Immunoglobulin Free Light Chains Research Articles

#2167 Polyclonal immunoglobulin free light chains: an evidence of cardiac remodeling in patients with glomerular diseases

Abstract Background and Aims It was previously shown, that polyclonal immunoglobulin free light chains (FLC) are the independent predictor of mortality in patients with decompensated heart failure, chronic kidney disease (CKD) and also in general population. Mechanisms of this phenomenon are still unknown. The aim of our study is to reveal the possible relationships between FLC and cardiac remodeling in patients with glomerular diseases. Method We included 97 patients with biopsy-proven diagnosis of different immune-mediated glomerular diseases, 53% were men, mean age was 48 ± 14.6 years. Serum Freelite FLC levels were measured as well as standard clinical and laboratory investigation. At the time of biopsy transthoracic echocardiography was also performed. Glomerular filtration rate (eGFR) was estimated by “2021 CKD-EPI Creatinine” equation. Results Median of eGFR was 46.8 ml/min/1.73 m² (28.7-80.0), FLC-kappa level was 27.4 mg/l (16.8- 48.4), FLC-lambda level was 28.2 mg/l (20.8-43.3). FLC-kappa levels were above reference range in 65%, FLC-lambda levels – in 54%. LV diastolic dysfunction (DD) was present in 68%. Comparison in groups showed that the elevation of FLC-kappa more than 19.4 mg/l was associated with bigger LV mass index (LVMI) – 138.3 ± 30.2 g/m² vs 99.9 ± 31.2 g/m² р = 0.022, the parallel correlation was found between LVMI and levels of FLC-lambda higher than 26.3 mg/l (136.2 ± 64.8 g/m² vs 104.0 ± 36.6 g/m², р = 0.023). Spearman analysis has shown statistically significant correlations between FLC levels and E/e’- Rs = 0.501 p = 0.0001 for FLC-kappa, Rs = 0.343 p = 0.012 for FLC-lambda. Combined FLC level higher than 52.9 mg/l was a significant predictor of DD with area under the ROC curve (AUC) 0.703 (sensitivity 76.5%, specificity 62.5%). Conclusion Elevation of serum pFLC level could be a promising marker of cardiac remodeling, especially, of DD in patients with glomerular diseases. Possible pathogenic role of FLC in cardiorenal relationships may be revealed in further studies.

Immunoglobulin repertoire sequencing and de novo sequencing – Powerful tools for identifying free light chains from patients with light chain cast nephropathy

In patients with light chain cast nephropathy (LCCN), abundantly produced monoclonal immunoglobulin free light chains (FLCs) play a vital role in pathogenesis. Determining the precise sequences of patient-derived FLCs is therefore highly desirable. Although immunoglobulin repertoire sequencing (5′ RACE-seq) has been proven to be sensitive enough to provide full-length V(D)J region (variable, diversity and joining genes) of FLCs using bone marrow samples, an invasive and bone marrow independent method is still in demand. Here a de novo sequencing workflow based on the bottom-up proteomics for patient-derived FLCs was established. PEAKS software was used for the de novo sequencing of peptides that were further assembled into full-length FLC sequences. This de novo protein sequencing method can obtain the full-length amino acid sequences of FLCs, and had been shown to be as reliable as 5′ RACE-seq. The two LCCN sequences derived from above the two methods were identical, and they possessed more hydrophobic or nonpolar amino acids compared with the corresponding germline, which may be associated with the pathogenesis.

Expert Center for cardiac amyloidosis: reality and perspectives

Aim. To evaluate the features of diagnosis of amyloid cardiomyopathy (ACMP), differential diagnosis of different types of amyloidosis and its clinical manifestations. Materials and methods. Were analyzed 150 cases of patients who consulted at the Expert Center for Amyloidosis with suspicion of the presence of ACMP. 63 patients were diagnosed with ACMP: 25 (39.7%) – women, 38 (60.3%) – men, with an average age of 64.1±1.5. 36 (57.1%) patients had AL-amyloidosis (immunoglobulin amyloid light-chain amyloidosis), 25 (39.7%) – ATTR-amyloidosis (transthyretin amyloidosis), 2 (3.2%) – AA-amyloidosis with heart failure (reactive systemic amyloidosis caused by hypersecretion of á-globulin). The analysis of clinical manifestations depending on the type of amyloidosis, data of laboratory and instrumental methods of diagnosis is carried out. Results. In most cases, 53 (84.1%) patients, amyloidosis manifested as signs of heart failure. Among cardiac manifestations, shortness of breath (95.2%), general weakness (93.7%), lower limb edema (76.2%) were the most common. To confirm the diagnosis, despite the high accuracy of the speckle-tracking echocardiography and magnetic resonance imaging of the heart with gadolinium, in rare cases a biopsy is required (e.g. there is a combination of clinical signs of several types of amyloidosis). Biopsy of the affected organ was performed in 31 (49.2%) patients. The strategy for further pathogenetic treatment depends on the determination of the type of amyloidosis. Free light chains of immunoglobulins were detected in 57.1% of cases, which allowed diagnosis of AL-amyloidosis. In 17 (38.6%) patients myocardial scintigraphy with 99mTc-pyrophosphate showed signs of ATTR-amyloidosis, which with a negative result of immunochemical studies allows non-invasive diagnosis of it. Conclusion. ACMP is a disease with an extremely adverse prognosis. Raising the awareness of specialists about ACMP is an important goal. With timely diagnosis, pathogenetic therapy can be started early, which will improve the quality of life and prognosis of patients with ACMP.

