Increased PK11195-PET Binding in the Normal Appearing White Matter in Clinically Isolated

Original Article

Title

Increased PK11195-PET binding in the normal appearing white matter in clinically isolated syndrome

Paolo Giannetti, MD,1 Marios Politis, MD, PhD,1,2 Paul Su, MSc,1 Federico Turkheimer,3

PhD, Omar Malik, MD, PhD,1 Shiva Keihaninejad, PhD,1 Kit Wu, MD,1 Adam Waldman,

PhD1, Richard Reynolds, PhD,1 Richard Nicholas*, MD, PhD,1 and Paola Piccini*, MD,

PhD1.

* These senior authors contributed equally to this study

1Centre for Neuroinflammation and Neurodegeneration, Faculty of Medicine, Imperial Col- lege London, Hammersmith Campus, Du Cane Road, London, W12 0NN, United Kingdom.

2Neurodegeneration Imaging Group, Department of Clinical Neuroscience, King’s College

London, De Crespigny Park, London, SE5 8AF, United Kingdom.

3Centre for Neuroimaging, Institute of Psychiatry, King's College London, De Crespigny

Park, London, SE5 8AF, United Kingdom.

Correspondence to: Dr. Paolo Giannetti, 4th Floor, Burlington Danes Building,

Hammersmith Campus, Du Cane Road, London, W12 0NN, UK. Tel.: +44-(0)20 7594 6657;

Fax: +44-(0)20 7594 6548; email: [email protected]

1 Running title: PK11195-PET in NAWM in CIS

Number of words in abstract: 295 words

Number of words in main text: 3,294 words

Number of figures: 6

Number of tables: 2

2 Summary

The most accurate predictor of the subsequent development of multiple sclerosis in clinically isolated syndrome is the presence of lesions at MRI. We used in vivo PET with [11C]-(R)-

PK11195, a biomarker of activated microglia, to investigate the normal appearing white matter and grey matter of subjects with clinically isolated syndrome to explore its role in the development of multiple sclerosis. Eighteen clinically isolated syndrome and 8 healthy control subjects were recruited. Baseline assessment included: history, neurological examination, expanded disability status scale, MRI and PK11195-PET scans. All assessments except the PK11195-PET scan were repeated over 2 years. SUPERPK methodology was used to measure the binding potential relative to the non-specific volume, BPND. We show a global increase of normal appearing white matter PK11195 BPND in clinically isolated syndrome subjects compared to healthy controls (p=0.014). Clinically isolated syndrome subjects with

T2 MRI lesions had higher PK11195 BPND in normal appearing white matter (p=0.009) and their normal appearing white matter PK11195 BPND correlated with the expanded disability status scale (p=0.007; r=0.672). At two years those who developed dissemination in space or multiple sclerosis, had higher PK11195 BPND in normal appearing white matter at baseline

(respectively p=0.007 and p=0.048). Central grey matter PK11195 BPND was increased in clinically isolated syndrome subjects compared to healthy controls but no difference was found in cortical grey matter PK11195 BPND. Microglial activation in clinically isolated syndrome normal appearing white matter is diffusely increased compared to healthy controls and is further increased in those who have MRI lesions. Furthermore microglial activation in clinically isolated syndrome normal appearing white matter is also higher in those subjects who developed multiple sclerosis at 2 years. Our finding, if replicated in a larger study, could be of prognostic value and aid early treatment decisions in clinically isolated syndrome.

3 Keywords: multiple sclerosis; clinically isolated syndrome; microglia; normal appearing white matter; PK11195-PET.

Abbreviations: CIS = Clinically Isolated Syndrome; CNS = Central Nervous System; MS =

Multiple Sclerosis; WM = White Matter; NAWM = Normal Appearing White Matter; GM =

Grey Matter; PK11195 = [(11)C](R)-PK11195; HC = Healthy Control; EDSS = Expanded

Disability Status Scale; ROI = Region-Of-Interest; BPND = Binding Potential of the specifically bound radioligand relative to the Non-Displaceable radioligand in tissue; SD =

Standard deviation; MAPER = Multi-Atlas Propagation with Enhanced Registration; K-S =

Kolmogorov-Smirnov test; DIS = Dissemination In Space; DIT = Dissemination In Time;

TSPO = Translocator Protein.

