggesting the decrease in functional tetrameric TTR concentration may serve as an indicator for disease age of onset in D39Y gene carriers. The study described a Chinese family with hATTR-CA due to the TTR variant D39Y with its destabilizing effect in both kinetic and thermodynamic stabilities.

593 PRESENTATION AND OUTCOME OF PATIENTS WITH COEXISTING CARDIAC AL AND ATTR AMYLOIDOSIS

Abstract Background Incidence of ATTRwt amyloidosis increased in the last years, especially thanks to the possibility of a non-bioptic diagnosis. However, if a monoclonal component (MC) is detected, amyloid typing is mandatory to rule out AL amyloidosis. Differential diagnosis is even more important now that we have labelled treatment for both types of amyloidosis. In some cases, typing on endomyocardial biopsy (EMB) revealed the presence of both light chains (LC) and transthyretin (TTR) in the heart of the same patient (Sidiqi, et al, Blood Cancer J. 2019). We present a series of patients with coexisting AL and ATTR in the heart. In these cases, we elected to administer treatment for AL amyloidosis due to the more rapid course of this disease and because in Italy and in Germany tafamidis is not prescribable for patients with coexisting AL amyloidosis. Objective: To evaluate the outcome of patients with coexisting AL and ATTR amyloidosis in the heart after treatment for AL amyloidosis. Material &amp; Methods: We identified 17 patients with coexisting AL and ATTR amyloidosis evaluated in a German (n=5) or Italian (n=12) referral center. Typing was performed by immunohistochemistry with custom made antibodies (IHC; N=5) and immunoelectron microscopy (IEM; N=8). In 4 cases, EBM samples were subjected to protein extraction following a new procedure (patent n EP3417295). SDS-PAGE/Western Blot analysis was performed for the main amyloidogenic proteins and its results were validated by mass spectrometry. Cardiac responses and progression were assessed according to AL amyloidosis criteria. Results 3 patients were excluded because of post-mortem diagnosis and 1 because amyloid deposits were characterized on bone marrow biopsy and it was not possible to unequivocally conclude for the coexistence of LC and TTR in the heart. Thirteen patients were included in the analysis. Four had a positive abdominal fat pad (AFP). Three underwent EMB because typing by IHC was not possible on AFP by IHC. In 1 IEM was positive for TTR only, but given the presence of an abnormal free LC ratio and albuminuria, the patient underwent EBM that was positive both for LC and TTR at IEM. Perugini score 1 was observed in 3 patients, of whom 1 was positive for a TTR mutation (Val40Ile). In 1 case, bone marrow plasma cell infiltrate was ≥60%, in absence of other multiple myeloma defining events. All patients received treatment for AL amyloidosis and 10 had response data. None was treated for ATTR. At 6 months 9 achieved a hematologic response (HR) and 3 a cardiac response. Six patients had a cardiac progression, despite 5 had achieved a HR (complete response [CR] in 2, very good partial response [VGPR] in 2 and partial response in 1 case). After a median follow-up of 33.8 months, 2 patients died of worsening of heart failure after 15 and 48 months from diagnosis, despite the maintenance of CR and VGPR. Both had AL cardiac progression at 6 months. Summary &amp; Conclusion This is the largest series of patients with coexisting AL and ATTR amyloid deposits in the heart and the first reporting outcome to AL treatment. The AFP was commonly negative and when positive typing was not possible by IHC. Moreover, IEM did not find AL deposits in 1 case. This was unexpected and may reflect a selection bias: patients with a MC and AL amyloidosis in the AFP are unlikely to be further tested for ATTR. This finding warrants further studies and a possible revision of diagnostic algorithms. Treatment for AL amyloidosis may not be able to stop progression in these patients, who should be granted access to anti-ATTR therapies.