Abstract 6170: Risk stratification of smoldering multiple myeloma using fourier transform infrared spectroscopy of serum

Abstract Background: Smoldering multiple myeloma (SMM) is a state where patients have more than 10% clonal plasma cells or a serum monoclonal protein > 3g/dl but no evidence of end organ damage. Patients with SMM are at risk of developing active multiple myeloma (defined by the CRAB criteria). The best stratification system with respect to the risk of progression to active myeloma is the 20/2/20 (20% plasma cells, M protein >2.0 g/dl, and involved to uninvolved immunoglobulin free light chain ratio >20). The presence of ≥2 criteria predicts a 50 - 60% risk of progression within 2 years. Thus, many patients considered ‘high-risk’ may not progress in this time frame, and patients with ≤1 risk factor can progress to active myeloma. Therefore, improved risk stratification methods are needed to correctly identify patients at high risk of progression. We hypothesized that Fourier Transform InfraRed (FTIR) spectroscopic analysis of serum combined with artificial intelligence (AI) can correctly risk stratify patients with SMM. Methods: We identified 192 SMM patients: 96 who progressed and 96 who did not progress within 2 years of diagnosis. We used stored sera from the time of diagnosis to perform FTIR to determine differences in the spectra of patients who did/did not progress and train an AI algorithm. A randomized 10-fold cross-validation analysis was performed using 172 samples (86 from each group) for training and the remaining 20 for testing to assess the model across 10 independent iterations. Subsequently, we blindly tested the trained model against an independent set of serum samples (N=24) from patients who progressed ≤3 years of diagnosis. Receiver operator characteristic (ROC) analysis was used to assess the FTIR AI algorithm output compared to the clinical outcome data. Results: From the 96 patients who progressed, 48 had complete data for the 20/2/20 risk stratification. 11 patients (23%) had ≤1 of the criteria and so would have been considered at low likelihood to progress. Of the 96 who did not progress within 2 years, 49 had complete data for risk stratification. Of these, 27 (55.1%) met the high-risk criteria of the 20/2/20 classification and would have been predicted to progress within 2 years. FTIR/AI model correctly identified the patients at risk of progression (95% accuracy) with AUC of 0.96. Similarly, it was able to classify patients who did not progress within 2 years of diagnosis (94% accuracy) with an AUC of 0.96. FTIR/AI correctly predicted the patients at risk of progression within 2 years (90%) and 3 years (87.5%) in the blinded validation group with an AUC of 0.89 and 0.82, respectively. Conclusion: Analysis of serum in patients with newly diagnosed SMM with FTIR is able to correctly identify those at risk of progression within 2 or 3 years of diagnosis with an accuracy of ~90% in a blinded external validation study. FTIR may, therefore, be used to assess SMM patient risk before committing to potentially toxic therapy. Citation Format: David Dingli, Darlyna Khonkhammy, Caden Gunnarson, Grant Schlauderaff, Dan Q. Pham, Ali Khammanivong. Risk stratification of smoldering multiple myeloma using fourier transform infrared spectroscopy of serum [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6170.

Immunoglobulin free light chains in severe asthma patient: Could they be a new biomarker?

Increasing evidence is available about the presence of increased serum concentration of immunoglobulin (Ig) free light chains (FLCs) in both atopic and non-atopic inflammatory diseases, including severe asthma, providing a possible new biomarker of disease. We analyzed clinical and laboratory data, including FLCs, obtained from a cohort of 79 asthmatic subjects, clinically classified into different GINA steps. A control group of 40 age-matched healthy donors (HD) was considered. Particularly, HD have been selected according to the absence of monoclonal components (in order to exclude paraproteinemias), were tested for total IgE (that were in the normal ranges) and were negative for aeroallergens specific IgE. Moreover, no abnormality of common inflammatory markers (i.e., erythrocyte sedimentation rate and C-reactive protein) was detectable. FLC-k levels were significantly increased in the asthmatic population, compared to the control group. Despite the absence of statistically significant differences in FLC-λ levels, the FLC-k/FLC-λ ratio displayed remarkable differences between the two groups. A positive correlation between FLC-κ and FLC-λ levels was found. FLC- λ level displayed a significant negative correlation with the FEV1 value. Moreover, the FLC-κ /FLC- λ ratio was negatively correlated with the SNOT-22 score and a positive correlation was observed between FLCs and Staphylococcus Aureus IgE enterotoxins sensitization. Our findings confirmed the role of FLCs in asthma as a potential biomarker in an inflammatory disease characterized by different endotypes and phenotypes. In particular, FLC-κ and FLC-k/FLC-λ ratio could be a qualitative indicator for asthma, while FLC-λ levels could be a quantitative indicator for clinical severity parameters.

Immunoglobulin light chain amyloidosis: 2024 update on diagnosis, prognosis, and treatment.

Immunoglobulin light chain amyloidosis is a clonal, nonproliferative plasma cell disorder in which fragments of immunoglobulin light or heavy chain are deposited in tissues. Clinical features depend on organs involved but can include heart failure with preserved ejection fraction, nephrotic syndrome, hepatic dysfunction, peripheral/autonomic neuropathy, and "atypical smoldering multiple myeloma or MGUS." Tissue biopsy stained with Congo red demonstrating amyloid deposits with apple-green birefringence is required for the diagnosis of AL amyloidosis. Organ biopsy is not required in 85% of patients. Verification that amyloid is composed of immunoglobulin light chains is mandatory. The gold standard is laser capture mass spectroscopy. N-terminal pro-brain natriuretic peptide (NT-proBNP or BNP), serum troponin T(or I), and difference between involved and uninvolved immunoglobulin free light chain values are used to classify patients into four stages; 5-year survivals are 82%, 62%, 34%, and 20%, respectively. All patients with a systemic amyloid syndrome require therapy to prevent deposition of amyloid in other organs and prevent progressive organ failure. Current first-line therapy with the best outcome is daratumumab, bortezomib, cyclophosphamide, and dexamethasone. The goal of therapy is a ≥VGPR. In patients failing to achieve this depth of response options for consolidation include pomalidomide, stem cell transplantation, venetoclax, and bendamustine. Delayed diagnosis remains a major obstacle to initiating effective therapy prior to the development of end-stage organ failure. Trials of antibodies to deplete deposited fibrils are underway.

