Внутренняя плазменная активность клеток, измеренная индексом свободной цепи света Каппы, ассоциируется с атрофией ретинального слоя в ранних множественных склерозах.

Introduction

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS) and the leading cause of permanent disability in young adults [ 1 ]. Despite recent advances in treatment, the disease course remains highly heterogeneous and difficult to predict [ 2 ]. Consequently, biomarkers to stratify patients according to their risk of future disease activity and disability accumulation are urgently needed.

Among others, the kappa free light chain (κ‐FLC) index and retinal layer thickness have been recently identified as prognostic markers. The κ‐FLC index is a surrogate of intrathecal plasma cell activity and, thus, of the extent of neuroinflammation [ 3 ]. The κ‐FLC index has demonstrated high diagnostic accuracy comparable to oligoclonal bands [ 4 , 5 ], and is associated with occurrence of relapses [ 6 ], magnetic resonance imaging (MRI) activity [ 7 ] and the accumulation of disability [ 8 , 9 ].

In addition to CSF markers, optical coherence tomography (OCT), a non‐invasive, economic procedure, enables quantification of retinal layer thickness, for MS most relevant the peripapillary measured retinal nerve fiber layer (pRNFL) and the macular ganglion cell plus inner plexiform layer (GCIPL) [ 10 , 11 ]. pRNFL and GCIPL thicknesses are able to identify previous (subclinical) optic neuritis (ON) [ 12 ], and have predictive value for physical and cognitive worsening in both short‐ [ 13 ] and long‐term follow‐up [ 14 , 15 ]. Therefore, both the κ‐FLC index as well as retinal layer thickness have been proposed to predict treatment failure in MS [ 8 , 9 , 16 ].

The κ‐FLC index and OCT are both incorporated into the 2024 revision of the McDonald criteria in different roles: the κ‐FLC index is used as a substitute for oligoclonal bands, while OCT serves as a tool to demonstrate involvement of the newly added fifth anatomical region, the optic nerve [ 17 , 18 , 19 , 20 ]. Although well investigated separately, data on the interplay of the κ‐FLC index and retinal layer thickness in MS is missing.

The present study aimed to close this knowledge gap and investigated whether κ‐FLC index is associated with pRNFL and GCIPL thickness.

Methods

Study Design and Patients

Patients from the Austrian MS cohort (AMSC) [ 21 ] presenting with a first CNS demyelinating event suggestive of MS were included in this prospective, observational, multicentre study at the Medical University of Innsbruck, the Medical University of Vienna, and the Hospital of St. John of God Linz. For the present study, we included patients diagnosed with clinically isolated syndrome (CIS) or relapsing MS (RMS) according to the 2017 revision of the McDonald criteria [ 22 ], in whom cerebrospinal fluid (CSF) analysis and OCT scan at baseline were available. An overview of patient inclusion is given in Figure 1 .

We assessed demographic (age, sex), clinical (Expanded Disability Status Scale [EDSS] score, history of relapses, history of ON, disease‐modifying therapy [DMT]), and MRI metrics at baseline visit.

Magnetic Resonance Imaging

The number of T2‐hyperintense lesions (T2L) was determined using automated lesion segmentation with the Lesion Segmentation Toolbox LST‐AI [ 23 ]. All segmentations were visually inspected and manually corrected by two experienced neuro‐radiologists, and lesion counts were derived from the final corrected masks. Contrast‐enhancing lesions (CEL) were counted manually by the neuro‐radiologists.

κ‐ FLC Detection

CSF was collected via lumbar puncture for routine diagnostic purposes. Peripheral venous blood was drawn concurrently followed by a clotting period of at least 30 min. Samples were centrifuged at 2000 g for 10 min at room temperature [ 24 ]. Routine CSF parameters including WBC count, total protein, CSF/serum albumin quotient ( Q alb ), and oligoclonal banding (OCB) were determined as previously reported [ 25 , 26 ].

κ‐FLC concentrations and albumin in CSF and serum were measured at the Neuroimmunology Laboratory of the Medical University of Innsbruck using nephelometry (Atellica; Siemens, Erlangen, Germany) and the N Latex FLC kappa and the N Albumin assay according to the manufacturer instructions [ 27 , 28 ].

Intrathecal synthesis of κ‐FLC was assessed by calculating the κ‐FLC index [ 29 ]: κ − FLC index = κ − FLC CSF / κ − FLC Serum Albumin CSF / Albumin Serum κ‐FLC index was considered positive in case of values ≥ 6.1. Vice versa, κ‐FLC index below this cut‐off was classified as negative [ 4 , 18 ].

