No association between variations in extracranial venous anatomy and clinical outcomes in multiple sclerosis patients over 5 years

This 5-year follow-up is a part of an ongoing single-center, longitudinal, observational, case-control Cardiovascular, Environmental and Genetic risk factor in MS (CEG-MS) study that is recruiting subjects in Buffalo, NY [20]. The study was approved by the local Institutional Review Board of the University of Buffalo, USA and University of Naples, Italy, and written informed consent was obtained from each participant. The inclusion criteria for this follow-up CEG sub-study of variations in extracranial venous anatomy and clinical outcomes are: a) subjects originally enrolled in the CEG-MS study in years 2009 through 2013 [5, 20], b) age 18–75 years at baseline, and c) being MS patients or HI with unknown history of neurological disease. Exclusion criteria are: a) presence of relapse and steroid treatment within 30 days preceding their enrollment in the study (for MS patients), b) pre-existing medical conditions known to be associated with cerebral pathology (cerebrovascular disease, positive history of alcohol abuse), c) evidence of brain ischemic or hemorragic infarcts, or space-occupying lesions on MRI exam performed within 30 days of physical/neurologic examination with the standardized study protocol, and d) pregnancy. In total, 128 consecutive subjects consisting of 90 MS patients (52 with relapsing-remitting, RR and 38 with secondary-progressive, SP disease subtype) and 38 HI with unknown history of neurological disease were enrolled in this CEG-MS sub-study by November 2016, when the first database lock occurred. Seven MS patients enrolled in the study had undergone percutaneous venous angioplasty at external centers for the treatment of their CCSVI at independent time points unrelated to the current study.

MS was defined using the revised McDonald criteria [21] and all subjects were examined with physical and neurological examination by experienced neurologists, who were blinded to the results of the DS examination. Expanded Disability Severity Scale (EDSS) score was obtained for each MS patient at baseline and at follow-up. An absolute change in EDSS (ΔEDSS) was calculated, and development of disability progression (DP) was documented as a clinical measure of irreversible neurological disability. DP was defined as an absolute change in EDSS over 5 years with an increase in EDSS of ≥1.5 point if the baseline EDSS was 0, or ≥ 1.0 point if the baseline was between 1.0–5.5, or ≥ 0.5 point if the baseline EDSS was ≥6.0. The medical history and relapses were documented during the 5-year follow-up study assessment.

Doppler assessment was performed at baseline and at follow-up on all study participants using the same echo color Doppler machine, MyLab 25 Gold (Esaote-Biosound, Genoa, Italy) equipped with 2.5 MHz and 7.5–10 MHz transducers, and by a single Doppler sonographer who was blinded to the disease status of the subjects. The baseline blinding was achieved, by instructing subjects not to reveal their disease status during the examination, including patients with no disability or walking difficulties to ensure blinding between non-disabled MS patients and HIs [5]. At the follow-up, the blinding was achieved by positioning subjects supine in a tilt chair by a non-blinded personnel, draping from neck down and removing any assistive device used by the patients from the room before the Doppler sonographer entered, to avoid any visual cues of the presence of disease. The reproducibility of our approach was reported previously [5].

Detailed description of the baseline DS assessment of the extracranial (IJV and vertebral veins, VV) and intracranial venous system was previously reported [5]. Briefly, each subject was examined on a tilt chair in supine (0°) and sitting position (90°) allowing a minimum of 2 min post positional change for vascular flow redistribution prior to DS assessment. At the follow-up, the subjects were also examined on a tilt chair in supine and sitting position. All baseline and follow-up exams were examined in a blinded manner by a certified centralized reader (MM) at the University of Naples, Italy. The detection of extracranial venous variations in extracranial venous anatomy focused on the detection of 5 anomalous venous hemodynamic (VH) criteria [5], with presence of ≥2 VH criteria indicating the fulfillment of CCSVI criteria, according to the International Society for Neurovascular Disease (ISNVD) consensus statement [22]. Due to the technical discrepancy in the assessment of the VH criteria at baseline and at the follow-up, the VH criteria 1 and 2 were not comparable between the two time-points [22].

