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Systemic AL amyloidosis is one of the most frequently diagnosed forms of systemic amyloidosis. It arises from mutational changes in immunoglobulin light chains. To explore whether these mutations may affect the structure of the formed fibrils, we determine and compare the fibril structures from several patients with cardiac AL amyloidosis. All patients are affected by light chains that contain an IGLV3-19 gene segment, and the deposited fibrils differ by the mutations within this common germ line background. Using cryo-electron microscopy, we here find different fibril structures in each patient. These data establish that the mutations of amyloidogenic light chains contribute to defining the fibril architecture and hence the structure of the pathogenic agent., (© 2024. The Author(s).)

Lenalidomide and dexamethasone (RD) is a standard treatment in relapsed/refractory immunoglobulin light chain (AL) amyloidosis (RRAL). We retrospectively investigated toxicity, efficacy and prognostic markers in 260 patients with RRAL. Patients received a median of two prior treatment lines (68% had been bortezomib-refractory; 33% had received high-dose melphalan). The median treatment duration was four cycles. The 3-month haematological response rate was 31% [very good haematological response (VGHR) in 18%]. The median follow-up was 56·5 months and the median overall survival (OS) and haematological event-free survival (haemEFS) were 32 and 9 months. The 2-year dialysis rate was 15%. VGHR resulted in better OS (62 vs. 26 months, P < 0·001). Cardiac progression predicted worse survival (22 vs. 40 months, P = 0·027), although N-terminal prohormone of brain natriuretic peptide (NT-proBNP) increase was frequently observed. Multivariable analysis identified these prognostic factors: NT-proBNP for OS [hazard ratio (HR) 1·71; P < 0·001]; gain 1q21 for haemEFS (HR 1·68, P = 0·014), with a trend for OS (HR 1·47, P = 0·084); difference between involved and uninvolved free light chains (dFLC) and light chain isotype for OS (HR 2·22, P < 0·001; HR 1·62, P = 0·016) and haemEFS (HR 1·88, P < 0·001; HR 1·59, P = 0·008). Estimated glomerular filtration rate (HR 0·71, P = 0·004) and 24-h proteinuria (HR 1·10, P = 0·004) were prognostic for renal survival. In conclusion, clonal and organ biomarkers at baseline identify patients with favourable outcome, while VGHR and cardiac progression define prognosis during RD treatment., (© 2021 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Systemic antibody light chains (AL) amyloidosis is characterized by deposition of amyloid fibrils derived from a particular antibody light chain. Cardiac involvement is a major risk factor for mortality. Using MAS solid-state NMR, we studied the fibril structure of a recombinant light chain fragment corresponding to the fibril protein from patient FOR005, together with fibrils formed by protein sequence variants that are derived from the closest germline (GL) sequence. Both analyzed fibril structures were seeded with ex-vivo amyloid fibrils purified from the explanted heart of this patient. We find that residues 11-42 and 69-102 adopt β-sheet conformation in patient protein fibrils. We identify arginine-49 as a key residue that forms a salt bridge to aspartate-25 in the patient protein fibril structure. In the germline sequence, this residue is replaced by a glycine. Fibrils from the GL protein and from the patient protein harboring the single point mutation R49G can be both heterologously seeded using patient ex-vivo fibrils. Seeded R49G fibrils show an increased heterogeneity in the C-terminal residues 80-102, which is reflected by the disappearance of all resonances of these residues. By contrast, residues 11-42 and 69-77, which are visible in the MAS solid-state NMR spectra, show 13 Cα chemical shifts that are highly like patient fibrils. The mutation R49G thus induces a conformational heterogeneity at the C terminus in the fibril state, whereas the overall fibril topology is retained. These findings imply that patient mutations in FOR005 can stabilize the fibril structure., Competing Interests: Conflict of interest—The authors declare no competing financial interest., (© 2020 Pradhan et al.)

