Introduction

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system, characterized by inflammation, demyelination, gliosis, and axonal loss.1 While the inflammatory aspect is routinely monitored in clinical practice using conventional magnetic resonance imaging (MRI), easily accessible biomarkers of neurodegeneration are needed to monitor disease progression and assess the efficacy of putative neuroprotective drugs.2, 3 One such potential candidate is an imaging biomarker that is found in the retina, namely, peripapillary retinal nerve fiber layer (pRNFL).4-7 Because the retina is unmyelinated, measurements of retinal layer thinning are not confounded by myelin and are thereby ideal for assessing neuroaxonal degeneration. Both the pRNFL, containing unmyelinated axons emerging from retinal ganglion cell neurons, and the combined macular ganglion cell and inner plexiform layers (mGCIPL), containing retinal ganglion cell bodies (i.e., from which the axons forming pRNFL originate) and dendrites may show abnormal thinning over time and their atrophy is a sequelae of optic neuritis and optic neuropathy.8 Several MS-related studies have shown that thinning of pRNFL and mGCIPL is associated with increased clinical disability and brain volume loss.5, 9-15

Optical coherence tomography (OCT) noninvasively captures cross-sectional images of the retina to generate three-dimensional high-resolution maps of the retinal architecture. One significant advantage of OCT compared with MRI is that it can be more sensitive in detecting small increments of neurodegeneration over time owing to its spatial resolution on the order of 5 microns, approximately 2–3 orders of magnitude better than MRI.

OCT may detect, and follow longitudinally, the degeneration of retinal axons and neurons by measuring change in pRNFL and mGCIPL thickness in patients with MS.16, 17 OCT technology therefore has the potential to provide a reliable and convenient tool to monitor disease progression, quantify the rate of neurodegeneration, and assess the potential neuroprotective treatment effect of MS therapies.

Although it is acknowledged as an easily applicable technique, OCT repeat measurement variability must be considered when assessing within-subject longitudinal changes or when applied in multicenter settings.18 Factors that influence the quality of scans and affect measurements, including signal strength, location of blood vessels in the inner retinal layers, acquisition procedures, etc., have been well described.19, 20 Post-acquisition segmentation of healthy and thinned retinal layers also influences reproducibility, and can be affected by the robustness of the software algorithm. It is therefore essential to assess the test-retest variability, that is, the intersession measurement difference in pRNFL that reflects the technical and/or operator-based limitations of the method but is not attributable to the underlying disease pathology.

The OCTiMS (Optical Coherence Tomography in Multiple Sclerosis) study is the first large prospective multicenter study designed to determine whether OCT imaging is sufficiently sensitive to detect progression in retinal neuroaxonal loss over 3 years in patients with relapsing-remitting MS (RRMS) compared with healthy participants in a multicenter trial-like setting. Changes in average pRNFL and mGCIPL thickness were assessed, along with their potential association with brain volume loss, Expanded Disability Status Scale (EDSS) score, and other clinical and paraclinical measures.

Methods Participants

The OCTiMS study (NCT02907281) was a 36-month, prospective, multicenter, noninterventional study, which enrolled 397 participants (333 RRMS and 64 reference participants) across 28 sites in Australia, Europe and North America. The study was approved by respective institutional review boards and all participants provided written informed consent before enrollment. All data were captured and monitored by an independent clinical research organization (CRO) using electronic case record forms (eCRF). As pRNFL thinning may also occur as a result of MS-associated optic neuritis (MS-ON), patients were categorized into those with prior history of MS-ON and those without MS-ON (non-MS-ON) and assessed accordingly. For patients with a history of MS-ON in one eye, the eye with prior MS-ON was studied. For patients with a history of MS-ON in both eyes, or no optic neuritis (ON) at all, the eye with the lowest respective pRNFL thickness at baseline was included. Healthy, age-matched, control participants were included as the reference group in all models. Participants who experienced an adverse event of MS-ON during the study were re-categorized to the MS-ON subgroup.