Abstract B112: SNAP23-dependent SNARE complex is a novel molecular target in AL Amyloidosis and Multiple Myeloma by blocking free light chain secretion and triggering a terminal unfolded protein response

Abstract The goal of this study was to investigate whether SNARE proteins are necessary for immunoglobulin (Ig) free light chain (FLC) secretion, thus representing a yet unexplored therapeutic target in AL amyloidosis (AL) and Multiple myeloma (MM). Both these disorders are incurable, and in the former, pathogenesis is invariably related to deposition of Ig FLC fibrils in target organs. The molecular mechanisms underlying FLC folding/secretion have not been studied, and no therapies directly target this intrinsic vulnerability. We hypothesize that FLC secretion is at least in part mediated by SNARE proteins, and their inhibition would block FLC exocytosis, causing retention of FLC-loaded vesicles, leading to activation of a terminal unfolded protein response (UPR) and apoptosis. We therefore hypothesized that novel, effective targets for MM and AL therapy could be identified within SNAREs. As a tool to study SNARE function, we stably expressed 7 distinct, tetracycline-inducible botulinum neurotoxin (BoNT) serotypes (BoNT/A-F) in FLC-secreting AL (ALMC1, ALMC2) and MM (U266, KMS11) cell lines. To monitor BoNT expression, a bicistronic lentiviral vector with a P2A-GFP was used. Upon doxycycline administration, we performed growth competition assay in polyclonally transduced cells and assessed kinetics of GFP expression over time and apoptosis via flow cytometry in monoclones. Western blot (WB) was used to assess cleavage of SNAREs upon expression of each BoNT. We used WB and ELISA to assess FLC secretion on a per-cell base upon BoNT expression. We used TurboID proximity labeling followed by mass spectrometry to identify cytotoxic BoNT interacting partners and CRISPR-Cas9 gene editing to validate putative BoNT targets. By querying the IFM170 gene expression database of newly diagnosed MM patients, we identified VAMP2, VAMP3, SYNTAXIN4, and SNAP23 as the top expressed SNARE and confirmed their high expression in a panel of AL and MM cell lines. Expression of all BoNT serotypes, except BoNT B, led to rapid apoptosis of AL and MM cells, with BoNT F standing out for extent and rapidity of cytotoxicity. Concurrent cleavage of SNAP23, VAMP3, and SYNTAXIN4 correlated with cytotoxicity of BoNT in AL and MM cell lines. Mechanistically, we showed that cytotoxic BoNTs, but not BoNT B, blocked FLC secretion and triggered the PERK-mediated terminal UPR with induction of CHOP and GADD34 followed by massive apoptosis. We identified SNAP23 as an interacting partner of cytotoxic BoNT F, but not BoNT B. Single SNAP23 KO did not completely recapitulate cytotoxic BoNT phenotype, as it was only partially cytotoxic. Based on our preliminary data, we hypothesize SYNTAXIN4 and VAMP3 to be synthetic lethal with SNAP23. Our data provide proof of concept that blocking FLC secretion in AL and MM cells is feasible and results in rapid cell apoptosis by triggering a terminal UPR, thus representing a promising, novel therapeutic approach. Using BoNT as a biological tool, we identified SNAP23 as a critical component of FLC exocytosis, paving the way for the design of novel therapeutics. Citation Format: Emre Karayol, Maria Moscvin, Tianzeng Chen, Peter G. Czarnecki, Annamaria Gulla, Kenneth C. Anderson, Giada Bianchi. SNAP23-dependent SNARE complex is a novel molecular target in AL Amyloidosis and Multiple Myeloma by blocking free light chain secretion and triggering a terminal unfolded protein response [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B112.

Teclistamab Induces Rapid Responses in Patients with Relapsed/Refractory AL Amyloidosis

Introduction Systemic light chain (AL) amyloidosis is a rare disorder characterized by tissue deposition of amyloid fibrils derived from immunoglobulin free light chains. While daratumumab in combination with cyclophosphamide, bortezomib and dexamethasone (Dara-CyBorD) is the standard for upfront treatment of AL, management of relapsed/refractory (R/R) disease is often challenging, particularly in context of multiorgan dysfunction. Teclistamab was recently approved for treatment of R/R multiple myeloma (MM) and showed an overall response rate (ORR) of 63% (N Engl J Med 2022;387:495). Notable adverse effects (AEs) included cytokine release syndrome (CRS), neurologic, renal and hematologic toxicities, and infections. Efficacy of teclistamab in AL has not been previously reported. Here, we present a series of three patients who received treatment with teclistamab for R/R AL and MM. Methods In this retrospective study, we included all patients with R/R AL and MM who received teclistamab at two US academic centers between October 2022 and July 2023. We recorded all standard metrics, including serologic and biomarker data. Hematologic and organ responses were evaluated based on standard consensus criteria. Toxicity assessment was performed in accordance with the NCI CTCAE v5.0, except for CRS and neurotoxicity, which were graded according to the 2019 ASTCT criteria (BBMT 2019;25:625). Results We identified 3 patients; baseline characteristics are provided in Table 1 and treatment data in Table 2. Median age was 67 years (range, 54-70). All patients were male, and 2 had AL λ-type. At the time of teclistamab initiation, the median number of prior therapies was 7 (5-10), with all patients being refractory to a bortezomib, lenalidomide and daratumumab. Median iFLC was 228 mg/L (45.5-1272) with 10% median bone marrow (BM) plasmacytosis. In one patient with rapidly progressive multiple myeloma, BM FISH showed high-risk cytogenetic changes, including gain 1q, del 1p and del 17p. All patients were hospitalized for treatment initiation and received escalating doses of 0.06 mg/kg, 0.3 mg/kg and 1.5 mg/kg during the ramp-up phase. Subsequent treatment with target dose of 1.5 mg/kg was continued in the outpatient setting on a weekly basis. Monitoring for cytokine release syndrome (CRS) and neurologic toxicity was performed in accordance with institutional protocols. At a median follow-up of 49 days (44-58), 2 patients achieved hematologic responses (HR), with 1 complete hematologic response (CR) and 1 low dFLC PR. Both hematologic responses occurred promptly within 3 weeks of treatment initiation, were deep with iFLC <10 mg/L, and were ongoing at the last follow-up. One patient with rapid renal progression prior to teclistamab also enjoyed a renal response by day 42. The most common toxicity was hematologic, with 1 patient experiencing grade 4 lymphopenia, grade 2 thrombocytopenia and hypogammaglobulinemia. There were no infectious complications or instances of febrile neutropenia, CRS or neurologic AEs. All patients received routine prophylaxis against VZV and PJP, as well as prophylactic IVIg. One patient with heavily pre-treated, high-risk MM had an aggressive clinical relapse with widespread osseous involvement within 6 months of an autologous stem cell transplant (ASCT) and experienced progressive disease as best response to teclistamab, necessitating discontinuation after 29 days in favor of cytotoxic chemotherapy. Conclusion In this series of 3 patients with heavily pre-treated, R/R AL and MM, teclistamab showed an impressive hematologic response rate of 67%. These responses were deep and occurred rapidly within 3 weeks of treatment initiation. One patient also enjoyed a renal response, whereas cardiac responses were non-evaluable in 2 patients. While the most common treatment-related toxicity was hematologic in nature, infections or febrile neutropenia were not observed, likely due to the institution of aggressive prophylactic measures. Importantly, no new safety signals were identified in AL patients. To our knowledge, this is the first report providing preliminary evidence for efficacy of teclistamab in R/R AL, highlighting the need to investigate this strategy in clinical trials.

