No association between peripheral serotonin-gene-related DNA methylation and brain serotonin neurotransmission in the healthy and depressed state.

BACKGROUND: Methylation of serotonin-related genes has been proposed as a plausible gene-by-environment link which may mediate environmental stress, depressive and anxiety symptoms. DNA methylation is often measured in blood cells, but little is known about the association between this peripheral epigenetic modification and brain serotonergic architecture. Here, we evaluated the association between whole-blood-derived methylation of four CpG sites in the serotonin transporter (SLC6A4) and six CpG sites of the tryptophan hydroxylase 2 (TPH2) gene and in-vivo brain levels of serotonin transporter (5-HTT) and serotonin 4 receptor (5-HT4) in a cohort of healthy individuals (N = 254) and, for 5-HT4, in a cohort of unmedicated patients with depression (N = 90). To do so, we quantified SLC6A4/TPH2 methylation using bisulfite pyrosequencing and estimated brain 5-HT4 and 5-HTT levels using positron emission tomography. In addition, we explored the association between SLC6A4 and TPH2 methylation and measures of early life and recent stress, depressive and anxiety symptoms on 297 healthy individuals. RESULTS: We found no statistically significant association between peripheral DNA methylation and brain markers of serotonergic neurotransmission in patients with depression or in healthy individuals. In addition, although SLC6A4 CpG2 (chr17:30,236,083) methylation was marginally associated with the parental bonding inventory overprotection score in the healthy cohort, statistical significance did not remain after accounting for blood cell heterogeneity. CONCLUSIONS: We suggest that findings on peripheral DNA methylation in the context of brain serotonin-related features should be interpreted with caution. More studies are needed to rule out a role of SLC6A4 and TPH2 methylation as biomarkers for environmental stress, depressive or anxiety symptoms.

Oral contraceptives and the serotonin 4 receptor: a molecular brain imaging study in healthy women.

OBJECTIVE: Sex steroid hormones potently shape brain functions, including those critical to maintain mental health such as serotonin signaling. Use of oral contraceptives (OCs) profoundly changes endogenous sex steroid hormone levels and dynamics. Recent register-based studies show that starting an OC is associated with increased risk of developing depression. Here, we investigate whether use of OCs in healthy women is associated with a marker of the serotonin system in terms of serotonin 4 receptor (5-HT4R) brain imaging. METHODS: [11C]SB207145-PET imaging data on 53 healthy women, of whom 16 used OCs, were available from the Cimbi database. We evaluated global effects of OC use on 5-HT4R binding in a latent variable model based on 5-HT4R binding across cortical and subcortical regions. RESULTS: We demonstrate that OC users have 9-12% lower global brain 5-HT4R binding potential compared to non-users. Univariate region-based analyses (pallidostriatum, caudate, hippocampus, amygdala, anterior cingulate cortex, and neocortex) supported the global effect of OC use with the largest difference present in the hippocampus (-12.8% (95% CI [-21.0; -3.9], Pcorrected = 0.03). CONCLUSION: We show that women who use OCs have markedly lower brain 5-HT4R binding relative to non-users, which constitutes a plausible molecular link between OC use and increased risk of depressive episodes. We propose that this reflects a reduced 5-HT4R gene expression, possibly related to a blunted ovarian hormone state among OC users.

BDNF val66met association with serotonin transporter binding in healthy humans.

The serotonin transporter (5-HTT) is a key feature of the serotonin system, which is involved in behavior, cognition and personality and implicated in neuropsychiatric illnesses including depression. The brain-derived neurotrophic factor (BDNF) val66met and 5-HTTLPR polymorphisms have predicted differences in 5-HTT levels in humans but with equivocal results, possibly due to limited sample sizes. Within the current study we evaluated these genetic predictors of 5-HTT binding with [11C]DASB positron emission tomography (PET) in a comparatively large cohort of 144 healthy individuals. We used a latent variable model to determine genetic effects on a latent variable (5-HTTLV), reflecting shared correlation across regional 5-HTT binding (amygdala, caudate, hippocampus, midbrain, neocortex, putamen and thalamus). Our data supported a significant BDNF val66met effect on 5-HTTLV such that met-carriers showed 2-7% higher subcortical 5-HTT binding compared with val/val individuals (P=0.042). Our data did not support a BDNF val66met effect in neocortex and 5-HTTLPR did not significantly predict 5-HTTLV. We did not observe evidence for an interaction between genotypes. Our findings indicate that met-carriers have increased subcortical 5-HTT binding. The small difference suggests limited statistical power may explain previously reported null effects. Our finding adds to emerging evidence that BDNF val66met contributes to differences in the human brain serotonin system, informing how variability in the 5-HTT level emerges and may represent an important molecular mediator of BDNF val66met effects on behavior and related risk for neuropsychiatric illness.