Optical Coherence Tomography

A comprehensive methodological description of our OCT protocol has been published previously [ 14 , 20 ]. In brief, OCT scans were conducted on both eyes using spectral‐domain OCT without pharmacologic pupil dilatation. pRNFL thickness was measured using a 12° circular scan (diameter 3.4 mm) centered on the optic disc. For assessment of the GCIPL, a macular cube scan covering a 20° × 20° field (512 A‐scans, 257 B‐scans, vertical alignment, ART 16) centered on the fovea was acquired. GCIPL thickness was defined as the mean layer thickness of the four inner (3 mm ring) and outer quadrants (6 mm ring) of the circular grid centered around the foveola as defined by the Early Treatment Diabetic Retinopathy Study [ 30 ].

Image segmentation was performed semi‐automatically, with manual adjustments applied where necessary. Quality control was ensured by adherence to the OSCAR‐IB criteria, and results were reported in line with APOSTEL recommendations [ 31 , 32 ].

By default, thicknesses of GCIPL and pRNFL, respectively, were calculated as the mean of the values for both eyes. Eyes with a history of symptomatic unilateral ON < 6 months before baseline were excluded from the total cohort (and were evaluated separately). To identify subclinical ON, we used established cut‐offs for inter‐eye asymmetry (IED; ≥ 4 μm for GCIPL and ≥ 5 μm for pRNFL) [ 12 , 33 ]. In these cases, we used only the eye with the higher value for layer thinning calculations. Thus, all parameters used for statistical analyses do not underlie inter‐eye interactions.

This approach provides a global estimate of retinal neuroaxonal loss—analogous to whole‐brain atrophy—while preserving independence of residuals in subject‐level regression models. The investigators performing the OCT were blinded to clinical parameters and vice versa.

Statistical Analysis

Statistical analysis was conducted using R [ 34 ]. Data are shown as frequencies and percentages for categorical variables, and as median and 25th–75th percentile for continuous variables.

To evaluate the association of κ‐FLC index and retinal layer thickness (pRNFL, GCIPL), we applied multivariable linear regression analyses with pRNFL or GCIPL thickness as dependent variable and the κ‐FLC index (continuous) as independent variable, adjusted for age (continuous), sex (dichotomous), disease duration (continuous), T2L (continuous), CEL (continuous), and center (categorical). The κ‐FLC index was log‐transformed to improve the approximation of normality of the residuals. Missing data were handled using complete case analysis.

All analyses were done using GCIPL and pRNFL thicknesses (i) across eyes in case of no ON history or inter‐eye asymmetry ( n = 100) and (ii) from eyes with clinically manifest ON ( n = 28).

Additionally, we performed sensitivity analyses (i) in the eyes with IED, (ii) in the subgroup of patients with a time from CSF sampling to OCT of > 7 days and (iii) in κ‐FLC index positive patients.

Statistical significance was defined as a two‐sided p ‐value < 0.05.

Ethics

The study was approved by the Ethics Committees of the Medical University of Innsbruck, Linz (approval number: 1050/2023) and the Medical University of Vienna (2323/2019). Prior to participation, all individuals provided written informed consent. All procedures involving human participants were conducted in accordance with the ethical principles outlined in the Declaration of Helsinki and complied with applicable national guidelines and regulatory requirements.

Results

A total of 100 patients at a median age of 33 (25th–75th percentile: 25–39) years and a female predominance (58%) were included in the study. The median disease duration was 12 (6‐43) days, and the median EDSS was 0 (0–1.5). Brain MRI showed a median of 13 (5‐30) T2L, with 37% of patients displaying CEL. None of the patients was on DMT. Optic neuritis was the initial manifestation in 28% of patients. OCT was performed a median of 7 (1–71) days after CSF sampling.

Eyes without ON showed a median pRNFL thickness of 99 μm (92–106) and a GCIPL thickness of 77 μm (71–83), while in ON eyes median pRNFL and GCIPL thicknesses were 102 μm (89–116) and 74 μm (68–79), respectively.

Median κ‐FLC index was 44 (13–105). The κ‐FLC index was positive (≥ 6.1) in 85% of patients. A comprehensive overview of baseline clinical, MRI, CSF and OCT findings is provided in Table 1 .

Higher κ‐ FLC Index Is Associated With Thinner pRNFL and GCIPL

In multivariable linear regression analysis adjusted for age, sex, disease duration, T2L, CEL and center, higher κ‐FLC index was significantly associated with thinner pRNFL (log‐transformed κ‐FLC index: β : −2.51; 95% CI: −4.01, −1.01; p = 0.001; Figure 2A ; Table 2A ) and GCIPL (log‐transformed κ‐FLC index: β : −1.24; 95% CI: −2.32, −0.17; p = 0.024; Figure 2B ; Table 2A ).

