CD138 as a Specific CSF Biomarker of Multiple Sclerosis

Abstract

Background and objectives: The aim of this study was to identify novel biomarkers for multiple sclerosis (MS) diagnosis and prognosis, addressing the critical need for specific and prognostically valuable markers in the field.

Methods: We conducted an extensive proteomic investigation, combining analysis of (1) CSF proteome from symptomatic controls, fast and slow converters after clinically isolated syndromes, and patients with relapsing-remitting MS (n = 10 per group) using label-free quantitative proteomics and (2) oligodendrocyte secretome changes under proinflammatory or proapoptotic conditions using stable isotope labeling by amino acids in cell culture. Proteins exhibiting differential abundance in both proteomic analyses were combined with other putative MS biomarkers, yielding a comprehensive list of 87 proteins that underwent quantification through parallel reaction monitoring (PRM) in a novel cohort, comprising symptomatic controls, inflammatory neurologic disease controls, and patients with MS at various disease stages (n = 10 per group). The 11 proteins that passed this qualification step were subjected to a new PRM assay within an expanded cohort comprising 158 patients with either MS at different disease stages or other inflammatory or noninflammatory neurologic disease controls.

Results: This study unveiled a promising biomarker signature for MS, including previously established candidates, such as chitinase 3-like protein 1, chitinase 3-like protein 2, chitotriosidase, immunoglobulin kappa chain region C, neutrophil gelatinase-associated lipocalin, and CD27. In addition, we identified novel markers, namely cat eye syndrome critical region protein 1 (adenosine deaminase 2, a therapeutic target in multiple sclerosis) and syndecan-1, a proteoglycan, also known as plasma cell surface marker CD138 and acting as chitinase 3-like protein 1 receptor implicated in inflammation and cancer signaling. CD138 exhibited good diagnostic accuracy in distinguishing MS from inflammatory neurologic disorders (area under the curve [AUC] = 0.85, CI 0.75-0.95). CD138 immunostaining was also observed in the brains of patients with MS and cultured oligodendrocyte precursor cells but was absent in astrocytes.

Discussion: These findings identify CD138 as a specific CSF biomarker for MS and suggest the selective activation of the chitinase 3-like protein 1/CD138 pathway within the oligodendrocyte lineage in MS. They offer promising prospects for improving MS diagnosis and prognosis by providing much-needed specificity and clinical utility.

Classification of evidence: This study provides Class II evidence that CD138 distinguishes multiple sclerosis from other inflammatory neurologic disorders with an AUC of 0.85 (95% CI 0.75-0.95).

Figure 1. Schematic Representation of the Workflow Used in the Study

CTRL: symptomatic controls; CIS: clinically isolated syndrome; FC-CIS: fast conversion to MS (<1 year) after a CIS; SC-CIS: slow conversion to MS (>2 years) after CIS; RRMS: relapsing-remitting multiple sclerosis patients.

Figure 2. Results of Label-Free Proteomic Analysis of Human CSF Samples From the Discovery Cohort

(A) Hierarchical clustering of the 12 proteins exhibiting difference in abundance in the CSF from patients with RRMS and CTRLs. (B) Hierarchical clustering of the 6 proteins exhibiting difference in abundance in the CSF from FC-CIS and SC-CIS.

Figure 3. PRM Analysis of Peptides From 4 Candidate Protein Biomarkers in the Verification Cohort

Intensity (light transition area in arbitrary units [A.U.]) of peptides showing difference in abundance in CSF samples of the verification cohort (Table 2) is shown for NGAL (A), CECR1 (B), CD27 (C), and CD138 (D). The LOQ (limit of quantification) is indicated (red dotted line) for each peptide. Statistical analyses of group comparisons are provided in Table 3. CTRL: symptomatic controls (n = 30); CIS: clinically isolated syndrome; SC-CIS: slow converter CIS (n = 15); FC-CIS: fast converter CIS (n = 15); RRMS: relapsing-remitting multiple sclerosis (n = 30); PPMS: primary progressive multiple sclerosis (n = 14); INDC: inflammatory neurologic disease control (n = 13); ON: isolated optic neuritis (n = 15); NINDC: noninflammatory neurologic disease control (n = 13), PINDC: peripheral inflammatory neurologic disease control (n = 13).

Figure 4. Diagnostic Value of Candidate MS Biomarkers

(A) ROC curves showing the diagnostic values of CD27 (AUC = 0.98), IGKC (AUC = 0.92), and CECR1 (AUC = 0.91) to discriminate RRMS from CTRL. Multivariate analysis indicated that no combination has better diagnostic value than CD27 alone. (B) ROC curves showing the diagnostic values of CD138 (AUC = 0.85), IGKC (AUC = 0.75), CD27 (AUC = 0.76), and NGAL (AUC = 0.69) to discriminate MS from INDC. Multivariate analysis indicated that no combination has better diagnostic value than CD138 alone. (C) ROC curves showing the diagnostic values of CECR1 (AUC = 0.78) and NGAL (AUC = 0.64) to discriminate RRMS from PPMS. Multivariate analysis indicated that no combination has better diagnostic value than CECR1 alone. (D) CSF CD138 and CD27 levels measured by ELISA in CTRL (n = 14), patients with RRMS (n = 20), and patients with PPMS (n = 9) and compared with the nonparametric Mann-Whitney test. **, p value <0.01; ***, p value <0.001; ****, p value <0.0001. ROC = receiver operating characteristic.

Figure 5. Expression of CD138 in Control and RRMS Human Brain and Rat Primary Cultures

(A) Immunohistochemistry of brain tissue showing predominant expression of CD138 in cells located in the perivascular spaces (arrowheads) and a sparse expression in brain parenchyma (arrows) of RRMS brain but not in the CTRL brain. CHI3L1 is strongly expressed in astrocytes (pink) from RRMS brain but shows no colocalization with CD138 (brown). Scale bar: 100 µm. (B) Immunofluorescence labeling showing a high expression of GFAP in activated astrocytes of the RRMS brain when compared with the CTRL brain. There is no colocalization of CD138 (arrows) and GFAP in the RRMS brain. Scale bar: 100 µm. (C) Immunostaining of CD138 (arrows) in rat primary cultures of OPCs at 6 DIV showing a stronger expression of CD138 in immature (O4+) than in mature (MBP+) oligodendrocytes and its absence in astrocytes (GFAP+) (arrowheads). Scale bar: 100 µm.