Objectives

The primary study objective was to evaluate change in average pRNFL thickness using OCT in patients with RRMS over a 36-month follow-up period and compare it with the findings from a group of healthy participants to determine whether the technology is sufficiently sensitive to detect disease-related pRNFL changes over time. Secondary objectives were as follows: to evaluate short-term reproducibility of pRNFL thickness measurements over a 4-week interval by test re-test estimation; to evaluate the correlation between change in pRNFL thickness with change in brain volume and disability progression in patients with RRMS. In addition, we evaluated change in mGCIPL thickness over 36 months in patients with RRMS and reference participants, as well as the relationship between change in mGCIPL thickness and change in brain volume and disability progression in patients with RRMS. pRNFL and mGCIPL thickness changes were also compared between subgroups according to duration of MS diagnosis determined at the time of enrollment. Finally, we explored the correlation of change in pRNFL thickness with change in other clinical and paraclinical measures and patient-reported outcomes in patients with RRMS including visual acuity, MRI measures, cognitive performance, and quality of life.

Key inclusion/exclusion criteria

All participants (aged 18–65 years inclusive) were enrolled and followed up between 29 May 2012 and 24 July 2017. Key inclusion criteria for the MS group were as follows: a diagnosis of RRMS as defined by the 2005 revision to the McDonald criteria21; MS disease duration of more than 1 year (from diagnosis of MS) before study entry (screening). The list of exclusion criteria is included in Table S1.

Optical coherence tomography

OCT scans in all participants were performed on Spectralis OCT1 machines (Heidelberg Engineering, Heidelberg, Germany), software version 1.6.2.0 or higher, with the eye tracking function enabled for best accuracy and the use of the baseline scan for alignment of subsequent scans on the same area of the retina. Data were collected from a peripapillary ring, a macular volume scan, the optic nerve head volume scan, and the papillomacular bundle volume scan (see supportive information Data S1 for more details on techniques used). All OCT scans underwent quality control (QC) assessment by the central reading center (Vienna Reading Center [VRC]). Figure S1 gives an overview of the scan patterns and the evaluation fields for each pattern used in the analysis.

OCT scans were performed at baseline and at every visit (1, 6 months, and every 6 months thereafter) for both groups. All operators at participating sites were certified by the VRC for the use of the correct scanning protocol, correct pseudonymization of study-specific image labelling, and image transfer before patient inclusion according to standardized certification processes to obtain images for the above-described acquisitions.

Automated segmentation was performed with the manufacturer's software (HEYEX version 1.9.10.0, Viewing Module version 6.0.9.0) and subsequently corrected by certified and trained graders of the VRC, supervised by a retina specialist, according to a predefined reading protocol. All graders were masked to the clinical status of the study participants (MS or healthy control group) and any other diagnostic information. Studies were performed and described in accordance to the QC criteria at the VRC, OSCAR-IB, and the Advised Protocol for OCT Study Terminology and Elements (APOSTEL) criteria.20, 22 The detailed OCT scanning protocol is provided as supporting information.

Ophthalmology examination

An ophthalmologic examination was performed at screening and every 12 months thereafter. The examination included ophthalmic medical history, slit lamp examination, dilated ophthalmoscopy, best corrected visual acuity testing at low 2.5% contrast and high contrast (number of letters read correctly on an Early Treatment Diabetic Retinopathy Study scale), and perimetry testing (optional at sites where required technical equipment was available). If, during the study, worsening of visual acuity or any clinically acute ON were suspected, an unscheduled ophthalmology examination was performed.