Introduction

Clinically isolated syndrome (CIS) is a single episode of central nervous system (CNS) dysfunction suggestive of focal demyelination ( Miller et al. , 2008). In those diagnosed with multiple sclerosis (MS) CIS represents the disease onset in ~80% ( Richards et al. , 2002).

However, only 61% of CIS subjects developed clinical symptoms consistent with MS after 20 years, but those that do develop MS by 5 years will already have a higher disability ( Chard et

al. , 2011). This outcome indicates that there are likely underlying factors in those who will get further symptoms consistent with MS and subsequently develop disability.

The most accurate predictor of MS development in CIS is the presence of brain lesions in the white matter (WM) on MRI ( Brex et al. , 2002). In 107 CIS patients followed up for ~20 years 82% with an abnormal baseline MRI scan versus 21% with a ‘normal’ baseline MRI

4 developed MS ( Fisniku et al. , 2008). This suggests that a more general effect is active in areas without lesions – the normal appearing WM (NAWM), which might act as a trigger to the development of lesions ( Linker et al. , 2009). In CIS grey matter (GM) atrophy is also reported ( Dalton et al. , 2004, Calabrese et al. , 2011) both in cortical regions and deep GM.

Neuropathological, biochemical and imaging studies have shown abnormalities within the

NAWM in MS ( Bjartmar et al. , 2001, Graumann et al. , 2003, Wheeler et al. , 2008, Aboul-

Enein et al. , 2010), the most prominent of which is the occurrence of microglial clusters around microvessels associated with increased expression of immune markers ( De Groot et

al. , 2001) and axonal changes ( Howell et al. , 2010), not visible on conventional MRI scans.

Significantly lower total N-acetyl-aspartate concentrations have been demonstrated in the

NAWM and were predictive of conversion to MS ( Wattjes et al. , 2008, Stromillo et al. ,

2013), whereas other MRI measures thus far have not been consistently abnormal or independently predictive of the development of MS ( Brex et al. , 1999, Fernando et al. , 2005).

[11C]-(R)-PK11195 (PK11195) PET scanning offers a method to visualise in vivo tissue changes, predominantly microglial and macrophage activation ( Banati et al. , 2000), derived either from increased cell number or activation level, in the brain. PK11195-PET has been used in MS to show inflammation within lesions ( Banati et al. , 2000, Debruyne et al. , 2003,

Versijpt et al. , 2005, Vas et al. , 2008) and cortical GM PK11195-PET activity correlates with disability ( Politis et al. , 2012). Given the possibility of generalised NAWM dysfunction and the potential importance of microglial clusters in MS, we utilised PK11195-PET scanning to identify whether there were any abnormalities within the NAWM in CIS and, given the prior evidence of GM PK11195-PET in MS, whether there was any activity in the GM.

Furthermore, if present, we were interested to understand whether any changes could potentially help refine the identification of subjects presenting with CIS who are at high risk of MS and future disability.

5 Materials and methods

Study population and assessment

The study received Hammersmith and Queen Charlotte’s Research Ethics Committee ethical approval and Administration of Radioactive Substances Advisory Committee permission to administer PK11195. On receiving fully informed consent according to the Declaration of

Helsinki, subjects with a diagnosis of CIS defined as a subject presenting with a single clinical episode suggestive of CNS demyelination, as well as healthy controls (HCs) were recruited. All participants attended for a PK11195-PET scan and a co-localizing gadolinium enhancing MRI scan within 7 days of each other. Patients were followed up yearly for 2 years. At each visit subjects were interviewed and the expanded disability status scale

(EDSS) (Kurtzke, 1983) was performed, and conversion to McDonald defined MS was assessed ( Polman et al. , 2011).

