PhD Nigel C Jones
email: ncjones@unimelb.edu.au

Maya Reddy

Paul Anderson

Michael R Salzberg

Terence J O'brien

Didier Pinault

Keywords: antipsychotic, gamma power, ketamine, schizophrenia, EEG

Acute administration of typical and atypical antipsychotics reduces EEG γ power, but only the

preclinical compound LY379268 reduces the ketamine-induced rise in γ power.

Acute administration of typical and atypical antipsychotics reduce EEG gamma power, but only the preclinical compound LY379268 reduces the ketamineinduced rise in gamma power.

Submitted as a regular research article.

Introduction

High-frequency neuronal network oscillations in the brain, including those in the gamma band , represent collections of neurons firing in synchrony linking different brain regions or local circuits [START_REF] Buzsaki | Neuronal oscillations in cortical networks[END_REF]. The functional role of high-frequency gamma oscillations has been linked to a diverse range of higher order brain function, including cognition [START_REF] Engel | Dynamic predictions: oscillations and synchrony in top-down processing[END_REF], sensory binding of information [START_REF] Joliot | Human oscillatory brain activity near 40 Hz coexists with cognitive temporal binding[END_REF], working memory tasks [START_REF] Howard | Gamma oscillations correlate with working memory load in humans[END_REF][START_REF] Tallon-Baudry | Induced gammaband activity during the delay of a visual short-term memory task in humans[END_REF] sensory perception [START_REF] Gross | Gamma oscillations in human primary somatosensory cortex reflect pain perception[END_REF]. Schizophrenia, a complex heterogeneous psychiatric disorder, is characterised by disturbances in many of such higher order brain functions, including perception and distortions of reality. The proposed role of these gamma oscillations in conscious integration, sensory perception and cognition has led to the conceptualization that abnormalities in gamma frequency activity may underlie many aspects of schizophrenia symptomatology (Lee et al., 2003b), and indeed the pathophysiology of the disease [START_REF] Herrmann | Human EEG gamma oscillations in neuropsychiatric disorders[END_REF]Lee et al., 2003a).

Ketamine and other NMDA receptor (NMDAr) antagonists at subanaesthetic doses induce perceptual distortions and visual hallucinations in healthy humans, and exacerbate psychotic symptoms in schizophrenic patients [START_REF] Krystal | Subanesthetic effects of the noncompetitive NMDA antagonist, ketamine, in humans. Psychotomimetic, perceptual, cognitive, and neuroendocrine responses[END_REF][START_REF] Lahti | Subanesthetic doses of ketamine stimulate psychosis in schizophrenia[END_REF][START_REF] Malhotra | Ketamine-induced exacerbation of psychotic symptoms and cognitive impairment in neuroleptic-free schizophrenics[END_REF]. These observations informed the NMDAr hypofunction hypothesis of schizophrenia, whereby reduced functionality at this receptor in some way manifests as schizophrenic-like symptoms. We [START_REF] Hakami | NMDA Receptor Hypofunction Induces Generalized and Persistent Aberrant  Oscillations Independent of Hyperlocomotion and Conscious Sensorimotor Processing[END_REF][START_REF] Pinault | N-methyl d-aspartate receptor antagonists ketamine and MK-801 induce wake-related aberrant gamma oscillations in the rat neocortex[END_REF] and others [START_REF] Ehrlichman | N-methyl-d-aspartic acid receptor antagonist-induced frequency oscillations in mice recreate pattern of electrophysiological deficits in schizophrenia[END_REF][START_REF] Lazarewicz | Ketamine Modulates Theta and Gamma Oscillations[END_REF][START_REF] Ma | The supramammillo-septal-hippocampal pathway mediates sensorimotor gating impairment and hyperlocomotion induced by MK-801 and ketamine in rats[END_REF] previously demonstrated that, in rodents, NMDAr antagonists dosedependently increase the power of ongoing gamma frequency oscillations. We also demonstrated that this effect is correlated with the hyperlocomotor response induced by these compounds [START_REF] Hakami | NMDA Receptor Hypofunction Induces Generalized and Persistent Aberrant  Oscillations Independent of Hyperlocomotion and Conscious Sensorimotor Processing[END_REF], a commonly employed model of acute psychosis (van den [START_REF] Van Den Buuse | Importance of animal models in schizophrenia research[END_REF]. These correlative findings linking druginduced hyperlocomotor activity to increases in gamma power in rats, and the clinical observations implicating gamma power abnormalities to psychotic symptoms in schizophrenic patients [START_REF] Baldeweg | Gamma-band electroencephalographic oscillations in a patient with somatic hallucinations[END_REF][START_REF] Gordon | Symptom profile and 'gamma' processing in schizophrenia[END_REF][START_REF] Spencer | Left auditory cortex gamma synchronization and auditory hallucination symptoms in schizophrenia[END_REF]Spencer et al., 2008b), suggest that NMDAr antagonist-induced increases in gamma power may represent an electrophysiological correlate of acute psychosis (Lee et al., 2003a).

The current study aimed to test the hypothesis that typical and atypical antipsychotic medications, both clinical and preclinical, acutely inhibit the increase in ongoing gamma power induced by the NMDAr antagonist ketamine, thereby presenting this as a predictive marker of antipsychotic activity. Our experimental design also allowed assessment of the effects of the antipsychotics alone on gamma power. Three compounds were chosen to test these hypotheses, each possessing a different spectrum of receptor binding and therapeutic mechanism of action, to gauge the effect of different classes of antipsychotic drugs: (i) haloperidol, a typical neuroleptic agent with potent antagonist activity at dopamine D 2 receptors [START_REF] Miyamoto | Treatments for schizophrenia: a critical review of pharmacology and mechanisms of action of antipsychotic drugs[END_REF]; (ii) clozapine, the classic atypical antipsychotic with a diverse pharmacological profile including antagonism at 5-HT receptors (particularly 5-HT 2A receptors), muscarinic and histamine receptors, and also interacts with the glycine site of NMDA receptors [START_REF] Schwieler | Clozapine interacts with the glycine site of the NMDA receptor: electrophysiological studies of dopamine neurons in the rat ventral tegmental area[END_REF], but with weaker binding affinity for dopamine D 2 receptors [START_REF] Miyamoto | Treatments for schizophrenia: a critical review of pharmacology and mechanisms of action of antipsychotic drugs[END_REF]; and (iii) LY379268, a preclinical drug which is an agonist at metabotropic (mGluR 2/3 ) glutamate and dopamine D 2/3 receptors [START_REF] Seeman | Glutamate agonists for treating schizophrenia have affinity for dopamine D2High and D3 receptors[END_REF], and possesses efficacy in animal models of psychosis [START_REF] Cartmell | Attenuation of specific PCP-evoked behaviors by the potent mGlu2/3 receptor agonist, LY379268 and comparison with the atypical antipsychotic, clozapine[END_REF][START_REF] Imre | The preclinical properties of a novel group II metabotropic glutamate receptor agonist LY379268[END_REF]. The primary electrophysiological outcome was baseline, or resting-state, gamma power as measured before and after drug administration, in keeping with our previous studies. This is in contrast with evoked or induced gamma power, such as those measured in response to an auditory stimulus [START_REF] Uhlhaas | Abnormal neural oscillations and synchrony in schizophrenia[END_REF]. Ongoing gamma power is an important, but commonly overlooked, measurement, since many studies examining evoked/induced gamma responses incorporate some form of 'baseline' correction.

METHODS

Animals

Male Wistar rats (total n=26; 250-350g) were bred and group-housed (2-3 per cage) in the Biological Research Facility of the Department of Medicine, Royal Melbourne Hospital, The University of Melbourne. The facility was kept on a 12 hour light/dark cycle, with lights on at 6am, and food (standard rat chow) and water available to animals ad libitum. At all times, care was taken to minimize pain and discomfort of the animals, and all experimental procedures were approved by the University of Melbourne Animal Ethics Committee (#0701821).

EEG Recording Electrode Implantation

Animals were anaesthetized by inhalation of isoflurane in equal parts of medical air and oxygen (5% induction, 1.5-2.5% maintenance) and positioned in a stereotaxic frame as described previously [START_REF] Hakami | NMDA Receptor Hypofunction Induces Generalized and Persistent Aberrant  Oscillations Independent of Hyperlocomotion and Conscious Sensorimotor Processing[END_REF][START_REF] Pinault | N-methyl d-aspartate receptor antagonists ketamine and MK-801 induce wake-related aberrant gamma oscillations in the rat neocortex[END_REF]. Briefly, a single midline incision was made over the scalp and six holes were drilled through the skull for stereotaxic implantation [START_REF] Paxinos | The Rat Brain in Stereotactic Coordinates[END_REF] of recording brass electrodes (2mm anterior and 2mm lateral to bregma bilaterally (active electrodes); 2mm posterior and 2mm lateral to bregma bilaterally (ground electrodes); and 2mm posterior and 2mm lateral to lambda bilaterally (reference electrodes). Brass electrodes were then screwed into the skull without breaching the dura, and dental cement applied to the skull to fix the electrodes in place. After recovery from anaesthesia, animals were placed in separate cages for at least 7 days prior to further experimentation.

