Effect of disease-modifying drugs on cortical lesions and atrophy in relapsing remitting multiple sclerosis

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1 Research Paper Effect of disease-modifying drugs on cortical lesions and atrophy in relapsing remitting multiple sclerosis Multiple Sclerosis Journal 0(00) 1 7! The Author(s) 2011 Reprints and permissions: sagepub.co.uk/journalspermissions.nav DOI: / msj.sagepub.com M Calabrese, V Bernardi, M Atzori, I Mattisi, A Favaretto, F Rinaldi, P Perini and P Gallo Abstract Objective: To measure the effects of disease-modifying drugs (DMDs) on the development of cortical lesions (CL) and cortical atrophy in patients with relapsing remitting multiple sclerosis (RRMS). Methods: RRMS patients (n ¼ 165) were randomized to subcutaneous (sc) interferon (IFN) beta-1a (44 mcg three times weekly), intramuscular (im) IFN beta-1a (30 mcg weekly) or glatiramer acetate (GA; 20 mg daily). The reference population comprised 50 untreated patients. Clinical and MRI examinations were performed at baseline, 12 months and 24 months. Results: One hundred and forty-one treated patients completed the study. After 12 months, 37/50 (74%) of untreated patients developed 1 new CL (mean 1.6), compared with 30/47 (64%) of im IFN beta-1a-treated patients (mean 1.2, p ¼ 0.021), 24/48 (50%) of GA-treated patients (mean 0.8, p ¼ 0.001) and 12/46 (26%) of sc IFN beta-1a-treated patients (mean 0.4, p < 0.001). After 24 months, 1 new CL was observed in 41/50 (82%) of untreated (mean 3.0), 34/47 (72%) of im IFN beta-1a-treated (mean 1.6, p < 0.001), 30/48 (62%) of GA-treated (mean 1.3, p < 0.001) and 24/46 (52%) of sc IFN beta-1a-treated patients (mean 0.8, p < 0.001). Mean grey matter fraction decrease in DMD-treated patients at 24 months ranged from 0.7 to 0.8 versus 1.0 in untreated patients (p ¼ 0.023). Conclusions: Disease-modifying drugs significantly decreased new CL development and cortical atrophy progression compared with untreated patients, with faster and more pronounced effects seen with sc IFN beta-1a than with im IFN beta-1a or GA. Keywords cortical lesions, disease-modifying drugs, double-inversion recovery, grey matter atrophy, magnetic resonance imaging, multiple sclerosis Date received: 16th September 2010; revised: 6th November 2010; 25th November 2010; accepted: 26th November 2010 Introduction The disease-modifying drugs (DMDs) interferon (IFN) beta and glatiramer acetate (GA) have been shown to reduce the frequency of relapses and the accumulation of white matter (WM) inflammation in patients with relapsing remitting multiple sclerosis (RRMS) in numerous studies, 1 6 particularly when therapy is initiated early in the disease course Nevertheless, abnormalities in WM alone cannot explain the full array of clinical symptoms in MS. 12 Histopathological studies have shown that grey matter (GM) lesions occur frequently in MS, in both the cortex and deep GM Double-inversion recovery (DIR) MRI has allowed cortical inflammatory lesions (CLs) to be detected The Multiple Sclerosis Centre of the Veneto Region, Department of Neurology, University Hospital of Padova, Padova, Italy. Corresponding author: Massimiliano Calabrese, Multiple Sclerosis Centre, University Hospital of Padova, via Giustiniani 5, Padova, Italy calabresem@hotmail.it

2 2 Multiple Sclerosis Journal 0(00) in vivo, 17 and recent data suggest that CLs may have a major role in determining disability in MS In large cohorts of patients with MS we have previously detected CLs in the early stages of disease, 17 including at clinical onset in patients with no WM lesions identified by conventional MRI sequences. 