Patient-derived amyloidogenic light chain triggers cardiovascular toxicity In Vivo and reveals ICAM-1 as a novel potential biomarker in patients with AL amyloidosis

Abstract

Abstract Background and aims: In light chain (AL), the circulating immunoglobulin light chains (LCs) have been suggested to provoke cardiotoxicity. We aimed to characterize the underlying mechanisms of LC-mediated cardiotoxicity in vivo to identify novel circulating biomarkers in AL with a bench to bedside approach. Methodology: We have previously described an amyloidogenic full length LC deriving from a patient with AL amyloidosis and cardiac involvement (AL_2718LV2 LC). NOD-scid IL2Rγnull (NSG) male and female mice (n=40) received 200 μg of AL_2718LV2 LC or vehicle (daily, intraperitoneally) for one or two months. Echocardiography, histology, assessment of circulating markers of toxicity, proteomic and molecular analysis was performed in the animal model. Plasma samples from 97 consecutive patients with AL were analyzed. ELISA was performed to measure Intercellular Adhesion Molecule 1 (ICAM-1) levels. Overall survival (OS) was from the time of initial diagnosis until the time of death or the time of last follow-up, if it occurred first. Optimal cutoffs for ICAM-1 levels were determined using maximally selected rank statistics. Kaplan-Meier survival curves and Univariate Cox proportional hazards models assessed survival differences overall and within Mayo stages. Results: LC-treated NSG mice had prolonged isovolumic contraction time, a marked increase of NT-proBNP and significantly elevated urea and creatinine at 2 months compared to controls. LC treated mice demonstrated microvascular damage and perivascular fibrosis. Proteomic analysis on the myocardium of NSG mice revealed that Vascular Cell Adhesion Molecule-1 (VCAM-1) increased along with NF-kB-p38- ICAM-1 pathway which was confirmed at the molecular level. None of the animals had amyloid deposits, indicating that ICAM-1 could relate to direct LC-mediated toxicity. We then explored the potential of ICAM-1 as a prognostic biomarker in AL patients. The median age of the cohort at diagnosis was 65 years (range 40–84); 45 (46.4%) were females, 70 (72.3%) and 69 (71.1%) presented with cardiac and renal involvement respectively. Median involved free light chain (iFLC) was 189.0 mg/L (range 14.3–7240), median difference between involved and uninvolved FLC (dFLC) was 170.7 mg/L (range 1.7–7228), and median ICAM-1 level was 10,309 pg/mL (range 1615–31,131). Per mayo stage (European modification), 11.5%, 53.1%, 21.9% and 13.5% were stage 1, 2, 3A & 3B respectively. ICAM-1 levels correlated with iFLC (Spearman r = 0.216, p = 0.034) and alkaline phosphatase (ALP) (r = 0.225, p = 0.027). The median follow-up duration was 2.8 years (range 0.04–16.8). Using an optimal ICAM-1 cutoff of 12,455.9 pg/mL, overall survival analysis showed a hazard ratio (HR) of 1.73 for high versus low ICAM-1 levels, with a 95% confidence interval (CI) of 1.03 to 2.89 and a p-value of 0.037, indicating a statistically significant association between high ICAM-1 levels and inferior survival. No significant survival differences were observed in Mayo stages I (HR = 1.54; 95% CI: 0.14–17.19; p = 0.72), Mayo stage II (HR = 1.62; 95% CI: 0.77–3.37; p = 0.20), or Mayo stage III (HR = 1.63; 95% CI: 0.77–3.45; p = 0.20). In the multivariate Cox regression model, which included age, systolic blood pressure (SBP), Mayo stage, dFLC, iFLC, and ICAM-1 group, high ICAM-1 levels remained independently associated with worse overall survival (HR = 1.74; 95% CI: 1.01–2.99; p = 0.0457). These results suggest that ICAM-1 may serve as a prognostic biomarker beyond established clinical risk factors. Conclusions: Our findings support that ICAM-1 is increased in murine hearts treated with amyloidogenic LC and correlates with iFLC in AL patients. ICAM-1 can have prognostic values for survival of AL patients independently of Mayo stage, age, dFLC, iFLC, and SBP. Our results merit further validation in larger cohorts.