MRI and PK11195-PET imaging

A clinical 1.5-Tesla MRI system (Siemens MAGNETOM Avanto) was used. Volumetric T1 weighted sequences (coronal and axial T1-spin echo: TR=635 ms, TE=17 ms, 5 mm slice thickness); T1 volumetric magnetization-prepared rapid acquisition with gradient echo

(MPRAGE, TR=1900 ms, TE=3.53 ms, TI=1100 ms, flip angle 15°, 1 mm isotropic voxels) pre- and post intravenous administration of gadobutrol (7.5 mmol) and T2-weighted sequences (axial T2-spin echo: TR=4540 ms, TE=97 ms, 5 mm slice thickness; Axial FLAIR:

TR=9000 ms, TE=114 ms, TI=2500 ms, 5 mm slice thickness) were acquired for image registration and to define regions of interest (ROIs). MRI images were re-orientated with the hori-

6 zontal line defined by the anterior – posterior commissure line and the sagittal planes parallel to the midline.

For the PET scanning a Discovery RX PET/CT scanner was used. Images were reconstructed with the ramp filter using the reprojection algorithm, acquiring a spatial resolution at 1 cm for

2D and 3D respectively of 4.8 mm and 5.8 mm FWHM, at 10 cm from the centre of respectively 6.3 mm and 6.5 mm ( Kemp et al. , 2006). The CT images were used for attenuation correction.

PK11195 was administered as an intravenous bolus; the tracer was injected over 10 s then flushed with saline solution over 20 s. Emission data generated 18 time frames of tissue data over 60 min (30 s background frame, 1 x 15 s frame, 1 x 5 s frame, 1 x 10 s frame, 1 x 30 s frame, 4 x 60 s frames, 7 x 300 s frames, and 2 x 600 s frames). PK11195 tracer was supplied by Hammersmith Imanetplc, London.

PK11195-PET analyses

Quantification of PK11195-PET data was carried out adopting the simplified reference region model ( Gunn et al. , 1997) that uses a reference tissue input function and applies a simplified one tissue compartment model to each pixel of the dynamic volume to generate a parametric map of binding potential relative to the non-specific volume (BPND) ( Innis et al. , 2007). The tissue reference input was extracted from the emission dynamic using the SUPERPK software (Imperial Innovations) ( Turkheimer et al. , 2007, Yaqub et al. , 2012). The reference region is selected by modelling each pixel kinetic as the weighted sum of 4 tissue classes, normal grey and white matter, vascular and microglia binding, the latter identified from the stri- atum and globus pallidum of patients with clinically manifest Huntington’s disease. The reference region is then calculated as the weighted average of the pixel grey matter indexes

7 across the whole brain. The method has been extensively validated against the gold-standard plasma input function and across centres ( Turkheimer et al. , 2007, Yaqub et al. , 2012).

PK11195-PET and MRI images were then automatically co-registered using the SPM2 software package (Functional Imaging Laboratory, Wellcome Department of Imaging Neuros- cience, UCL).

Analysis of Regions of Interest

ROIs were manually drawn within lesional WM and NAWM using the ANALYZE software

(version 8.1, Mayo Foundation) to generate maps for each subject. WM lesions were MRI- defined as T2-FLAIR hyperintensity identified by an experienced neuroradiologist (AW) and a neurologist (PG). NAWM was defined as non-lesional WM, ROIs were drawn as far as possible from WM lesions (>1cm). NAWM ROIs were sampled in all subjects with a mean

(±SD) volume of 1.75 (±0.88) cm3. GM was automatically segmented (SPM2 software) and

ROIs maps generated using the multi-atlas propagation with enhanced registration (MAPER) approach ( Heckemann et al. , 2010). Manually drawn and MAPER generated maps were then used to calculate the volume and the BPND of PK11195 for each ROI.

