Dr Martina Di Simplicio
email: martina.disimplicio@psych.ox.ac.uk

MD Raymond Norbury

PhD Catherine J Harmer

Prefrontal Cortex in Subjects at Risk for Depression

Keywords: depression, SSRI, emotion, neuroticism, prefrontal cortex, self-referential

scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

There is increasing evidence to suggest that major depression is characterised by alterations in orbitomedial and ventromedial prefrontal cortex (omPFC; vmPFC) both at a structural and functional level, with decreased grey matter volume and increased metabolism in anterior cingulate (ACC) and ventromedial frontopolar cortex, which may correlate with symptom severity [START_REF] Price | Neurocircuitry of mood disorders[END_REF]. These areas participate in a limbic-cortical-striato-pallido-thalamic circuit implicated in the processing of emotional stimuli [START_REF] Phillips | Neurobiology of emotion perception I: The neural basis of normal emotion perception[END_REF][START_REF] Phan | Functional neuroanatomy of emotion: a meta-analysis of emotion activation studies in PET and fMRI[END_REF] and may be specifically involved in assessing salience and value, and regulating response to emotional information [START_REF] Rolls | The orbitofrontal cortex and beyond: from affect to decision-making[END_REF][START_REF] Ochsner | The cognitive control of emotion[END_REF].

At a behavioural level, depression has also been correlated with mood-congruent aberrant processing of emotional information such as increased attention and memory for negative verbal stimuli [START_REF] Leppanen | Emotional information processing in mood disorders: a review of behavioral and neuroimaging findings[END_REF][START_REF] Bradley | Implicit memory bias in clinical and non-clinical depression[END_REF]. Such biases have also been associated with increased neural activation in omPFC areas such as the rostral ACC [START_REF] Fu | Neural responses to sad facial expressions in major depression following cognitive behavioral therapy[END_REF][START_REF] Yoshimura | Rostral anterior cingulate cortex activity mediates the relationship between the depressive symptoms and the medial prefrontal cortex activity[END_REF]. Thus, emotion processing biases and their neural correlates may reinforce negative thought distortions that maintain affective symptoms according to cognitive theories of depression [START_REF] Beck | The evolution of the cognitive model of depression and its neurobiological correlates[END_REF].

Interestingly, different therapies such as antidepressants [START_REF] Davidson | The neural substrates of affective processing in depressed patients treated with venlafaxine[END_REF][START_REF] Chen | Functional coupling of the amygdala in depressed patients treated with antidepressant medication[END_REF][START_REF] Mayberg | Regional metabolic effects of fluoxetine in major depression: serial changes and relationship to clinical response[END_REF][START_REF] Fu | Attenuation of the neural response to sad faces in major depression by antidepressant treatment: a prospective, event-related functional magnetic resonance imaging study[END_REF][START_REF] Eugene | Neural correlates of inhibitory deficits in depression[END_REF], vagus nerve [START_REF] Pardo | Chronic vagus nerve stimulation for treatment-resistant depression decreases resting ventromedial prefrontal glucose metabolism[END_REF] or deep brain stimulation [START_REF] Mayberg | Deep brain stimulation for treatment-resistant depression[END_REF], and CBT [START_REF] Fu | Neural responses to sad facial expressions in major depression following cognitive behavioral therapy[END_REF][START_REF] Goldapple | Modulation of cortical-limbic pathways in major depression: treatment-specific effects of cognitive behavior therapy[END_REF][START_REF] Dichter | The effects of Brief Behavioral Activation Therapy for Depression on cognitive control in affective contexts: An fMRI investigation[END_REF] have been reported to normalise resting metabolism and activation to emotional stimuli in medial PFC areas. These convergent observations suggest that omPFC and vmPFC malfunction could be a central feature of major depression but do not clarify whether its correction occurs as a direct effect of treatment or as a secondary effect consequent to symptoms remission and mood change.

Recent studies suggest that antidepressant drugs may start modifying neuropsychological biases in emotional information processing early on in treatment; such changes could allow depressed subjects to experience environmental stimuli more positively over time and thus correct their negative cognitive biases and lead to symptom remission that is manifest within the usual 2-3 weeks of delay from beginning of treatment [START_REF] Harmer | Serotonin and emotional processing: does it help explain antidepressant drug action?[END_REF][START_REF] Harmer | Efficacy markers in depression[END_REF]. Data from healthy volunteers support this model, showing early effects of antidepressants directly on the neural correlates of emotional information processing, namely decreasing fronto-limbic response to negative emotional stimuli and increasing the signal to positive ones [START_REF] Harmer | Antidepressant drug treatment modifies the neural processing of nonconscious threat cues[END_REF][START_REF] Norbury | Short-term antidepressant treatment modulates amygdala response to happy faces[END_REF][START_REF] Murphy | Effect of a single dose of citalopram on amygdala response to emotional faces[END_REF]. For example, repeated dose reboxetine administration has been shown to potentiate prefrontal cortical response to positive selfdescriptors and diminish the neural memory workload in mPFC and other areas necessary for correct recognition of the same stimuli [START_REF] Norbury | The effects of reboxetine on emotional processing in healthy volunteers: an fMRI study[END_REF]. However, it remains unclear whether antidepressants produce the same early changes in the neural substrates of emotion processing in clinical populations as these seen in healthy volunteers.

Interpretation of results from clinical samples is often problematic due to the confounding presence of concentration deficits and other general cognitive symptoms. Moreover, it is difficult to separate medication effects on mood state from those on emotional information processing during acute depression. Hence, the current study investigated the neural substrates of antidepressant action on subjects that present biases in emotional processing related to increased risk for depression and similar to those shown during low mood states, but without clinical symptoms of affect dysregulation. In particular, subjects with high neuroticism present a well-established vulnerability to affective and anxiety disorders [START_REF] Kendler | Personality and major depression: a Swedish longitudinal, population-based twin study[END_REF][START_REF] Kendler | The genetic and environmental relationship between major depression and the five-factor model of personality[END_REF]. They also show negative biases in facial expression processing and in categorisation and memory for selfdescriptors (28) that appear to be underpinned by alterations in a network of brain areas including the medial PFC [START_REF] Chan | Risk for depression is associated with neural biases in emotional categorisation[END_REF][START_REF] Canli | Toward a neurogenetic theory of neuroticism[END_REF][START_REF] Haas | Stop the sadness: Neuroticism is associated with sustained medial prefrontal cortex response to emotional facial expressions[END_REF] and could be particularly relevant to their vulnerability to depression. We therefore hypothesised that seven days antidepressant administration would reduce the dysfunctional response to negatively valenced self-descriptors present in high neuroticism in brain areas related to evaluation and control of emotional stimuli (e.g. vmPFC).

Materials and Methods

Thirty-four healthy volunteers (17 females) were initially recruited from the general population into the study with a mean age of 25.8 (SD±7.1). Written informed consent was provided prior to entry into the study, as approved by the local Ethics Committee. All subjects presented a score above 16/24 on the Neuroticism scale of the Eysenck Personality Questionnaire [START_REF] Eysenck | Manual of the EPQ (Eysenck Personality Questionnaire)[END_REF] (mean=17.82±2.1) and were screened with a Structured Clinical Interview for DSM-IV I (33) for current or past Axis I disorders. All subjects were right-handed, in good physical health and free of any medication, excluding the contraceptive pill, and had no contraindications for fMRI scanning. Subjects also completed the State and Trait Anxiety Inventory (34), Beck Depression Inventory [START_REF] Beck | An inventory for measuring depression[END_REF], Dysfunctional Attitudes Scale [START_REF] Weisman | Development and validation of the Dysfunctional Attitudes Scale: A preliminary investigation[END_REF] and Life Events Rating Scale [START_REF] Goodyer | Short-term outcome of major depression: II. Life events, family dysfunction, and friendship difficulties as predictors of persistent disorder[END_REF].