Sex hormone manipulation slows reaction time and increases labile mood in healthy women.

BACKGROUND: Women show increased risk of depressive symptoms in life phases where ovarian steroid hormone levels fluctuate or decline rapidly. The risk mechanisms may include changes in mental state and affective cognition possibly mediated by serotonergic neurotransmission. METHODS: In a randomized controlled double-blinded trial, 61 healthy women (mean age 24.3±4.9 years) were tested with measures of affective verbal memory, reaction time, mental distress, and serotonin transporter binding at baseline and at follow-up after receiving gonadotropin-releasing hormone agonist (GnRHa) or placebo intervention. Women also reported daily mood profiles during intervention. We tested direct effects of intervention and indirect effects through changes in serotonin transporter binding on verbal affective memory, simple reaction time and self-reported measures of mental distress, and further effects of GnRHa on daily mood. RESULTS: GnRHa induced an increase in simple reaction time (p=0.03) and more pronounced fluctuations in daily self-reported mood in a manner dependent on baseline mood (p=0.003). Verbal affective memory recall, overall self-perceived mental distress, and serotonin transporter binding were not affected. CONCLUSIONS: In healthy women transient sex-steroid hormone fluctuations decrease speed of information processing and further produce more labile mood only in women with elevated levels of mood disturbances at baseline.

Central 5-HT neurotransmission modulates weight loss following gastric bypass surgery in obese individuals.

The cerebral serotonin (5-HT) system shows distinct differences in obesity compared with the lean state. Here, it was investigated whether serotonergic neurotransmission in obesity is a stable trait or changes in association with weight loss induced by Roux-in-Y gastric bypass (RYGB) surgery. In vivo cerebral 5-HT2A receptor and 5-HT transporter binding was determined by positron emission tomography in 21 obese [four men; body mass index (BMI), 40.1 ± 4.1 kg/m(2)] and 10 lean (three men; BMI, 24.6 ± 1.5 kg/m(2)) individuals. Fourteen obese individuals were re-examined after RYGB surgery. First, it was confirmed that obese individuals have higher cerebral 5-HT2A receptor binding than lean individuals. Importantly, we found that higher presurgical 5-HT2A receptor binding predicted greater weight loss after RYGB and that the change in 5-HT2A receptor and 5-HT transporter binding correlated with weight loss after RYGB. The changes in the 5-HT neurotransmission before and after RYGB are in accordance with a model wherein the cerebral extracellular 5-HT level modulates the regulation of body weight. Our findings support that the cerebral 5-HT system contributes both to establish the obese condition and to regulate the body weight in response to RYGB.

Central 5-HT4 receptor binding as biomarker of serotonergic tonus in humans: a [11C]SB207145 PET study.

Identification of a biomarker that can inform on extracellular serotonin (5-HT) levels in the brains of living humans would enable greater understanding of the way brain circuits are modulated by serotonergic neurotransmission. Substantial evidence from studies in animals and humans indicates an inverse relationship between central 5-HT tonus and 5-HT type 4 receptor (5-HT4R) density, suggesting that 5-HT4R receptor density may be a biomarker marker for 5-HT tonus. Here, we investigated whether a 3-week administration of a selective serotonin reuptake inhibitor, expected to increase brain 5-HT levels, is associated with a decline in brain 5-HT4R binding. A total of 35 healthy men were studied in a placebo-controlled, randomized, double-blind study. Participants were assigned to receive 3 weeks of oral dosing with placebo or fluoxetine, 40 mg per day. Brain 5-HT4R binding was quantified at baseline and at follow-up with [(11)C]SB207145 positron emission tomography (PET). Three weeks of intervention with fluoxetine was associated with a 5.2% reduction in brain 5-HT4R binding (P=0.017), whereas placebo intervention did not change 5-HT4R binding (P=0.52). Our findings are consistent with a model, wherein the 5-HT4R density adjusts to changes in the extracellular 5-HT tonus. Our data demonstrate for the first time in humans that the imaging of central 5-HT4R binding may be used as an in vivo biomarker of the central 5-HT tonus.

FKBP5 and emotional neglect interact to predict individual differences in amygdala reactivity.