Assessment of drug effects on EEG gamma power and locomotor activity

On the day of experimentation, rats were brought into the Behavioural Testing Facility in the Department of Medicine at least 30 minutes prior to the start of the study to allow for habituation to the environment. Rats were then individually placed into an open arena (1 m diameter) with each recording electrode attached to a cable suspended from the ceiling to facilitate continuous recording of the EEG. Each rat was allowed to explore the arena for 30 minutes, at which time they were injected subcutaneously (sc) with either haloperidol (0.025 -0.25 mg/kg; n=8), clozapine (1-5 mg/kg sc, n=7), LY379268 (0.3 -3 mg/kg sc; n=5) or the appropriate vehicles. Following a further 30 minutes, rats were then injected with ketamine (5mg/kg sc) or vehicle (0.9% saline) and returned to the arena for a further 60 minutes EEG recording. This procedure was repeated on subsequent days (with at least 2 days in between trials) with a different dose of antipsychotic until each rat had received each dose of its assigned antipsychotic. During the 120 minute recording period, the animal's locomotor activity was video-tracked and objectively assessed using Ethovision Software (Noldus®). Total distance traveled was calculated for every 2 minute interval.

A subsequent study was performed to assess the effects of a single administration of antipsychotic over a 24 hour period. These studies were performed in the home cages of the animals in the Biological Research Facility of the Department of Medicine, Royal Melbourne Hospital, The University of Melbourne, and were started between noon and 4pm. Animals (n=6) were connected to the EEG cables, and allowed a 30 minute habituation period prior to being injected with either haloperidol (0.25 mg/kg sc), clozapine (5 mg/kg sc), LY379268 (3 mg/kg sc) or vehicle (0.9% saline) in a randomised fashion, with at least 2 days in between experiments. Animals were left undisturbed for a further 24 hours of EEG recording following drug treatment.

EEG data acquisition processing

The EEG recordings from each hemisphere were acquired using a MacLab amplifier and A-D converter, and Chart V 3.5 software (ADInstruments, Bella Vista, NSW, Australia) with a bandpass of 1-1000 Hz. The line power 50 Hz noise was eliminated from the signal using selective eliminators (Humbugs; Digitimer, Letchworth Garden City, UK). The gamma frequency activity on the EEG recordings were analyzed using NEUROSCAN® software (Compumedics, Melbourne, Australia) as previously described [START_REF] Hakami | NMDA Receptor Hypofunction Induces Generalized and Persistent Aberrant  Oscillations Independent of Hyperlocomotion and Conscious Sensorimotor Processing[END_REF]. Briefly, using Fast Fourier Transformations (FFT), average power in the gamma frequency band (30-80 Hz) was determined for each 2.05 sec epoch for the entire recording period, and average power was then calculated for each 2 min interval. For both gamma power and locomotor activity data, the 15 values obtained during the 30 min baseline pre-treatment period were averaged for each individual animal, and all recorded values then expressed as a percent change of this baseline response. For the 24 hour recordings, the average power was calculated for each 5 minutes for the first 3.5 hours, and then for each 30 minutes thereafter, using the same 2.05 sec epoch determination as above, this time for all power bands, including gamma (30-80Hz), delta (1-4Hz), theta (4-8 Hz), alpha (8-13 Hz) and beta (13-30Hz) frequency oscillations. The first 6 values, representing the 30 minute baseline, were averaged for each individual animal, and all recorded values then expressed as a percent change of this baseline response, also as above.

Drugs and vehicles

Isoflurane was purchased from Abbott Pharmaceuticals (IL, USA). Clozapine (Sigma, St Louis, USA) was dissolved using pure acetic acid and then diluted with a 10% acetic acid solution that was neutralised to a pH of 6.0 using 10M NaOH. LY379268 (TOCRIS, Bristol, UK) was diluted in sterile water. Haloperidol (Sigma, St Louis, USA) and ketamine (Parnell Laboratories, Australia) were diluted in 0.9% sterile saline.

Statistical analyses

For both the gamma power and locomotor activity data, the values obtained during the 30 min pre-injection period were averaged for each individual animal, and all recorded values then expressed as a percent change from this baseline value. Two statistical analyses were designed for this study to answer the two separate study aims. The first determined the effect of exposure to antipsychotics on spontaneous ongoing EEG gamma power and locomotor activity. This was statistically assessed separately for each antipsychotic compound using two-way ANOVA with repeated measures (time after injection), with Bonferroni's post hoc analysis when appropriate. The second analysis was designed to assess the effect of antipsychotic drugs on ketamineinduced changes in gamma power and locomotor activity. This was also performed using two-way ANOVA with repeated measures (time after injection). However, since the antipsychotic drugs influenced the baseline gamma power, the trough-topeak rises of both gamma power and locomotor activity following ketamine administration were compared using a one-way ANOVA with repeated measures (dose of drug). Trough values corresponded to the 2 minute interval immediately prior to ketamine injection (i.e., t=58 min after trial initiation). Peak values were calculated as the point of greatest gamma power or locomotor response following ketamine injection, and were calculated for each individual animal and each drug dose. In ~88% of all cases, the peak response occured between 6-10 mins after ketamine injection. The trough-to-peak values were then determined by subtracting the value of the trough from the peak for each individual animal, and then averaged per group. It should be noted here that both the trough and the peak values of each recording comprised of the averages of 119 x 2.05 sec epochs, ensuring that sporadic experimental noise of a single epoch did not overly affect the trough or peak values of the group mean. Statistical comparisons were performed using GraphPad Prism®, and statistical significance was set at p<0.05 for all tests.

RESULTS

The antipsychotics haloperidol, clozapine and LY379268 reduce spontaneous ongoing cortical gamma power.

All three antipsychotics tested had a clear and rapid effect to decrease the power of spontaneous gamma power in the EEG, compared to vehicle treatments. Haloperidol significantly reduced gamma power in a time-and dose-dependent manner (F (1, 8) =6. 94,p=0.001;Fig 1A insert) with a maximal reduction to 68% of control levels. Clozapine also rapidly reduced ongoing gamma power, with all three doses achieving this to the same extent (F (1, 8) =11.63,p<0.001;Fig 2A insert). The maximal reduction induced by clozapine was to 74% of control. LY379268 also induced an abrupt and dose-dependent drop in ongoing gamma power (F (1, 5) =39. 35,p<0.001;Fig 3A insert), with the highest dose (3 mg/kg) reducing power to 41% of control. The experimental design of these studies only allowed assessment of the effects of antipsychotic alone for 30 minutes post-injection. Therefore, a subsequent set of studies were performed to investigate these effects over a 24 hr period. In these studies, the statistically significant reductions in gamma power induced of all antipsychotics were replicated (Haloperidol (reduced to 61% of control): F (1, 6) =16.39, p=0.003; Clozapine (66%): F (1, 6) =19.86, p=0.001; LY379268 (36%): p<0.001;Fig 4). Furthermore, the effects of all antipsychotics were still significantly reduced for at least 12 hr following administration compared to vehicle (using Bonferroni's post-hoc analyses).

F (1, 6) =180.4,
We also examined the acute impact of antipsychotics in other ongoing EEG activities, which could be distinguished from their frequency bands, including delta (1-4Hz), theta (4-8 Hz), alpha (8-13 Hz) and beta (13-30Hz. No significant change in delta or theta was observed after treatment with any of the tested drugs (p>0.05 for all drugs and all powers, compared to vehicle). However, the power of both alpha (F (1, all analyses), suggesting that their action specifically targeted spontaneously occurring gamma oscillations.

Although some heavy sedation was seen following administration of the highest doses of clozapine (5 mg/kg) and LY379268 (3 mg/kg), and ataxia without sedation with the highest dose of haloperidol (0.25 mg/kg), these effects did not result in any difference in total locomotor activity during the 30 minute pre-treatment window, compared with vehicle treatment (Haloperidol: F (1, 8) =1.14, p=0.352; Clozapine: F (1, 7) =0.13, p=0.940; LY379268: F (1, 5) =3.611, p=0.062). Locomotor activity was not measured during the long-term recordings performed in home cages.

Pretreatment with LY379268, but not haloperidol or clozapine, reduces the trough-to-peak ketamine-induced increase in gamma power.

In keeping with our previous studies [START_REF] Hakami | NMDA Receptor Hypofunction Induces Generalized and Persistent Aberrant  Oscillations Independent of Hyperlocomotion and Conscious Sensorimotor Processing[END_REF][START_REF] Pinault | N-methyl d-aspartate receptor antagonists ketamine and MK-801 induce wake-related aberrant gamma oscillations in the rat neocortex[END_REF], administration of ketamine (5 mg/kg sc) induced an acute increase in gamma power which peaked 8-12 minutes after injection (peak response: haloperidol study -231 ± 17% compared to baseline; clozapine study -260 ± 21%; LY379268 study -252 ± 39%). Pretreatment with haloperidol (0.05 -0.25mg/kg) significantly reduced the ketamine response on gamma power (F (1, 8) =13.43,p<0.001;Fig 1A). Further, this attenuating effect of haloperidol did not demonstrate dose-dependence. Clozapine pretreatment (1-5 mg/kg) appeared to reduce the gamma power response of ketamine, but this was not a dose-dependent effect, and failed to achieve statistical significance (F (1, 7) =2.18,p=0.117;Fig 2A). Pretreatment with LY379268 (0.3-3 mg/kg) dramatically and dose-dependently reduced the ketamine-induced increase in gamma power (F (1, 5) =95.28, p<0.001; Fig 3A ) with the highest dose preventing any rise in gamma power above the original baseline level.

The attenuating effect of the antipsychotics on the ketamine response appeared to be influenced by the ability of these compounds to reduce the peak power of gamma oscillations themselves. To take this into account, 'trough-to-peak' values were calculated, thereby controlling for the 'antipsychotic alone' effect on gamma power. Neither haloperidol (F (1, 8) =1.51, p=0.241; Pretreatment with antipsychotics inhibits the ketamine-induced hyperlocomotor response.