A double-blind, placebo-controlled, randomised design was used, with subjects randomly allocated to either 7 days of citalopram 20 mg/day (n=19) or placebo (n=15). During treatment daily mood, anxiety and side effects ratings were collected with the Befindlichskeit Scale [START_REF] Von Zerssen | Evaluation of depressive states, especially in longitudinal studies[END_REF] (BFS), Visual Analogue Scales (VAS) measuring happiness, sadness, hostility, alertness, anxiety and calmness and a side effects questionnaire.

On the last day of treatment subjects participated in a single 12 minute experiment employing rapid event-related fMRI while performing a word categorisation task, using positive and negative self-descriptors. For this task, a list of 90 personality adjectives was constructed, of which half were unambiguously positive and half negative [START_REF] Anderson | Likeableness ratings of 555 personality-trait words[END_REF]. Positive and negative words were matched on length, frequency [START_REF] Francis | Frequency Analysis of English Usage: Lexicon and Grammar[END_REF] and meaningfulness. Ten presentations each of 'left' and 'right' words were included as a baseline control words condition. The words 'left' and 'right' were selected in order to control for the sensorimotor aspects of the task without requiring subjective assessment of the positive or negative aspects of the word. Thus, 110 words were presented in total, with the order randomised across subjects. Stimuli were presented on a personal computer using E-Prime (version 1.0; Psychology Software Tools Inc., Pittsburgh, PA) and projected onto an opaque screen behind the scanner bore, which subjects viewed using angled mirrors. During the categorisation task subjects were instructed to characterise the words as 'like' or 'dislike' in a self-referential fashion as quickly and accurately as possible.

Specifically, subjects were asked to imagine whether they would like or dislike it if they overheard someone referring to them as possessing each of these personality traits. This instruction was previously used in behavioural studies [START_REF] Chan | Highly neurotic never-depressed students have negative biases in information processing[END_REF][START_REF] Harmer | Increased positive versus negative affective perception and memory in healthy volunteers following selective serotonin and norepinephrine reuptake inhibition[END_REF] and other behavioural and neuroimaging studies [START_REF] Norbury | The effects of reboxetine on emotional processing in healthy volunteers: an fMRI study[END_REF][START_REF] Miskowiak | Single dose antidepressant administration modulates the neural processing of self-referent personality trait words[END_REF] with healthy unselected and high neurotic volunteers. Subjects indicated their decision as 'like' or 'dislike' by pressing a button with the right and left index or middle finger, respectively. For the control words 'right' and 'left', subjects responded with their right and left index or middle finger respectively. Subject responses were recorded using an MRI-compatible keypad. Like and dislike responses and reaction times were recorded by E-Prime. Each trial consisted of a fixation cross (500ms) immediately followed by a personality or control word (1000ms). The duration of the intertrial interval (ITI) varied between 4000 and 8000ms according to a Poisson distribution, with a mean ITI of 5000 ms.

Subjects also underwent a 12 minute resting state acquisition while cerebral blood flow (CBF) levels were measured using an single shot three-dimensional (3D) gradient and spin echo Arterial spin labelling technique (GRASE-ASL). During this time they were instructed to watch a black screen and remain awake.

Imaging data were acquired on a 3T TIM Trio scanner located at the Oxford Centre for Clinical and Magnetic Resonance Research (OCMR). FMRI data were acquired with a voxel resolution of 3x3x3 mm 3 , TR=3s, TE=30ms, Flip angle=87 o . Field maps were also acquired using a dual echo 2D gradient echo sequence with echos at 5.19 and 7.65 ms, and repetition time of 444ms (64x64x40 grid; voxel resolution of 3mm isotropic). T1-weighted structural images were acquired for subject alignment using an MPRAGE sequence with the following parameters: Voxel resolution 1x1x1 mm 3 on a 176x192x192 grid, Echo time(TE)= 4.53 ms, Inversion time(TI)= 900 ms, Repetition time (TR)= 2200 ms. For ASL acquisition parameters see [START_REF] Macintosh | Measuring the effects of remifentanil on cerebral blood flow and arterial arrival time using 3D GRASE MRI with pulsed arterial spin labelling[END_REF].

Due to rapid movement artefacts and/or registration problems of the perfusion MRI images, 5 subjects were excluded from the final analysis (n = 16, citalopram treated). Data were analysed using FSL version 4.1 [START_REF] Smith | Advances in functional and structural MR image analysis and implementation as FSL[END_REF]. Pre-processing included slice acquisition time correction, withinsubject image realignment [START_REF] Jenkinson | Improved optimisation for the robust and accurate linear registration and motion correction of brain images[END_REF], non-brain removal [START_REF] Smith | Fast and robust automated brain extraction[END_REF], spatial normalisation (to Montreal Neurological Institute [MNI] 152 stereotactic template), spatial smoothing, and high-pass temporal filtering to a maximum of 0.008 Hz. Susceptibility-related distortions were corrected as far as possible using FSL field-map correction routines. Given that the sample investigated was expected to present with high levels of baseline anxiety which could result in worse compliance to the scanner environment and increased motion during the experiment session, we included an additional pre-processing procedure to reduce motion related artefacts. Briefly, individual subject's data were subjected to an Independent Component Analysis (ICA) implemented within FSL. The resulting spatial maps were visually inspected and obvious motion /physiological artefacts were removed [START_REF] Beckmann | Probabilistic independent component analysis for functional magnetic resonance imaging[END_REF].

In the first-level analysis, individual activation maps were computed using the general linear model with local autocorrelation correction [START_REF] Woolrich | Temporal autocorrelation in univariate linear modeling of FMRI data[END_REF]. Three explanatory variables were modelled: 'negative', 'positive' and 'control' words. All variables were modelled by convolving the onset of each word with a haemodynamic response function, using a variant of a gamma function (i.e. a normalisation of the probability density function of the gamma function) with a standard deviation of 3 s and a mean lag of 6 s. In the second-level analysis, individual data were combined at the group level (citalopram vs. placebo) using a mixed-effects group cluster analysis across the whole brain corrected for multiple comparisons [START_REF] Woolrich | Multilevel linear modelling for FMRI group analysis using Bayesian inference[END_REF]. This mixed effects approach accounts for intra-subject variability and allows general population inferences to be drawn. Significant activations were identified using a cluster-based threshold of statistical images [height threshold of Z = 2.3 and a (corrected) spatial extent threshold of p < 0.05] (50).