Statistical analysis was performed using the IBM Statistical Package for the Social Sciences (SPSS version 24.0). Student t-test, chi-square test and Mann Whitney U-test were used testing the differences between study groups, as appropriate. Wilcoxon signed-rank test was used to compare the individual VH criteria within the study groups between the baseline and the follow-up. As there was no significant difference observed in age, gender or disease duration between the study groups (MS vs HIs), chi-square test or Wilcoxon signed-rank test was used to investigate the association between the CCSVI status or the individual venous hemodynamic criteria. The evolution of clinical outcome measures (annualized relapse rate, ARR, ∆EDSS and DP) were analyzed using independent-sample t-test, Mann Whitney U-test and chi-square test as appropriate. A nominal p-value of ≤0.05 was considered statistically significant, and p < 0.1 was considered a trend, using two-tailed test.

The demographics and clinical characteristics of the study subjects are presented in Table 1. The mean age at baseline was 45.1 years in HIs and 47.3 years in MS patients (p = 0.387). The average follow-up duration was similar in MS patients and HIs (p = 0.342). No significant differences were observed in the confounding variables between the study groups in terms of their gender, body mass index (BMI), prevalence of pre-existing comorbidities such as hypertension, hyperlipidemia or diabetes mellitus.

In the MS group, 7 (8%) of the patients had progressed from clinically isolated syndrome (CIS) to relapsing-remitting (RR) MS disease subtype, and 10 (11%) from RRMS to secondary-progressive (SP) MS (Table 1). The DP was observed in 25 (28%) of the patient population (Table 1). Eighty (89%) of MS patients were on disease-modifying treatment at the time of their follow-up visit (Table 1). Median ΔEDSS over the follow-up was 0.5, mean annualized ARR was 0.2 per year, and 61% of the patients remained relapse-free from baseline to follow-up.

Detailed demographic and clinical characteristics of the RRMS and SPMS patients at baseline are provided in the Additional file 1: Table S1. Demographic data was also compared between subjects fulfilling and not-fulfilling ISNVD CCSVI criteria at baseline, to understand the influence of these variables on the outcome measures over the follow-up (Table 2). No significant differences were observed between these groups in terms of age, disease duration, BMI, comorbidities or follow-up duration.

Table 3 shows individual VH criteria and frequency of subjects fulfilling ISNVD CCSVI criteria in HIs and MS patients at baseline, and at the follow-up. At baseline, 52 (58%) of MS patients and 14 (37%) of HIs (p = 0.03) fulfilled ISNVD CCSVI criteria. At the follow-up, the ISNVD CCSVI criteria were fulfilled in 55 (61%) of MS patients and 21 (56%) of HIs (p = 0.486).

At the baseline visit, there was a significantly higher prevalence of positive VH criterion 3 in MS patients compared to HIs (54 vs 14, p = 0.011). At the 5-year follow-up visit, there was a significantly higher prevalence of positive VH criterion 5 in MS patients compared to HIs (13 vs 0, p = 0.018), as none of the HIs presented with negative ΔCSA.

Wilcoxon signed-rank test showed no within group changes in VH criteria positivity or the fulfillment of the ISNVD CCSVI criteria in MS patients and HIs between baseline and the follow-up. A trend toward significance was observed in VH criterion 5 positivity in MS patients from baseline to follow-up (Z = − 1.732; p = 0.083). On further stratification of MS patients, 9 (24%) of SPMS had positive VH criterion 5 at the follow-up, which was significantly greater compared to baseline (Z = − 2.33; p = 0.02).

Table 4 shows evolution of clinical outcomes, according to the baseline fulfillment of the ISNVD CCSVI criteria. At baseline, there was a trend for higher EDSS in those who fulfilled, compared to those who did not fulfill the criteria (p = 0.08), which was not evident at the follow-up (p = 0.11). No differences between those who fulfilled ISNVD CCSVI criteria and those who did not were observed over the follow-up in ΔEDSS, development of DP, number of the relapses between baseline and follow-up, ARR or being relapse-free. No differences in evolution of clinical outcomes was found, according to the baseline fulfillment of the ISNVD CCSVI criteria when RRMS and SPMS patients were considered separately.

Of 7 MS patients who underwent percutaneous angioplasty, 3 (42.9%) were in RRMS and 4 (57.1%) in the SPMS group. Six (86%) of those fulfilled ISNVD CCSVI criteria at baseline and their median EDSS was 4.0. Median time period from the baseline to the date of procedure was 3 months. At their follow-up, 57% patients continued to have fulfilled ISNVD CCSVI criteria. The mean ∆EDSS was 0.2 in RRMS and 0.8 in SPMS patients, respectively over the follow-up. DP was detected in 2 of the 7 patients (33%) and the mean ARR of this subgroup was 0.13.