In the subgroup of ON eyes, the κ‐FLC index was not statistically significantly associated with retinal layer thinning (pRNFL: log‐transformed κ‐FLC index: β : −4.54; 95% CI: −11.08, 1.99; p = 0.160; GCIPL: log‐transformed κ‐FLC index: β : −0.90; 95% CI: −3.51, 1.71; p = 0.473; Table 2B ).

Sensitivity analysis revealed consistent directions of the estimates of pRNFL and GCIPL even for eyes ( n = 33) excluded from the main analysis due to IED (Figure S1 ). The κ‐FLC index did not differ between patients with IED (41 [10–105]) compared to patients without IED (43 [14–119]). Sensitivity analyses for patients with a time from CSF sampling to OCT of > 7 days (Figure S2 ) and for κ‐FLC index positive patients (Figure S3 ) also showed consistent direction of the estimates.

Discussion

In the present study, we evaluated the relationship between two biomarkers recently introduced in the diagnostic criteria of patients with suspected MS, namely κ‐FLC index, reflecting intrathecal plasma cell activity [ 18 ], and retinal layer thickness measured by OCT, reflecting neuroaxonal damage [ 19 ]. Our key finding is that at the time of disease onset the κ‐FLC index is associated with pRNFL and GCIPL thinning.

The prevailing concept of MS pathology is based on two main pathophysiological processes within the CNS: inflammation and neuroaxonal degeneration. While this duality was historically thought to be rather consecutive, nowadays, overwhelming evidence for their simultaneous nature has grown [ 35 , 36 ]. In this context, the κ‐FLC index can be viewed as a surrogate for the extent of neuroinflammation, resembling intrathecal plasma cell activity and intrathecal immunoglobulin production [ 3 ]. Retinal layer thinning assessed by pRNFL and GCIPL is well‐established as a marker of neuroaxonal degeneration reflecting physical disability accrual, cognitive decline, and brain atrophy [ 14 , 15 , 37 , 38 ].

Of note, pRNFL and GCIPL thinning are neither pathophysiologically nor clinically fully interchangeable: pRNFL consists of axonal projections of retinal ganglion cells and is sensitive to acute axonal processes [ 39 , 40 , 41 ]. In acute ON, pRNFL may initially show edema (pseudohypertrophy). Within 1–3 months after ON, retrograde axonal loss occurs due to inflammatory demyelination [ 39 , 40 , 41 ]. GCIPL, on the other hand, comprises the ganglion cell bodies and their dendrites. Contrary to pRNFL, GCIPL does not show edema even in acute ON, and GCIPL atrophy can be detected earlier after ON than pRNFL thinning. Both pRNFL and GCIPL serve as robust biomarkers for axonal and neuronal loss in MS, respectively, whereby GCIPL is more sensitive for early neurodegenerative changes and ongoing progression [ 42 ]. Against this background, we provide evidence that the κ‐FLC index is inversely associated with pRNFL and GCIPL thickness in patients with newly diagnosed MS.

The observation of more pronounced retinal layer thinning already at the time of disease manifestation in eyes unaffected by acute ON along with increased κ‐FLC index may reflect more intense and/or longer‐standing intrathecal plasma cell activity leading to subclinical neuroaxonal damage. This hypothesis is strengthened by reported associations of brain atrophy with both kFLC index and retinal layer thinning [ 37 , 38 ]. Additionally, thinner GCIPL thickness has been reported to be associated with increased intrathecal B cell activity, i.e., by higher CD19 + B cell frequencies and intrathecal immunoglobulin G synthesis as determined by IgG index [ 43 ]. The findings that κ‐FLC index, IgG index and CSF B cell frequency—all of them surrogates of intrathecal inflammation—correlate with retinal layer thickness show biological consistency. At this point, we would like to highlight that one of the advantages of the κ‐FLC index is its higher sensitivity over other quantitative CSF surrogates with a sensitivity of approximately 90% [ 4 ] (compared to e.g., the IgG index with a sensitivity of only 70% [ 26 ]). Furthermore, the κ‐FLC index captures also intrathecal IgM synthesis [ 44 ] which has been shown to occur in a subset of MS patients associated with worse prognosis. These differences might explain why some prior studies did not observe association of conventional CSF surrogates with retinal layer thickness [ 45 ].