To check for potential confounding effects of sub-threshold differences in blood perfusion levels, demeaned CBF values obtained during resting state GRASE-ASL were entered in the analysis as a voxel-wise covariate of no interest. Activation during the categorisation of self-referent emotional words was analysed in three separate contrasts: negative vs. control, positive vs. control and positive vs. negative words. Only trials where the 'like' or 'dislike' response corresponded to the word valence (i.e. 'like' for positive words and 'dislike' for negative words) were included in the analysis. Significant interactions were characterized by extracting percent blood oxygenation level-dependent (BOLD) signal change within the areas of significant difference, and then subjected to a repeated measures ANOVA (between subjects variable = group; within subjects variable = word valence) followed by appropriate post-hoc t-tests (SPSS v.14.0). Corresponding Brodmann Areas (BA) were identified by transforming MNI coordinates into Talairach space [START_REF] Talaraich | Co-planar stereotaxic atlas of the human brain: three-dimensional proportional system[END_REF].

Perfusion MRI data were analysed based on the method described in [START_REF] Chappell | Vascular territory image analysis using vessel encoded arterial spin labeling[END_REF]. A nonparametric permutation-based (5000 iterations)independent-samples t-test was used to test for whole brain voxel-wise between-group differences in CBF perfusion levels [START_REF] Nichols | Nonparametric permutation tests for functional neuroimaging: a primer with examples[END_REF].

Behavioural data ('correct words matching' and mean reaction time) were analysed using repeated measures ANOVA, with group as the between-subject variable and valence as the within-subjects variable (positive, negative and control words). Herein, 'correct matching' refers to the trials where participants categorised positive words as 'like' or negative words as 'dislike' and pressed the right button for the control word 'right' and left button for the control word 'left'. Independent samples t-tests were performed to clarify significant interactions, as well as to examine group differences in mood and personality ratings.

Results

Affect ratings and behavioural results

Groups did not differ in terms of state and trait anxiety, depressive symptoms, dysfunctional attitudes and life events, or in reported psychiatric family history. Citalopram did not affect subjective ratings of state or mood as measured by VAS and BFS (all P's >.050) (Table 1).

Subjects receiving citalopram reported significantly more side effects of nausea, particularly on the first three days of treatment (time x group Anova: main group effect F (1,30) =11.38, p=.002).

There were no significant differences between the groups in task performance ('correct matching': valence x group Anova F (2,64) =.269, p=.675; RT: valence x group Anova F (2,64) =.295, p=.745) during the words categorisation task (Table 2).

Perfusion results

In the analysis of cerebral blood perfusion GRASE-ASL data acquired during resting state, no differences were found in CBF levels across the whole brain between subjects treated with citalopram or placebo. Also, no significant correlations were found between cerebral perfusion and haemodynamic response to the different task conditions.

BOLD fMRI results

Main effect of task (across groups)

Relative to control words, response to negative words was characterised by significant activations in the bilateral occipital cortex (BA17,18,19), bilateral temporo-parietal cortex (BA20, BA22, BA39, BA40), right motor cortex (BA4), precuneus (BA7), right prefrontal cortex (BA6), medial prefrontal cortex (BA8), frontal pole (BA10), orbitofrontal cortex (BA11) and bilateral putamen and caudate. Positive words contrasted with control words also activated the following areas across the whole sample: bilateral occipital cortex (BA17,18,19), bilateral temporo-parietal cortex (BA20,22,39,40), left motor cortex (BA4), right prefrontal cortex (BA6,9), precuneus (BA7), medial prefrontal cortex (BA9), frontal pole (BA10), orbitofrontal cortex (BA11) and bilateral putamen and caudate. In addition, for negative vs. positive and positive vs. negative words a significant response cluster was present across the whole sample in the bilateral primary somatosensory and motor cortex (BA2,3,4), and bilateral cerebellum (Table 1 in supplementary material).

BOLD fMRI between-group differences

At the whole brain level, corrected for multiple comparisons, we observed a significant group x word valence interaction (negative vs. control words) in the ventromedial prefrontal cortex (cluster size (voxels) = 565, Z=3.41, p=.005, corrected; MNI x=0, y=40, z=16) (Figure 1a). Visual inspection of this cluster showed that it spanned rostral ACC (BA32), right orbitofrontal cortex (BA11) and frontal pole (BA10), also including the right caudate (Table 3). Post hoc analysis on BOLD signal change extracted from the mean activation voxel (BA32, MNI: 0, 40, 16) showed that the interaction was driven by a difference in response to negative words (word type x group interaction: F (1,27) =5.488, p=.027; negative words: t (27) =1.644, p=.112; control words: t (27) = -.883, p=.385). In particular, citalopram decreased the response to negative self-referred words showing a deactivation pattern in the ventromedial prefrontal cortex (Figure 1b).

No significant interaction effect was found neither in the contrast between positive and control words, nor in the contrast between negative vs. positive words. Exploratory analysis at a Z=2.8 uncorrected threshold for the negative vs. positive contrast are reported in Table 2 in supplementary material.

Discussion

The results from our study suggest that short term SSRI administration can modulate PFC responses to negative self-referential stimuli in individuals at high risk of depression. Such effects replicate those seen in healthy non-selected volunteers [START_REF] Norbury | The effects of reboxetine on emotional processing in healthy volunteers: an fMRI study[END_REF][START_REF] Miskowiak | Single dose antidepressant administration modulates the neural processing of self-referent personality trait words[END_REF] and translate them in to a clinically relevant sample. Such an action would be expected to normalise dysfunctional patterns of activation found in subjects with high neuroticism, and may be involved in the evolution of later clinical effects over time in clinically depressed subjects.

The cluster modulated by antidepressant administration falls within a relatively extended area in the vmPFC (Figure 1a). Lesions to the vmPFC produce recognised changes in a range of emotional and social behaviours and in decision-making [START_REF] Rudebeck | The contribution of distinct subregions of the ventromedial frontal cortex to emotion, social behavior, and decision making[END_REF]. Although it is not possible to extrapolate from our data whether any of the subregions encompassed within the cluster (anterior cingulate (ACC), orbitofrontal and frontopolar cortex) plays a more specific role, two main functions of the vmPFC could be related to the differential activation observed.

First, this area is widely interconnected with sub-cortical structures that regulate autonomic function and receives projections from temporal lobe areas of multimodal sensory association, processing elaborate stimuli such as facial expressions [START_REF] Carmichael | Sensory and premotor connections of the orbital and medial prefrontal cortex of macaque monkeys[END_REF]. These reciprocal projections are believed to mediate complex emotional responses, including integration and conflict monitoring of emotional and cognitive information via allocation of attentional resources, thus guiding choices and behaviour [START_REF] Bush | Cognitive and emotional influences in anterior cingulate cortex[END_REF]. In a previous study, using the same words categorisation task, we have shown that high neuroticism is correlated with higher activation to negative words in the ACC, close to the activation site highlighted in the present study [START_REF] Chan | Risk for depression is associated with neural biases in emotional categorisation[END_REF]. Similarly, ACC recruitment was also found to increase parallel to neuroticism scores during presentation of trials of emotional conflict in an emotional Stroop task combining words and facial stimuli [START_REF] Haas | Emotional conflict and neuroticism: personalitydependent activation in the amygdala and subgenual anterior cingulate[END_REF] and to relate to higher self-criticism tendencies [START_REF] Mannie | Affective modulation of anterior cingulate cortex in young people at increased familial risk of depression[END_REF][START_REF] Gotlib | Neural processing of reward and loss in girls at risk for major depression[END_REF][START_REF] Longe | Having a word with yourself: neural correlates of self-criticism and self-reassurance[END_REF]. These data suggest that altered ACC function may contribute to the biased attention towards negative stimuli and higher emotional conflict seen in vulnerability to depression, which mirror those seen in patients with major depression and are suggested to reflect the tendency to dwell excessively on negative stimuli typical of dysphoria [START_REF] Fu | Neural responses to sad facial expressions in major depression following cognitive behavioral therapy[END_REF][START_REF] Yoshimura | Rostral anterior cingulate cortex activity mediates the relationship between the depressive symptoms and the medial prefrontal cortex activity[END_REF][START_REF] Eugene | The impact of individual differences on the neural circuitry underlying sadness[END_REF]. Therefore our finding of a reduced activation in the vmPFC, including parts of the rostral ACC (BA32), to negative self-descriptors in high neuroticism following treatment is consistent with the idea that antidepressants reduce allocation of attentional resources to negatively valenced stimuli in subjects who usually show higher emotional conflict and a preference for negative attributions (Figure 1b).