19 Moreover, CLs were associated with critical symptoms such as epilepsy, 18 inversely associated with a benign disease course 20 and correlated with cortical atrophy in primary progressive MS 21 and with cognitive decline in RRMS. 22 To our knowledge, no previous studies have examined the effect of DMDs on CL development in RRMS. The objective of this study was to assess the effects of DMDs compared with no treatment on new CL development and cortical atrophy progression in patients with RRMS. The DMDs studied were subcutaneous (sc) IFN beta-1a (Rebif Õ, Merck Serono, Geneva, Switzerland), intramuscular (im) IFN beta-1a (Avonex Õ, Biogen Idec, Cambridge, MA, USA) and GA (Copaxone Õ, Teva Pharmaceutical Industries, Petach Tikva, Israel). Materials and methods Patients and treatment From 1 January 2007 to 30 June 2008, 165 RRMS patients were enrolled in this 2-year, prospective, single-centre, phase 4, randomized pilot study. Inclusion criteria were diagnosis of RRMS according to the McDonald/Polman diagnostic criteria for MS, 24,25 age range years and an Expanded Disability Status Scale (EDSS) score of 5.0. Patients were assigned randomly (1 : 1 : 1) to receive sc IFN beta-1a, 44 mcg three times weekly; im IFN beta-1a, 30 mcg once weekly; or sc GA, 20 mcg once daily. The random allocation sequence was computer generated. Fifty patients constituted the reference population, and comprised DMD-untreated patients with RRMS. Reasons for lack of treatment included both physicianand patient-driven factors, such as a relatively benign clinical course (low relapse rate and low WM lesion load on MRI scan), needle phobia, female patients who were pregnant or trying for pregnancy, and interruption of previous therapy attempts with DMDs owing to persistent adverse effects or poor treatment adherence (in this case, a wash-out period of 3 months from therapy interruption was required before study entry). Patients previously treated with immunosuppressive drugs were excluded from the study. Clinical disability (EDSS) and MRI scans were performed at baseline, and after 12 and 24 months. The local Ethics Committee approved the study, and written informed consent was obtained from all patients prior to entering the study. Assessments Image acquisition Images were acquired using a 1.5 T scanner (Achieva, Philips Medical Systems, Best, The Netherlands) with 33 mt/m power gradient and a 16-channel head coil. No major hardware upgrades of the scanner occurred during the study, and bimonthly quality-assurance sessions took place to guarantee measurement stability. The following images were acquired for each subject: (1) DIR: repetition time (TR) ¼ 15,631 ms; echo time (TE) ¼ 25 ms; inversion time (TI) ¼ 3400 ms; delay ¼ 325 ms; echo train length (ETL) ¼ 17; 50 contiguous axial slices with thickness ¼ 3 mm; matrix size ¼ ; and field of view (FOV) ¼ mm 2 ; (2) fast fluid attenuated inversion recovery (FLAIR): TR ¼ 10,000 ms; TE ¼ 120 ms; TI ¼ 2500 ms; ETL ¼ 23; 50 contiguous axial slices with thickness ¼ 3.0 mm; matrix size ¼ ; and FOV ¼ mm 2 ; (3) three-dimensional (3D) fast field echo (FFE) sequence: 120 contiguous axial slices with the offcentre positioned on zero, TR ¼ 25 ms; TE ¼ 4.6 ms; flip angle ¼ 30 ; slice thickness ¼ 1.2 mm; matrix ¼ ; and FOV ¼ mm 2 ; and (4) postcontrast T1-weighted spin echo: TR ¼ 618 ms; TE ¼ 10 ms; flip angle 90 ; 20 contiguous axial slices with thickness ¼ 5.5 mm; matrix size ¼ ; and FOV ¼ mm 2 ; acquired 5 min after intravenous administration of gadolinium (Gd)-EDTA acid (0.1 mmol/kg). Subjects were carefully repositioned according to published guidelines for serial MRI studies of MS. 26 Image analysis All imaging was carried out at a single imaging centre, and all images were assessed by the consensus of two experienced observers who were blinded to the patients identity and treatment. The number of CLs, the T2 hyperintense WM lesion volume (T2-WM-LV), the GM fraction (GMF) and the percentage of GM volume change between baseline and 24-month scans were calculated as previously described. 27 The number of Gd-enhancing lesions was measured on post-contrast Gd-enhanced T1-weighted images. Statistical analysis Between-group differences were assessed using analysis of variance, followed by the Tukey test 28 to account for multiple comparisons. Pearson chi-square was applied to test the effect of DMDs on the percentage of patients that developed new CLs compared with untreated patients.