Magnetic resonance imaging

All study participants underwent MRI scanning (1.5 or 3T) of the brain at screening and at yearly intervals. In the RRMS patient group, pulse-sequences included 3DT1 for brain volume measurement, proton density (PD)/T2 for new lesion count and volume, T1 for new hypointense lesion count and volume, and gadolinium-enhanced T1 sequences for lesion count and volume. Lesions were identified by expert radiology readers and quantified by trained technicians using MIPAV software with local thresholding. Brain volume measurements were performed using SIENA[X] (http://fsl.fmrib.ox.ac.uk). In the reference participant group, 3DT1 sequences for brain volume measurements (all MRI scans) and T2 lesions (screening scan only) were performed. All MRIs were performed in a standardized manner according to the MRI manual upon performance of a successful dummy-scan. Each MRI scan performed was previewed by the local neuroradiologist for incidental findings before the scans were sent to and further processed by the MRI central reading center at VU University Medical Center in Amsterdam.

Clinical assessments EDSS

Disability was assessed every 6 months by certified EDSS raters in clinical MS studies.23 Disability progression was defined as a one-point EDSS increase for those with a baseline score of ≤5.0, or a 0.5-point increase for those with a baseline score >5.0, with the EDSS progression confirmed after 6 months.

Symbol digit modalities test

The symbol digit modalities test (SDMT) was performed every 12 months to assess attention and information processing speed. During the test, subjects substituted symbols in a row by the corresponding number and responded verbally.24, 25

Patient-reported indices for multiple sclerosis

The patient-Reported Indices for Multiple Sclerosis (PRIMuS) instrument was used to assess patients’ quality of life (QoL) and included activities specifically designed for MS. The test was performed every 12 months.26

Statistical methodology

Statistical analyses were performed using SAS (version 9.4). All hypothesis tests were evaluated at a 0.05 level of significance. All confidence intervals (CIs) were two sided at a 95% confidence level.

Average pRNFL thickness was summarized by group and ON history at each visit, including change from baseline. A mixed-model repeated measures (MMRM) approach was applied to compare the change from baseline in average pRNFL thickness between groups, considering results from all available time points, and adjusting for age, gender, and baseline pRNFL thickness. The reference subjects were included as the reference group for both the MS-ON and non-MS-ON subgroups. The group difference in least squares (LS) mean of change from baseline in average pRNFL thickness (patients with MS reference subjects), which reflects the change from baseline for the MS group, after adjusting for the change in the reference group (e.g., to account for age-related change in healthy subjects), and the associated 95% CI and p-value are presented for each time point. The LS mean of pRNFL change from baseline within each group is presented for Month 36. The analysis of the primary variable was repeated for the subfield measures of pRNFL thickness: papillomacular bundle, overall temporal quadrant, and temporal fields 6.

An additional analysis was performed to evaluate the change in mGCIPL thickness at Month 36 based on a mixed effect model. Change from baseline in both pRNFL and mGCIPL thickness at Month 36 was stratified by disease duration [defined as time from MS diagnosis to study screening (≤3 years, >3 to ≤5 years, >5 years)] for patients with MS and subgroups of ON history (MS-ON, non-MS-ON) and groups of different disease duration were compared using an analysis of variance model (ANOVA).

Regression analyses were applied to assess the relationship between the change from baseline in EDSS/percentage change in brain volume and the change from baseline in average pRNFL/mGCIPL thickness at Month 36. All results are presented with nominal p-value with no adjustment for multiplicity due to the observational character of the study.

Results Baseline characteristics

A total of 333 patients with MS and 64 reference participants without neurologic disease provided a baseline and at least one post baseline pRNFL measurement in at least one eye. Thirty-nine (11.7%) patients with MS and eight (12.5%) reference participants discontinued the study (Fig. 1). Demographics were well balanced between patients with MS and reference participants, with a small difference found for age (38.9 vs. 39.4, p = 0.003), which was not deemed clinically relevant. Normalized brain volume was lower in the MS groups (p < 0.0001) (Table 1). Differences between MS-ON and non-MS-ON patients in terms of cognitive performance, normalized brain volume and disease characteristics including MS duration, EDSS, and T2 lesion volume were all statistically non-significant (Table 2). Ocular characteristics, including measures of visual acuity and pRNFL/mGCIPL thickness, were highest in eyes of heathy participants, lowest in eyes with past MS-ON, and intermediate in eyes of patients without MS-ON (Table 3). All differences between groups (reference vs. MS and MS-ON vs. non-MS-ON) were significant. Six patients developed MS-ON during the study period. Prior to the episode, these patients had been included in the non-MS-ON group, but they were re-categorized to the MS-ON group at follow-up (after the episode). While non-severe glaucoma was not part of the exclusion criteria, it should be noted that only two patients (0.6%) had glaucoma in the MS group and 1 (1.4%) in the reference group.