Second, the vmPFC is also implicated in the attribution of emotion to oneself and others with medial regions possibly more activated in response to self-judgements and ventral areas representing the affective value of the stimuli [START_REF] Ochsner | For better or for worse: neural systems supporting the cognitive down-and up-regulation of negative emotion[END_REF]. For example, frontopolar areas within the vmPFC have been proposed to play a role in regulation and inhibition of emotionally salient stimuli particularly if they are related to the distinction between self and others [START_REF] Cunningham | Implicit and explicit evaluation: FMRI correlates of valence, emotional intensity, and control in the processing of attitudes[END_REF]. These circuits are highlighted during resting state when subjects are more or less automatically engaged in baseline monitoring of internal states [START_REF] Raichle | A default mode of brain function: a brief history of an evolving idea[END_REF] and have therefore been referred to as a default mode network of the brain which appears to deactivate during the performance on cognitive tasks. Failure to deactivate these areas during various tasks has then been interpreted as a difficulty to disengage from self-reflection and self-monitoring or as a tendency to relate aspects of the task to the self. Recently it has been proposed that a general inability to regulate self-referential activity, underpinned by alterations in the whole default mode network during emotion processing, may contribute to depressive illness [START_REF] Sheline | Resting-state functional MRI in depression unmasks increased connectivity between networks via the dorsal nexus[END_REF][START_REF] Sheline | The default mode network and self-referential processes in depression[END_REF]. In particular neural alterations during specific processing of self-referential material, for example during response to personality traits [START_REF] Lemogne | Self-referential processing and the prefrontal cortex over the course of depression: a pilot study[END_REF] or judgments of self-relatedness [START_REF] Grimm | Increased self-focus in major depressive disorder is related to neural abnormalities in subcortical-cortical midline structures[END_REF], in the dorsomedial and dorsolateral prefrontal cortex seem to correlate with the severity of symptoms such as hopelessness. These signals could correspond to the neural representation of negative self-related rumination, a cognitive process considered to be one of the crucial mechanisms responsible for the development and maintenance of clinical depression [START_REF] Papageorgiou | Depressive ruminatio: nature, theory and treatment[END_REF].

Our task, which required volunteers to relate different personality characteristics to themselves, revealed a failure to deactivate vmPFC areas in particular during response to negative selfdescriptors in the placebo group (Figure 1b). This pattern is consistent with the evidence that high neurotic subjects present with a higher propensity to negative self-attributions and rumination levels [START_REF] Haas | Stop the sadness: Neuroticism is associated with sustained medial prefrontal cortex response to emotional facial expressions[END_REF]. Consistent with this, the deactivation signal to negative self-referential words induced by citalopram administration may correspond to decreased self-referent processing and a diminished perceived intensity of the negative valence of words potentially referring to the self. As such, short term antidepressant treatment could lead to less attentional resources being allocated to self-attribution of negative stimuli. Further studies are needed to explore whether such effects may with prolonged treatment help to resolve the difficulties in disengaging from self-focused rumination and negative emotions that predispose to and maintain depression.

Our study presents some limitations which should be taken into consideration when interpreting the results. First, we did not find any differences when directly comparing positive and negative self-descriptors, but only in the contrast between negative and control words (which does not account for arousal and repetition effects). Hence, the results will need to be replicated by future experiments to confirm a valence specific effect of antidepressant action on negative self-referential processing. A second limitation is that we did not include a direct control group with low neuroticism. Our study, however, was directly built upon data from previous research comparing individuals with high and low neuroticism scores, and the finding of an antidepressant modulatory effect on a region shown to be altered in high neuroticism (such as the ACC) suggest that this effect could be specific to this population at risk. Future studies should include both high and low risk samples in the design to allow for a more exact interpretation of the results and validate this hypothesis. Last, it has to be noted that neuroticism is an unspecific risk factor for anxiety and mood disorders. Nevertheless, the discussion of our results has been restricted within the framework of the neurobiological correlates of depression, because previous literature suggests that biases in processing of verbal stimuli are more likely to be linked to low mood rather than anxiety symptoms [START_REF] Harmer | Efficacy markers in depression[END_REF]. Notwithstanding these limitations, to our knowledge these are the first data that present antidepressant effects on BOLD haemodynamic response after having controlled for potential confounding effects on general haemoperfusion, hence strengthening the validity of the data presented. We believe that this approach could be an important way to increase the reliability of interpretations extrapolated from pharmaco-fMRI data.

In conclusion, the cognitive neuropsychological model of antidepressant drug action [START_REF] Harmer | Why do antidepressants take so long to work? A cognitive neuropsychological model of antidepressant drug action[END_REF] suggests that delayed symptom remission is built on early treatment effects, which modulate the neural correlates of emotional processing. Findings backing this hypothesis from healthy volunteers' samples [START_REF] Norbury | Short-term antidepressant treatment modulates amygdala response to happy faces[END_REF][START_REF] Murphy | Effect of a single dose of citalopram on amygdala response to emotional faces[END_REF][START_REF] Miskowiak | Single dose antidepressant administration modulates the neural processing of self-referent personality trait words[END_REF][START_REF] Rawlings | A single dose of mirtazapine modulates neural responses to emotional faces in healthy people[END_REF] have been recently extended to a clinical population study showing that at a behavioural level a single dose of reboxetine was able to decrease response latency and improve memory for positive words in depressed patients before the initiation of any subjective symptom change [START_REF] Harmer | Effect of acute antidepressant administration on negative affective bias in depressed patients[END_REF]. Although preliminary, our data then further support this model by translating evidence of early antidepressant effects on neural responses to emotional stimuli from healthy samples to a population at risk presenting subclinical features of depressive cognitive-emotional biases.

Further studies are needed to translate these findings to depressed patients, to explore the time course of this effect in relation to subjective modifications in affect and to establish the degree to which this normalisation can predict later symptom remission and relapse prevention.

Figure legends

Figure 1a

Significant cluster of activation depicting greater signal response in the ventromedial prefrontal cortex (vmPFC) in the placebo vs. citalopram treated group during recognition of negative selfdescriptors vs. control words. Images thresholded at Z> 2.3, P< 0.05, corrected.

Figure 1b

The group x emotion interaction in the ventromedial prefrontal cortex displaying deactivation to negative words (green bar) under citalopram. Percent signal change plotted on the y axis is extracted from the mean activation voxel taken from the whole brain analysis significant cluster in the negative vs. control words contrast. Signal from activation to positive words is included for illustration purposes. Regions showing increased activation in the placebo vs. citalopram group in the group x emotion interaction in the contrast of negative self-descriptor words vs. control words; MNI coordinates refer to the mean activation voxel of the cluster and the peak activation voxels of main sub-regions within the same cluster.