A SNAP23-Dependent SNARE Complex Mediates Free Light Chain Secretion in AL Amyloidosis and Multiple Myeloma and Its Blockade Triggers a Terminal Unfolded Protein Response and Massive Apoptosis

Background AL amyloidosis (AL) is an incurable plasma cell (PC) disorder with only one FDA-approved therapy. Deposition of immunoglobulin free light chain (FLC) fibrils in target organs is the sole pathogenic mechanism in AL. However, therapeutic strategies directly targeting FLC secretion are not available. SNARE proteins, the specific targets of botulinum neurotoxin (BoNT), mediate the docking and fusion of secretory vesicles. We hypothesized that targeting specific sets of SNAREs via BoNT will block FLC exocytosis, causing retention of FLC-loaded vesicles and triggering a terminal unfolded protein response (UPR) and apoptosis in AL cells. We postulate that SNAREs are potential novel targets in AL and other plasma cell disorders. Materials and Methods We stably expressed 7 distinct, inducible BoNT serotypes (BoNT/A-F) in FLC-secreting AL (ALMC1 and ALMC2) and multiple myeloma (MM, KMS11) cell lines via tetracycline-inducible (Tet-On) bicistronic lentiviral vectors with a P2A-GFP which allowed us to use GFP as a biomarker of BoNT expression. We established monoclones of successfully transduced cells for each BoNT. We performed flow cytometry to assess the kinetics of GFP induction and assess for apoptosis via AnnexinV/DAPI staining. Western blot (WB) was used to assess the cleavage of SNAREs upon expression of each BoNT serotype. We used WB and ELISA to assess FLC secretion per cell upon expression of each BoNT serotype. We used TurboID proximity labeling followed by mass spectrometry to identify cytotoxic BoNT interacting partners and used co-IP for confirmation. We used RNP-based CRISPR-Cas9 gene editing to knock out (KO) putative BoNT targets. Results By querying the IFM170 gene expression database of newly diagnosed MM patients, we identified VAMP2, VAMP3, SNAP23, and SYNTAXIN4 as the top expressed SNAREs. Their high expression was retained in a large panel of AL and MM cell lines. Expression of all BoNT serotypes, except for BoNT B, led to rapid apoptosis of AL and MM cells, with BoNT F standing out for the extent and rapidity of cytotoxicity. Dual cleavage of SNAP23 and VAMP3 correlated with cytotoxicity of BoNT in AL and MM cell lines. Mechanistically, we showed that cytotoxic BoNTs, but not non-toxic BoNT B, led to decreased FLC secretion and increased FLC retention and triggered the PERK pathway of the UPR with increased expression of CHOP and GADD34, preceding the onset of apoptosis. We identified SNAP23 as an interacting partner of highly cytotoxic BoNT F, but not non-toxic BoNT B. KO experiment showed single SNAP23 KO to be partially toxic for AL and MM cell lines but did not recapitulate the effects of cytotoxic BoNTs entirely. Based on our preliminary data, we hypothesize VAMP3 and SYNTAXIN4 to be synthetic lethal with SNAP23. Conclusions Our data provide proof of concept that blocking FLC secretion in AL and MM cells is feasible and results in rapid cell apoptosis by triggering a terminal UPR, thus representing a promising, novel therapeutic approach. By using BoNT as a biological tool, we identified SNAP23 as a critical component of FLC exocytosisin AL cell lines, thus representing a novel, promising molecular target in AL amyloidosis and multiple myeloma.

Applying Machine Learning to Support Early Diagnosis of Light-Chain Amyloidosis: A Combination of Knowledge-Based Approach with Data-Driven Approach

Introduction Immunoglobulin light chain (AL) amyloidosis is a rare disease involving the clonal proliferation of bone-marrow-residing plasma cell and resulting in overproduction of serum immunoglobulin free light chains that affects multiple organs. There are several available effective treatments including autologous stem cell transplant, bortezomib, anti-CD38 antibodies, and immunomodulatory drugs. However, because of the atypical symptoms and signs of this disease, diagnostic delays are still the major challenge resulting in poor prognosis. In recent years, machine learning (ML) models have been used to assist in early diagnosis. Therefore, to address this clinical unmet need, we aim to build ML algorithms from clinical data and assess their performance in differentiating AL amyloidosis from similar conditions. Methods Monocenter medical records data were collected from 49 patients with AL amyloidosis and 198 non-AL amyloidosis patients on a ratio of 1:4 in Peking University People's Hospital between January 1, 2013, and December 31, 2021. The non-AL amyloidosis group were patients with diseases of similar symptoms including autoimmune liver disease, myocarditis, and hypertrophic cardiomyopathy. Variables for model development were selected from 30 demographic characteristics and clinical features from routine clinical examination based on the results of recursive feature elimination and hematologists' knowledge. We proposed a four-step approach to develop and evaluate the diagnostic models. In the first step, all patients were randomly allocated into a training set and a testing set with a ratio of 4:1. Second, we derived five separate ML models including logistic regression, support vector machine (SVM), extreme gradient boosting (XGBoost), light gradient boosting (LightGBM), and CatBoost algorithms to differentiate AL amyloidosis from other diseases with similar symptoms and validated the models using five-fold cross validation methods. Third, parameters of model with the highest areas under the receiver operating characteristic curves (AUROC) were updated in the full training set. Finally, the performances of the selected model were evaluated by AUROC, sensitivity, specificity and F1-score in the testing set. Results Twelve features including alanine aminotransferase, troponin, albumin, aspartate aminotransferase, activated partial thromboplastin time, albumin and globulin (A/G) ratio, direct bilirubin, platelet, fibrinogen, blood urea nitrogen, body weight and age were selected to construct ML models. The AUROC values for AL amyloidosis differential diagnosis were 0.55 with logistic regression, 0.63 with SVM, 0.84 with XGBoost, 0.89 with LightGBM, and 0.88 with CatBoost. The LightGBM model, which achieved the highest AUROC, also achieved the best performance with a sensitivity of 0.92, a specificity of 0.60, a F1-score of 0.73, a negative predictive value of 0.97, a positive predictive value of 0.60, and an accuracy of 0.82. Conclusion Our results show that the LightGBM model has the best performance to identify patients with AL amyloidosis from patients with similar symptoms. This novel ML-based diagnostic model has potential to assist in the earlier diagnosis of AL amyloidosis in clinical settings. Further studies are needed to confirm these findings in different study populations.