Ketamine (5 mg/kg) induced an acute hyperlocomotor response which peaked 6-8 minutes after injection and persisted for up to 30 minutes (peak values: haloperidol study -736 ± 241% compared to baseline; clozapine study -989 ± 182%; LY379268 study -931 ± 388%). Pretreatment with haloperidol (reaching significance (p<0.05) at 0.1 mg/kg and 0.25 mg/kg) and LY379268 (reaching significance at 0.3 mg/kg, and further suppression with 3 mg/kg) prior to ketamine injection dosedependently inhibited this hyperlocomotor response (overall two-way ANOVA: haloperidol: F (1, 8) =4.01, p=0.017; LY379268: F (1, 5) =6.83, p=0.006), whereas the inhibitory effect of clozapine on locomotor activity did not reach statistical significance (F (1, 7) =0.677, p=0.575). When comparing trough-to-peak values, all three compounds significantly attenuated locomotor activity induced by ketamine (one-way ANOVA: Haloperidol: F (1, 8) =9.64, p<0.001; Clozapine: F (1, 7) =3.99, p=0.024; LY379268: F (1, 5) =10.60, p=0.012).

DISCUSSION

The current study describes a differential effect of antipsychotics on the ketamine-induced increase in the power of spontaneously occurring gamma oscillations: LY379268 potently suppresses this effect, in direct contrast to clozapine and haloperidol which were unable to modify the 'trough-to-peak' response of ketamine. LY379268, which is a preclinical compound which is being evaluated as a potentially novel treatment for patients with schizophrenia, exerted its effect in a clear and powerful dose dependent manner. We speculate that this differential effect to that of the two compounds in current clinical use as anti-psychotic drugs, may be due to its agonist effects on mGluR 2/3 receptors, a property not shared by the other two drugs. All three compounds were, however, reduced, at least to some extent, the peak in EEG gamma power induced by ketamine administration. An additional, and no less intriguing finding, was that all antipsychotic drugs tested reduced the baseline power of ongoing EEG gamma activity, an effect which is maintained for at least 12 hours. Given that this property was similar across all three drugs tested, including two wellestablished clinical compounds, despite their very different spectrum of pharmacological actions, this may represent an electrophysiological biomarker of clinical antipsychotic efficacy.

A number of previous studies in rodents examining the effect of psychotomimetic drugs, in particular NMDAr antagonists such as ketamine, have demonstrated increases in the power of ongoing gamma oscillations on the EEG [START_REF] Ehrlichman | N-methyl-d-aspartic acid receptor antagonist-induced frequency oscillations in mice recreate pattern of electrophysiological deficits in schizophrenia[END_REF][START_REF] Hakami | NMDA Receptor Hypofunction Induces Generalized and Persistent Aberrant  Oscillations Independent of Hyperlocomotion and Conscious Sensorimotor Processing[END_REF][START_REF] Lazarewicz | Ketamine Modulates Theta and Gamma Oscillations[END_REF][START_REF] Ma | The supramammillo-septal-hippocampal pathway mediates sensorimotor gating impairment and hyperlocomotion induced by MK-801 and ketamine in rats[END_REF][START_REF] Pinault | N-methyl d-aspartate receptor antagonists ketamine and MK-801 induce wake-related aberrant gamma oscillations in the rat neocortex[END_REF]. It is proposed that this abnormal gamma activity represents aberrant diffuse network noise and is a potential electrophsiological correlate of a psychotic-like state. In apparante conflict with this, many clinical studies describing abnormalities in gamma oscillatory power in schizophrenic patients have reported decreases in EEG gamma power responses to evoked stimuli (eg: [START_REF] Ford | Out-of-synch and out-of-sorts: dysfunction of motor-sensory communication in schizophrenia[END_REF][START_REF] Kwon | Gamma frequency-range abnormalities to auditory stimulation in schizophrenia[END_REF][START_REF] Spencer | Abnormal neural synchrony in schizophrenia[END_REF]Spencer et al., 2008a). However, most of these studies group do not take into account the influence of antipsychotic medication (see [START_REF] Hong | Evoked gamma band synchronization and the liability for schizophrenia[END_REF]). When examining subsets of schizophrenia patients, studies of evoked and induced gamma power responses describe positive correlations between hallucinatory symptoms and gamma power responses [START_REF] Gordon | Symptom profile and 'gamma' processing in schizophrenia[END_REF][START_REF] Spencer | Left auditory cortex gamma synchronization and auditory hallucination symptoms in schizophrenia[END_REF]Spencer et al., 2008b), suggesting that certain psychotic disease states may be associated with different levels gamma activity and reactivity. Further, a single case report measured ongoing gamma oscillatory power during acute psychotic episodes, and described significantly elevated gamma oscillatory power, most prominent at times when the symptoms were most severe [START_REF] Baldeweg | Gamma-band electroencephalographic oscillations in a patient with somatic hallucinations[END_REF] and other reports describe increases in basal ongoing gamma waves in patiennts with schizophrenia [START_REF] Turetsky | Persistent auditory-evoked gamma band oscillations in schizophrenia[END_REF]. These reports complement a study of first episode psychosis patients who displayed an excess of absolute gamma phase synchrony elicited by a selective attention task compared to controls [START_REF] Flynn | Increased absolute magnitude of gamma synchrony in first-episode psychosis[END_REF], and a study describing increases in auditory-evoked gamma band responses in humans following acute ketamine [START_REF] Hong | Gamma and delta neural oscillations and association with clinical symptoms under subanesthetic ketamine[END_REF]. Collectively, these findings suggest that increased gamma power responses, and indeed ongoing gamma oscillatory power, may be positively associated with psychotic symptoms in schizophrenia. If this were the case, then it may be expected that drugs that reduce psychotic symptoms may reduce the amount of EEG gamma power. This is supported by the results of this study, demonstrating a potent (and in some cases dose-dependent) suppression of the power of the spontaneous background gamma oscillations that was common to all antipsychotic drugs tested.

The generation of gamma oscillations is believed to be driven by populations of fast-spiking interneurons, providing feedback inhibition onto pyramidal cells [START_REF] Bartos | Synaptic mechanisms of synchronized gamma oscillations in inhibitory interneuron networks[END_REF][START_REF] Fisahn | Cholinergic induction of network oscillations at 40 Hz in the hippocampus in vitro[END_REF][START_REF] Fuchs | Recruitment of parvalbumin-positive interneurons determines hippocampal function and associated behavior[END_REF]. Systemic administration of NMDAr antagonists is thought to block NMDAr-mediated firing of these interneurons, resulting in enhanced firing rates of pyramidal neurons and therefore an elevation of the power of gamma rhythms [START_REF] Jackson | NMDA receptor hypofunction produces concomitant firing rate potentiation and burst activity reduction in the prefrontal cortex[END_REF][START_REF] Moghaddam | Activation of glutamatergic neurotransmission by ketamine: a novel step in the pathway from NMDA receptor blockade to dopaminergic and cognitive disruptions associated with the prefrontal cortex[END_REF]. Gamma frequency oscillations can also be influenced by many other receptor systems, including dopaminergic and cholinergic [START_REF] Lisman | Circuit-based framework for understanding neurotransmitter and risk gene interactions in schizophrenia[END_REF], receptor types which are targeted by the antipsychotics used in the current project. Taking into account the distinct mechanisms of action of the drugs tested here, the common property of the antipsychotics to reduce ongoing gamma power is probably not be mediated via a common receptor mechanism, but may represent a more fundamental effect against downstream psychotic mechanisms. In addition, the duration of effects of these compounds (lasting >12 hours following administration), is suggestive of a gene transcription-mediated effect, since brain levels, at least of clozapine and haloperidol [START_REF] Baldessarini | Tissue concentrations of clozapine and its metabolites in the rat[END_REF][START_REF] Campbell | Tissue levels of haloperidol by radioreceptor assay and behavioral effects of haloperidol in the rat[END_REF], would be expected to be low/negligible at this timepoint.