In systemic light chain amyloidosis, an overexpressed antibody light chain (LC) forms fibrils which deposit in organs and cause their failure. While it is well-established that mutations in the LC's V L domain are important prerequisites, the mechanisms which render a patient LC amyloidogenic are ill-defined. In this study, we performed an in-depth analysis of the factors and mutations responsible for the pathogenic transformation of a patient-derived λ LC, by recombinantly expressing variants in E. coli . We show that proteolytic cleavage of the patient LC resulting in an isolated V L domain is essential for fibril formation. Out of 11 mutations in the patient V L , only one, a leucine to valine mutation, is responsible for fibril formation. It disrupts a hydrophobic network rendering the C-terminal segment of V L more dynamic and decreasing domain stability. Thus, the combination of proteolytic cleavage and the destabilizing mutation trigger conformational changes that turn the LC pathogenic., Competing Interests: PK, MV, MP, LH, BW, UH, MZ, RK, SS, MG, JB No competing interests declared, (© 2020, Kazman et al.)

The difference between involved minus uninvolved serum free light chains (dFLC) has been established as an invaluable hematologic parameter in systemic amyloid light chain (AL) amyloidosis. However, patients with an initial dFLC level <50 mg/L are currently deemed not evaluable for response to therapy. Therefore, we aimed to characterize this subgroup of patients and to define novel hematologic response parameters. We retrospectively analyzed 783 AL patients newly diagnosed at our center between 2002 and 2016. Patients with a dFLC level <50 mg/L showed smaller bone marrow plasmacytosis compared to patients with a dFLC level ≥50 mg/L (7% vs 10%, P < .001), but no significant differences in all analyzed chromosomal aberrations. Cardiac involvement was less frequent (45% vs 80%, P < .001) and less severe (Mayo 2004 stage III: 18% vs 51%, P < .001), whereas kidney involvement was more prevalent (83% vs 53%, P < .001) and proteinuria was higher (7.3 g/L vs 5.0 g/L, P < .001). In multivariate analyses, a dFLC level <50 mg/L appeared to be an independent prognostic factor with respect to overall survival (hazard ratio [HR] = 0.50, P = .003) and renal survival (HR = 0.56, P = .020). Patients with a dFLC level <50 mg/L showed a higher proportion of complete hematologic response after first-line therapy compared to patients with a dFLC level ≥50 mg/L (39% vs 9%, P < .001). Receiver-operating characteristics analysis identified a low-dFLC partial response (dFLC <10 mg/L for patients with a dFLC between 20 and 50 mg/L), which predicted overall and renal survival already at 3 months after the start of therapy. Importantly, a parallel Italian study validated this new hematologic remission parameter. The outcome of prospective clinical trials might be adversely influenced by exclusion of the favorable clinical subgroup with an initial dFLC <50 mg/L. We propose the appreciation of dFLC in hematologic response assessment for all patients with a baseline dFLC >20 mg/L., (© 2017 by The American Society of Hematology.)

Immunoglobulin light chain (AL) amyloidosis is a rare plasma cell dyscrasia characterized by the deposition of abnormal amyloid fibrils in multiple organs, thus impairing their function. In the largest cohort studied up to now of 118 CD138-purified plasma cell samples from previously untreated immunoglobulin light chain amyloidosis patients, we assessed in parallel copy number alterations using high-density copy number arrays and interphase fluorescence in situ hybridization (iFISH). We used fluorescence in situ hybridization probes for the IgH translocations t(11;14), t(4;14), and t(14;16) or any other IgH rearrangement as well as numerical aberrations of the chromosome loci 1q21, 8p21, 5p15/5q35, 11q22.3 or 11q23, 13q14, 15q22, 17p13, and 19q13. Recurrent gains included chromosomes 1q (36%), 9 (24%), 11q (24%), as well as 19 (15%). Recurrent losses affected chromosome 13 (29% monosomy) and partial losses of 14q (19%), 16q (14%) and 13q (12%), respectively. In 88% of patients with translocation t(11;14), the hallmark chromosomal aberration in AL amyloidosis, a concomitant gain of 11q22.3/11q23 detected by iFISH was part of the unbalanced translocation der(14)t(11;14)(q13;q32) with the breakpoint in the CCND1/MYEOV gene region. Partial loss of chromosome regions 14q and 16q were significantly associated to gain 1q. Gain 1q21 detected by iFISH almost always resulted from a gain of the long arm of chromosome 1 and not from trisomy 1, whereas deletions on chromosome 1p were rarely found. Overall and event-free survival analysis found a potential adverse prognostic effect of concomitant gain 1q and deletion 14q as well as of deletion 1p. In conclusion, in the first whole genome report of clonal plasma cells in AL amyloidosis, novel aberrations and hitherto unknown potential adverse prognostic effects were uncovered., (Copyright© 2017 Ferrata Storti Foundation.)