Table 1 . Personality and affect ratings

 1 All questionnaires completed at baseline; Visual Analogue Scales (VAS) and Befindlichskeit Scale (BFS) scores reported refer to measures on day 7 prior to testing fMRI scan. EPQ= Eysenck personality Questionnaire, STAI= Spielberger Stait and Trait Anxiety Inventory, BDI=Beck depression Inventory, DAS=Dysfunctional Attitudes Scale, LES=Life Events Scale.

	Measure a	Placebo (n=16) Citalopram (n=18)	Analysis
	EPQ neuroticism	17.2 (1.6)	18.32 (2.3)	t (32) =-1.564, p=.128
	STAI Trait Anxiety	43.9 (5.7)	49.7 (10)	t (32) =-1.92, p=.064
	STAI State Anxiety	37.4 (9.7)	42.6 (10.4)	t (32) =-1.485, p=.147
	BDI	7 (5.7)	10 (5.9)	t (32) =-1.445, p=.158
	DAS total	138.3 (19)	141.5 (26)	t (32) = -.384, p=.703
	LES total	1 (1.1)	1.5 (1.5)	t (32) =-1, p=.323
	Day 7 -BFS	30.8 (25)	34.5 (22)	t (32) =-.432, p=.669
	Day 7 -VAS happiness	7.7 (1.5)	7.6 (2.3)	t (32) =.186, p=.853
	Day 7 -VAS sadness	3 (2.2)	1.9 (1.9)	t (32) =1.508, p=.142
	Day 7 -VAS hostility	2.5 (2)	1.3 (1.6)	t (32) =1.812, p=.080
	Day 7 -VAS alertness	6.2 (2.9)	6.5 (2.6)	t (32) =-.261, p=.796
	Day 7 -VAS anxiety	3.5 (2.6)	3.8 (2.8)	t (32) = -.331, p=.743
	Day 7 -VAS calmness	6.4 (2.6)	5.6 (2.7)	t (32) =.787, p=.437

a

Table 2 . Behavioural performance on self-descriptors categorisation task

 2

		Measure a	Placebo (n=16)	Citalopram (n=18)
	Negative self-	Correct matching	84.1 (15)	80.5 (22)
	descriptors	RT (ms)	1282.6 (282)	1285.9 (223)
	Positive self-	Correct matching	93.9 (7)	91.8 (13)
	descriptors	RT (ms)	946.6 (227)	967.7 (247)
		Correct matching	94.6 (5.8)	95.2 (11.6)
	Control words
		RT (ms)	887.2 (196)	862.4 (137)
	a Values show Mean (SD).

Table 3 . Areas of significant activation during categorisation of negative self-descriptors Brain Region a

 3

		Broadmann Area	Cluster size (voxels)	Z value	x	y	z
	Ventromedial Prefrontal Cortex		565	3.41	0	40	16
	Rostral Anterior Cingulate Cortex	32		3.29	30	36	-20
	Frontopolar Cortex	10		3.06	20	52	-12
	Orbitofrontal cortex	11		3.05	20	38	-20
	a

Acknowledgements

The study was supported by a Medical Research Council grant to Dr Catherine Harmer.

Conflict of Interest


Beatrix Elsberger

PhD Louise Lankston

BSc Donald C Mcmillan

PhD Mark A Underwood

MD Joanne Edwards
email: j.edwards@clinmed.gla.ac.uk

Presence of tumoural C-reactive protein correlates with progressive prostate cancer

Keywords: C-reactive protein, prostate cancer, immunohistochemistry, biochemical relapse, disease specific survival

C-reactive protein (CRP) is an acute phase protein implicated in the progression of cancer. A positive correlation between tumour stage and plasma CRP levels was demonstrated in prostate cancer, indicating a relationship between raised CRP levels and more aggressive disease suggesting a role for inflammatory response in tumour progression.

Aim of this study was to assess the tumoural presence and cellular location of CRP and establish if these are linked to clincopathological features of the cohort and patient survival.

Tissue microarray technology was employed to analyse 50 matched pairs of hormone sensitive and refractory prostate cancers. Immunohistochemistry was performed using antibody to CRP. CRP was assessed using the weighted histoscore method.

CRP presence was observed in the cytoplasm and nucleus of selected tumours.

Cytoplasmic CRP correlated positively with metastases at diagnosis (p=0.039), whereas nuclear CRP presence correlated with metastases at relapse (p=0.006). A trend towards an increase in cytoplasmic and nuclear CRP presence from hormone sensitive to hormone refractory tumours was noticed. No significant association between tumoural CRP presence, time to biochemical relapse or disease specific survival was observed.

Tumoural CRP is likely to play a role in progression of prostate cancer, as it is associated with increased presence of metastases at time of diagnosis and time of relapse. A larger powered study is necessary to establish if CRP presence is associated with disease specific survival.

Introduction

Prostate cancer has the highest incidence and the second highest mortality of any cancer affecting UK males. Whilst the 5 year relative survival for organ confined prostate cancer is over 90%, for locally advanced or metastatic disease it is only around 30% 1 . The mainstay of treatment for locally advanced or metastatic prostate cancer is androgen deprivation therapy 2 . Unfortunately this therapy is only effective for a median of 18 months, following which the patients relapse and the tumours are able to growth in a castrate environment [START_REF] Jin | The nuclear factor-kappaB pathway controls the progression of prostate cancer to androgen-independent growth[END_REF] . At this stage they are termed castrate resistant (CRPC). Currently there is no definitive curative treatment for castrate resistant prostate cancers 2 , however taxane therapy can extend survival for a median time period of 3 months. C-reactive protein (CRP) is an acute phase protein implicated in the progression of prostate cancer. CRP is a member of the pentraxin family and composed of five identical polypeptide units which confer a very high stability to the molecule. Plasma CRP is mainly produced by hepatocytes in response to inflammatory cytokines, particularly IL-6 and IL-1. Due to its high stability, CRP has a relatively long plasma half life of 19 hours, and is a naive systemic marker of inflammation [START_REF] Pepys | C-reactive protein: a critical update[END_REF][START_REF] Yang | Human C-reactive protein binds activating Fcgamma receptors and protects myeloma tumor cells from apoptosis[END_REF] . Elevated plasma CRP levels are found in many disease processes including infection, myocardial infarction and malignancies [START_REF] Yang | Human C-reactive protein binds activating Fcgamma receptors and protects myeloma tumor cells from apoptosis[END_REF] . Elevated circulating CRP levels have been shown to be linked to poor prognosis in several cancers including multiple myeloma, melanoma, lymphoma, ovarian, renal, pancreatic, lung and gastrointestinal tumours [START_REF] Beer | C-reactive protein as a prognostic marker for men with androgen-independent prostate cancer: results from the ASCENT trial[END_REF] . In metastatic and castrate resistant prostate cancer elevated plasma CRP levels have been recorded and shown to be independent predictors of poor survival and to be associated with a lower probability of response to chemotherapy [START_REF] Mcardle | Systemic inflammatory response, prostatespecific antigen and survival in patients with metastatic prostate cancer[END_REF][START_REF] Jabs | Expression of C-reactive protein by renal cell carcinomas and unaffected surrounding renal tissue[END_REF] .