3 Calabrese et al. 3 Results Patients A total of 215 patients were enrolled in the study. Of these, 165 were randomized to DMD treatment, with 55 patients in each treated group. One hundred and forty-one of the randomized patients completed the 2-year follow-up (46 in the sc IFN beta-1a group, 47 in the im IFN beta-1a group and 48 in the GA group); 24 (14.5%) randomized patients were lost to follow-up. Therefore, evaluable end-of-study data were available for the 141 treated patients who completed the study and for all 50 untreated patients. At baseline, the untreated and DMD-treated (pooled treatment groups) patients differed in EDSS score (p ¼ 0.001) and T2-WM-LV (p < 0.001) (Table 1): both variables were higher in DMD-treated patients. No differences were observed relative to disease duration or sex, although untreated patients were slightly older than DMD-treated patients (p ¼ 0.004). Effect of DMD treatment on CL and WM lesion development Therapy with DMD was highly effective not only in reducing WM pathology (new T2-WM lesions, p < 0.001; new T1 Gd-enhancing lesions, p < 0.001, Table 2), but also in preventing the appearance of new CLs. At the end of 12 months, 74% (37/50) of untreated patients had new CLs compared with 45% (63/141) of DMD-treated patients (p < 0.001; Figure 1). At the end of 24 months of therapy, 64% (90/141) of DMD-treated patients had new CLs compared with 82% (41/50) of untreated patients (p < 0.001; Figure 1). The number of new CLs in the treated group was also significantly lower than in the untreated group, at both 12 and 24 months (p < 0.001; Table 2). Effect of individual DMDs on CL lesion development After 12 months, at least one CL was observed in 74% (37/50) of DMD-untreated patients (mean [SD] number of new CLs: 1.6 [1.1], range 0 4), in 64% (30/47, p ¼ 0.27) of im IFN beta-1a-treated patients (mean [SD] number of new CLs: 1.2 [1.1], range 0 4, p ¼ 0.021); in 50% (24/48, p ¼ 0.014) of GAtreated patients (mean [SD] number of new CLs: 0.8 [1.0], range 0 3, p ¼ 0.001); and in 26% (12/46, p < 0.001) of sc IFN beta-1a-treated patients (mean [SD] number of new CLs: 0.4 [0.7], range 0 3, p < 0.001). The effect of sc IFN beta-1a in preventing the development of new CLs at 12 months was higher compared with both im IFN beta-1a (p < 0.001) and GA (p ¼ 0.07). After 24 months of therapy, new CLs were observed in 82% (41/50) of DMD-untreated patients (mean [SD]: 3.0 [1.6], range 1 8); 72% (34/47, p ¼ 0.25) of Table 1. Baseline demographic, clinical and MRI characteristics of DMD-treated and -untreated patients Characteristic sc IFN beta-1a im IFN beta-1a GA DMD-treated DMD-untreated N Women, n (%) 32 (69.5) 32 (68.0) 35 (72.9) 99 (70.2) 36 (72.0) p ¼ a Age (years) 35.9 (9.1; 18 55) 34.8 (9.6; 18 52) 38.9 (10.2; 21 50) 36.5 (9.9; 18 55) 39.6 (11.8; 18 55) p ¼ a Disease duration (years) 5.7 (4.9; 0 9) 5.3 (5.1; 0 8) 5.5 (6.1; 0 9) 5.6 (2.4; 0 9) 6.0 (4.8; 0 8) p ¼ a EDSS score 1.9 (1.0; ) 1.9 (0.8; ) 2.1 (1.1; ) 2.0 (1.1; ) 1.3 (0.9; 0 2.0) p ¼ a ARR 1.2 (0.6; 0 3) 1.2 (0.7; 0 4) 1.3 (0.7; 0 3) 1.2 (0.7; 0 4) 1.1 (0.8; 0 4) p ¼ Number of CLs 3.4 (4.0; 0 18) 3.5 (3.2; 0 15) 3.5 (3.6; 0 18) 3.5 (3.9; 0 18) 3.5 (3.8; 0 15) p ¼ T2-WM-LV (cm 3 ) 9.2 (9.0; ) 8.1 (7.2; ) 8.9 (10.1; ) 8.7 (12.1; ) 3.3 (6.2; ) p < a Grey matter fraction (%) 37.2 (2.9; ) 37.1 (3.0; ) 37.4 (2.8; ) 37.3 (2.9; ) 37.0 (2.6; ) p ¼ a Data are reported as mean (standard deviation; range) unless indicated otherwise. a p-value versus untreated patient. ARR: annualized relapse ate, CL: cortical inflammatory lesion, DMD: disease-modifying drug, EDSS: Expanded Disability Status Scale, GA: glatiramer acetate, IFN: interferon, im: intramuscular, sc: subcutaneous, WM-LV: white matter lesion volume.