Intervention into amyloid deposition with anti-amyloid agents like the polyphenol epigallocatechin-3-gallate (EGCG) is emerging as an experimental secondary treatment strategy in systemic light chain amyloidosis (AL). In both AL and multiple myeloma (MM), soluble immunoglobulin light chains (LC) are produced by clonal plasma cells, but only in AL do they form amyloid deposits in vivo We investigated the amyloid formation of patient-derived LC and their susceptibility to EGCG in vitro to probe commonalities and systematic differences in their assembly mechanisms. We isolated nine LC from the urine of AL and MM patients. We quantified their thermodynamic stabilities and monitored their aggregation under physiological conditions by thioflavin T fluorescence, light scattering, SDS stability, and atomic force microscopy. LC from all patients formed amyloid-like aggregates, albeit with individually different kinetics. LC existed as dimers, ∼50% of which were linked by disulfide bridges. Our results suggest that cleavage into LC monomers is required for efficient amyloid formation. The kinetics of AL LC displayed a transition point in concentration dependence, which MM LC lacked. The lack of concentration dependence of MM LC aggregation kinetics suggests that conformational change of the light chain is rate-limiting for these proteins. Aggregation kinetics displayed two distinct phases, which corresponded to the formation of oligomers and amyloid fibrils, respectively. EGCG specifically inhibited the second aggregation phase and induced the formation of SDS-stable, non-amyloid LC aggregates. Our data suggest that EGCG intervention does not depend on the individual LC sequence and is similar to the mechanism observed for amyloid-β and α-synuclein., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Cytogenetic aberrations detected by interphase fluorescence in situ hybridization (iFISH) of plasma cells are routinely evaluated as prognostic markers in multiple myeloma. This long-term follow-up study aimed to assess the prognosis of systemic light chain amyloidosis (AL) patients treated with high-dose melphalan (HDM) chemotherapy and autologous stem cell transplantation, depending on iFISH results. Therefore, we analyzed a consecutive cohort of 123 AL patients recruited from 2003 to 2014. HDM was safe, with only 1 of 123 patients dying as a result of treatment-related mortality, and effective, with a complete remission (CR) rate of 34%. Translocation t(11;14) as the most prevalent aberration (59%) led to an improved CR rate after high-dose therapy (41.2% vs 20.0%; P = .02), translating into a prolonged hematologic event-free survival (hemEFS; median, 46.1 vs 28.1 months; P = .05) and a trend for better overall survival (median, not reached vs 93.7 months; P = .07). In multivariate analysis, t(11;14) was confirmed as a favorable prognostic factor regarding hemEFS along with lower values for the difference between involved and uninvolved free light chains. Conversely, deletion 13q14, gain of 1q21, and hyperdiploidy had no significant prognostic impact. The high-risk cytogenetic aberrations t(4;14), t(14;16), and del(17p13) conferred an unfavorable prognosis, although statistical significance was reached only for univariate CR analysis in this small group of 9 patients. Thus, t(11;14) positivity in HDM-treated AL patients conferred superior CR rates and hemEFS. In view of the reduced response of t(11;14) to bortezomib, this highlights the impact of therapy on the prognostic role of cytogenetic aberrations., (© 2016 by The American Society of Hematology.)