CONSORT diagram.

Table 1. Baseline demographics and clinical characteristics. Characteristics MS group Reference group Group difference

All patients with MS (N = 333)

Patients without MS-ON1 (N = 235)

Patients with MS-ON (N = 98)

Healthy participants

(N = 64)

p-value* Age (years) Mean ± SD 38.9 ± 8.56 38.7 ± 8.56 39.4 ± 8.60 35.4 ± 9.73 0.003 Median (min, max) 39.0 (19.0, 55.0) 39.0 (19.0, 55.0) 39.0 (21.0, 55.0) 35.0 (21.0, 53.0) Sex, n (%) Female 233 (70.0) 166 (70.6) 67 (68.4) 45 (70.3) 0.956 Male 100 (30.0) 69 (29.4) 31 (31.6) 19 (29.7) Race, n (%) Caucasian 318 (95.5) 223 (94.9) 95 (96.9) 61 (95.3) 0.752 Black 4 (1.2) 3 (1.3) 1 (1.0) 0 Asian 5 (1.5) 4 (1.7) 1 (1.0) 2 (3.1) Other 6 (1.8) 5 (2.1) 1 (1.0) 1 (1.6) SDMT Mean ± SD 55.6 ± 13.37 55.2 ± 13.10 56.4 ± 14.02 n/a n/a Median (min, max) 58.0 (0.0, 90.0) 56.0 (16.0, 90.0) 59.0 (0.0, 81.0) n/a MRI normalized brain volume (cm3) Mean ± SD 1511 ± 103 1515 ± 99 1501 ± 112 1589 ± 101 <0.0001 Median (min, max) 1514.0 (1203.0, 1774.0) 1519.0 (1203.0, 1774.0) 1503.0 (1270.0, 1748.0) 1603.0 (1333.0, 1895.0) MRI, magnetic resonance imaging; MS, multiple sclerosis; MS-ON, multiple sclerosis-associated optic neuritis; n/a, not available; SD, standard deviation; SDMT, symbol digit modalities test. N is the total number of subjects in the analysis population (different from the number of subjects with data available). 1 Without previous MS-ON. * All p-values are two-side. For continuous variables, p-value is from two sample t-test comparing the MS and reference groups. For categorical variables, if any of the entries is <10, then p-value is from Fisher's exact test; if all entries are >=10, then p-value is from Chi-square test. Table 2. Baseline disease characteristics of the MS group. Characteristics

All patients with MS

(N = 333)

Patients without MS-ON1

(N = 235)

Patients with MS-ON

(N = 98)