Prevalence, Characteristics, and Clinical Impact of Clonal Hematopoiesis of Indeterminate Potential in AL Amyloidosis

Backgrounds: AL Amyloidosis is a plasma cell dyscrasia whose hallmark is the circulation and deposition of immunoglobulin free light chains in target organs. The extent and severity of cardiac involvement are the primary determinants of mortality in AL amyloidosis. Patients harboring clonal hematopoiesis of indeterminate potential (CHIP) have an increased cardiovascular risk compared to the general population. In the setting of multiple myeloma receiving autologous stem cell transplants, CHIP presence has been associated with decreased overall survival. Still, large studies looking at the incidence and outcome of patients with concurrent CHIP and AL amyloidosis are missing. Herby, we present the results of a single-center, retrospective cohort study, where we assessed the prevalence of CHIP in AL amyloidosis and associated disease characteristics and outcomes. Methods: We collected the demographics and clinical characteristics of patients with AL amyloidosis at Dana Farber Cancer Institute (DFCI) from January 2018 to April 2023 who underwent a bone marrow aspirate/biopsy with a rapid heme panel. Variables of interest included age, ethnicity, Mayo 2004 stage, Palladini renal stage, involved organs, FISH abnormalities, left ventricular ejection fraction (LVEF), documented coronary artery disease (CAD), orthostatic hypotension (OH), chemotherapy (CT) treatment received and related response. The primary outcome was major organ dysfunction progression-free survival (MOD-PFS), defined by Kastritis et al. The variant allele fraction (VAF) to define CHIP was set at 2%. Two samples t-test, Wilcoxon rank sum test, and Fisher's exact test were used to compare groups. Univariate and stratified multivariate Cox proportional hazard regression models were used to estimate the association between CHIP status and MOD-PFS. Analyses were carried out with the Stata statistical package. This study was approved by the DFCI Institutional Review Board and performed in accordance with the Declaration of Helsinki. Results: We identified 76 patients as meeting the inclusion criteria. The median age was 67 years. FISH was available for 54 patients. All patients received cytoreductive CT (CyborD 47%, DaraCyborD 37%). CHIP was identified in 16 patients (21%), a significantly higher figure as compared to historical age-matched healthy controls. DNMT3A was the most frequently involved gene (7/16, 44%), followed by GNB1, TET2, ATM (each 2/16, 12.5%), and SF3B1, ZRSR2, EZH2, BRCC3, PPM1D, ASXL1 (6%) (Figure 1). The presence of CHIP strongly associated with t(11;14) (11/13, 85% in patients with CHIP, versus 15/41, 37%, in patients without CHIP, p=0.004) and with a lower Palladini renal stage in patients with renal involvement (p=0.001). The median follow-up time for the entire cohort was 25 months (range 1 - 175 months). In a univariate analysis, CHIP presence was not associated with lower MOD-PFS (hazard ratio [HR] 0.998, 95% CI 0.38 - 2.64). In a univariate analysis, a Mayo stage >2 (HR 4.82, 95% CI 1.95 - 11.91, p = 0.001), the presence of CAD (HR 4.97, 95% CI 1.79 - 13.79, p=0.02), LVEF ≤40% (HR 3.18, 95% CI 1.17 - 8.66, p = 0.023), and OH requiring treatment (HR 2.65, 95% CI 1.74 - 5.97, p = 0.019) were associated with a decreased MOD-PFS. In a multivariate model stratified for age, only the presence of CAD and OH requiring treatment remained associated with worse MOD-PFS (HR 5.65, 95% CI, 1.33 - 23.99, p = 0.019; HR 4.57, 95% CI, 1.30 - 16.05, p = 0.018, respectively). In the multivariate Cox regression model (Figure 2), CHIP presence was not associated with a decreased MOD-PFS (HR 1.32, 95% CI, 0.44 - 3.98, p = 0.624). When focusing on the impact of only DNMT3A, a tendency towards a statistically significant association with worse MOD-PFS was noted in the multivariate model (HR 3.72, 95% CI 0.92 - 15.01, p = 0.065). Conclusion: We present the largest dataset exploring the prevalence of CHIP in AL amyloidosis patients and its clinical correlates. We noted an association between the presence of CHIP and t(11;14), the most frequent cytogenetic abnormality in AL amyloidosis, and adverse prognostic factors. Patients with CHIP were also found to have a lower Palladini renal risk score. Harboring CHIP was not associated with worse MOD-PFS; however, DNMT3A mutation tended to correlate with worse MOD-PFS. Larger prospective studies are warranted to validate these findings and better elucidate the impact of CHIP in patients with AL amyloidosis.

Quantification of Free Immunoglobulin Light Chains in Urine.