Statistical Analyses

Statistical analyses were performed using SPSS (Version 17.0) and PRISM (GraphPad

Software, Inc. Version 6.01 for Windows). For all the variables, the normality of the distribution was tested with the Kolmogorov-Smirnov test (K-S), while the Shapiro-Wilk test was used for small samples (n<5). If normality was satisfied, bivariate correlation was tested using Pearson correlation coefficient. If normality was not satisfied, the Spearman's rho correlation coefficient was used. Differences between two groups were tested using the

8 Independent Samples T-Test, corrected when equal variance was not assumed (Welch’s correction), according to the Levene’s test for the equality of variance. When the assumption of normality was not satisfied, the difference between two groups was tested using the

Independent Samples Mann-Whitney U Test. K-S was also used to compare population distributions. The general linear model for repeated measures was used to analyse the variance of within-subjects variables and between-subjects factors. The Greenhouse-Geisser correction was then used to test the ROIxGroup interaction (given the significant Mauchly’s test of sphericity).

Results

CIS: clinical onset, MRI T2 WM lesions and outcome at two years

Eighteen CIS subjects (Table 1) and 8 HCs (mean age of 30.2 (±5.5) years, 5 female) were recruited. The CIS clinical presentation was a spinal cord syndrome in 8, brainstem syndrome in 7, optic neuritis in 2 and one a hemispheric lesion. Sixteen subjects completed the 2 year follow-up, two patients withdrew because they felt they had no problems. At 2 years 13 patients presented with the radiological criteria for dissemination in space (DIS), 13 for dissemination in time (DIT) and 12 had MS, as defined in 2010 McDonald criteria (Polman

et al. , 2011). Five patients had a second relapse satisfying the criteria for clinically-defined

MS. As with previous reports ( Fisniku et al. , 2008, Chard et al. , 2011) baseline MRI T2 lesion number and volume in subjects who developed MS at 2 years was increased compared to those subjects who had no further symptoms (Table 2). This was significant for both the number of MRI T2 lesions (p=0.016) and the MRI T2 volume in our study population

(p=0.012). As expected, in T2 lesions the PK11195 BPND was higher than in NAWM, respectively 0.153 (±0.095) and 0.078 (±0.027) (p=0.0096). 9 The BPND of PK11195 is globally increased within the NAWM of CIS subjects

To assess whether there were any differences in the NAWM PK11195 BPND between CIS subjects and HCs (Fig. 1), we initially considered the mean NAWM PK11195 BPND for each subject, and found PK11195 BPND was significantly increased in CIS subjects compared to

HCs (p=0.024, Fig. 2a). Given that the PK11195 BPND is measured in multiple ROIs we compared the NAWM PK11195 BPND distribution over all the ROIs studied and found that in

CIS the NAWM PK11195 BPND was significantly different compared to HCs (p<0.0001, Fig.

2b). Using repeated measures with ROI as the repeated factor and Group as a between subject factor we found a significant increase in NAWM PK11195 BPND in the CIS Group compared to the HC group (p=0.014, Fig. 2c). The increased PK11195 BPND in NAWM did not show any significant ROI effect (p=0.414), implying a diffuse global change. There was no ROI effect evident when the clinical symptom onset was considered.

Presence of MRI T2 lesions in CIS is associated with increased global PK11195

BPND change in NAWM, which correlates with disability

Giving the known importance of T2 MRI lesions in prognosis of CIS, both in terms of developing MS and disability, we explored whether the presence of WM lesions was associated with higher PK11195 BPND in the NAWM. We found that mean NAWM PK11195

BPND per subject was significantly increased in those CIS subjects with lesions compared to those without lesions (p=0.017, Fig. 3a). To exclude any effect from the multiple ROIs measured, we again utilised repeated measure testing between two groups (group 1: CIS with lesions vs group 2: CIS without lesions) and found a significant increase in the CIS group with lesions compared to those without lesions (p=0.009, Fig. 3b), independent of the ROI

10 repeated factor (p=0.414) implying a diffuse global change. The relevance of T2 lesions is further supported by the finding that in CIS subjects with lesions there was a significant correlation (p=0.007; r=0.673) between the BPND of PK11195 in NAWM and disability score

(EDSS, Fig. 3c).