4 4 Multiple Sclerosis Journal 0(00) Table 2. Main clinical and MRI changes during the 2-year follow-up sc IFN beta-1a im IFN beta-1a GA DMD-treated DMD-untreated GMF decrease at 24 months (%) 0.8 (0.4; ) 0.8 (0.6; ) 0.8 (0.5; ) 0.8 (0.6; ) 1.0 (0.5; ) p ¼ NS p ¼ NS p ¼ NS p ¼ ARR 0.4 (0.6; 0 3) 0.5 (0.6; 0 3) 0.5 (0.4; 0 3) 0.5 (0.6; 0 3) 1.3 (0.7; 0 4) p < p < p < p < EDSS change 0.2 (0.5; 0 2.5) 0.2 (0.4; 0 2.0) 0.3 (0.5; 0 2.0) 0.3 (0.5; 0 2.5) 0.5 (0.6; 0 3.0) p ¼ NS p ¼ NS p ¼ NS p ¼ NS New CL at month (0.7; 0 3) 1.2 (1.1; 0 4) 0.8 (1.0; 0 3) 0.8 (1.1; 0 4) 1.6 (1.1; 0 4) p < p ¼ 0.04 p < p < New CL at month (0.8; 1 4) 1.6 (1.1; 1 6) 1.3 (1.1; 1 5) 1.2 (1.1; 1 6) 3.0 (1.6; 1 8) p < p < 0.01 p < p < New T2 WM lesion at month (1.0; 0 4) 1.3 (1.1; 0 5) 1.2 (1.0; 0 4) 1.2 (1.0; 0 5) 2.1 (1.9; 1 4) p < p < p < p < New Gd þ lesion at month (1.0; 0 3) 0.9 (0.9; 0 3) 1.1 (1.0; 0 2) 1.0 (1; 0 3) 1.9 (1.0; 0 4) p < p < p < p < p < Data are reported as mean (SD; range) and p-values are versus untreated patients. ARR: annualized relapse rate, CL: cortical inflammatory lesion, DMD: disease-modifying drug, EDSS: Expanded Disability Status Scale, GA: glatiramer acetate, GMF: grey matter fraction, IFN: interferon, im: intramuscular, NS: not significant, sc: subcutaneous, SD: standard deviation, WM: white matter. Figure 1. Proportion of patients with new cortical lesions at 12 and 24 months. DMD: disease-modifying drug, GA: glatiramer acetate, IFN: interferon, im: intramuscular, sc: subcutaneous. im IFN beta-1a-treated patients (mean [SD]: 1.6 [1.2], range 1 6, p < 0.001); 62% (30/48, p ¼ 0.03) of GA-treated patients (mean [SD]: 1.3 [1.1], range 1 5, p < 0.001); and 52% (24/46, p ¼ 0.002) of sc IFN beta-1a-treated patients (mean [SD]: 0.8 [0.7], range 1 4, p < 0.001). At month 24, the effect of sc IFN beta-1a in preventing the development of new CLs was higher compared with both im IFN beta-1a (p ¼ 0.034) and GA (p ¼ 0.05). Effect of DMD therapy on cortical atrophy progression Baseline GMF was similar across all patient groups (Table 1). At month 24, the mean percentage (SD) decrease in GMF was 1.0% (0.5%) in untreated patients, 0.7% (0.3%) for sc IFN beta-1a, 0.8% (0.6%) for im IFN beta-1a and 0.8% (0.5%) for GA (p ¼ for all DMD-treated vs untreated patients;

5 Calabrese et al. 5 Table 2). GMF decrease did not differ significantly among the treatment groups. Effect of DMD therapy on clinical efficacy outcomes Untreated patients had a mean annualized relapse rate (ARR) of 1.3 (SD: 0.7, range 0 4) as assessed over the 2-year study, a figure very similar to that observed at baseline in the RRMS patients included in the most recent Phase III clinical trials on disease-modifying agents for MS, such as fingolimod, cladribine and daclizumab The ARR was 67% lower in patients treated with