A positive correlation between tumour stage and plasma CRP levels has been demonstrated in both renal cell carcinoma and prostate cancer [START_REF] Mcardle | Systemic inflammatory response, prostatespecific antigen and survival in patients with metastatic prostate cancer[END_REF][START_REF] Jabs | Expression of C-reactive protein by renal cell carcinomas and unaffected surrounding renal tissue[END_REF] . This indicates a relationship between raised CRP levels and more aggressive disease suggesting a role for inflammatory response in tumour progression. But as yet the mechanism for this is unexplained.

It is hypothesised that CRP exerts its effect via activation of NF-кB pathway. NF-кB presence has been linked with invasion and metastasis in prostate cancer and has been shown to be upregulated in castrate resistant prostate cancer [START_REF] Edwards | The role of HER1-HER4 and EGFRvIII in hormone-castrate resistant prostate cancer[END_REF][START_REF] Jin | The nuclear factor-kappaB pathway controls the progression of prostate cancer to androgen-independent growth[END_REF] . NF-кB is also believed to have a proinflammatory role; its activation in tumour associated inflammatory cells has been shown to contribute to tumour growth by inducing synthesis of tumour promoting proinflammatory mediators [START_REF] Huang | Blockade of NF-kappaB activity in human prostate cancer cells is associated with suppression of angiogenesis, invasion, and metastasis[END_REF] . In vitro evidence from human hepatoma cell lines also suggests a link between NF-кB and CRP, suggesting that NF-кB participates in C-reactive protein production. Other work done in hepatoma cell lines and coronary artery smooth muscle cells looking at IL-17 stimulation of C-reactive protein production again showed this relationship between NF-кB and CRP [START_REF] Li | Inflammation-associated cancer: NF-kappaB is the lynchpin[END_REF] .

Recent work has shown that CRP levels are not only increased systemically but also locally within the tumour. Jabs et al showed that circulating CRP levels correlated with CRP mRNA expression in renal cell carcinoma and Nozoe has provided immunohistochemical evidence of CRP production by squamous cell carcinoma of the oesophagus [START_REF] Jabs | Expression of C-reactive protein by renal cell carcinomas and unaffected surrounding renal tissue[END_REF][START_REF] Nozoe | Immunohistochemical expression of Creactive protein in squamous cell carcinoma of the esophagus -significance as a tumor marker[END_REF] . The origin of this "tumour C-reactive protein" is poorly explained.

This study seeks to determine the relationship between tumoural CRP presence, interrelationship to clinicopathological features of the patient cohort and cancer specific survival in the progression of prostate cancer from hormone naive to castrate resistant tumours.

Material and Methods:

Patient Cohort

Ethics approval was granted to this study by the Scottish multi-centre research ethics committee.

Patients with matched hormone naive and castrate resistant tumour samples were retrospectively selected for analysis. To be selected for the study, patients had to have initially responded to hormone deprivation therapy, defined as a decrease of PSA level of 50%, and then gone on to relapse, described as two consecutive rises in PSA level of over 10%. A pre and post sample had to be available for analysis. Pre-hormone naive samples were obtained from patients diagnosed with locally advanced or metastatic prostate cancer who received surgery followed by androgen deprivation or maximum androgen blockade therapy. The hormone naive samples were obtained from a TURP or TRUS guided biopsy.

Patients then had to relapse. Post-hormone samples could only be collected if patients required TURP to relieve clinical symptoms (e.g. bladder outflow obstruction). This tissue was then utilised to construct a tissue microarray (TMA).

Tissue microarray construction

Tissue microarrays were already available for use in the current study. Two 0.6mm 2 cores of lowest and highest Gleason grade prostate cancer tissue, identified by the pathologist, were removed from representative areas of the tumour taken from prostate cancer patients. All TMA blocks were constructed in duplicate. For missing samples and samples not on the TMA due to insufficient tissue 4 µm sections taken from the original paraffin blocks were used.

Immunohistochemistry-anti C-Reactive Protein

Sections were dewaxed and rehydrated through a series of xylene and alcohol washes. Heat mediated antigen retrieval was performed in Tris EDTA buffer (pH 8) for 20 minutes in a DAKO microwave. Sections were incubated in 5% blocking solution at 25°C for 20 minutes before being incubated in 1:100 dilution of primary antibody at 25°C for 2 hours. The primary antibody for detecting C-reactive protein was rabbit monoclonal [Y284] to C-reactive protein (Abcam plc., Cambridge, UK). Antibody antigen binding was detected using the peroxidasebased Envision (Dako, Cambridgeshire, UK) technique using a diaminobenzidine chromogenic substrate system. Sections were then counterstained using Haematoxylin, dehydrated and mounted in DPX. In each run a positive and negative isotype matched control was included to ensure no false positive staining.

Immunohistochemistry-Scoring

Protein presence of each core (4 per tumour specimen) was assessed using the weighted histoscore method (H score method). The weighted histoscore method grades staining intensity as negative (0), weak (1), moderate (2) and strong (3) and then multiplies the percentage of tumour cells within each category by the number assigned to that category (0, 1, 2 or 3 respectively) [START_REF] Kirkegaard | Observer variation in immunohistochemical analysis of protein expression, time for a change?[END_REF] . The histoscore ranges from zero (minimum) to 300 (maximum).

All TMAs were scored independently by two observers for their nuclear and cytoplasmic protein staining within each tumour core. Agreement between observers was good and measured in interclass correlation coefficient (ICCC). All ICCC scores were above 0.7.

Western Blot

Western blots were performed to ensure the specificity of the antibody used in the immunohistochemistry study.

Purified Heat-Shock Protein (HSP) 90 (Abcam plc, Cambridge), C-Reactive Protein (Abcam plc, Cambridge), and colorectal cell lysates (Abnova, Taiwan) were resolved by 4-12% gradient Bis-Tris gel electrophoresis (Invitrogen, UK); proteins were transferred to nitrocellulose membranes (Millipore, UK), blocked for 1 hour in 5% Marvel in TTBS and probed with primary antibody, anti-CRP (1:1000 Abcam, UK) or anti-HSP90 (1:750 AbCam, UK at 4°C overnight. Membranes were incubated with secondary antibodies (anti-rabbit 1:2000, Cell Signalling Technologies, UK) and 10ul of anti-biotin (Amersham, UK) and visualized with ECL kit (Amersham, UK). The membranes were stripped by incubating with Re-Blot Plus stripping buffer (Chemicon, UK) before re-probing with primary antibody, anti-HSP-90 (1:25 Abcam, UK) at 4°C overnight. Membranes were incubated with secondary antibodies (anti-mouse 1:10000, Cell Signalling Technologies, UK) and 10ul of anti-biotin (Amersham, UK) and visualized with ECL kit (Amersham, UK) (figure 1).

Statistical Analysis

Wilcoxon Signed Rank Tests were used to compare CRP presence between androgen naive and androgen innaive tumours. Kaplan-Meier curves generated time to relapse, time to death from relapse and disease specific survival rates. The log rank test was used to compare significant differences between subgroups using univariate analysis. A p-value of 0.05 was deemed to be significant. Data are expressed as median and range. The statistical analyses were performed using a statistical software package (SPSS 15.0 Inc., Chicago, IL, USA).

Results:

Clinicopathological details of patient cohort

The cohort consisted of 50 patients. Matched data pairs were available for 42 patients.

