The combination of CSF neurofilament light chain and glial fibrillary acidic protein improves the prediction of long-term confirmed disability worsening in multiple sclerosis

Abstract

Our objective was to evaluate the individual and combined prognostic attributes of baseline serum and CSF measurements of Neurofilament light chain (sNfL, cNfL) and glial fibrillary acidic protein (sGFAP, cGFAP) on long term clinical outcomes in MS. In this retrospective single center study, patients with serum and CSF stored at first MS presentation and > 15-years of follow-up were analyzed. NfL and GFAP were quantified from cryopreserved samples using a digital immunoassay and analyzed as predictors of confirmed disability worsening (CDW). Sixty patients (70% female) underwent baseline tandem CSF and serum sampling and were followed for a mean of 17.8 years (SD 2.5). 32 developed CDW. By logistic regression, while sNfL cNfL and cGFAP showed prognostic merit (AUC 0.72, 0.70, 0.62 respectively), sGFAP did not (AUC 0.5). The combination of cNfL and cGFAP improved CDW prediction compared to either measure considered in isolation (AUC 0.72). The optimal predictive cut-off for CDW (Youden's index) for cNfL was 596 pg/mL and for cGFAP was 8160 pg/mL. Kaplan-Meier analysis of the cutoff-defined 'high-high' and 'low-low' combined cNfL and cGFAP groupings improved prediction of CDW compared to either marker individually (Hazard ratio 4.5 (95% CI 2.7-18.3), Logrank P < 0.0001). Cox Proportional Hazards regression demonstrated that high baseline cNfL and cGFAP were independently prognostic of subsequent CDW after adjusting for baseline age, sex, EDSS score and subsequent treatment exposure. Each unit increase in Ln(cNfL) and Ln(cGFAP) was respectively associated with an additional hazard of 2.36 (95% CI 1.12-5.52) and 2.26 (95% CI 1.03-5.21). CSF NfL and GFAP are independently prognostic of long-term clinical worsening in MS, and may represent a complementary pairing.

Fig. 1

Patient selection from the Ottawa MS biobank for inclusion in this study.

Fig. 2

Spearman correlation between serum and CSF levels of NfL and GFAP measured at baseline. NfL but not GFAP correlated well between serum and CSF. Only CSF GFAP correlated with NfL levels in both compartments. In our cohort, neither NfL or GFAP correlated with age. Legend: c: CSF GFAP: glial fibrillary acidic protein, NfL: neurofilament light chain, s: serum.

Fig. 3

Receiver operating characteristics (ROC) curves analysis to assess the diagnostic accuracy of unadjusted serum and CSF (A) NfL, (B) GFAP and (C) the combination of cNfL and cGFAP to predict CDW at 15 years of follow-up. sNfL and cNfL conferred similar diagnostic accuracy (A), while cGFAP but not sGFAP was contributory (B). The combination of cNfL and cGFAP improved the AUC compared to either variable considered in isolation (C). Legend: AUC: area under curve (95% confidence interval), c: CSF, CDW: confirmed disability worsening, GFAP: glial fibrillary acidic protein, NfL: neurofilament light chain, s: serum.

Fig. 4

Kaplan Meier survival curves and median survival times of MS patients to develop CDW based on cutoff-defined high (red curves) versus low (blue curves) biomarker levels of (A) cNfL, (B) cGFAP and (C) the combination of cNfL + cGFAP over the follow-up period. High versus low cNfL (A) or cGFAP (B) were individually prognostic of time-to-CDW. The combination of both high cNfL and high cGFAP (as opposed to low levels of both markers) showed a more marked separation in the survival curves (C). P-values and hazard ratios represent the logrank comparison between high and low groupings. The black dotted line denotes the survival of the total cohort. Legend: CDW: confirmed disability worsening, cGFAP: CSF glial fibrillary acidic protein, cNfL: CSF neurofilament light chain, HR: logrank hazard ratio.

Fig. 5

Cox proportional hazard models of CDW prognostication based on baseline biomarker measurements, age, sex, EDSS score and subsequent DMT exposure with cNfL and cGFAP evaluated as (A) continuous Ln-transformed biomarker concentrations, (B) dichotomous variables (1/0) based of Youden’s index-defined high/low cutoffs, and (C) combined dichotomous model based on ‘high-high’ versus ‘low-low’ biomarker groupings. High concentrations of either cNfL or cGFAP was independently prognostic of CDW in both the continuous (A) and dichotomous (B) models after adjusting for age, sex, EDSS score and DMT exposure. In the combined dichotomous model, patients with high (as opposed to low) levels of both biomarkers had a greater hazard of developing CDW (C). Legend: CDW: confirmed disability worsening, cNfL: CSF neurofilament light chain, cGFAP: CSF glial fibrillary acidic protein, EDSS: expanded disability status scale; DMT: disease-modifying treatment.