sc IFN beta-1a (0.4) and 63% lower (0.5) in patients treated with im IFN beta-1a or GA (p < for each treated group vs untreated patients). Mean EDSS score increased by 0.5 points in untreated patients and by 0.3 points in DMD-treated patients (p ¼ 0.1). Changes in EDSS score did not differ significantly among the treatment groups (p-values ranging between and 0.759). Discussion This longitudinal observational study shows for the first time, to our knowledge, that DMD therapy is highly effective at preventing the accumulation of demyelinating lesions in the cortex. Indeed, DMD treatment was found to prevent the development of new CLs and reduce the progression of cortical atrophy during a follow-up of 2 years in patients aged 18 to 55 years with RRMS. Furthermore, after 1 year of therapy, the effect of sc IFN beta-1a was significantly more pronounced compared with that of either im IFN beta-1a or GA, suggesting that high-dose, high-frequency IFN beta-1a therapy may control the cortical inflammatory process more rapidly in patients with MS. At 24 months, the treatment effect was still in favour of high-dose, highfrequency sc IFN beta-1a compared with the other two DMDs, although the difference among treatments was less significant. Our findings suggest that sc IFN beta-1a has a faster onset of immunomodulatory action, so that it exerts a greater effect on CLs during year 1 than in year 2, whereas the other DMDs have a slower onset of effect and thus exert a greater effect in year 2. However, the modest significance observed at year 2 may be explained, at least partly, by the small number of patients in each of the three treatment arms. Indeed, a power calculation indicated that at least 37 patients were needed in each arm to detect, over 2 years, a significant difference between sc IFN beta-1a and im IFN beta-1a or GA. In line with previous studies, all treatments were effective in reducing WM inflammation in terms of both T2 WM lesion accumulation and the appearance of new T1 Gd-enhancing lesions. Several longitudinal structural imaging studies have shown robust evidence of progressive brain atrophy in patients with MS, especially in the fronto-temporal cortices. In MS, brain atrophy occurs significantly faster than in healthy subjects 38,39 and is viewed as being the result of extensive demyelination and axonal loss in both WM and GM. 40 Previous studies have indicated that 3-year IFN beta-1a treatment significantly slowed progression of whole-brain and GM atrophy in patients with RRMS, 41 although this effect was not significant after only 1 year of treatment. 42 Reduced whole-brain atrophy in IFN beta-1a-treated patients has also been shown in a larger sample of patients with RRMS. 42 Our study is in line with these observations, since 2 years of DMD therapy was shown to have significantly slowed progression of cortical atrophy compared with that in untreated patients. Inasmuch as the accumulation of CL was found to predict cortical atrophy, 30,43 it seems likely that the effect of DMDs in reducing cortical atrophy may, at least partly, be the consequence of the DMD-induced reduction of CL accumulation. Recent studies indicate a strong relationship between cortical pathology and some clinical aspects of MS, especially cognitive impairment. 