Systemic amyloid light chain (AL) amyloidosis is a life-threatening protein deposition disorder; however, effective therapy can dramatically improve the prognosis of AL patients. Therefore, accurate diagnosis of the underlying hematologic disease is important. Multi-parameter flow cytometry (MFC) is a reliable method to analyze lymphatic neoplasias and to detect even a small lymphatic clone. We analyzed the presence of clonal plasma cell (PC) and B cells in the bone marrow of 63 patients with newly diagnosed AL amyloidosis by MFC. We compared the results with the levels of monoclonal protein, the histopathology and cytogenetic results. As reference of light chain restriction, we used the immunohistochemical results of κ or λ positive amyloid deposits in various tissues. MFC identified underlying clonal lymphatic cells in all but two patients (61 of 63, 97%). Sixty-one patients harbored malignant PCs, whereas B-cell lymphomas were identified in two patients. Furthermore, MFC indicated at least one putative immunotherapeutical target (CD20, CD38, CD52, or SLAMF7) on malignant PCs in all but one patient. These results demonstrate that MFC is a reliable tool for an accurate diagnosis of the underlying hematologic disease and the detection of potential immunotherapeutical targets in patients with AL amyloidosis., (© 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Purpose: Light chain (AL) amyloidosis is caused by the accumulation of misfolded proteins, which induces the dysfunction of vital organs. NEOD001 is a monoclonal antibody targeting these misfolded proteins. We report interim data from a phase I/II dose-escalation/expansion study of NEOD001 in patients with AL amyloidosis and persistent organ dysfunction (NCT01707264)., Patients and Methods: Patients who had completed at least one previous anti-plasma cell-directed therapy, had partial hematologic response or better, and had persistent organ dysfunction received NEOD001 intravenously every 28 days. Dose levels of 0.5, 1, 2, 4, 8, 16, and 24 mg/kg were evaluated (3 + 3 study design). Primary objectives were to determine the maximum tolerated dose and the recommended dose for future studies and to evaluate safety/tolerability. Secondary and exploratory objectives included pharmacokinetics, immunogenicity, and organ responses on the basis of published consensus criteria., Results: Twenty-seven patients were enrolled in seven cohorts (dose-escalation component). No drug-related serious adverse events (AEs), discontinuations because of drug-related AEs, dose-limiting toxicities, or antidrug antibodies were reported. The most frequent AEs were fatigue, upper respiratory tract infection, cough, and dyspnea. Recommended dosing was 24 mg/kg. Pharmacokinetics support intravenous dosing every 28 days. Of 14 cardiac-evaluable patients, eight (57%) met the criteria for cardiac response and six (43%) had stable disease. Of 15 renal-evaluable patients, nine (60%) met the criteria for renal response and six (40%) had stable disease., Conclusion: Monthly infusions of NEOD001 were safe and well tolerated. Recommended future dosing was 24 mg/kg. Organ response rates compared favorably with those reported previously for chemotherapy. A phase II expansion is ongoing. A global phase III study (NCT02312206) has been initiated. Antibody therapy targeting misfolded proteins is a potential new therapy for the management of AL amyloidosis., (© 2016 by American Society of Clinical Oncology.)

The kidney is involved in 70% of patients with immunoglobulin light-chain (AL) amyloidosis, but little is known on progression or reversibility of renal involvement, and criteria for renal response have never been validated. Newly diagnosed patients from the Pavia (n = 461, testing cohort) and Heidelberg (n = 271, validation cohort) centers were included. Proteinuria >5 g/24 h and estimated glomerular filtration rate (eGFR) <50 mL/min predicted progression to dialysis best. Proteinuria below and eGFR above the thresholds indicated low risk (0 and 4% at 3 years in the testing and validation cohorts, respectively). High proteinuria and low eGFR indicated high risk (60% and 85% at 3 years). At 6 months, a ≥25% eGFR decrease predicted poor renal survival in both cohorts and was adopted as criterion for renal progression. A decrease in proteinuria by ≥30% or below 0.5 g/24 h without renal progression was the criterion for renal response, being associated with longer renal survival in the testing and validation populations. Hematologic very good partial or complete remission at 6 months improved renal outcome in both populations. We identified and validated a staging system for renal involvement and criteria for early assessment of renal response and progression in AL amyloidosis that should be used in clinical practice and trial design., (© 2014 by The American Society of Hematology.)