CIS subjects who develop MS at 2 years have increased NAWM BPND of PK11195 at baseline

Given that those with CIS have an increased risk of developing MS, we tested the difference in mean NAWM PK11195 BPND in each subject between those who developed MS at 2 years and those who did not. Mean NAWM PK11195 BPND per subject was increased in those who developed MS at 2 years (p=0.04), whereas those who did not develop MS had PK11195

BPND signal levels similar to HC (Fig. 4a). Further analysis found that this significant increase in mean PK11195 BPND was evident in the group who fulfilled the criteria for DIS

(p=0.014) but not for the group who fulfilled the criteria for DIT (p=0.207). Again we confirmed that these findings were a diffuse global change by utilising repeated measures with ROI as the repeated factor and Group as a between subject factor in the NAWM

PK11195 BPND in CIS who developed MS at the 2-year follow-up. We found that NAWM

PK11195 BPND was increased in the CIS group who developed MS (p=0.048, ROI effect p=0.612) and that this was related to the group who satisfied the DIS criteria (DIS + vs DIS- p=0.007, ROI effect p=0.369) and not the DIT criteria (p=0.308). Thus, the baseline

PK11195 BPND activity in NAWM was significantly higher in CIS subjects who subsequently developed MS and who satisfied the DIS criteria but not DIT criteria (Fig. 4b).

GM central structures show a higher PK11195 BPND in CIS

11 In contrast to the findings in the WM, there were no differences in the GM PK11195 BPND between CIS subjects and HCs in the mean GM PK11195 BPND (CIS 0.112 ±0.080 and HCs

0.121 ±0.079), its distribution within the ROIs studied and in the cortical GM. However, in the central GM structures (Fig. 5) PK11195 BPND was significantly higher in CIS than HC, both using repeated measures for Group effect (p=0.028, with no ROI effect p=0.136) than the subjects’ average (p=0.006, Fig. 6).

The presence of MRI T2 lesions were not associated with any differences in cortical GM

PK11195 BPND (with MRI T2 lesions 0.092 (±0.064); without MRI T2 lesions 0.100

(±0.062), p=0.588)) but MRI T2 lesions were associated with increased central GM structure

PK11195 BPND (with MRI T2 lesions 0.208 (±0.059); without MRI T2 lesions 0.173

(±0.051), group effect p=0.049, with no ROI effect p=0.635). However, there was no associated GM PK11195 BPND difference in those who were diagnosed with MS at 2 years compared to those who were not.

Discussion

In this study PK11195-PET has been used to investigate a surrogate marker of microglia activation, TSPO expression, in the NAWM and the GM of CIS subjects. NAWM PK11195

BPND is globally increased in CIS compared to HCs at baseline and interestingly this increase was concentrated in those who had MRI T2 lesions at baseline imaging. Consistent with the known association of T2 lesions and the increased subsequent risk of MS, those who went on to develop McDonald defined MS at 2 years had higher global NAWM PK11195 BPND at baseline. In the cortical GM there was no difference in PK11195 BPND between CIS and HCs.

However, the central GM PK11195 BPND was increased in CIS compared to HCs.

12 PK11195 has proved particularly useful as a PET tracer because of its favourable dynamics and kinetics; it is a high affinity TSPO ligand and has the ability to cross the blood brain barrier ( Schweitzer et al. , 2010) with a well established methodology for quantification

( Turkheimer et al. , 2007, Yaqub et al. , 2012) and its binding is not affected by the known polymorphism in the TSPO gene that affects other ligands ( Owen et al. , 2011). In both MS and experimental autoimmune encephalomyelitis (EAE) tissues PK11195 binding was found to be consistently and strongly coupled with microglial activation ( Shah et al. , 1994,

Vowinckel et al. , 1997, Banati et al. , 2000, Venneti et al. , 2008). Given the potential role of areas of activated microglial clustering ( van Horssen et al. , 2012) that are not visible to MR imaging, we utilised PK11195-PET as a biomarker to determine if there was any evidence of microglial activation in the NAWM of CIS subjects.