22,23,43 In this study, however, the small number of patients included, the relatively short period of observation and the intrinsic limits of Rao s Brief Repeatable Battery (the low test retest reliability of which is not optimal for monitoring drug efficacy in clinical trials) did not permit the analysis of the effect of DMD on cognition. We are aware that our study has some limitations. First, it is reasonable to assume that a portion of the patients who had not been given DMD treatment may have had a relatively benign clinical course, and therefore lower EDSS scores, smaller mean numbers of CLs and lower volumes of T2 WM lesions at baseline than other patients enrolled in this study. However, if the control group had a more favourable disease course, the development of new CLs in this group during the study strongly reinforces the potential for DMDs to reduce cortical pathology in MS. Secondly, it should be noted that all treatment groups in this study had a relatively small sample size. Overall, our study confirms the inflammatory origin of CLs and strongly supports the importance of immunomodulatory treatment for MS. We have shown that treatment with DMDs can significantly reduce the accumulation of CLs in RRMS, with a trend in favour of high-dose, high-frequency IFN beta-1a treatment. Since literature data suggest that CLs constitute a relevant aspect of MS, 17,19,21,27,43 the impact of DMDs in preventing their accumulation should be further investigated in multicentre clinical trials in a larger number of patients.

6 6 Multiple Sclerosis Journal 0(00) Acknowledgements The authors thank Clare McNulty of, and Reza Sayeed on behalf of, Caudex Medical, Oxford, UK (supported by Merck Serono S.A. Geneva, Switzerland, an affiliate of Merck KGaA, Darmstadt, Germany), for their assistance in editing the manuscript for non-intellectual content. Funding This study was funded by an independent medical grant from Merck Serono S.A. Geneva, Switzerland, an affiliate of Merck KGaA, Darmstadt, Germany. Conflict of interest M Calabrese has received consultancy and speaking honoraria from Biogen-Elan/Dompe Italy, Merck Serono and sanofi-aventis. P Perini has received consultancy and speaking honoraria from Biogen-Elan/Dompe Italy and sanofi-aventis. P Gallo has received research support from the Veneto Region Government - Ricerche Regionali Finalizzate, the University of Padova, Bayer-Schering Pharma, Biogen- Elan/Dompe Italy, Merck Serono and sanofi-aventis, and speaking honoraria from Bayer-Schering Pharma, Biogen- Elan/Dompe Italy, Merck Serono, sanofi-aventis and Novartis. V Bernardi, M Atzori, I Mattisi, A Favaretto and F Rinaldi have nothing to disclose. References 1. Goodin DS, Frohman EM, Garmany Jr GP, Halper J, Likosky WH, Lublin FD, et al. Disease modifying therapies in multiple sclerosis: report of the Therapeutics and Technology Assessment Subcommittee of the American Academy of Neurology and the MS Council for Clinical Practice Guidelines. Neurology 2002; 58: IFNB Multiple Sclerosis Study Group, University of British Columbia MS/MRI Analysis Group. Interferon beta-1b in the treatment of multiple sclerosis: Final outcome of the randomized controlled trial. 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