Purpose: To identify the criteria for hematologic and cardiac response to treatment in immunoglobulin light chain (AL) amyloidosis based on survival analysis of a large patient population., Patients and Methods: We gathered for analysis 816 patients with AL amyloidosis from seven referral centers in the European Union and the United States. A different cohort of 374 patients prospectively evaluated at the Pavia Amyloidosis Research and Treatment Center was used for validation. Data was available for all patients before and 3 and/or 6 months after initiation of first-line therapy. The prognostic relevance of different criteria for hematologic and cardiac response was assessed., Results: There was a strong correlation between the extent of reduction of amyloidogenic free light chains (FLCs) and improvement in survival. This allowed the identification of four levels of response: amyloid complete response (normal FLC ratio and negative serum and urine immunofixation), very good partial response (difference between involved and uninvolved FLCs [dFLC] < 40 mg/L), partial response (dFLC decrease > 50%), and no response. Cardiac involvement is the major determinant of survival, and changes in cardiac function after therapy can be reliably assessed using the cardiac biomarker N-terminal natriuretic peptide type B (NT-proBNP). Changes in FLC and NT-proBNP predicted survival as early as 3 months after treatment initiation., Conclusion: This study identifies and validates new criteria for response to first-line treatment in AL amyloidosis, based on their association with survival in large patient populations, and offers surrogate end points for clinical trials.

AL amyloidosis (AL) results from the misfolding of immunoglobulin light chains (IG LCs). Aim of this study was to comprehensively analyse kappa LC sequences from AL patients in comparison with multiple myeloma (MM). We analysed IGKV/IGKJ usage and associated organ tropism and IGKV1/D-33 in terms of mutational analysis and theoretical biochemical properties. cDNA and bulk RNA sequencing of the LCs of AL and MM patients. We studied 41 AL and 83 MM patients showing that IGKV1 was most expressed among kappa AL and MM, with higher frequency in AL (80% vs. 53%, p =.002). IGKV3 was underrepresented in AL (10% vs. 30%, p =.014). IGKJ2 was more commonly used in AL than in MM (39% vs. 29%). Patients with IGKV1/D-33 were associated with heart involvement (75%, p =.024). IGKV1/D-33-segments of AL had a higher mutation count (AL = 12.0 vs. MM = 10.0). FR3 and CDR3 were most frequently mutated in both, with a median mutation count in FR3 being the highest (AL = 4.0; MM = 3.5) and one mutation hotspot (FR3 (83I)) for IGKV1/D-33/IGKJ2 was associated with cardiac involvement. This study confirmed that germline usage has an influence on AL amyloidosis risk and organ involvement. [ABSTRACT FROM AUTHOR]

Light chain amyloidosis (AL) is a rare disease caused by the generalized deposition of misfolded free light chains. Patients with immunoglobulin M gammopathy (IgM) and indolent B-cell lymphoma such as marginal zone lymphoma (MZL) may in some instances develop AL amyloidosis. So far, CAR T cells for AL amyloidosis have only been reported utilizing the B cell maturation antigen as target, while CD19 has so far not been used in AL amyloidosis. We report the case of a 71-year-old male, diagnosed with systemic AL kappa amyloidosis and MZL, receiving third-generation CAR T cell therapy targeting CD19. Prior treatment included bendamustine/rituximab and cyclophosphamide/ dexamethasone with subsequent autologous stem cell transplantation. CAR T application was well tolerated despite heart and kidney amyloid manifestations, and only early low-grade procedure-specific toxicities were observed. A continuous decrease in IgM, kappa light chains and kappa-to-lambda light chain difference was observed in the patient from day + 30 on, resulting in a deep hematological response six months after treatment. In summary, we present a novel case of CAR T cell treatment with third generation CD19 directed infusion for AL amyloidosis with an underlying secretory active B cell lymphoma, showing that this is an effective treatment modality and can be applied to patients with subsequent AL amyloidosis. [ABSTRACT FROM AUTHOR]

Light chain amyloidosis (AL) is one of the most common forms of systemic amyloidosis and is caused by the deposition of insoluble fibrils derived from misfolded and aggregated immunoglobulin light chains (LC). To uncover the causes leading to this aggregation, we compared AL LC sequences with those of patients with the related disease multiple myeloma (MM), which do not aggregate in insoluble fibrils in vivo. IGLV2‐14 is one of the most common AL‐associated IGLV subfamilies. Here, we analysed IGLV2‐14 LC sequences of 13 AL and eight MM patients in detail. We found that AL‐associated LCs presented a lower median mutation count (7.0 vs. 11.5 in MM; P = 0.045), as well as an overall composition of less charged amino acids than MM LCs. However, we did not find a mutation that was present in ≥ 50% of the AL and not in the MM sequences. Furthermore, we did not find a significant difference in the isoelectric point (pI) in general, suggesting similar stability of the LCs in AL and MM. However, the subgroup of patients without a detectable heavy chain stood out. Surprisingly, they are characterized by an increase in mutation count (median 7.0 vs. 5.5) and pI (median 7.82 vs. 6.44, P = 0.043). In conclusion, our data suggest that the amount of mutations and the introduction of charges play a crucial role in AL fibril formation, as well as the absence or presence of a potential heavy chain binding partner. [ABSTRACT FROM AUTHOR]