Time since MS diagnosis Years, mean (SD) 7.28 ± 5.75 n/a n/a 1–≤3 years, n (%) 100 (30.0) 69 (29.4) 31 (31.6) >3–≤5 years, n (%) 44 (13.2) 34 (14.5) 10 (10.2) >5 years, n (%) 189 (56.8) 132 (56.2) 57 (58.2) Previous DMT, n (%) Fingolimod 45 (13.5) n/a n/a Natalizumab 60 (18.0) n/a n/a iDMT (interferons or glatiramer acetate) 162 (48.6) n/a n/a DMF or teriflunomide 14 (4.2) n/a n/a Other 5 (1.5) n/a n/a No treatment 61 (18.3) n/a n/a EDSS score Mean ± SD 1.9 ± 1.43 1.9 ± 1.42 2.1 ± 1.44 Median (min, max) 2.0 (0.0, 7.0) 1.5 (0.0, 7.0) 2.0 (0.0, 6.0) MRI PD/T2 lesion volume (mm3) Mean ± SD 3663 ± 5095 3523 ± 4659 4037 ± 6036 Median (min, max) 1922.5 (0.0, 48514.0) 1880.0 (0.0, 34627.0) 2206.0 (66.0, 48514.0) DMF, dimethyl fumarate; iDMT, injectable disease modifying therapy; EDSS, expanded disability status scale; max, maximum; min, minimum; MRI, magnetic resonance imaging; MS, multiple sclerosis; MS-ON, multiple sclerosis-associated optic neuritis; n/a, not available; PD, proton density; SD, standard deviation. N is the total number of subjects in the analysis population (different from the number of subjects with data available). 1 Without previous MS-ON. Table 3. Ocular baseline characteristics. Number of patients MS group Reference group Group difference All patients with MS Patients without MS-ON1 Patients with MS-ON Healthy participants p-value* N = 333 N = 235 N = 98 N = 64 Retinal thickness (μm) pRNFL 89.0 ± 15.30 91.7 ± 14.21 82.5 ± 15.85 100.2 ± 9.23 <0.0001 mGCIPL 79.3 ± 14.22 82.2 ± 12.91 72.5 ± 14.94 91.9 ± 8.42 <0.0001 Visual acuity (ETDRS letters) High contrast 56.1 ± 9.49 57.0 ± 8.25 53.8 ± 11.69 59.6 ± 5.42 <0.0001 Low contrast 27.8 ± 11.65 29.8 ± 10.44 22.9 ± 12.95 34.3 ± 7.28 <0.0001 Values are mean ± SD. N is the total number of subjects in the analysis population. ETDRS, early treatment diabetic retinopathy study; mGCIPL, combined macular ganglion cell-inner plexiform layer; MS, multiple sclerosis; MS-ON; multiple sclerosis-associated optic neuritis; ON, optic neuritis; pRNFL, peripapillary retinal nerve fiber layer; SD, standard deviation. 1Without previous MS-ON * All p-values are two-sided from two sample t-test comparing the MS and reference groups. p-values for the subgroup comparison MS-ON versus non-MS-ON were also calculated and were all <0.0001. Changes in inner retinal layers thicknesses

The decrease in pRNFL thickness from baseline (unadjusted for differences in baseline thickness at the time of enrollment) over time was greater for patients with RRMS compared with the reference group (Fig. 2). The mean (standard deviation [SD]) average pRNFL thickness decrease from baseline at Month 36 was 1.61 (4.60) µm in patients with RRMS compared with 0.09 (2.98) µm in reference participants. Decreases in pRNFL thickness over time were similar in the non-MS-ON and MS-ON subgroups.

Mean change from baseline in pRNFL thickness over time by ON status. The change from baseline has been overlaid with the known physiological variation between measurements and is shown for months 1 to 36 1(Balk et al. Invest Ophthalmol Vis Sci 2012;53:1251–1257). SE, standard error; M, month; MS, multiple sclerosis; MS-ON; multiple sclerosis-associated optic neuritis; ON, optic neuritis; pRNFL, peripapillary retinal nerve fiber layer; n, number of eyes observed per time point.

After adjusting for potential imbalance between groups (i.e., MS vs. reference subjects) in baseline covariates including age, gender, and baseline RNFL score, the least square mean of change from baseline in RNFL at Month 36 was −1.74 with 95% CI (−2.04, −1.45) in the MS group versus 0.12 with 95% CI (−0.51, 0.74) in the reference group (Table 4). The LS mean difference (95% CI) between patients with RRMS and reference participants for change in average pRNFL thickness at Month 36 was −1.86 (−2.54, −1.17) μm (p < 0.0001; Table 4); this thus represents the change from baseline for the MS group after adjusting for changes (including age-related change) in healthy subjects. The difference between the two groups for change in average pRNFL thickness were statistically significant from Month 18 onward: Month 18: −1.13 (−1.75, −0.52) μm (p = 0.0003); Month 24: −1.11 (−1.76, −0.46) μm (p = 0.0008); and Month 30: −1.16 (−1.82, −0.49) μm (p = 0.0007). At Month 6 and Month 12, the group differences were −0.10 (−0.65, 0.46) μm (p = 0.7289), and −0.55 (−1.13, 0.03) μm (p = 0.0621), respectively. The LS mean group differences in change in pRNFL thickness were similar regardless of MS-ON history (Table 4).