At initial diagnosis the median age of patients was 70 years (Interquartile range (IQR) 65.5-73.0 years). Median Gleason score was 7.0 (IQR 6.5-9.0) and 15 patients (35.7%) had metastases. Median time to biochemical relapse from diagnosis was 2.59 years (IQR 1.67-4.05 years) and median overall survival was 4.39 years (IQR 3.34-6.74). Patients with locally advanced (66%) or metastatic prostate cancer (34%) subsequently received surgery and androgen deprivation therapy (14 sub capsular bilateral orchidectomy, 27 GnRH analogue, 1 had both). Twenty-nine of the 42 patients also received anti androgen therapy and this included all those who received GnRH analogues. On univariate analysis tumoural PSA expression (p<0.001) was associated with shorter time to biochemical relapse and overall survival was associated with presence of metastases at relapse (p=0.013).

During the period of follow up, 36 patients died of their cancer, a further 10 patients died of intercurrent disease, 1 patient was lost to follow. At the end of the follow up period 3 patients were still alive. Correlations between the clinicopathological characteristics of this cohort are shown in table 1a and 1b.

CRP presence in hormone naive prostate tumours

Due to tissue limitation CRP presence was successfully assessed in 86% of hormone naive tumour samples (43/50). From this 86%, 79.1% (34/43) exhibited cytoplasmic and 27.9% (12/43) nuclear CRP presence (figure 2a). Tumours were subdivided into high or low presence of CRP according to median values of the protein being assessed. Chi square analysis was then performed to assess correlation between clinical parameters and protein presence (table 1a). Presence of CRP in the cytoplasm correlated positively with metastases at diagnosis (p=0.039, table 1a). Interestingly, cytoplasmic CRP presence was not shown to correlate with nuclear CRP presence.

CRP presence in castrate resistant prostate tumours

Due to tissue limitation CRP presence was successfully assessed in 82% of castrate resistant tumour samples (41/50). From this 82%, 75.6% (31/41) displayed cytoplasmic and 42.9 %(18/42) nuclear CRP presence (figure 2b). Again tumours were subdivided into high or low CRP presence according to median values of the protein being assessed. Chi square analysis was then performed to assess correlation between clinical parameters and protein presence (table1b). Nuclear CRP presence correlated with metastases at relapse (p=0.006, table 1b).

Change in CRP presence in the transition from hormone naive to castrate resistant disease

In order to determine if levels of CRP presence changed in the transition from hormone naive prostate cancer to castrate resistant disease, boxplots were constructed and levels were compared hormone naive tumours to castrate resistant tumours using the Wilcoxon Signed Ranks Tests. Whilst these results did not reach significance, there was a trend towards an increase in presence of cytoplasmic and nuclear C-reactive protein noted (figure 3a and3b).

As the hormone naive and castrate resistant tumour samples are matched in this cohort, it was possible to analyse changes of CRP levels in pre and post hormone escape on an individual patient basis. In order for a change in CRP presence to be deemed significant, the difference had to be greater than 50. This value was chosen as it is the threshold used to judge acceptable levels of interobserver variability and is unlikely to be a chance finding.

This information can then be used to divide patients into those with a decrease in CRP presence, those with no change and those with an increase.

Survival Analysis

Kaplan-Meier survival curves were generated and Log rank tests performed to assess if presence of CRP within the tumour was related to time to relapse or disease specific survival in hormone naive prostate cancer or time to death from relapse or disease specific survival in castrate resistant prostate cancer. Again, CRP presence was divided into high or low according to median values. No significant association was seen in the hormone naïve prostate cancer patient cohort between CRP presence, time to biochemical relapse (cyto CRP p=0.392, nuc CRP p=0.998; figure 4a and4b) and disease specific survival (cyto CRP p=0.0.830, nuc CRP p=0.0.481; figure 4c and4d). In the castrate resistant patient cohort no significant associations with time to death from relapse (cyto CRP p=0.946, nuc CRP p=0.783; figure 4e and4f) and disease specific survival (cyto CRP p=0.917, nuc CRP p=0.139; figure 4g and4h) was observed either.

Increased CRP presence in the cytoplasm was noticed in 4 patients, whereas 2 patients showed a decrease in cytoplasmic CRP. However the majority of patients (n=26) had no significant changes CRP presence in the cytoplasm. None of patients showed a significant change in presence of CRP in the nucleus. Therefore due to the low patient numbers demonstrating significant changes in tumoural CRP presence, survival analysis was not performed.

Discussion:

Currently it is hypothesised that CRP plays an important role in the progression of prostate cancer and is associated with a more aggressive and invasive phenotype. The results of the current study demonstrate that cytoplasmic CRP in hormone naive tumours significantly correlates with presence of metastases at cancer diagnosis. Serum levels of CRP have previously been linked with metastases in prostate cancer [START_REF] Lehrer | C-reactive protein is significantly associated with prostate-specific antigen and metastatic disease in prostate cancer[END_REF] . This present study also established a correlation between metastases at relapse and nuclear CRP presence.

To our knowledge, there is currently no literature available examining the cellular location of CRP in prostate tumour specimens. It is not established if cellular location of CRP influences clinical outcome of prostate cancer patients. Multiple proteins (Akt, NF-kappa-B, IKK alpha) are known to have diverse roles in different locations of the cell. Src kinase, when activated at tyrosine site 419, and highly expressed in breast cancer tissue at cellular membrane is associated with shorter disease specific survival [START_REF] Elsberger | Is expression or activation of Src kinase associated with cancer-specific survival in ER-, PR-and HER2-negative breast cancer patients?[END_REF] .

In order to study the role of CRP in the progression of prostate cancer, CRP presence was assessed before and after progression of the tumour. There was no statistically significant difference between CRP presence before and after castrate resistance. However there was a trend towards increased CRP presence in castrate resistant prostate cancer specimens noted. This could be likely due to low patient numbers and warrants further work in expanding the patient cohort.

There are limitations of examining the biology of the primary tumour versus its metastases. It would be of interest to investigate CRP presence and expression profile of primary tumours with matched metastasis to provide insight regarding change of CRP. Recent literature suggests that the cancer's biology at its metastatic sites differs from the primary tumour. Erin et al. found altered gene expression in breast cancer liver metastasis [START_REF] Erin | Altered gene expression in breast cancer liver metastases[END_REF] , whereas Koo et al.

established different immunohistochemical breast cancer phenotypes expression according

to metastatic site [START_REF] Koo | Metastatic breast cancer shows different immunohistochemical phenotype according to metastatic site[END_REF] . Not just the potential dissimilar biology of the primary to the metastasis needs to be considered, but also the possibility that metastatic sites may be even more predictive of prostate cancer patients' clinical outcome than primary tumours.

No significant association was seen between CRP presence within the tumour and disease specific survival. This is most likely due to this study being underpowered to detect significant associations with survival. The majority of available literature relating high Creactive protein expression with decreased survival uses serum levels of CRP to correlate with patient survival [START_REF] Nozoe | Preoperative elevation of serum C-reactive protein as an independent prognostic indicator of colorectal carcinoma[END_REF] .