Although we observed common features of the different antipsychotics tested (i.e.: all reduce baseline gamma power and all inhibit the ketamine-induced hyperlocomotor response), only the preclinical mGluR 2/3 agonist was able to suppress the trough-to-peak effect of ketamine on gamma power. LY379268 has also been shown to effectively block behavioural effects of NMDAr antagonists, such as disruptive effects on working memory [START_REF] Moghaddam | Reversal of phencyclidine effects by a group II metabotropic glutamate receptor agonist in rats[END_REF] and hyperlocomotor activity (Imre et al., 2006a), effects promoting this as a potential treatment for patients with schizophrenia. The unique ability of this compound to block ketamine-induced increases in gamma EEG power reported here may reflect a effect of this drug more directly on the neuronal mechanisms controlling gamma oscillations in the brain than either clozapine or haloperidol. Group II mGluRs (i.e.: mGluR 2 and mGluR 3 ) are generally located presynaptically and inhibit the release of glutamate from the presynaptic terminal [START_REF] Moghaddam | Reversal of phencyclidine effects by a group II metabotropic glutamate receptor agonist in rats[END_REF], providing a negative feedback loop on pyramidal cell firing. MK801, which like ketamine, is another NMDAr antagonist which we have previously demonstrated to increase EEG gamma power [START_REF] Hakami | NMDA Receptor Hypofunction Induces Generalized and Persistent Aberrant  Oscillations Independent of Hyperlocomotion and Conscious Sensorimotor Processing[END_REF][START_REF] Pinault | N-methyl d-aspartate receptor antagonists ketamine and MK-801 induce wake-related aberrant gamma oscillations in the rat neocortex[END_REF], has also been shown to increase the firing rate of cortical neurons in awake rats [START_REF] Jackson | NMDA receptor hypofunction produces concomitant firing rate potentiation and burst activity reduction in the prefrontal cortex[END_REF]. Activation of mGluR 2/3 with LY354740 dose-dependently suppresses this effect [START_REF] Homayoun | Activation of metabotropic glutamate 2/3 receptors reverses the effects of NMDA receptor hypofunction on prefrontal cortex unit activity in awake rats[END_REF][START_REF] Homayoun | Orbitofrontal cortex neurons as a common target for classic and glutamatergic antipsychotic drugs[END_REF]. This may well be relevant to the observations in this study showing that a drug that activates mGluR 2/3 receptor subtypes inhibits the ability of ketamine to increase cortical gamma activity, in contrast to the two other antipsychotic drugs, suggesting a direct cellular interaction between ionotropic (NMDA) and metabotropic (mGluR 2/3 ) receptors. While this observation raises the possibility that the effect of LY379268 to inhibit ketamineinduced elevations in gamma power is mediated via its agonist activity at mGluR 2/3 receptors, this needs to be confirmed by other studies specifically investigating this speculation. Indeed, it is further confounding the understanding of the mechanism of this effect is a previous study that reported that subchronic pretreatment with the related compound, LY354740 which is purported to be a more potent agonist of mGluR 2/3 [START_REF] Monn | Synthesis, pharmacological characterization, and molecular modeling of heterobicyclic amino acids related to (+)-2-aminobicyclo[3.1.0] hexane-2,6-dicarboxylic acid (LY354740): identification of two new potent, selective, and systemically active agonists for group II metabotropic glutamate receptors[END_REF], was unable to block ketamine-induced hyperlocomotor activity (Imre et al., 2006b). Although evidence exists for direct interactions between mGluR 5 receptors and NMDA receptors [START_REF] Awad | Activation of metabotropic glutamate receptor 5 has direct excitatory effects and potentiates NMDA receptor currents in neurons of the subthalamic nucleus[END_REF][START_REF] Lecourtier | Positive allosteric modulation of metabotropic glutamate 5 (mGlu5) receptors reverses N-Methyl-D-aspartate antagonist-induced alteration of neuronal firing in prefrontal cortex[END_REF], further studies are required to exact the precise cellular mechanisms linking mGluR 2/3 and NMDA receptors.

The current study also supports the findings of our previous research [START_REF] Hakami | NMDA Receptor Hypofunction Induces Generalized and Persistent Aberrant  Oscillations Independent of Hyperlocomotion and Conscious Sensorimotor Processing[END_REF][START_REF] Pinault | N-methyl d-aspartate receptor antagonists ketamine and MK-801 induce wake-related aberrant gamma oscillations in the rat neocortex[END_REF] that, although NMDAr antagonist-induced increases in hyperlocomotor activity and in gamma power often occur together, the hyperlocomotor response does not, in itself, drive the elevations in gamma power. Here we showed that although both haloperidol and clozapine both inhibited the hyperlocomotor response of ketamine, they did not completely reverse the rise in gamma EEG power. In addition, animals under anaesthesia still demonstrate a gamma power response to ketamine, and other psychotomimetic compounds such as amphetamines which potently elicit a hyperlocomotor response [START_REF] Jones | A genetic epilepsy rat model displays endophenotypes of psychosis[END_REF] have minimal effects on cortical or hippocampal gamma power [START_REF] Ehrlichman | N-methyl-d-aspartic acid receptor antagonist-induced frequency oscillations in mice recreate pattern of electrophysiological deficits in schizophrenia[END_REF][START_REF] Ma | The supramammillo-septal-hippocampal pathway mediates sensorimotor gating impairment and hyperlocomotion induced by MK-801 and ketamine in rats[END_REF][START_REF] Pinault | N-methyl d-aspartate receptor antagonists ketamine and MK-801 induce wake-related aberrant gamma oscillations in the rat neocortex[END_REF].

Although all three compounds were able to reduce the amount of ongoing gamma oscillations, only LY379268 also influenced EEG activity in other frequency bands. Both alpha and beta power were significantly reduced by this drug in a similar abrupt and enduring fashion as for its effect on gamma power. It is not clear what ramifications this would have clinically, but beta power also has been strongly linked to cognitive processing [START_REF] Uhlhaas | Abnormal neural oscillations and synchrony in schizophrenia[END_REF].

In conclusion, the data from this study demonstrate prominent and long-lasting reductions in ongoing gamma EEG power following treatment with a range of antipsychotics, both clinical and preclinical, with differing pharmacological mechanisms of action. The wealth of research investigating gamma power abnormalities in schizophrenia have so far largely neglected to address the effect of antipsychotic medications when assessing disease-induced changes in gamma power, but this should be taken into account in future studies. Indeed, this property may be related to the clinical efficacy against positive symptoms of schizophrenia, since both clozapine and haloperidol were able to reduce the baseline gamma power, and may therefore provide an electrophysiological biomarker of antipsychotic activity. These compounds also mitigated the maximum increase in gamma power in response to acute systemic administration of ketamine, which may also be an important and potentially clinically relevant predictive outcome in its own right. Although the trough-to-peak response of ketamine was not influenced by the currently used antipsychotics, we have not here examined the effects of chronic antipsychotic treatment, nor the effects of other clinically used agents possessing different spectra of clinical effects (i.e. including those efficacious against cognitive deficits). This measure may yet prove to have added value to the predictive models currently used in the pre-clincial anti-psychotic drug development, including drug-induced hyperlocomotor activity. Table 1. Descriptive summary of effects of antipsychotics on gamma power and locomotor activity. Upper panel describes effects of antipsychotics on baseline and ketamine-induced changes in gamma power; lower panel describes effects of drugs on locomotor activity. Percentage change of ketamine-induced effects (middle columns) were calculated 6-8 mins after ketamine injection at the peak of the ketamine response. * in this instance, 100% inhibition of response was observed with the lowest dose of LY379268, so no dose-dependence was attained. n.s. non-significant effect.

  Fig 5A) nor clozapine (F (1, 7) =1.26, p=0.317; Fig 5B) had any statistically significant effect on the ketamine-induced trough-to-peak increases in gamma power. However, LY379268 significantly attenuated the effect of ketamine (F (1, 5) =5.97, p=0.026; Fig 5C), demonstrating a clear differential effect compared with the other antipsychotics.

Figure

  Figure and table:

Figure 1 .

 1 Figure 1. Effects of haloperidol on ketamine-induced elevations in gamma power and locomotor activity in rats. (A -insert) Haloperidol (0.05 -0.25 mg/kg sc) dosedependently depressed ongoing gamma power by up to 30% during the 30 min pretreatment phase of the recordings, compared to vehicle treatment. (A) Haloperidol significantly but incompletely reduced the rise in gamma power induced by ketamine (5mg/kg sc), compared with vehicle pretreatment. This effect was not dosedependent. (B) Pretreatment with haloperidol dose-dependently suppressed ketamine induced hyperlocomotor activity, conmpared with vehicle pretreatment. Data represent mean ± S.E.M.; n=8. Open arrow indicates timing of haloperidol administration, and closed arrow indicates ketamine administration.

Figure 2 .

 2 Figure 2. Effects of clozapine on ketamine-induced elevations in gamma power and locomotor activity in rats. (A -insert) Clozapine depressed ongoing gamma power by up to 25% during the 30 min pretreatment phase of the recordings, compared to vehicle treatment, although this was not dose-dependent for the doses used. (A) Clozapine did not significantly affect the rise in gamma power induced by ketamine (5mg/kg sc), compared with vehicle pretreatment. (B) Pretreatment with clozapine (1 -5 mg/kg sc) induced a dose-dependent but incomplete suppression of ketamine (5 mg/kg sc) induced hyperlocomotor activity conmpared with vehicle pretreatment. Data represent mean ± S.E.M.; n=7. Open arrow indicates timing of clozapine administration, and closed arrow indicates ketamine administration.

Figure 3 .

 3 Figure 3. Effects of LY379268 on ketamine-induced elevations in gamma power and locomotor activity in rats. (A -insert) LY379268 potently and dose-dependently depressed ongoing gamma power by up to 60% during the 30 min pretreatment phase of the recordings, compared to vehicle treatment. (A) LY379268 significantly and completely reduced the rise in gamma power induced by ketamine (5mg/kg sc), compared with vehicle pretreatment. This effect was dose-dependent. (B) Pretreatment with LY379268 (0.3 -3 mg/kg sc) induced a dose-dependent and complete suppression of ketamine (5 mg/kg sc) induced hyperlocomotor activity conmpared with vehicle pretreatment. Data represent mean ± S.E.M.; n=5. Open arrow indicates timing of LY379268 administration, and closed arrow indicates ketamine administration.

Figure 4 .

 4 Figure 4. Extended effects of antipsychotics on ongoing gamma power. When administered at high doses, all three antipsychotics produced a robust and significant decrease in spontaneous ongoing gamma power for 12 hours, as compared to vehicle. Data represent mean ± S.E.M.; n=6. This effect persisted for at least 24 hours.

Figure 5 .

 5 Figure 5. Effects of antipsychotics on 'peak-to-trough' gamma power increase induced by ketamine (5 mg/kg sc). Neither haloperidol (Hal) (A) nor clozapine (Cloz) (B) influenced the net increase (expressed as max % change from trough) of ongoing gamma power induced by ketamine at any pretreatment dose. In contrast, pretreatment with LY379268 (LY) (C) was able to significantly suppress the 'peakto-trough' gamma power rise induced by ketamine (*p<0.05). Data represent mean + S.E.M.; sample sizes as for Figs 1-3.