In subgroup analysis of patients with clinically manifest ON, linear regression models of retinal layers and κ‐FLC index revealed also a negative estimate (Table 2B ). Even though the association between retinal layers and κ‐FLC index in patients with clinically manifest ON seems reasonable from a pathophysiological point of view, failing statistical significance might be due to the limited statistical power due to the small number of only 28 patients, this observation should be interpreted with caution given also the wide CI range and considered as hypothesis‐generating observation which should be addressed by future studies.

From a clinical point of view, these findings highlight the complementary nature of pRNFL, GCIPL, and the κ‐FLC index in the diagnostic assessment of patients with a first clinical presentation suggestive of MS. Each biomarker captures a distinct, yet interrelated aspect of early MS pathology. Incorporating all three measures at the time of diagnosis may therefore provide a more comprehensive characterization of inflammatory and neurodegenerative disease components. Moreover, markedly elevated κ‐FLC index in combination with low pRNFL and GCIPL thickness may be considered useful indicators for treatment decision making towards starting early high‐efficacy DMT. Additionally, due to its non‐invasive nature, high reproducibility, and economical advantages, OCT offers an easy‐to‐perform opportunity for longitudinal monitoring of neuroaxonal damage [ 46 , 47 , 48 , 49 , 50 ]. However, it has to be stated that the prognostic value of both κ‐FLC index and retinal layer thinning has not been investigated yet and should be addressed by future research.

Some limitations of the present study have to be pointed out. First, the sample size remains modest, thus, statistically nonsignificant findings may not be interpreted as proof of no impact, e.g., in subgroup analyses in eyes with acute ON as discussed above. Second, although all centers employed OCT platforms with identical scanning and quality control protocols, we cannot exclude some variability due to center [ 46 ]. Third, the interval between CSF sampling and OCT was 7 (range 1–71) days. We cannot fully exclude that subclinical acute inflammatory changes occurring close to symptom onset may have influenced OCT measurements in patients with very short intervals. Sensitivity analysis revealed estimates in the same directions. Furthermore, this is a cross‐sectional analysis. Longitudinal (and re‐baseline) data were not available. As a result, we can neither assess dynamic changes of retinal layer thickness over time nor evaluate if κ‐FLC index predicts retinal atrophy in the future. Although focal inflammation of the optic nerve precedes secondary axonal degeneration and, consequently, retinal layer thinning from a pathophysiological perspective, our cross‐sectional data do not allow to establish a causal relationship between the κ‐FLC index, reflecting generalized intrathecal inflammation, and subclinical optic nerve involvement. This seems a promising research question for further studies.

In conclusion, the present study reveals—for the first time—an association of increased κ‐FLC index with pRNFL and GCIPL thinning. Therefore, we substantiate evidence that retinal layer atrophy in early MS is associated with intrathecal plasma cell activity.

Author Contributions

Klaus Berek: conceptualization, investigation, writing – original draft, methodology, validation, visualization, software, formal analysis, project administration, data curation. Martin Schmidauer: conceptualization, investigation, methodology, validation, visualization, project administration, data curation. Fabian Föttinger: writing – review and editing, project administration, data curation, investigation. Michael Auer: writing – review and editing, project administration, data curation, investigation. Thomas Berger: writing – review and editing, supervision, resources, investigation. Franziska Di Pauli: writing – review and editing, supervision, resources, investigation. Luisa Velez Escola: writing – review and editing, investigation. Thomas Hofer: investigation, writing – review and editing. Barnabas Kremser: investigation, writing – review and editing. Stephanie Mangesius: investigation, writing – review and editing, data curation, formal analysis, software, methodology. Jan P. Nolte: investigation, writing – review and editing, data curation, project administration, formal analysis, software. Marie‐Christine Pali: investigation, writing – review and editing, data curation, methodology. Berthold Pemp: investigation, writing – review and editing. Katharina Stock: investigation, writing – review and editing, data curation, formal analysis, software, methodology. Markus Tiefenthaler: investigation, writing – review and editing, data curation, formal analysis, software, methodology. Janette Walde: writing – review and editing, software, formal analysis. Anne Zinganell: investigation, writing – review and editing, project administration, data curation. Florian Deisenhammer: investigation, writing – review and editing, supervision, resources. Gabriel Bsteh: investigation, writing – review and editing, supervision, resources. Harald Hegen: funding acquisition, writing – review and editing, data curation, supervision, resources, software, formal analysis, investigation, validation, visualization.

Funding

This study was funded by Novartis.