Recent work has shown that C-reactive protein levels are not only increased systemically but also locally within the tumour. Jabs et al showed that circulating C-reactive protein levels correlated with CRP mRNA expression in renal cell carcinoma and Nozoe has provided immunohistochemical evidence of CRP production by squamous cell carcinoma of the oesophagus [START_REF] Jabs | Expression of C-reactive protein by renal cell carcinomas and unaffected surrounding renal tissue[END_REF][START_REF] Nozoe | Immunohistochemical expression of Creactive protein in squamous cell carcinoma of the esophagus -significance as a tumor marker[END_REF] . The origin of this "tumour C-reactive protein" is unclear. It has been suggested that the tumour is responsible for increased IL-6 production which correlates with CRP production [START_REF] Nozoe | Preoperative elevation of serum C-reactive protein as an independent prognostic indicator of colorectal carcinoma[END_REF][START_REF] Mcardle | The relationship between T-lymphocyte subset infiltration and survival in patients with prostate cancer[END_REF] . However McArdle et al propose an alternative explanation, suggesting that the source of the IL-6 is the host inflammatory cells rather than the tumour [START_REF] Mcardle | The relationship between T-lymphocyte subset infiltration and survival in patients with prostate cancer[END_REF] . Indeed, increased CD4+ T-lymphocyte infiltration in prostate cancer has been associated with poor cancer specific survival, independent of stage [START_REF] Huang | Blockade of NF-kappaB activity in human prostate cancer cells is associated with suppression of angiogenesis, invasion, and metastasis[END_REF] . We have performed RT-PCR for CRP in 10 clinical prostate specimens and have used liver tissue as a positive control in this study mRNA CRP was undetectable in prostate tumour samples (unpublished results). This therefore suggests that the tumour cells are taking up CRP from their surrounding environment rather producing it.

The role of tumoural presence of CRP or even tumour expression of CRP, which was predicted to be linked to survival, is a relatively novel concept. Low patient numbers and small range of histoscores made classification of patients into sufficiently large subgroups for statistically analysis difficult and could be possible reasons why this study did not show a significant association between relapse, survival and CRP presence in prostate tumours.

Conclusions:

This study demonstrates that tumoural CRP presence is likely to play a role in progression of prostate cancer, being associated with presence of metastases at time of diagnosis and time of relapse. However this study does not show a significant association between tumoural CRP presence, biochemical relapse and patient survival. CRP presence in this cohort fails to predict time to relapse or disease specific survival. Despite of this, CRP remains key potential target as driver of cancer progression. Further work is required in a larger cohort to definitively determine the association of tumoural presence and location of CRP, biochemical relapse and survival, to explore the relationship between serum CRP and presence of CRP within the tumour itself and to elucidate the possible roles of involved signalling pathways.

Figure legend Figure 1 :

 legend1 Figure legendFigure 1: Western Blots for CRP and HSP90 antibody specificity Figure 1a: Panel 1 shows a western blot probed with antibody to C-reactive protein, against purified HSP 90 lane 1 and purified C-reactive protein. Panel 2 shows a western blot probed with antibody to HSP-90, against purified HSP 90 lane 1 and purified C-reactive protein. These western blots show that the C-reactive protein antibody only detects C-reactive protein and does not cross react with HSP 90.

Figure 1b :

 1b Figure 1b: Panel 1 shows a western blot probed with antibody to C-reactive protein, against; lane 1 purified C-reactive protein, lane 2 nuclear fraction of cell lysates extracted from colon tumour cells, lane 3 cytoplasmic fraction of cell lysates extracted from colon tumour cells and lane 4 whole cell lysates extracted from colon tumour cells. Panel 2 shows a western blot probed with anti body to HSP-90, against; lane 1 purified C-reactive protein, lane 2 nuclear fraction of cell lysates extracted from colon tumour cells, lane 3 cytoplasmic fraction of cell lysates extracted from colon tumour cells and lane 4 whole cell lysates extracted from colon tumour cells. These western blots show that the C-reactive protein antibody is not expressed in colon tumour cells.

Figure 2 :

 2 Figure 2: IHC pictures demonstrating the distribution of CRP in hormone naive (Fig 2a) and hormone castrate resistant (Fig 2b) prostate cancer samples.

Figure 3 :

 3 Figure 3: Boxplots illustrating the difference of cytoplasmic CRP (Fig 3a) and nuclear CRP (Fig 3b) in hormone naive and hormone castrate resistant prostate cancer specimens.

Figure 4 :

 4 Figure 4: Recurrence and Survival graphs for hormone naive and castrate resistant prostate cancer cohorts. Bold line: patients with high CRP presence; dotted line: patients with low CRP presence; cyto CRP= cytoplasmic CRP; nuc CRP= nuclear CRP.

Figure

  Figure 4a and 4b: No significant association noted between CRP presence in the cytoplasm and nucleus and time to biochemical relapse in hormone naïve prostate cancer patients.

Figure 4c and 4d :

 4d Figure 4c and 4d: No significant association observed between CRP presence in the cytoplasm and nucleus and disease specific survival of hormone naïve prostate cancer patients.

Figure 4e and 4f :

 4f Figure 4e and 4f: No significant association was seen between CRP presence in the cytoplasm and nucleus of castrate resistant prostate cancer specimens and time to death from biochemical relapse.

Figure 4g and 4h :

 4h Figure 4g and 4h: No significant association was observed CRP presence in the cytoplasm and nucleus of castrate resistant prostate cancer specimens and disease specific survival.

Table 1a : The interrelationships between the clinicopathological characteristics of patients with hormone naive prostate cancer

 1a

	Variables	Gleason	Metastases at	Metastases at	PSA at	CRP	CRP
		score	Diagnosis	Relapse	Relapse	cyto	nuclear
		<7 / ≥7	Yes/no	Yes/No	≤4 / >4	High/Low	High/Low
	Age
	<70yrs / >70yrs	NS	NS	0.012	0.010	NS	NS
	Gleason score
	<7 / ≥7		0.005	NS	NS	NS	NS
	Metastases at
	Diagnosis			0.041	NS	0.039	NS
	Yes/No
	Metastases at
	Relapse				NS	NS	NS
	Yes/No
	PSA at Relapse
	≤4 / >4					NS	NS
	CRP cyto
	High/Low						NS

Table 1b : The interrelationships between the clinicopathological characteristics of patients with hormone castrate resistant prostate cancer

 1b Table1ashows the interrelationship between CRP presence at different cellular levels and clinicopathological characteristics of patients with hormone naive prostate cancer; Table1bdemonstrates the interrelationship between CRP presence at different cellular levels and

	clinicopathological characteristics of patients with hormone castrate resistant prostate
	cancer; CRP cyto= cytoplasmic CRP presence. p-values are deemed non-significant (NS) if
	Variables above 0.05	Gleason	Metastases at	Metastases at	PSA at	CRP	CRP
		score	Diagnosis	Relapse	Relapse	cyto	nuclear
		<7 / ≥7	Yes/No	Yes/No	≤4 / >4	High/Low	High/Low
	Age
	<70yrs / >70yrs	NS	NS	0.012	0.010	NS	NS
	Gleason score
	<7 / ≥7		0.005	NS	NS	NS	NS
	Metastases at
	Diagnosis			NS	NS	NS	NS
	Yes/No
	Metastases at
	Relapse				NS	NS	0.006
	Yes/No
	PSA at Relapse
	≤4 / >4					NS	NS
	CRP cyto
	High/Low						NS

Acknowledgement:

This study was supported by the Glasgow Royal Infirmary Endowment Grant.