Abstract 15218: Hereditary Transthyretin Cardiac Amyloidosis in a Patient at High Risk for Plasma Cell Dyscrasias: A Complex Diagnostic Work-Up

Background: Cardiac amyloidosis (CA) is most commonly caused by the infiltration of misfolded amyloid transthyretin (ATTR) or light chain (AL) molecules in the myocardium. The workup for CA requires serum assessment to exclude the rapidly progressive AL subtype before imaging to assess the more common and indolent ATTR form. The diagnostic algorithm has the potential to become complex as in this case about a woman with Sjogren’s syndrome which predisposes to plasma cell dyscrasias. Case: A 68-year-old female with congenital long QT syndrome requiring implantable cardioverter-defibrillator (ICD), bilateral carpal tunnel, and recent diagnosis of Sjogren’s syndrome was found to have new systolic heart failure during workup for interstitial lung disease. Labs revealed pancytopenia, abnormal kappa/lambda ratio (2.25), and polyclonal gammopathy on immunofixation. Echocardiogram showed moderately reduced systolic function with an ejection fraction of 35-40% and moderate concentric left ventricular hypertrophy. Decision-making: Cardiology obtained cardiac magnetic resonance imaging (CMR) that returned indeterminate due to ICD artifact. Hematology performed a bone marrow biopsy that was negative for plasma cell dyscrasia, though stained positive for amyloid on congo red with mass spectrometry revealing the valine 142 isoleucine (Val142Ile) amino acid substitution consistent with hereditary ATTR-CA. To definitively assess for endomyocardial ATTR involvement the patient was sent for cardiac scintigraphy using 99mTc-labeled pyrophosphate which returned negative with a cardiac to contralateral lung uptake ratio of 1.001 and visual grade of 0. With suspicion still high, an endomyocardial biopsy was pursued and stained positive for congo red with mass spectrometry confirming hereditary Val142Ile ATTR-CA, allowing for tafamidis initiation. Conclusion: We present a complex diagnostic case of ATTR amyloid in a patient who is at high risk for plasma cell dyscrasias with an abnormal kappa/lambda ratio, non-diagnostic CMR, and negative nuclear scan. Despite significant advances in imaging techniques allowing for less invasive diagnostics, this case highlights the role of endomyocardial biopsy in complicated cases.

Extracellular Volume Fraction by Computed Tomography Predicts Long-Term Prognosis Among Patients With Cardiac Amyloidosis

BackgroundLight chain (AL) and transthyretin (ATTR) amyloid fibrils are deposited in the extracellular space of the myocardium, resulting in heart failure and premature mortality. Extracellular expansion can be quantified by computed tomography, offering a rapid, cheaper, and more practical alternative to cardiac magnetic resonance, especially among patients with cardiac devices or on renal dialysis. ObjectivesThis study sought to investigate the association of extracellular volume fraction by computed tomography (ECVCT), myocardial remodeling, and mortality in patients with systemic amyloidosis. MethodsPatients with confirmed systemic amyloidosis and varying degrees of cardiac involvement underwent electrocardiography-gated cardiac computed tomography. Whole heart and septal ECVCT was analyzed. All patients also underwent clinical assessment, electrocardiography, echocardiography, serum amyloid protein component, and/or technetium-99m (99mTc) 3,3-diphosphono-1,2-propanodicarboxylic acid scintigraphy. ECVCT was compared across different extents of cardiac infiltration (ATTR Perugini grade/AL Mayo stage) and evaluated for its association with myocardial remodeling and all-cause mortality. ResultsA total of 72 patients were studied (AL: n = 35, ATTR: n = 37; median age: 67 [IQR: 59-76] years, 70.8% male). Mean septal ECVCT was 42.7% ± 13.1% and 55.8% ± 10.9% in AL and ATTR amyloidosis, respectively, and correlated with indexed left ventricular mass (r = 0.426; P < 0.001), left ventricular ejection fraction (r = 0.460; P < 0.001), N-terminal pro–B-type natriuretic peptide (r = 0.563; P < 0.001), and high-sensitivity troponin T (r = 0.546; P < 0.001). ECVCT increased with cardiac amyloid involvement in both AL and ATTR amyloid. Over a mean follow-up of 5.3 ± 2.4 years, 40 deaths occurred (AL: n = 14 [35.0%]; ATTR: n = 26 [65.0%]). Septal ECVCT was independently associated with all-cause mortality in ATTR (not AL) amyloid after adjustment for age and septal wall thickness (HR: 1.046; 95% CI: 1.003-1.090; P = 0.037). ConclusionsCardiac amyloid burden quantified by ECVCT is associated with adverse cardiac remodeling as well as all-cause mortality among ATTR amyloid patients. ECVCT may address the need for better identification and risk stratification of amyloid patients, using a widely accessible imaging modality.