6) =11.58, p=0.007) and beta (F (1, 6) =24.05, p<0.001) was significantly reduced by LY379268. The other drugs did not significantly alter these bands either (p>0.05 for

Acknowlegdments:

This work was supported by a University of Melbourne Joint Projects grant (NJ and DP). NJ is supported by an NHMRC Career Development Award.

Disclosures/Conflicts of Interest:

All authors declare no conflicts of interest, financial or otherwise, associated with this work.


Dr Tajinder Singhrao

Elizabeth Higham

Patrick French
email: patrick.french@camdenpct.nhs.uk

Dr Patrick

LYMPHOGRANULOMA VENEREUM PRESENTING AS PERIANAL ULCERATION: AN EMERGING CLINICAL PRESENTATION?

Keywords: Lymphogranuloma venereum, Ulcer, Homosexuality, Male, Chlamydia trachomatis

Key Messages

1. These cases, to our knowledge are the first described LGV infections presenting with isolated perianal ulcer disease with normal proctoscopy and other causes excluded.

2.

LGV typically presents with proctitis symptoms amongst HIV positive MSM within the UK.

3. We recommend testing for LGV infection in HIV positive MSM presenting with isolated perianal ulcer disease using chlamydia nucleic acid amplification tests (NAATs).

A positive rectal

LGV Chlamydia NAAT with isolated perianal ulcer disease may provide insight into the biology and transmission of LGV, including subclinical proctitis and unrecognised ulceration.

Introduction

LGV, first identified in the UK in 2004, has become endemic among MSM. The prevalence of LGV amongst MSM (symptomatic and asymptomatic) attending London GU clinics in 2009 was estimated at 0.9% rectal and 0.04% urethral.[1] HPA surveillance from 2003-2008 found 849 cases of LGV, the majority in London (72%). All cases were male, predominantly white MSM (99%), co-infected with HIV(75%).[2] Risk factors for acquisition include co-infection with an STI, unprotected receptive anal intercourse, fisting, group and anonymous sex.

Historically

LGV infection was an inguinal syndrome [3] including a transient primary ulcerative lesion with thrombolymphangitis and perilymphangitis resulting in tender inguinal lymphadenopathy. However an anorectal LGV syndrome among MSM is well recognised [4] and is the commonest presentation of LGV in the UK [5] with genital ulcers and inguinal symptoms rarely encountered.[6] HPA data since 2004 has consistently shown >75% of patients identified with LGV infection presented with proctitis.[7]

Case Report

A 28 year old white British male with HIV presented with a two week history of a single painful ulcer at the anal margin without rectal or inguinal symptoms and no proctitis on proctoscopy. This patient had been diagnosed with LGV and rectal gonorrhoea four months previously, presenting with proctitis, inguinal lymphadenopathy and an indurated perianal ulcer. LGV test of cure was negative two months following previous treatment.

A 45 year old white British male with HIV presented with a three day history of a single painful 0.5cm indurated perianal ulcer associated with tender unilateral inguinal lymphadenopathy without proctitis on proctoscopy. He subsequently developed fevers.

Chlamydia trachomatis specific DNA of an LGV associated serovar was identified on rectal (not ulcer) swabs taken from both patients. PCR tests for HSV1/HSV2/T.pallidum taken from the ulcers in both patients were negative. At presentation patient 1 was RPR negative (having been treated for primary syphilis 1 year previously) and patient 2 had negative T.pallidum serology. Urethral Chlamydia NAATs were negative in both patients. Patient 1 was diagnosed with hepatitis C at presentation.

Both reported unprotected receptive and insertive anal intercourse in the last 3 months. At presentation patient 1 was treated for an anal fissure and patient 2 for presumed herpes simplex virus (HSV) infection. Both patients received a three week course of doxycycline once found to be Chlamydia positive. Their symptoms resolved completely on treatment and both had a negative test of cure at 6 weeks.

Discussion

From 2004 to March 2010 we have seen 146 presentations of LGV at our centre accounting for 17% of cases reported to the HPA (unpublished data). Whilst largely amongst the HIV positive population, 22 /146 (15%) presentations affected HIV negative patients. Only 11/146 (7.5%) did not have proctitis symptoms, of these 2 were asymptomatic, 2 had isolated ulcer disease (the patients we report here) and 7 had genital symptoms, 4 with inguinal syndrome, some with systemic symptoms. We have seen 2 other patients with LGV and perianal ulcers who had concurrent proctitis. Both had negative PCR tests for HSV from the ulcer and T.pallidum serological testing was negative (T.pallidum PCR testing was not undertaken). Both had ulcer resolution with doxycycline. A case control study showed association between anorectal ulceration and LGV but it is not clear whether these patients had peri-anal, anal canal disease or concomitant proctitis. [8] We are not aware of any other published reports of patients with isolated perianal ulceration from LGV infection in which other causes of ulceration were excluded.

LGV associated genital ulcer disease is rare in the UK, explaining why our patients were not initially treated for LGV infection. A cohort of patients presenting to London GU clinics with inguinal syndrome and ulceration described 4 patients with genital ulceration, one with multiple genital and perianal ulcers.[9] Primary LGV erosions typically affect the genitalia and are classically painless. [6] Neither of our patients had genital ulceration or proctitis. Both had positive rectal LGV chlamydia NAATs (HSV and syphilis were excluded on PCR testing). The validity of ulcer Chlamydia NAATs is uncertain and this paper suggests it may be a useful test. It raises questions regarding the biology and transmission of LGV, including possibilities of sub-clinical proctitis and ulceration.

Health promotion and behavioural intervention paramount in preventing and identifying cases of LGV infection and addressing sexual health needs of risk groups. [5,10] The HPA recommend LGV testing for all symptomatic HIV positive MSM.[2] There is a strong argument for rectal Chlamydia NAATs to be used in MSM with proctitis and this report provides evidence that rectal LGV testing should be extended to patients with anogenital ulcers.

Acknowledgements:

Both patients provided written consent.

We would like to thank our colleagues at the UCLH Medical Microbiology laboratory and the Sexually Transmitted Bacteria Reference Laboratory at the HPA.

Competing Interests: None

Funding: None Word Count 750 words (excluding key points, abstract, title page, acknowledgments, references and headings) (Abstract 150 words) "The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd to permit this article (if accepted) to be published in STI and any other BMJPGL products and sublicences such use and exploit all subsidiary rights, as set out in our licence http://group.bmj.com/products/journals/instructions-for-authors/licence forms." TS wrote the first draft of the paper. EH analysed the clinic data. EH and TS both contributed to subsequent drafts of the paper. PF contributed to subsequent revisions of the case report and is guarantor of this paper.

Reference


Annick Borquez
email: annick.borquez06@imperial.ac.uk

Timothy B Hallett

Gabriela B Gomez

Geoffrey P Garnett

Annick Bórquez

Condom use by female sex workers and their clients in Mexico: who decides and does it matter?

Keywords: HIV, mathematical model, condom, sex work (prostitution), sexual behaviour 2

published or not. The documents may come

Condom use by female sex workers and their clients in

Mexico: who decides and does it matter?

INTRODUCTION

In Mexico and other countries with concentrated HIV epidemics, targeting high risk groups has been a focus of STI control efforts. [START_REF] Gayet | Prácticas sexuales de las poblaciones vulnerables a la epidemia de VIH/SIDA en México[END_REF][START_REF] Aral | Behavioral aspects of sexually transmitted diseases: core groups and bridge populations[END_REF] An important determinant of the extent of HIV spread will be the heterogeneity in risk behaviour within these groups. Mathematical models using data on behaviour allow us to predict the risks of HIV spread and the impact of interventions. Whilst information on individuals' own behaviour can be recorded, information on partner's risks is less readily captured. In fact, in many cases, several different patterns of behaviour can be consistent with the same individual-level behavioural reports. In this paper, we test the hypothesis that patterns of mixing and condom use among sex workers and their clients are a determinant of the potential extent of epidemic spread.

METHODS

A deterministic mathematical model was developed that represents heterosexual transmission of HIV between FSWs, their clients and non-commercial partners (Figure S-1). We focused on the heterogeneity in behaviour between FSWs categorising them into eight mutually exclusive sexual activity classes defined by: (i) condom use at last sex; (ii) number of clients per week; and (iii) having a non-commercial partner (Table S- S-2).The model is described in detail in the supplementary material (Model equations S-1).

Based on those data, it was assumed that some FSW always use condoms (84%) and some never do (16%). Then, the effect of the mixing pattern between clients and FSWs was explored by simulating an HIV epidemic under two scenarios. In one scenario (Figure 1A) "homogeneous client population", it was assumed that condom use was determined by FSWs only (i.e. clients chose FSWs randomly, such that clients used condom in some transactions and not others). In the alternative scenario (Figure 1B) "heterogeneous client population", it was assumed that condom use also depended on clients' preferences (i.e. some clients always used condoms and some never did, and each type of client sought FSWs who matched their behaviour). The contribution of each sub-population to the HIV epidemic was also evaluated through sensitivity analyses by measuring changes in incidence according to the frequency of condom use with both clients and partners, and the number of clients.

RESULTS

HIV epidemics were simulated under the two scenarios. In the homogenous client scenario (Figure 1C), the incidence in the total population was greater than in the heterogeneous client scenario (Figure 1D), peaking at 2 versus ~0.4 cases per 100 personyears at risk (pyar). Figures 1E andF compare the incidence at equilibrium among FSWs, their clients and non-commercial partners. In the homogenous client scenario, the most affected groups were FSWs who did not use condoms and their stable partners as well as all clients (Figure 1E). Among FSWs who used condoms with clients, HIV incidence was 15 times lower than among FSW who did not. In the heterogeneous client scenario (Figure 1F), FSWs and clients that used condoms did not get infected. Incidence among FSWs and clients that did not use condoms was similar to the rate in the homogenous client scenario.

