CSF sphingomyelin: a new biomarker of demyelination in the diagnosis and management of CIDP and GBS

Abstract

Objective: To validate sphingomyelin (SM) dosage in the cerebrospinal fluid (CSF) of patients affected by chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and Guillain-Barré syndrome (GBS) as a reliably assessable biomarker.

Methods: We prospectively enrolled 184 patients from six Italian referral centres, in whom CSF SM levels were quantified by a fluorescence-based assay optimised and patented in our laboratory.

Results: We confirmed increased levels of SM in the CSF of patients affected by typical CIDP (n=35), atypical CIDP (n=18) and acute inflammatory demyelinating polyradiculoneuropathy, AIDP (n=12) compared with patients affected by non-demyelinating neurological diseases, used as controls (n=85) (p<0.0001, p=0.0065 and p<0.0001, respectively). In patients with CIDP classified for disease stage, SM was higher in active CIDP compared with both controls and stable CIDP (p<0.0001), applying for a selective tool to treatment tailoring or withdrawal. SM was also increased in AIDP compared with axonal GBS, discerning the demyelinating from axonal variant of the disease. SM did not correlate with CSF protein levels, stratifying patients independently from commonly used CSF indexes, and displaying high specificity to avoid potential misdiagnosis. Finally, SM correlated with the main clinical scores and some neurophysiological parameters in patients with CIDP and AIDP.

Conclusions: CSF SM is a diagnostic and staging wet biomarker for acquired demyelinating neuropathies and may effectively improve the management of patients affected by GBS and CIDP.

Figure 1

CSF SM levels in patients affected by CIDP and GBS. (A) Patients affected by acquired demyelinating neuropathies displayed increased levels of SM in the CSF, compared with patients affected by non-demyelinating diseases. In particular, patients with both typical and atypical CIDP showed increased levels of CSF SM compared with controls (ie, OND) (1.44±0.18 and 0.97±0.14 vs 0.41±0.03 pmol/µL). Consistently, SM levels in the CSF of patients with AIDP were increased compared with both OND and patients affected by axonal forms of GBS (1.34±0.23 vs 0.41±0.03 and 0.30±0.03 pmol/µL). (B) Patients with CIDP, independently from the clinical form, displayed increased levels of CSF SM when in the active stage of the disease compared with OND and stable CIDP (1.63±0.17 vs 0.41±0.03 and 0.59±0.07 pmol/µL); conversely, no difference in terms of SM content was found between clinically stable patients with CIDP and OND (0.59±0.07 vs 0.41±0.03 pmol/µL). (C) A ROC curve analysis was performed to define the characteristics of SM levels as a CSF biomarker of active demyelination. A cohort of patients affected by active CIDP and AIDP was compared with the OND cohort. SM levels significantly increased in patients compared with OND (1.56±0.14 vs 0.41±0.03 pmol/µL). We found that AUC for SM was 0.9447, indicative of a very good discriminatory biomarker. CSF SM testing exhibited high sensitivity (80.85%) and specificity (98.82%) in the identification of patients affected by chronic and acute demyelinating polyradiculoneuropathy. The SM cut-off for optimum sensitivity and specificity was 0.9819 pmol/µL. (D) SM testing displayed a 100% specificity in the identification of patients with CIDP in the active stage of the disease and patients with AIDP from a cohort of patients with axonal neuropathies (1.56±0.14 vs 0.63±0.05 pmol/µL). Data were presented as mean±SEM. Unpaired two-tailed t-test was used for statistical comparison between two groups. Holm-Sidak multiple comparison test after one-way analysis of variance was used for statistical comparison of multiple groups. *p<0.05; **p<0.01; ****p<0.0001; ns=not significant. Specificity and sensitivity of SM assay were tested by ROC curve analysis. A cut-off value for SM was also calculated. All statistical analysis was performed using the Graph Pad V.7.0 (Prism) software. AIDP, acute inflammatory demyelinating polyradiculoneuropathy; AUC, area under the curve; CIDP, chronic inflammatory demyelinating polyradiculoneuropathy; CSF, cerebrospinal fluid; GBS, Guillain-Barré syndrome; OND, other neurological diseases; ROC, receiver-operating characteristic; SM, sphingomyelin.

Figure 2

CSF protein and SM levels in potential misdiagnosed CIDP. (A) CSF protein concentration was tested for reliability in the identification of potential misdiagnosed patients with CIDP (also referred to as ‘no EFNS/PNS CIDP’) from patients affected by definite active CIDP (also referred to as ‘EFNS/PNS CIDP’). As expected, we found a significant increase of CSF protein in ‘EFNS/PNS CIDP’ compared with ‘no EFNS/PNS CIDP’ (1.02±0.09 vs 0.44±0.04 g/L). Using the URL of 0.45 g/L, CSF protein concentration displayed just a 50% specificity to correctly identify patients with CIDP. (B) SM levels in the CSF of the same patients showed a significant increase in ‘EFNS/PNS CIDP’ compared with ‘no EFNS/PNS CIDP’ (1.63±0.16 vs 0.41±0.04 pmol/µL). Of note, SM dosage demonstrated 100% specificity to correctly recognise the cohort affected by definite CIDP, allowing to exclude patients affected by other neuropathies and also potential misdiagnosed CIDP. Data were presented as mean±SEM. Unpaired two-tailed t-test was used for statistical comparison between two groups. ****p<0.0001. Specificity and sensitivity of CSF proteins and SM were tested by ROC curve analysis. All statistical analysis was performed using the Graph Pad V.7.0 (Prism) software. CIDP, chronic inflammatory demyelinating polyradiculoneuropathy; CSF, cerebrospinal fluid; EFNS, European Federation of Neurological Societies; PNS, Peripheral Nerve Society; ROC, receiver-operating characteristic; SM, sphingomyelin; URL, upper reference limit.

Figure 3

Correlation of CSF SM levels with clinical scales in CIDP and AIDP. (Panel A) SM levels were correlated with clinical scales, including INCAT and ONLS to grade disease severity, and MRC sum score to grade muscle strength in the CSF of patients affected by active CIDP. Patients displayed a low but significant correlation of SM with all clinical scales. (Panel B) SM levels were correlated with clinical scales, including GBS disability scale and ONLS to grade disease severity, and MRC sum score to grade muscle strength in the CSF of patients affected by AIDP. Patients displayed a high and significant correlation of SM with GBS disability scale and a moderate but also significant correlation with ONLS and MRC sum score. Spearman’s rank correlation test was used for statistical analysis. *p<0.05; ***p<0.001. All statistical analysis was performed using the Graph Pad V.7.0 (Prism) software. AIDP, acute inflammatory demyelinating polyradiculoneuropathy; CIDP, chronic inflammatory demyelinating polyradiculoneuropathy; GBS, Guillain-Barrésyndrome; INCAT, inflammatory neuropathy cause and treatment; MRC, Medical Research Council; ONLS, Overall Neuropathy Limitations Scale; SM, sphingomyelin.