Longitudinal high-frequency blood biomarkers of axonal injury and astrocytic activation after immune reconstitution in multiple sclerosis

Background .Serum neurofilament light chain (sNfL) and glial fibrillary acidic protein (sGFAP) reflect axonal damage and astrocytic injury. Their clinical role in longitudinal real-world monitoring after immune reconstitution therapy (IRT) in multiple sclerosis (MS) remains insufficiently defined. We evaluated longitudinal sNfL and sGFAP in people with multiple sclerosis (pwMS) treated with alemtuzumab (ATZ) as a model of IRT to determine their prognostic and monitoring value in real-world care. Methods PwMS initiating ATZ were prospectively followed up every three months for up to five years. sNfL and sGFAP levels were measured using single molecule array (Simoa) and converted to age- and BMI-adjusted Z scores based on healthy control datasets. Longitudinal trajectories were analyzed with generalized linear mixed models. ROC analysis with Youden’s index identified optimal cut-offs for evidence of disease. Logistic and Cox regression models assessed predictive values. Event-related analyses examined biomarker changes around relapses, MRI activity, EDSS worsening, and retreatment. Results .Eighty-four pwMS (mean age 36.5 ± 9.0 years, 76% female) were included. Baseline sNfL Z scores were significantly higher in males and in those with recent MRI activity or treatment failure. sNfL rose transiently one month after the first ATZ course, declined by month 3, and remained stably reduced thereafter. Youden’s index-derived baseline sNfL Z scores ≥ 0.75 predicted disease activity during year 1 (OR: 5.10, 95% CI 1.79–14.49), and Z scores > 1.0 predicted disease activity after the second ATZ course (HR: 2.96, 95% CI 1.04–8.44). Event-related analyses showed significant sNfL elevations around relapses ( p  = 0.008), EDSS worsening ( p  = 0.037), MRI activity ( p  = 0.036), and retreatment ( p  = 0.004), compared to matched pwMS without disease activity. sGFAP levels remained overall stable over follow-up ( p  = 0.677) and showed no consistent associations with clinical or MRI activity. Discussion sNfL provided both predictive and monitoring value in pwMS treated with ATZ, supporting its integration into individualized follow-up strategies. In contrast, sGFAP remained overall stable and did not associate with inflammatory events, reflecting distinct biomarker biology. These findings demonstrate the real-world clinical utility of high-frequency sNfL monitoring for early detection of breakthrough disease activity after IRT and support its integration as a complementary tool alongside clinical and radiological follow-up.