The higher overall incidence under the homogenous client scenario is largely because the risk of infection experienced by clients who do not use condoms in the heterogeneous scenario is shared with all the clients in the homogenous client scenario, and, secondly that the clients in the homogenous scenario act as a bridge to distribute infections to FSW that do use condoms.

In sensitivity analyses decreases in condom use were associated with increased HIV incidence in the homogeneous client scenario but not in the heterogeneous client scenario until a threshold level of unprotected sex allowed HIV spread. The same pattern was observed for increases in contact rates among FSW (Figure S-2). These results suggest that continued epidemic spread is more sensitive to changes in behaviour when there is a homogenous client population.

DISCUSSION

This modelling exercise highlights the importance of mixing patterns with regard to condom use in determining the scale of HIV epidemics and the impact of changes in risk behaviour on transmission. Different prevention strategies may be appropriate to control HIV infection depending on these patterns. For a heterogeneous client population, interventions could be efficiently targeted to those particular FSWs and clients who do not use condoms while in the homogenous mixing scenario, interventions would have to include strong campaigns for all clients to adopt condom use in order to reduce the bridging of transmission between the two groups of FSWs. The only available data that could allow us making hypotheses about which mixing scenario would better approximate the sex industry in Mexico come from U.S. border cities. These suggest there is a group of clients that refuses to use condoms and generally buys sex from FSWs who are also more likely not to use condoms. [START_REF] Bucardo | A qualitative exploration of female sex work in Tijuana, Mexico[END_REF][START_REF] Goldenberg | Correlates of Unprotected Sex With Female Sex Workers Among Male Clients in Tijuana, Mexico[END_REF][START_REF] Larios | An exploration of contextual factors that influence HIV risk in female sex workers in Mexico: The Social Ecological Model applied to HIV risk behaviors[END_REF][START_REF] Patterson | Correlates of HIV, sexually transmitted infections, and associated high-risk behaviors among male clients of female sex workers in Tijuana, Mexico[END_REF] However, this is a very particular context making it difficult to generalise the findings.

KEY MESSAGES

Reported levels of condom use among FSW could translate in different HIV epidemics depending on the clients' patterns of use and mixing

Consistent condom use by particular clients generates lower HIV prevalence than if the same inconsistent usage of condom is shared by all clients.

Information on condom use negotiation among clients of FSW would be valuable for modelling, surveillance and programmatic purposes

  1). We parameterised the model using data from a behavioural survey undertaken in Morelos and Michoacán, Mexico, where 2401 FSW participated in a validation study part of the second generation surveillance implemented by the INSP (Instituto Nacional de Salud Pública) and CENSIDA (Centro Nacional para la Prevención y el Control del VIH/SIDA) in 2001 (Table

Figure 1 -

 1 Figure 1 -HIV prevalence and incidence among all groups and incidence at equilibrium by population sub-group.

Figures C

  Figures C and D show the HIV prevalence in light gray and incidence in black among FSWs, their partners and their clients for the Het-CP and the Hom-CP scenario respectively. Figures E and Fshow the incidence at equilibrium by sub-population assuming two mixing patterns between FSWs and clients: E -Homogenous client population, and F -Heterogeneous client population. 'FSW-C': FSWs who consistently use condoms; 'FSW-NC': FSWs who never use condoms; 'Clients': clients of FSWs who use condoms only with FSWs who use condoms (homogeneous client scenario only); 'Client-C': clients who consistently use condoms and only have sex with FSWs who use condoms (heterogeneous client scenario only), 'Clients-NC': Clients who never use condoms and only have sex with FSWs who do not use condoms (heterogeneous client scenario only); 'Partners-C': non-paying partners of FSWs who sometimes use condoms; 'Partners-NC': non-paying partners of FSWs who never use condoms. All model parameters are provided in the online appendix.

The classification of FSWs and the mixing patterns simulated in this report are extreme ends of a continuous range of behavioural patterns. Amongst other complex aspects of sexual behaviour that are only partially accessible through ego-centric survey data[START_REF] Ghani | Patterns of sex worker-client contacts and their implications for the persistence of sexually transmitted infections[END_REF][START_REF] Ghani | The role of sexual partnership networks in the epidemiology of gonorrhea[END_REF], these differences in mixing patterns may be an important reason why behavioural interventions do not always translate to substantial changes in STI incidence, and why impact may vary between settings. Collecting information on within-person variation in condom use in addition to that more usually collected on between-individual variation, could lead to a better understanding of transmission in these populations, whilst a further characterisation of clients' negotiation of condom use could strengthen interventions.

ACKNOWLEDGEMENTS

We thank the Instituto Nacional de Salud Pública (INSP) and the Centro Nacional para la Prevención y el Control del VIH/SIDA (CENSIDA), México for sharing this data with us as well as all the participants in the behavioural survey for their time and collaboration.

FUNDING

AB thanks the Medical Research Council, UK. GBG is funded by the Wellcome Trust Foundation (#WPIA-P30479).TBH and GPG also thank The Wellcome Trust.

COMPETING INTERESTS

All authors declare no conflicts of interest exist.

ETHICS APPROVAL

The study received approval from the scientific, ethical and bioethical committees of the INSP. All participants gave their informed consent and provided answers to the questionnaire anonymously.

CONTRIBUTORS

AB, GPG designed the study. AB, TBH and GBG performed the analyses. All authors contributed to the interpretation of results and write up.

COPYRIGHT LICENCE STATEMENT

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive license (or non-exclusive for government employees) on a worldwide basis to the BMJ Group and co-owners or contracting owning societies (where published by the BMJ Group on their behalf), and its Licensees to permit this article (if accepted) to be published in Sexually Transmitted Infections and any other BMJ Group products and to exploit all subsidiary rights, as set out in our license.


MD L Livi

MD Icro Meattini
email: icro.meattini@unifi.it

MD C Saieva

MD S Borghesi

V Scotti

MD A Petrucci

MD A Rampini

MD L Marrazzo

V Di Cataldo

MD S Bianchi

MD L Cataliotti

MD G Biti

The impact of young age on breast cancer

Keywords: breast cancer, young patients, radiation therapy, anthracyclines, mastectomy, breastconserving surgery

AIMS: we conducted a retrospective analysis in order to evaluate the impact of age on women aged less than 35 years affected by breast cancer.

MATERIALS AND METHODS

: between January 1972 and December 2006, 346 patients aged less than 35 years underwent adjuvant treatment at Florence University. The mean age of the patient population was 32 years (range 22-35): 76 patients were under 30 years old, the remaining were above 30 years old. RESULTS: in our series, 215 patients received adjuvant radiotherapy to whole breast after conservative surgery, 131 patients underwent mastectomy without subsequent radiation therapy and 323 patients had lymphadenectomy. 191 patients received adjuvant chemotherapy, 73 with anthracyclines-containing regimen. With a median time of 2.5 years (range 6 months to 27.6 years)

local relapses were observed in 67 cases (19.4%). At the multivariate analysis of local disease free survival, ductal and ductal plus lobular histotypes, having more than 3 positive nodes and age emerged as independent significant relapse predictors (p =0.018, p=0.0005, p=0.003 and p=0.024, respectively). For the DSS analysis, the median follow-up was 6.8 years (range 0.6-36.7 years). At the multivariate analysis, age (p=0.0038), positive nodes (p=0.0035) and distant metastases (p<0.0001) resulted to be independent death predictors. Patients younger than 30 had a worse prognosis. At the univariate analysis also local relapse resulted to be statistically significant (p=0.0004).

CONCLUSIONS: anthracyclines-based chemotherapy seems to improve the outcome of these patients. However there is an urgent need for tailored treatment investigations within the framework of randomized, controlled clinical trials.

INTRODUCTION

About 25% of cases of breast cancer (BC) occur before the menopause, and 15% of women are diagnosed in the reproductive age group [START_REF] Jemal | Cancer statistics[END_REF][START_REF] Zekri | Epirubicin/Vinorelbine adjuvant chemotherapy in young women with breast cancer is associated with preservation of menstrual function[END_REF], with approximately 2% of cases developing in young women aged 20 to 34 years and 11% between 35 and 44 [START_REF] Ries | SEER Cancer Statistics Review, 1975-2005[END_REF].

BC at young age has a more aggressive biological behaviour and is associated with a more unfavourable prognosis compared with the disease arising in older premenopausal patients [START_REF] Hickey | Breast cancer in young women and its impact on reproductive function[END_REF].

Systemic adjuvant therapy with hormonal treatment, chemotherapy, or both are undoubtedly related to the improved rates of mortality from BC noted over the last decade. In a large population-based Canadian study the use of adjuvant systemic therapy was directly related to improved survival rates in women with early BC. Overall survival improved by 10% for women younger than 50 years of age between 1974 and 1984, and by 4% for women aged 50 to 89 years between 1980 and 1984 [START_REF] Olivotto | Adjuvant systemic therapy and survival after breast cancer[END_REF].

We conducted a retrospective analysis in order to evaluate the disease specific survival (DSS) and the local disease free survival (LDFS) in a series of young women with BC treated with integrated adjuvant therapeutic modalities.

PATIENTS AND METHODS

From January 1972 to December 2006, 346 patients aged less than 35 years with BC underwent adjuvant treatment at the Radiotherapy Unit of the University of Florence.