Conflicts of Interest

K.B. has participated in meetings sponsored by and received travel funding or speaker honoraria from Roche, Teva, Merck, Biogen, Sanofi and Novartis. He is associate editor of Frontiers in Immunology/Neurology, Section Multiple Sclerosis and Neuroimmunology. M.S. has participated in meetings sponsored by or received travel grants from Novartis and Sanofi‐Genzyme. F.F. has participated in meetings sponsored by, received speaker honoraria or travel funding from Novartis, Biogen, and Merck. M.A. received speaker honoraria and travel grants and participated in meetings sponsored by Biogen, Merck, Novartis, Sanofi, and Horizon Therapeutics. T.B. has participated in meetings sponsored by and received honoraria (lectures, advisory boards, consultations) from pharmaceutical companies marketing treatments for M.S.: Allergan, Bayer, Biogen, Bionorica, BMS/Celgene, Genesis, GSK, GW/Jazz Pharma, Horizon, Janssen‐Cilag, MedDay, Merck, Novartis, Octapharma, Roche, Sandoz, Sanofi‐Genzyme, Teva, and UCB. His institution has received financial support in the past 12 months by unrestricted research grants (Biogen, Bayer, BMS/Celgene, Merck, Novartis, Roche, Sanofi‐Genzyme, Teva) and for participation in clinical trials in multiple sclerosis sponsored by Alexion, Bayer, Biogen, Merck, Novartis, Octapharma, Roche, Sanofi‐Genzyme, and Teva. F.D.P. has participated in meetings sponsored by, received honoraria (lectures, advisory boards, consultations) or travel funding from Amgen, Biogen, Celgene, Horizon, Janssen, Merck, Novartis, Sanofi‐Genzyme, Roche and Teva. L.V.E. has nothing to disclose. T.H. has participated in meetings sponsored by, received honoraria (lectures, advisory boards, consultations) or travel funding from Biogen, BMS, Ipsen, Janssen, Lundbeck, Merck, Novartis, Sanofi‐Genzyme, and Teva‐Ratiopharm. B.K. has nothing to disclose. S.M. has nothing to disclose. J.P.N. has participated in meetings sponsored by, received speaker honoraria or travel funding from Novartis, Biogen, Merck, and Neuraxpharm. M.‐C.P. has nothing to disclose. B.P. has received speaker honoraria and travel funding from Novartis, Chiesi, and Santen and honoraria for consulting and/or advisory board meetings from Chiesi and GenSight. K.S. has nothing to disclose. M.T. has nothing to disclose. J.W. has nothing to disclose. A.Z. has participated in meetings sponsored by, received speaking honoraria or travel funding from Biogen, Merck, Novartis, Sanofi‐Genzyme, Janssen, Bristol Myers Squibb, and Teva. F.D. has participated in meetings sponsored by or received honoraria for acting as an advisor/speaker for Alexion, Almirall, Biogen, BMS, Sanofi, Amgen, Janssen, Laurea Group, Medwhizz, Merck, Novartis Pharma, Neuraxpharm, Roche, Sandoz, and Teva. He is review editor of Frontiers Neurology. G.B. has received travel funding from Biogen, Merck, Novartis, Roche, Sanofi, and Teva; has received speaker honoraria from Biogen, BMS, Heidelberg Engineering, Janssen, Lilly, Medwhizz, Merck, Neuraxpharm, Novartis, Roche, Sanofi, Teva, and Zeiss; has received honoraria for consulting with Adivo Associates, Biogen, BMS, Janssen, Merck, Novartis, Roche, Sanofi, and Teva. He has received unrestricted research grants from BMS, Merck, and Novartis. He serves on the Executive Committee of the European Committee for Treatment and Research in Multiple Sclerosis (ECTRIMS) and the Board of Directors of the International Multiple Sclerosis VisualSystem Consortium (IMSVISUAL). H.H. has participated in meetings sponsored by, received speaker honoraria or travel funding from Alexion, Amgen, Bayer, Biogen, Bristol Myers Squibb, Janssen, Merck, Novartis, Sanofi‐Genzyme, Siemens, Roche, and Teva, and received honoraria for acting as consultant for Biogen, Bristol Myers Squibb, Novartis, Roche, Sanofi‐Genzyme, and Teva. His institution has received research grants from Amgen, Novartis, and Roche.

Supporting information

Figure S1: Sensitivity analysis of eyes with IED compared to the total cohort.

Figure S2: Sensitivity analysis of patients with an interval between CSF sampling and OCT of more than 7 days compared to the total cohort.

Figure S3: Sensitivity analysis of κ‐FLC positive patients compared to the total cohort.

Acknowledgements

Open Access funding provided by Medizinische Universitat Innsbruck/KEMÖ.

Data Availability Statement

Data supporting the findings of this study are available from the corresponding author upon reasonable request by a qualified researcher and upon approval by the ethics committees and the data‐clearing committees of the Medical Universities of Innsbruck and Vienna.

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