Table 4. Change from baseline in pRNFL and mGCIPL thickness and comparison between groups at Month 36.

ON history

Visit group

n

LS mean of change from baseline

(95% CI)

Group difference (95% CI) in change from baseline (Patients with MS – Reference participants, adjusted for baseline) p-value pRNFL thickness (µm) 1 All Patients with MS (N = 333) 284 −1.74 (−2.04, −1.45) −1.86 (−2.54, −1.17) <0.0001 Reference participants (N = 64) 56 0.12 (−0.51, 0.74) Non-MS-ON Patients with MS (N = 235) 200 −1.79 (−2.12, −1.45) −1.88 (−2.59, −1.16) <0.0001 Reference participants (N = 64) 56 0.09 (−0.55, 0.72) MS-ON Patients with MS (N = 98) 84 −1.61 (−2.25, −0.96) −1.80 (−2.71, −0.88) 0.0001 Reference participants (N = 64) 56 0.19 (−0.41, 0.79) mGCIPL thickness (µm) 2 All Patients with MS (N = 333) 285 −0.54 (−0.85, −0.24) −2.03 (−2.78, −1.28) <0.0001 Reference participants (N = 64) 55 1.49 (0.80, 2.18) Non-MS-ON Patients with MS (N = 235) 201 −0.47 (−0.83, −0.12) −1.77 (−2.51, −1.03) <0.0001 Reference participants (N = 64) 55 1.30 (0.63, 1.97) MS-ON Patients with MS (N = 98) 84 −1.58 (−2.32, −0.85) −3.89 (−5.17, −2.61) <0.0001 Reference participants (N = 64) 55 2.31 (1.37, 3.25) N is the total number of participants in the analysis population, n is the number of participants with data available. The average pRNFL thickness was the mean of the circular scan subfields 1 to 8. Eye was nested within subject, and both were included as random effects. For participants with a history of ON, the unaffected eye was excluded. The reference participants were included as the reference group in all models. Participants who experienced an adverse event of ON during the study were re-categorized and included in the ON history subgroup. CI, confidence interval; mGCIPL, combined macular ganglion cell and inner plexiform layers; LS, least squares; MS, multiple sclerosis; MS-ON, MS- associated optic neuritis; pRNFL, peripapillary retinal nerve fiber layer. 1 Mixed model for repeated measures (MMRM) included terms for group, visit, age, gender, and the interaction of group and visit with baseline average pRNFL thickness as a covariate. 2 Mixed model includes terms for group, age, gender, and baseline mGCIPL thickness as a covariate.

The mixed effect model analysis conducted on mGCIPL measurements yielded similar results at Month 36, demonstrating greater thinning of mGCIPL in the MS group compared with control participants over the 3 years. The mean (SD) average mGCIPL thickness at Month 36 decreased from baseline by 0.39 (3.096) μm in patients with MS and increased by 1.02 (5.063) μm in reference participants. The LS mean difference (95% CI) between patients with RRMS and reference participants for change in mGCIPL thickness at Month 36 (i.e., rate of thinning corrected with healthy age-related attrition) was −2.03 (−2.78, −1.28) (p < 0.0001; Table 4). Results were similar regardless of MS-ON history.

Comparison of change in papillomacular bundle, temporal quadrant pRNFL thickness, and temporal field 6 pRNFL thickness between groups at Month 36 are outlined in Table 5 (see Fig. S1 for the description of the fields). While there were no significant differences between groups for temporal field 6 RNFL thickness, there was a significant decrease in the papillomacular bundle and temporal