This group, followed-up clinically and with MRI for 2 years, had a high rate of conversion to

McDonald confirmed MS (12 converted vs 4 who did not convert). However, because two were lost to follow-up as they felt well this would make them a typical CIS population (Chard

et al. , 2011). The finding that in CIS there is a global increase in NAWM microglial activation, would be consistent with the presence of underlying areas of the NAWM that might be predisposed to lesion formation in which clusters of activated microglia are present

( van Horssen et al. , 2012) together with diffuse changes to axons and nodes of Ranvier

( Howell et al. , 2010). The increase is more prominent in those who had MRI T2 lesions at baseline and this group is known to have a higher risk of developing MS than those with no

MRI T2 lesions ( Fisniku et al. , 2008, Chard et al. , 2011). Consistent with this, in our population higher levels of NAWM PK11195 BPND were associated with a higher risk of developing MS at 2 years predominantly through subsequent dissemination in space. This could suggest, if confirmed in further studies, that the underlying change in the NAWM increases the risk of a WM lesion developing.

13 Alterations in brain homeostasis can cause microglial activation in normal appearing brain tissue ( van Horssen et al. , 2012) and does not necessarily indicate irreversible damage or even impaired function of underlying brain tissue. In addition, it is possible that microglial activation in the NAWM is a protective response to inflammation ( Graumann et al. , 2003,

Heppner et al. , 2005). However, the increase in NAWM PK11195 BPND seen in subjects with

MRI T2 WM lesions was correlated with the EDSS which might suggest, in our limited population that in subjects with WM lesions the NAWM is already affected and associated with an impaired neuronal function. The association of higher microglia activity in NAWM and clinical disability was not present at 2 years. This could indicate that any impaired neuronal function was temporary, however due to the exploratory nature of the study

PK11195-PET was not repeated at this point, thus this interpretation represents only one possible explanation. Is well known that microglial activation in the WM is associated with more aggressive MS ( Kutzelnigg et al. , 2005) and it is possible to inhibit microglial activation at the early stages of EAE, and this has a beneficial effect on the final clinical outcome ( Heppner et al. , 2005, Bhasin et al. , 2007).

In CIS patients studied here, the GM in the central structures showed higher PK11195 BPND compared to HCs. Thalamic atrophy is known to be present in CIS and early MS ( Cifelli et

al. , 2002, Calabrese et al. , 2011, Langkammer et al. , 2013) and increased PK11195-PET activity in the thalamus has been reported ( Banati et al. , 2000). However, the increased

PK11195-PET activity in the thalamus has not been reported in CIS before, but would be consistent with the MRI thalamic atrophy seen in CIS. Cortical GM PK11195 BPND was not increased in CIS compared to HCs. We have previously shown using the same methods that there was increased cortical GM PK11195 BPND in MS, and that this correlated with disability ( Politis et al. , 2012). The lack of any elevation in cortical GM PK11195 BPND in

CIS could be because PK11195-PET is not sensitive enough to detect any changes despite

14 our detection of changes in deep GM PK11195 BPND. Cortical GM atrophy has been found on MRI ( Dalton et al. , 2004) in CIS and this could indicate that cortical GM atrophy might precede microglial activation and with microglial activation becoming more prominent in the cortex as the condition progresses.

In conclusion, we report widespread microglial activation in the NAWM in CIS. Higher levels of microglial activation were seen in those CIS subjects with WM lesions on MRI at baseline and in those who subsequently who developed MS. However, the relatively small population and the lack of a follow-up PK11195-PET scan represent limitations and further work is required to understand the persistence of the abnormal NAWM activation and how

NAWM activation compares to MRI in predicting future development of MS.

Figure Legends

Figure 1

Normal appearing white matter (NAWM) on PK11195-PET images co-registered and fused with MRI of three subjects of our population. The first subject is a HC (A) with PK11195

BPND in NAWM of 0.028; the second is a CIS subject without T2 MRI lesions (B), an expanded disability status scale (EDSS) score of 3.5 and PK11195 BPND in NAWM of 0.037; the third is a CIS subject with T2 MRI lesions (C), EDSS=2.0 and PK11195 BPND in NAWM of 0.136. The colour scale bar represents the BPND of PK11195.