Our series included patients without clinical and radiological evidence of local or distant recurrence after breast surgery at the time of the first evaluation in our Radiotherapy Unit. None of them had prior malignant disease. The mean age of the patient population was 32 years (SD±2.90; range 22-35): 76 patients (22.0%) were under 30 years old, the remaining were above 30 years old.

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

Statistical Analyisis

The survival time was calculated from the date of surgery to the date of death or the date of the last follow-up for the patients resulted to be alive. In our survival analysis we considered as events the deaths for BC disease (DSS). LDFS was calculated from the date of surgery to the date of local relapse (LR) occurrence. We used the term cumulative incidence to specify the occurrence, as percentage, of local relapse at well-defined follow-up points. Likewise we used the term survival, to specify the percentage of patients still alive, and thus at risk, at well-defined follow-up points. The crude probability of death or LR occurrence was estimated by using the Kaplan-Meier method and differences between patient groups were assessed by the log-rank test. Estimated relative risks of dying or LR occurrence were expressed as hazard ratios (HR) and their corresponding 95% confidence intervals (95% CI). Univariate Cox regression models were used to evaluate the effect of each specific parameter. Multivariate Cox regression models with stepwise selection were performed to identify the major significant death or LR occurrence predictors. All the patients are included in all the analyses. Statistical results were considered significant at a p-value <0.05. All statistical tests were performed by the SAS software.

RESULTS

Patients' characteristics are shown in Table 1. Most cases showed a pT 1 (56%), a ductal histotype (64.2%), and none positive lymph node (56.1%).

According to the protocol followed in our Institute, all patients received radiotherapy (RT) to whole breast only after breast conserving surgery. In our series, 215 patients received adjuvant RT to whole breast, 131 patients underwent mastectomy without subsequent RT and 323 patients had lymphadenectomy (93.3%).

The mean RT dose delivered was 50 Gy (range 46-52 Gy), in 2 Gy daily fractions. A dose boost on the tumour bed was administered by electrons. At the discretion of the radiation oncologist

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

the total boost dose (2 Gy daily fraction) ranged between 6 and 10 Gy for patients with negative surgical margins and between 14 and 16 Gy for patients with positive margins.

One hundred and ninety-one patients received adjuvant chemotherapy (CT), that consisted of 6 courses of iv CMF 1-8,28 (cyclophosphamide 600 mg/m 2 , methotrexate 40 mg/m 2 and 5fluorouracil 600 mg/m 2 ) for 98 patients; 4 courses of epirubicin (100 mg/m 2 every 21 days) followed by 4 cycles of iv CMF for 43 women; 4 courses of AC 1,21 (doxorubicin 60 mg/m 2 and cyclophosphamide 600 mg/m 2 ) for 11 patients; 4 AC followed by 4 cycles of paclitaxel 1,21 (175 mg/m 2 ) for 19 cases, and other schedules in 20 patients.

Adjuvant hormonal treatment was prescribed for 74 patients, 22 of them received Tamoxifen, and 52 Tamoxifen associated with GnRH agonist. Ten patients were treated with bilateral oophorectomy.

With a median time of 2.5 years (range 6 months to 27.6 years) LR were observed in 67 cases (19.4%). Twenty patient had supraclavicular fossa relapse (5.8%), 14 developed single chest wall relapse (4.0%), 13 had multiple chest wall relapse (3.8%), 10 had multicentric relapse (2.9%), 7 developed internal mammary nodal chain relapse (2.0%) and three patients had axillary relapse (0.9%).

At the univariate regression analysis for LDFS (Table 2), histotype and positive axillary lymph nodes emerged as significant LR occurrence predictors (p=0.0002 and p=0.0008 respectively). Particularly, the lobular and the ductal plus lobular histotypes showed a significant increased risk of LR in comparison to the ductal histotype (p=0.02 and p<0.0001, respectively).

Patients with a number of positive axillary lymph nodes greater than 3 showed an almost three-fold increase in risk of LR when compared with patients with negative axillary lymph nodes (p=0.0004).

CT did not emerge as a significant relapse predictor (p=0.66), although a protective effect was found. Even though not statistically significant (p=0.068) age was shown to have some effect. At the multivariate analysis with stepwise regression lobular and ductal plus lobular histotypes,

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

positive nodes >3 and age emerged as independent significant relapse predictors (p=0.018, p=0.0005, p=0.003 and p=0.024 respectively).

After a median time of 2.6 years (range 0.5-22.7 years), distant metastases occurred in 120 patients (34.7%).

For the DSS analysis, the median follow-up period was 6.78 years (range 0.6-36.7 years). At the time of the present analysis 215 patients (62.1%) were still alive. At the regression univariate analysis age (p<0.0001), pathological tumour size (p<0.0001), histotype (p=0.028), positive nodes (p<0.0001), CT (p=0.0001), LR (p=0.0004) and distant metastases (p<0.0001) all emerged as significant death predictors, as shown in Table 3. Surgical treatment (mastectomy versus breast conservation) did not turn out to be a significant LR predictor at the statistical analysis (data not shown).

In the subgroup of patients with positive axillary lymph nodes we evaluated whether a different regimen of CT influenced DSS. We found that there was not a significant statistical difference in DSS between CMF and anthracyclines (p=0.71) although anthracyclines showed a better DSS (40% versus 21%). At the multivariate analysis with stepwise regression performed on the whole series, age (p=0.0038), positive nodes (p=0.0035) and distant metastases (p<0.0001) resulted to be independent death predictors. DSS curves of the whole series by number of positives lymph-nodes and age groups (age ≤30 versus 30-35 years) are shown in Figure 1 and2.

We analyzed whether survival rates have changed over the 30 year time span of the study, by splitting the whole period in four intervals (before 1980, 1981-1990, 1991-2000, after 2000). As expected, a statistically significant improvement in terms of overall survival emerged in recent years (p=0.008), but no effect is observed on the results of the multivariate analysis.

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DISCUSSION

Survival following BC is improving, with over 88% of patients alive at 5 years [START_REF] Pagani | Breast cancer in young women: climbing for progress in care and knowledge[END_REF] although the prognosis appears to be worse in young women under 35 years at diagnosis [START_REF] Aebi | Is chemotherapy alone adequate for young women with oestrogen-receptor-positive breast cancer?[END_REF]. In our series of BC cases younger than 35 years at diagnosis, age younger than 30 years, positive nodes and distant metastases lead to a worse outcome in terms of DSS. In particular, age lower than 30 and positive nodes resulted to be independent prognostic factors at multivariate analysis for LDFS.

Similarly, Henderson reported lymph node involvement in BC to be the dominant prognostic indicator for later systemic disease [START_REF] Henderson | The relationship between prognostic and predictive factors in the management of breast cancer[END_REF]. Concerning age, Bernstein et al found that, if the HR of 1.0 describes the risk for women aged 40 to 49, then it is 1.8 for women under 30 years, 1.7 for those 30 to 34 years, and 1.5 for those 35 to 39 years [START_REF] Bernstein | How young is too young? The impact of age on premenopausal breast cancer prognosis[END_REF].

The observed difference in prognosis may relate to differences in the biology of BC in younger women. In our study, as in other reported series, the majority of BCs presenting in young women are invasive cancers and most are ductal infiltrating. Younger women are more likely to show larger tumours [START_REF] Anders | Young age at diagnosis correlates with worse prognosis and defines a subset of breast cancers with shared patterns of gene expression[END_REF]. Moreover it is now recognized that 'triple negative' (ER-ve, PR-ve and HER2-ve) and basal-like BCs, more common in younger women, have an aggressive clinical behaviour and are more likely to relapse within the first 5 years [START_REF] Rakha | Basal-like breast cancer: a critical review[END_REF].

In our series, patients younger than 30 years with positive nodes and specific histotypes (lobular or ductal plus lobular) had an higher rate of LR occurrence. Vicini et al. suggested that young patients have a significantly greater risk of local recurrence that is independent of other previously defined risk factors [START_REF] Vicini | Impact of young age on outcome in patients with ductal carcinoma-in-situ treated with breast-conserving therapy[END_REF] .

In our study patients who underwent adjuvant CT had a better outcome in terms of LDFS at univariate analysis although with no significant results. Although the results of our study should be interpreted cautiously due to the paucity of the population and the lack of a randomized design, they suggest that, in this subset of patients, anthracycline-based CT reduced disease relapse.

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Adjuvant CT is known to decrease the LR and to improve the survival rate of women with node-positive BC [13]. In women under 50 years old, the mean absolute improvement in 10-year survival after adjuvant chemotherapy is 7-11% [START_REF] Breast | Polychemotherapy for early breast cancer: an overview of the randomised trials[END_REF].

It has been demonstrated that 4 courses of AC are equivalent to 6 courses of CMF [START_REF] Fisher | Two months of doxorubicin-cyclophosphamide with and without interval reinduction therapy compared with 6 months of cyclophosphamide, methotrexate, and fluorouracil in positive-node breast cancer patients with tamoxifen-nonresponsive tumors: results from the National Surgical Adjuvant Breast and Bowel Project B-15[END_REF]. However, it is known that anthracycline-containing regimens yield superior results, in terms of both recurrence-free survival and OS, either in node-positive or in node-negative BC patients [START_REF] Levine | Randomized trial of intensive cyclophosphamide, epirubicin, and fluorouracil chemotherapy compared with cyclophosphamide, methotrexate, and fluorouracil in premenopausal women with node-positive breast cancer. National Cancer Institute of Canada Clinical Trials Group[END_REF][START_REF] Hutchins | Randomized, controlled trial of cyclophosphamide, methotrexate, and fluorouracil versus cyclophosphamide, doxorubicin, and fluorouracil with and without tamoxifen for high-risk, node-negative breast cancer: treatment results of Intergroup Protocol INT-0102[END_REF]. Similarly, our results showed a lower risk of LR in patients who underwent anthracyclinebased CT. A recent meta-analysis reported that in young women (under 50 years old), 6 cycles of an anthracycline-based combination CT (e.g. with FEC or FAC) are associated with a reduction in mortality of about 38% which approximates to a 5-15% absolute improvement in survival at 15 years of follow-up [START_REF] Group | Adjuvant chemotherapy in oestrogen-receptor-poor breast cancer: patient-level meta-analysis of randomised trials[END_REF].