The serum-free immunoglobulin light chain assay has been recommended as a screening test for monoclonal gammopathy. We evaluated the usefulness of urine free immunoglobulin light concentration for selection of specimens for immunofixation electrophoresis. Using kits from The Binding Site for Freelite ®, we validated examination of urine for measuring free κ and λ light chains. The results of urine free light chain concentrations were evaluated to ascertain if the results could be used to reduce the number of specimens requiring urine protein immunofixation electrophoresis. In the 515 specimens examined, there was no evidence of monoclonal gammopathy or history of monoclonal gammopathy in 331. Monoclonal κ or λ light chains were detectable in 42 and 30 specimens, respectively. There was history of κ or λ chain associated monoclonal gammopathy in 62 and 50 patients, respectively. In the 38 monoclonal κ positive urine specimens, with light chain data, κ/λ ratio was >5.83 in all specimens. In 27 specimens positive for monoclonal λ light chains, with light chain data, the urine λ/κ ratio was > 0.17 in 24 of 27 specimens and > 0.041 in all specimens. In patients without monoclonal gammopathy all specimens had a κ/λ ratio of >5.83 or λ/κ ratio >0.17. The Freelite ® assay from The Binding Site is suitable for quantification of free light chains in urine. In patients with known history of monoclonal gammopathy, urine immunofixation electrophoresis may be omitted in specimens with κ/λ ratio of <5.83 for κ associated lesions and λ/κ ratio of <0.041 for λ associated lesions. However, the results do not support using this test for first-time urine testing for monoclonal light chains as it is not predictive of positive result, nor does it exclude a monoclonal light chain in urine.

Serological and Molecular Characterization of Hepatitis C Virus-Related Cryoglobulinemic Vasculitis in Patients without Cryoprecipitate.

Prolonged B cells stimulation due to the Hepatitis C virus (HCV) can result in autoimmunity, stigmatized by rising levels of cryoglobulins (CGs), the rheumatoid factor (RF), and free light chains (FLC) of immunoglobulins (Ig) associated with a range of symptoms, from their absence to severe cryoglobulinemic vasculitis and lymphoma. Here, we aimed to identify an immunological signature for the earliest stages of vasculitis when cryoprecipitate is still not detectable. We firstly analyzed the IgG subclasses, FLC, and RF in 120 HCV-RNA-positive patients divided into four groups according to the type of cryoprecipitate and symptoms: 30 asymptomatic without cryoprecipitate (No Cryo), 30 with vasculitis symptoms but without CGs that we supposed were circulating but still not detectable (Circulating), 30 type II and 30 type III mixed cryoglobulinemia (Cryo II and Cryo III, respectively). Our results revealed that patients with supposed circulating CGs displayed a pattern of serological parameters that closely resembled Cryo II and Cryo III, with a stronger similarity to Cryo II. Accordingly, we analyzed the groups of Circulating and Cryo II for their immunoglobulin heavy chain (IgH) and T-cell receptor (TCR) gene rearrangements, finding a similar mixed distribution of monoclonal, oligoclonal, and polyclonal responses compared to a control group of ten HCV-RNA-negative patients recovered from infection, who displayed a 100% polyclonal response. Our results strengthened the hypothesis that circulating CGs are the origin of symptoms in HCV-RNA-positive patients without cryoprecipitate and demonstrated that an analysis of clonal IGH and TCR rearrangements is the best option for the early diagnosis of extrahepatic complications.

Elevated serum polyclonal immunoglobulin free light chains in patients with severe asthma.

Background: Inflammation plays a pivotal role in the pathophysiology of asthma. Free light chains (FLC) can cause inflammation by mast cell antigen-activation. Serum immunoglobulin (Ig) FLC κ, but not λ, were shown elevated in adult males with asthma. We sought to investigate if serum Ig FLC concentrations are affected by asthma severity and their relationships with inflammatory outcomes. Methods: By using immunoassays, we measured serum κ and λ Ig FLCs in 24 severe persistent asthma patients, 15 patients with moderate persistent asthma, 15 steroid-naïve mild persistent asthma patients and 20 healthy control subjects in a cross-sectional observational study. Total and specific serum IgE concentrations, fractional exhaled nitric oxide (FENO), lung function, peripheral blood eosinophils and neutrophils, and C reactive protein (CRP) were also measured. Results: Serum κ FLC concentrations were elevated in severe asthma patients compared mild asthma patients (p < 0.05) and healthy subjects (p < 0.05). Serum λ FLCs were higher in severe asthma patients than in healthy subjects (p < 0.05) and correlated with blood eosinophil counts (percentage, κ: r = 0.51, p = 2.9678-6; λ: r = 0.42, p = 1.7377-4; absolute values, κ: r = 0.45, p = 6.1284-5; λ: r = 0.38, p = 7.8261-4), but not with total or specific serum IgE. In severe asthma patients, serum Ig FLC correlated with serum CRP (κ: r = 0.33; p = 0.003; λ: r = 0.38, p = 8.8305-4) and blood neutrophil cell counts (percentage, κ: r = 0.31; p = 0.008; λ: r = 0.29, p = 0.01; absolute values, κ: r = 0.40; p = 3.9176-4; λ: r = 0.40, p = 4.5479-4), were elevated in subjects with blood eosinophilia (≥300 cells/µL) (n = 13) compared with non-eosinophilic subjects (n = 10) (κ: 19.2 ± 1.2mg/L versus 12.1 ± 1.3mg/L, p < 0.001; λ: 27.2 ± 2.6mg/L versus 16.8 ± 2.5mg/L, p < 0.01), but were similar in atopic (n = 15) versus nonatopic subjects (n = 9) (κ: p = 0.20; λ: p = 0.80). Serum FLC were negatively correlated with lung function tests, including forced expiratory volume in one second (FEV1) (κ: r = -0.33; p = 0.0034; λ: r = -0.33; p = 0.0035), and FEV1/forced vital capacity ratio (κ: r = -0.33; p = 0.0034; λ: r = -0.33; p = 0.0036). Conclusion: Serum Ig FLCs are elevated in severe asthma adults and might represent new surrogate markers of inflammation. The pathophysiological implications of these findings require further research. This study was approved by the ethics committee of the University Hospital Agostino Gemelli Foundation and Catholic University of the Sacred Heart (approval number P/1034/CE2012).

Nuclear Factor-κB is a Prime Candidate for the Diagnosis and Control of Inflammatory Cardiovascular Disease.

Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is responsible for the regulation of genes involved in inflammation and immune responses. NF-κB may play an important role in cardiovascular diseases (CVDs), atherosclerosis and diabetes. Several therapeutic agents used for the treatment of CVDs and diabetes, such as pimobendan and sodium-glucose cotransporter 2 inhibitors, exert anti-inflammatory effects by inhibiting NF-κB activation; anti-inflammatory therapy may have beneficial effects in CVDs and diabetes. Several pharmacological agents and natural compounds may inhibit NF-κB, and these agents alone or in combination may be used to treat various inflammatory diseases. Immunoglobulin-free light chains could be surrogate biomarkers of NF-κB activation and may be useful for evaluating the efficacy of these agents. This review discusses recent advances in our understanding of how the NF-κB signalling pathway controls inflammation, metabolism and immunity, and how improved knowledge of these pathways may lead to better diagnostics and therapeutics for various human diseases.

P326 FALSE POSITIVE BONE–SCINTIGRAPHY IN ELDERLY HYPERTROPHIC CARDIOMYOPATHY

Abstract Differential diagnosis of cardiomyopathies with hypertrophic phenotype is crucial because some secondary forms of cardiac hypertrophy can be susceptible of specific treatment aimed to revert and/or stabilize the increase in wall thickness.A positive nuclear scintigraphy with hydroxy bisphosphonate bone tracer (99mTc–HPD)is believed to have high sensitivity and specificity for the diagnosis of transthyretin (TTR) amyloid cardiomyopathy. An 85–year–old man with systemic arterial hypertension was admitted because of pre–syncope associated to evidence at Holter monitoring of both bradiarrhythmias(1st degree AV block with PR interval of 300 ms and several episodes of 2nd degree Mobitz type 1 AV block with electrical pause &amp;gt;3 s) and tachyarrhythmias.He suffered from a bilateral carpal tunnel syndrome that underwent surgery 2 years earlier.Routine laboratory tests were normal except for elevated cardiac troponin HS(0.295 mcg/L,nv &amp;lt; 0.014 mcg/L) and N–terminal pro–BNP (384.3 pg/mL,nv = 0–254 pg/mL).Because of clinical suspicion of senile amyloidosis,nuclear scintigraphy with hydroxy bisphosphonate bone tracer(99mTc–HPD)was performed,that revealed a grade 3 myocardial uptake.Serum–urine immunofixation and immunoglobulin free light–chain was negative for monoclonal gammopathy.Neurological examination was unremarkable Genetic test did not show TTR mutation.Thus,a diagnosis of wild–type TTR cardiac amyloidosis was suggested.The patient,in view of Tafamidis treatment,underwent a LV endomyocardial biopsy.Six specimens were drawn from the LV,cut and processed for histology,immunohistochemistry,and transmission electron microscopy. Unexpectedly,histology failed to confirm amyloid deposition but revealed the presence of severely hypertrophied disarrayed cardiomyocytes fragmented in short runs by interstitial and replacement fibrosis,denoting a sarcomeric hypertrophic cardiomyopathy.Congo red staining at polarized light scored negative for areas of apple–green florescence.Immunohistochemistry form transthyretin,kappa/lambda light chain and SAA was negative.Transmission electron microscopy failed to demonstrate the presence of rigid unbranched amyloid fibrils.Thus,a final diagnosis of hypertrophic cardiomyopathy was reached. Coclusion:A positive cardiac signal of bone scintigraphy can occur in other pathologic settings.Future studies are needed to investigate the incidence of false–positive TTR cardiac amyloidosis to bone scintigraphy compared with endomyocardial biopsy.

Method Comparison and Workflow Differences Using the Same Free Light Chain Assay on 2 Analyzer Platforms.

The Freelite assay (The Binding Site) is utilized to quantify serum immunoglobulin free light chains (sFLC), which is crucial for diagnosing and monitoring plasma cell dyscrasias (PCDs). Using the Freelite test, we compared methods and evaluated workflow differences across two analyzer platforms. sFLC concentrations were measured in 306 fresh serum specimens (cohort A) and 48 frozen specimens with documented sFLC >20 mg/dL (cohort B). Specimens were analyzed on the Roche cobas 8000 and Optilite analyzers using the Freelite κ and λ assays. Performance was compared using Deming regression. Workflow was compared by assessing turnaround time (TAT) and reagent usage. For cohort A specimens, Deming regression revealed a slope of 1.04 (95% CI, 0.88-1.02) and an intercept of -0.77 (95% CI, -0.57 to 1.85) for sFLCκ and a slope of 0.90 (95% CI, -0.04 to 1.83) and intercept of 1.59 (95% CI, -3.12 to 6.25) for sFLCλ. Regression of the κ/λ ratio revealed a slope of 2.44 (95% CI, 1.47-3.41) and intercept of -8.13 (95% CI, -16.82 to 0.58) with a concordance kappa of 0.80 (95% CI, 0.69-0.92). The proportion of specimens with TAT >60 min was 0.33% and 8% for the Optilite and cobas, respectively (P < 0.001). The Optilite required 49 (P < 0.001) and 12 (P = 0.016) fewer tests for sFLCκ and sFLCλ relative to the cobas. Cohort B specimens showed similar but more dramatic results. Analytical performance of the Freelite assays was comparable on the Optilite and cobas 8000 analyzers. In our study, the Optilite required less reagent, had a slightly reduced TAT, and eliminated manual dilutions for samples with sFLC concentrations >20 mg/dL.