Figure 2

15 Global changes in normal appearing white matter (NAWM) in clinically isolated syndromes

(CIS) subjects compared to healthy controls (HC). Inset A shows the average of PK11195

BPND for each subject represented by a black dot (HC PK11195 BPND average in NAWM is

0.042, while is 0.071 in CIS subjects). The inset B shows the significant difference in

NAWM regions frequency distribution (Y axis, percentage) according to their PK11195 BPND

(X axis) between HC and CIS. There is a higher percentage of NAWM regions with low

PK11195 BPND in HC (in blue, left side of the graph) than CIS (in red, right side of the graph)

Inset C shows the significant group effect studied using repeated measures with NAWM regions of interest as the repeated factor and Group (HC in blue; CIS in red) as a between subject factor.

Figure 3

Global changes in normal appearing white matter (NAWM) in clinically isolated syndromes

(CIS) subjects without T2 white matter (WM) lesions at MRI scan compared to CIS subjects with T2 WM lesions. Inset A shows the average of PK11195 BPND for each subject represented by a black dot (CIS without WM lesions PK11195 BPND average in NAWM is

0.032, while is 0.078 in CIS subjects with WM lesions). The inset B shows the significant group effect studied using repeated measures with NAWM regions of interest as the repeated factor and Group (CIS without WM lesions in green; CIS with WM lesions in red) as a between subject factor. Inset C shows the significant correlation between PK11195 BPND (Y axis) and Expanded Disability Status Scale (EDSS, X axis) in CIS with WM lesions

(r=0.673); dotted lines represent error bars.

16 Figure 4

Baseline PK11195 BPND in normal appearing white matter (NAWM) of healthy controls

(HC) and clinically isolated syndromes (CIS) who remained CIS or were diagnosed with multiple sclerosis (MS) at the 2-year follow-up (FU). Inset A shows the average of PK11195

BPND for each subject represented by a black dot (HC PK11195 BPND average in NAWM is

0.042, CIS PK11195 BPND average in NAWM is 0.045, while is 0.080 in CIS subjects who developed MS at 2-year FU). On the Y axis of inset B are listed the 16 subjects who completed the 2-year FU ordered according to their NAWM PK11195 BPND value (X axis) at the baseline PK11195-PET scan. The green colour represent the dissemination in space (DIS, present in 13 subjects); in red the dissemination in time (DIT, present in 13 subjects). Patients who developed both DIS and DIT, hence diagnosed with multiple sclerosis (MS, 12 subjects) according to the 2010 revision of McDonald criteria for MS diagnosis, are therefore MS labelled on the Y axis. In blue the subjects who remained at the CIS stage and did not develop both DIS and DIT.

Figure 5

Coronal section of central structures on PK11195-PET images coregistred and fused with

MRI of two subjects of our population. The first subject is a healthy volunteer (A) with

PK11195 BPND in GM central structures of 0.163; the second is a clinically isolated syndrome

(CIS) subject (B), EDSS=4.0 and PK11195 BPND in GM central structures of 0.248. The colour scale bar represents the BPND of PK11195.

Figure 6 17 Baseline PK11195 BPND in central structure of healthy controls (HC) and clinically isolated syndromes (CIS) subjects. This figure shows the average of PK11195 BPND for each subject represented by a black dot (HC PK11195 BPND average in central structures is 0.167, while is

0.197 in CIS subjects).

Funding

PG thanks the EFNS (European federation of neurological Societies) for a Scientific Fellow- ship in 2009, the FISM (Fondazione Italiana Sclerosi Multipla) for a training (research) fel- lowship, (Cod. 2010/B/7) and MSTC (Multiple Sclerosis Trials Collaboration).

FT received a MRC PET Methodology Grant G1100809/1. RN was supported by the

National Institute for Health Research (NIHR) Imperial Biomedical Research Centre. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health.

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