Cytogenetic abnormalities are common in patients with amyloid light-chain (AL) amyloidosis; some are associated with poorer outcomes. This post hoc analysis of ANDROMEDA evaluated the impact of certain cytogenetic abnormalities on outcomes in this patient population. Patients with newly diagnosed AL amyloidosis were randomised 1:1 to daratumumab, bortezomib, cyclophosphamide, and dexamethasone (D-VCd) or VCd. Outcomes were evaluated in the intent-to-treat (ITT) population and in patients with t(11;14), amp1q21, del13q14, and del17p13. Overall, 321 patients had cytogenetic testing (D-VCd, n = 155; VCd, n = 166); most common abnormalities were t(11;14) and amp1q21. At a median follow-up of 20.3 months, haematologic complete response rates were higher with D-VCd vs VCd across all cytogenetic subgroups and organ response rates were numerically higher with D-VCd vs VCd across most subgroups. Point estimates for hazard ratio of major organ deterioration-PFS and -EFS favoured D-VCd over VCd for all cytogenetic subgroups. Deep haematologic responses (involved minus uninvolved free light chains [FLC] <10 mg/L or involved FLC ≤20 mg/L) were seen in more patients with D-VCd than VCd in all ITT and t(11;14) cohorts. These results support the use of D-VCd as standard of care in patients with newly diagnosed AL amyloidosis regardless of cytogenetic abnormalities. [ABSTRACT FROM AUTHOR]

Light chain amyloidosis is one of the most common forms of systemic amyloidosis. The disease is caused by the misfolding and aggregation of immunoglobulin light chains to insoluble fibrils. These fibrils can deposit in different tissues and organs such as heart and kidney and cause organ impairments that define the clinical presentation. In this study, we present an overview of IGLV-IGLJ and IGLC germline utilization in 85 patients classified in three clinically important subgroups with dominant cardiac, renal as well as cardiac and renal involvement. We found that IGLV3 was the most frequently detected IGLV-family in patients with dominant cardiac involvement, whereas in renal patients IGLV1 were most frequently identified. For patients with dominant heart and kidney involvement IGLV6 was the most frequently detected IGLV-family. In more detailed analysis IGLV3-21 was observed as the most dominant IGLV-subfamily for patients with dominant heart involvement and IGLV1-44 as the most frequent IGLV-subfamily in the group of patients with dominant kidney involvement. For patients with dominant heart and kidney involvement IGLV6-57 was the most frequently detected IGLV-subfamily. Additionally, we were able to show an exclusive linkage between IGLJ1 and IGLC1 as well as between IGLJ2 and IGLC2 in the fully assembled IGL mRNA. [ABSTRACT FROM AUTHOR]

Several studies recently showed that ex vivo fibrils from patient or animal tissue were structurally different from in vitro formed fibrils from the same polypeptide chain. Analysis of serum amyloid A (SAA) and Aβ-derived amyloid fibrils additionally revealed that ex vivo fibrils were more protease stable than in vitro fibrils. These observations gave rise to the proteolytic selection hypothesis that suggested that disease-associated amyloid fibrils were selected inside the body by their ability to resist endogenous clearance mechanisms. We here show, for more than twenty different fibril samples, that ex vivo fibrils are more protease stable than in vitro fibrils. These data support the idea of a proteolytic selection of pathogenic amyloid fibril morphologies and help to explain why only few amino acid sequences lead to amyloid diseases, although many, if not all, polypeptide chains can form amyloid fibrils in vitro. [ABSTRACT FROM AUTHOR]