Diagnostic value of the saliva immunoglobulin free light chains concentrations measured in primary Sjцgren's syndrome

Proliferation and hyperactivation of B-lymphocytes in the salivary glands is a feature of primary Sjцgren's syndrome (pSS). Detection in saliva of proteins synthesized by B-lymphocytes may be important in the diagnosis of this disease.Objective: to evaluate the diagnostic value of measuring the concentration of immunoglobulin free light chains (FLC) in saliva in patients with pSS.Material and methods. The cross-sectional study included 24 patients with pSS over the age of 18 years. PSS was diagnosed according to the 2016 ACR/EULAR classification criteria. The control group consisted of 11 healthy volunteers. Blood-salivary glands histohematic barrier permeability ratio for albumin, FLC was measured. Quantitative determination of FLC and in blood and saliva was performed by enzyme immunoassay. An immunohistochemical study of biopsies of minor salivary glands (MSG) was carried out with a quantitative assessment of CD3+, CD4+, CD8+, CD20+, CD21+, CD68+, CD138+ cells. The Mann–Whitney U-test was used to compare quantitative traits. Identification of diagnostic thresholds for the concentration of FLC in saliva for the diagnosis of pSS was carried out using the ROC analysis method. An operating characteristic curve was plotted, the area under the curve, indicators of diagnostic specificity, diagnostic sensitivity, and diagnostic accuracy were calculated.Results and discussion. The obtained values corresponded to the low permeability of the histohematic barrier of the salivary glands for albumin and FLC in patients with pSS and healthy individuals. The median concentrations of FLC ê and ë in the saliva of patients with pSS and healthy volunteers were 1.08 [0.58; 1.91], 1.038 [0.55; 2.03] mg/l and 0.36 [0.32; 0.54], 0.35 [0.21; 0.52] mg/l, respectively. The concentration of FLC in the saliva of patients with pSS was statistically significantly higher than in the control group (p&lt;0.01). The amount of FLC ê and ë in saliva correlated with the rate of unstimulated saliva flow: rs=-0.483 (p=0.02), rs=-0.491 (p=0.017), respectively.A relationship was found between the concentration of ê-chains in saliva and the specific number of CD138+ cells: rs=0.733 (p=0.025). Statistically significant correlations between the concentration of ë-chains and the number of mononuclear cells in the MSG have not been established.Based on the results of ROC analysis, diagnostic thresholds for FLC concentrations in the saliva of patients with pSS were determined. Concentrations of ê- and ë-type FLC in saliva of 0.56 and 0.68 mg/l correspond to area under the curve values of 0.84 (95% confidence interval, CI 0.69–0.98) and 0.83 (95% CI 0.71–0.97), sensitivity 79.2% (95% CI 59.5–90.8) and 75% (95% CI 55.1–88), specificity 81.8% (95% CI 52.3–96.8) and 90.9% (95% CI 62.3–99.5), respectively.Salivary FLC concentrations were compared in patients with pSS receiving and not receiving glucocorticoids (GC). The groups did not differ in a statistically significant way in terms of clinical and laboratory parameters. The median daily dose of GC was 10 [5; 10] mg in prednisolone equivalent. There were no significant differences between the concentrations of saliva FLC in patients of these groups.Conclusion. Salivary-fixed FLCs are most likely produced by cells localized in the stroma of the salivary glands. Determination of the concentration of FLC in saliva can be proposed as a diagnostic test for the pSS. The concentration of free ê-chains in saliva can be considered as a surrogate marker of benign B-cell proliferation in the MSG. Therapy with low and medium doses of GC in pSS does not affect the concentration of FLC in saliva.

The potential role of serum polyclonal free light chains as markers of immune activation in psoriatic patients

Polyclonal free light chains (FLCs) of immunoglobulins include κ and λ chains and represent a sensitive marker of activation and/or dysfunction of the immune system. The aim of this study was to investigate the role of FLCs as markers of immune activation in the management of psoriatic patients treated with biologics. The overall study population included 45 patients affected by mild-to-severe psoriasis with either ongoing biological treatment or without any current systemic therapy. Peripheral blood samples were taken from all patients and 10 healthy subjects in order to determine immunoglobulins, light chains and FLCs by quantitative nephelometric assay. Moreover, antinuclear antibodies (ANA) were detected by immunofluorescence. Psoriatic patients showed significant increased levels of κ and λ FLCs compared to healthy controls. Interestingly, κ and λ FLCs values were significantly increased only in psoriatic patients with ongoing biological treatment and, in particular, in responder subjects. Furthermore, both κ and λ FLCs significantly correlated with duration of therapy. For patients with FLC levels above normal range and under biological treatment for more than 12 months, the odds of being ANA+ was greater relative to patients with FLC levels above normal range but under biological treatment for less than 12 months. Increased FLC levels may represent a marker of immune reactivation in psoriatic patients treated with biologic agents. We suggest that determining FLC levels has clinical relevance, with a cost/benefit ratio justifying such evaluation in the clinical management of psoriasis.

Distinct phenotype of neutrophil, monocyte, and eosinophil populations indicates altered myelopoiesis in a subset of patients with multiple myeloma.

Hematologic malignancies, including multiple myeloma (MM), promote systemic immune dysregulation resulting in an alteration and increased plasticity of myeloid cell subsets. To determine the heterogeneity of the myeloid cell compartment in the peripheral blood of patients with MM, we performed a detailed investigation of the phenotype and function of myeloid subpopulations. We report that a subset of MM patients exhibits a specific myeloid cell phenotype indicative of altered myelopoiesis characterized by significant changes in the properties of circulating granulocytic, monocytic, and eosinophilic populations. The subset, referred to as MM2, is defined by a markedly elevated level of CD64 (FcγRI) on the surface of circulating neutrophils. Compared to healthy controls or MM1 patients displaying intermediate levels of CD64, neutrophils from MM2 patients exhibit a less differentiated phenotype, low levels of CD10 and CXC chemokine receptor 2 (CXCR2), increased capacity for the production of mitochondrial reactive oxygen species, and an expansion of CD16neg immature neutrophil subset. Classical and patrolling monocytes from MM2 patients express elevated levels of CD64 and activation markers. MM2 eosinophils display lower levels of C-C Chemokine receptor 3 (CCR3), Toll-like receptor 4 (TLR4, CD284), and tissue factor (TF, CD142). The MM2 (CD64high) phenotype is independent of age, race, sex, and treatment type. Characteristic features of the MM2 (CD64high) phenotype are associated with myeloma-defining events including elevated involved/uninvolved immunoglobulin free light chain (FLC) ratio at diagnosis. Detailed characterization of the altered myeloid phenotype in multiple myeloma will likely facilitate the identification of patients with an increased risk of disease progression and open new avenues for the rational design of novel therapeutic approaches.