Can the degree of meningeal inflammation and cortical pathology be used to stratify early progressive MS patients? R. Magliozzi, M. Calabrese, C. Cruciani, S. Rossi, M. Castellaro, C. Romualdi, R. James, A. Gajofatto, MD Benedetti, A. Bertoldo, M. Pitteri, F. Facchiano, S.Monaco, O. Howell, R. Reynolds.

The changing clinical course of multiple sclerosis is a matter of gray matter: cortical lesion load associates with progression of disability in multiple sclerosis Calabrese M et al., Brain. 2012; Geurts et al., Lancet Neurol 2012; Calabrese M et al., Ann Neurol The combination of grey and white matter parameters gives a more comprehensive view of multiple sclerosis pathology Better understanding of the progressive phase of the disease

MOG * * * * The immune response becomes compartmentalised as MS progresses Grey matter atrophy and the accumulation of cortical lesions plays a major role in the progression of clinical deficit in MS and correlates with the rate of progression CD20 Subpial cortical demyelinated lesion Meningeal infiltrates MOG Serafini et al., JNEN 2004; Magliozzi et al., Brain 2007

Compartmentalized meningeal inflammation is associated with increased cortical demyelination Organised meningeal inflammation and increased cortical demyelination are associated with early neurological disability and aggressive disease course in a large proportion of secondary progressive MS cases 32 yrs 26 yrs 21 yrs31 yrs 62y 48y 51y 36y 35 yrs 44 yrs Howell et al, 2011 Brain Compartmentalized meningeal inflammation is associated with more severe disease outcome

TNF Meninges CSF Gardner, Magliozzi et al., Brain 2013 Meningeal inflammation is associated with increase in inflammatory gene expression in the meninges and protein expression in the CSF of post-mortem SPMS

Hypothesis: Can the degree of meningeal inflammation and cortical pathology be used to stratify a subgroup of MS patients with rapid progression? Specific Aim: To find a combination of CSF biomarkers, neuropathological and early neuroimaging correlates of disease progression in order to predict onset and rate of MS progression. Main tasks Combined analysis of patient’s CSF and MRI profiling to be correlated with cortical damage as visualized by neuropathology (post-mortem) and by MRI

10 F+ SPMS RNA CSF Inflammatory protein profiling 10 F- SPMS

Significant changes in meningeal gene expression * -Significant increases in TLO/lymphoid stimulating chemokines (CXCL13, CXCL10, CXCL9) were seen in the meninges of F+ MS cases -Significant increases in pro-inflammatory cytokine gene expression are seen in the meninges of F+ MS cases (CXCL20, CCL22, IL22, IL2) -Significant increase in regulatory molecules (IL17a and IL9) in the meninges of F- MS cases

Inflammatory molecules over-expressed in the CSF of post-mortem SPMS cases Highly intrathecal pro-inflammatory profile, including several molecules linked to B-cell activity, in F+ MS cases

3D Double Inversion Recovery MRI profiling at disease onset - On the basis of grey matter lesion (GML blue arrows ) number and volume and of the global and regional cortical thickness, the selected MS patients have been classified as group MS1 or MS2 group - No difference in the of white matter lesion WML (yellow arrows) <10 GML< MS1: 15 MS Low GML load MS2: 21 MS High GML load Combined analysis of CSF and MRI profiling of 36 MS patients

Cluster analysis of the levels of 69 inflammatory mediators examined in the CSF of MS patients at disease onset (Immuno-assay Bio-Plex System) IL-16 CXCL12 LIGTH CCL19 MIF Chitinase 3 -like 1 CXCL10 CCL21 CXCL13 GM-CSF TNF-alpha sTNF-R1 sTNF-R2 TWEAK APRIL IL-6 IL-10 sIL-6 alpfa IL-12 IL-19 sCD163 IL-22 IL-26 IL-27(p28) IL-35 MMP-1 MMP-2 Osteocalcin Osteopontin (OPN) Pentraxin -3 TSLP CCL2 CCL8 CCL15 CCL23 CXCL16 CCL20 CX3CL1 CXCL1 IL-8 IFN-gamma CCL11 CCL24 CCL1 BAFF sCD30 CXCL9 IFN-lambda1 gp130 IL-20 IL-11 CXCL11 CCL26 IL-12(p40) IL-32 IL-34 IL-1beta IL-2 IL-4 CXCL5 CCL25 INF-alfa 2 INF-beta IFNlambda2 CCL7 CCL13 CCL22 CXCL2 CCL3 MS1 MS2 MS patients with different cortical lesion load are characterized by specific correspondent CSF inflammatory profile since early stage of the disease

Significant increase in the levels of anti-inflammatory molecules in the CSF of MS patients with lower cortical lesion load (MS1 group) Over-expressed molecules in MS1 compared to MS2; p value:* < 0,05; **<0,01; *<0,001 (Mann-Whitney). *** * ** * Th2 regulatory chemokines Anti-viral immune response Anti-inflammatory and regulatory intrathecal inflammatory profile is associated to lower cortical lesion load

Significant increase in the levels of inflammatory molecules in the CSF of MS patients with higher cortical lesion load (MS2 group) *** * * Lymphoid chemokines **** ** **** Plasmablast differentiation Intrathecal inflammatory profile linked to B-cell activation and lymphoid neogenesis associated in MS patients with higher cortical lesion load

Overexpression of other TNF family molecules in the CSF of MS patients with higher cortical lesion load (MS2 group) ** *** ** *** **** Pro-inflammatory mediators Highly intrathecal pro-inflammatory profile is associated with higher cortical lesion load

Conclusions Meningeal infiltrates may represent the main source of intrathecal inflammatory activity mediating the gradient of cortical tissue injury since early disease stages and in progressive MS. Molecular CSF and cortical MRI profiling of MS patients: useful tools for disease prognosis, monitoring and identify early treatments and combination therapy CXCL13, CXCL12, CXCL19, CXCL21 IL6, IL10 TNF, APRIL, BAFF, LIGHT IFNG GM-CSF MMP2 Pentraxin-III - On-going more detailed longitudinal assessment - Validation study on second cohort of MS patients

M. Calabrese S. Monaco M. Castellaro M. Pitteri G. Farina A. Gajofatto A.Bertoldo B.MD Benedetti Dept. of Neurological and Movement Sciences, University of Verona, Italy C. Romualdi Biotechnology Centre and Department of Biology, University of Padova F. Facchiano S. Rossi Istituto superiore di Sanità, Rome, Italy ACKNOWLEDGEMENTS R. Reynolds Carolina Cruciani Rachel Janes Centre for Neuroinflammation and Neurodegeneration, Division of Brain Sciences, Imperial College London, UK O. Howell School of Life Sciences, Swansea University, Swansea, United Kingdom