It was suggested that CMF might not be enough to treat very young premenopausal patients [START_REF] Papaldo | Is anthracycline-based chemotherapy alone adequate for young women with estrogen receptor-positive breast cancer?[END_REF]. The anthracyclines and taxanes are considered the most effective drugs in the adjuvant setting and CMF is currently given to young patients in only few selected occasions. In our series the difference in DSS between CMF and anthracyclines was not statistically significant, most probably due to a small number of patients receiving CT. At least two randomised trials showed that 4 cycles of AC followed by paclitaxel improved OS compared with AC alone in patients with node-positive BC [START_REF] Henderson | Improved outcome from adding sequential paclitaxel but not escalating doxorubicin dose in an adjuvant chemotherapy regimen for patients with node positive primary breast cancer[END_REF][21].

Endocrine treatment effects had been considered of secondary importance for younger women presenting with a node-positive BC. However, the value of chemo-endocrine therapy for pre-menopausal patients has been defined recently. Meta-analysis of multiple randomized trials confirms that combining anthracyclines-based CT with 5 years of Tamoxifen, in women with HR+ve disease, reduces the risk of dying from BC by about 57% [22]. The International Breast Cancer Study Group Trial 13-93 results showed that Tamoxifen after adjuvant CT significantly improved treatment outcome in premenopausal patients with endocrine-responsive disease (HR for Tamoxifen vs no Tamoxifen 0.59; p <0.0001) [23].

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The impact of LR on survival is still a debated topic. Our data show that LR decreases breast cancer specific survival, but only in the univariate analysis (p=0.0004). In our Institute, all patients received RT to whole breast only after breast-conserving surgery. According to a recent paper [24], the local disease control does not uniformly improve survival; the largest absolute reduction in 5year LR probability after post-mastectomy RT was seen for the poor prognosis group (>3 positive nodes, tumor size >5 cm, Grade 3), but this large LR reduction did not translate into any reduction in 15-year BC mortality.

CONCLUSIONS

Young age, number of positive lymph nodes and distant metastases are associated with a worse DSS. LR resulted a statistically significant parameter at DSS univariate analysis. Patients younger than 30 years at the diagnosis of BC with more than 3 positive axillary lymph nodes and specific histotypes resulted to be associated to an increased risk of LR.

There is an urgent need for tailored treatment investigations with anthracyclines-based CT regimens, and young women with BC should be considered the optimal candidates for a combined anti-cancer strategy within the framework of randomized, controlled clinical trials.

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  JA, Wang M, Martino S, et al. Weekly paclitaxel in the adjuvant treatment of breast cancer. N Engl J Med 2008; 358:1663-71. 22) Early Breast Cancer Trialists' Collaborative Group. Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials. Lancet 2005; 365:1687-717. 23) International Breast Cancer Study Group. Tamoxifen after adjuvant chemotherapy for premenopausal women with lymph node-positive breast cancer: International Breast Cancer Study Group Trial 13-93. J Clin Oncol 2006; 24:1332-41. 24) Kyndi M, Overgaard M, Nielsen HM, et al. High local recurrence risk is not associated with large survival reduction after postmastectomy radiotherapy in high-risk breast cancer: a subgroup analysis of DBCG 82 b&c. Radiother Oncol 2009; 90:74-9.

Table 1 .

 1 Distribution of 346 breast cancer according to selected individual characteristics.

	Variable	N	%
	Age (years) < 30	76	22.0
	>30 and <35	270	78.0
	Surgical treatment
	Mastectomy	131	37.9
	Breast conservation	215	62.1
	Lymphadenectomy	323	93.3
	pT
	1a-1b	49	14.2
	1c	110	31.8
	2	148	42.8
	3	23	6.6
	4	16	4.6
	Histotype
	Ductal	222	64.2
	Lobular	31	9.0
	Ductal plus Lobular	37	10.7
	Other	56	16.1
	Positive lymph nodes
	0	194	56.1
	1-3	90	26.0
	>3	62	17.9
	Oestrogen receptor
	Negative	79	22.8
	Positive	113	32.7
	NA	154	44.5
	Progesterone receptor
	Negative	81	23.4
	Positive	110	31.8
	NA	155	44.8
	Total	346	100.0
	NA: not available

Table 2 .

 2 Local Disease Free Survival analysis* of 346 breast cancer cases according to selected individual characteristics: number of patients at risk, number of relapses, cumulative incidence (C.I.), log rank test and Hazard Risk (HR) with 95% Confidence Interval (Univariate regression analysis).

	Variable	Patients Relapses	C.I.	log rank	HR
		at risk	(n)	(%)	test	(95%CI)
		(n)
	Age (years) < 30	76	18	40
	>30 and <35	270	49	32	0.068	-
	pT
	1a-1b	49	7	23
	1c	110	18	35
	2	148	34	30
	3	23	5	28
	4	16	3	35	0.64	-
	Histotype
	Ductal	222	33	21		1^
	Lobular	31	9	31		2.37 (1.13-4.95)
	Ductal plus Lobular	37	13	60		3.79 (1.98-7.26)
	Other	56	12	38	0.0002	1.42 (0.73-2.75)
	Pos. lymph nodes
	0	194	34	33		1^
	1-3	90	16	26		1.16 (0.64-2.10)
	>3	62	17	40	0.0008	2.93 (1.62-5.30)
	Chemotherapy
	No	155	37	36
	EPI+CMF	43	6	17
	CMF	98	21	30
	AC	11	-	-
	EPI+TAX	19	1	6
	Other regimens	20	2	11	0.66	-
	Total	346	67	34
	1^= reference category for univariate regression analysis.
	* The multivariate regression analysis included all parameters listed in the table 2 (age, pT, histotype,
	number of positive lymph nodes, chemotherapy). Only four parameters emerged as independent significant
	relapse predictors at stepwise selection (age: HR: 0.53, 95%CI 0.31-0.92, p=0.024; lobular histotype:
	HR: 2.38, 95%CI 1.16-4.90, p=0.018; lobular+ductal histotype : HR: 3.18, 95%CI 1.66-6.08, p=0.0005;
	positive lymph nodes >3: HR: 2.39, 95%CI 1.33-4.27, p=0.008).

Table 3 .

 3 Disease specific survival* of 346 breast cancer cases according to selected individual characteristics: number of patients at risk, number of deaths, survival, log rank test and Hazard Risk with 95% Confidence Interval (Univariate regression analysis). DSS curves of 346 breast cancer cases by number of lymph-nodes positives. DSS curves of 346 breast cancer cases by study period.

	Variable	Patients Deaths Survival log rank	HR
		at risk	(n)	(%)	test	(95%CI)
		(n)
	Age (years) < 30 >30 and <35	76 270	39 92	33.2 50.5	<0.0001	1^ 0.48 (0.33-0.69)
	pT
	1a-1b	49	10	65.6		1^
	1c	110	20	66.6		0.78 (0.37-1.67)
	2	148	76	39.5		2.13 (1.10-4.13)
	3	23	13	36.0		2.73 (1.20-6.23)
	4	16	12	14.6	<0.0001	4.54 (1.96-10.5)
	Histotype
	Ductal	222	83	50.4		1^
	Lobular	31	15	25.5		1.46 (0.84-2.53)
	Ductal plus Lobular	37	17	18.5		1.84 (1.09-3.12)
	Other	56	16	54.1	0.028	0.74 (0.44-1.27)
	Pos. lymph nodes
	0	194	44	68.7		1^
	1-3	90	45	30.1		2.57 (1.69-3.89)
	>3	62	42	8.2	<0.0001	6.05 (3.92-9.30)
	Chemotherapy
	No	155	52	60.6		1^
	EPI+CMF	43	15	45.8		1.84 (1.02-3.19)
	CMF	98	43	20.1		1.68 (1.12-2.52)
	AC	11	-	-		-
	EPI+TAX	19	6	51.2	<0.0001	2.16 (1.13-6.27)
	Other regimens	20	15	16.8		3.67 (1.06-6.54)
	Local relapses
	No	279	87	55.8		1^
	Yes	67	44	20.1	0.0004	1.90 (1.32-2.73)
	Distant Metastases
	No	226	13	91.6	<0.0001	1^
	Yes	120	118	0.9		33.8 (18.8-60.8)
	Total	346	131	46.8
	1^= reference category for univariate regression analysis
	*The multivariate regression analysis included all parameters listed in the table 3 (age, pT, histotype, number
	of positive lymph nodes, chemotherapy, local relapses, distant metastases). Only three parameters emerged
	as independent significant death predictors at stepwise selection (age: HR: 0.57, 95%CI 0.39-0.83,
	p=0.0038; positive lymph nodes: HR: 1.42, 95%CI 1.12-1.79, p=0.0035; distant metastases: HR: 26.98,
	95%CI 14.75-49.34, p<0.0001).