Individual variation in physiological responsiveness to stress mediates risk for mental illness and is influenced by both experiential and genetic factors. Common polymorphisms in the human gene for FK506 binding protein 5 (FKBP5), which is involved in transcriptional regulation of the hypothalamic-pituitary-adrenal (HPA) axis, have been shown to interact with childhood abuse and trauma to predict stress-related psychopathology. In the current study, we examined if such gene-environment interaction effects may be related to variability in the threat-related reactivity of the amygdala, which plays a critical role in mediating physiological and behavioral adaptations to stress including modulation of the HPA axis. To this end, 139 healthy Caucasian youth completed a blood oxygen level-dependent functional magnetic resonance imaging probe of amygdala reactivity and self-report assessments of emotional neglect (EN) and other forms of maltreatment. These individuals were genotyped for 6 FKBP5 polymorphisms (rs7748266, rs1360780, rs9296158, rs3800373, rs9470080 and rs9394309) previously associated with psychopathology and/or HPA axis function. Interactions between each SNP and EN emerged such that risk alleles predicted relatively increased dorsal amygdala reactivity in the context of higher EN, even after correcting for multiple testing. Two different haplotype analyses confirmed this relationship as haplotypes with risk alleles also exhibited increased amygdala reactivity in the context of higher EN. Our results suggest that increased threat-related amygdala reactivity may represent a mechanism linking psychopathology to interactions between common genetic variants affecting HPA axis function and childhood trauma.

Linking variability in brain chemistry and circuit function through multimodal human neuroimaging.

Identifying neurobiological mechanisms mediating the emergence of individual differences in behavior is critical for advancing our understanding of relative risk for psychopathology. Neuroreceptor positron emission tomography (PET) and functional magnetic resonance imaging (fMRI) can be used to assay in vivo regional brain chemistry and function, respectively. Typically, these neuroimaging modalities are implemented independently despite the capacity for integrated data sets to offer unique insight into molecular mechanisms associated with brain function. Through examples from the serotonin and dopamine system and its effects on threat- and reward-related brain function, we review evidence for how such a multimodal neuroimaging strategy can be successfully implemented. Furthermore, we discuss how multimodal PET-fMRI can be integrated with techniques such as imaging genetics, pharmacological challenge paradigms and gene-environment interaction models to more completely map biological pathways mediating individual differences in behavior and related risk for psychopathology and inform the development of novel therapeutic targets.

Accelerated radical radiotherapy for non-small cell lung cancer using two common regimens: a single-centre retrospective study of outcome.

AIMS: A variety of radical radiotherapy regimens are in use for non-small cell lung cancer. Continuous hyperfractionated accelerated radiotherapy (CHART: 54 Gy in 36 fractions over 12 days) and accelerated hypofractionated radiotherapy using 55 Gy in 20 fractions over 4 weeks are standard fractionations in our centre. The primary aim of this retrospective study was to evaluate survival outcome seen in routine clinical practice. MATERIALS AND METHODS: All case notes and radiotherapy records of radically treated patients between 1999 and 2004 were retrospectively reviewed. Basic patient demographics, tumours, characteristics, radiotherapy and survival data were collected. RESULTS: In total, 277 patients received radical radiotherapy: 137 and 140 patients received CHART and hypofractionated radiotherapy, respectively. There were differences noted in the demographics between the two treatment schedules: median age 65 years (range 41-83) vs 73 years (range 33-87); histological confirmation rates 90% vs 76%; prior chemotherapy 34% vs 19% for CHART and hypofractionated treatment, respectively. For CHART patients, stages I, II, III and unclassified were 12, 8, 68 and 12% and the staging for the hypofractionated regimen was 54, 11, 34 and 2%, respectively. The median overall survival from the time of diagnosis was 20.4 months with a 40% 2-year survival rate. For the two fractionations the median survival was 16.6 months vs 21.4 months and 34% vs 45% of patients were alive at 2 years in the CHART and hypofractionated groups, respectively. On multivariate analysis, stage was the only factor affecting overall survival - no difference was seen according to radiotherapy regimen. CONCLUSION: This single-centre study reflects the outcome of unselected consecutively treated non-small cell lung cancer patients. Adjusting for stage, there was no significant difference in survival seen according to regimen. Encouragingly, CHART outcome shows reproducibility with the original CHART paper. Our hypofractionated outcome is similar to that previously reported, but despite this being the UK's most common regimen, 55 Gy in 20 daily fractions remains unvalidated by phase III trial data.

Capacity for 5-HT1A-mediated autoregulation predicts amygdala reactivity.

We examined the contribution of 5-HT1A autoreceptors (with [11C]WAY100635 positron emission tomography) to amygdala reactivity (with blood oxygenation level-dependent functional magnetic resonance imaging) in 20 healthy adult volunteers. We found a significant inverse relationship wherein 5-HT1A autoreceptor density predicted a notable 30-44% of the variability in amygdala reactivity. Our data suggest a potential molecular mechanism by which a reduced capacity for negative feedback regulation of 5-HT release is associated with increased amygdala reactivity.

The use of lung dose-volume histograms in predicting post-radiation pneumonitis after non-conventionally fractionated radiotherapy for thoracic carcinoma.

AIMS: To assess the use of lung dose-volume histogram (DVH) parameters (specifically V20Gy) in the prediction of radiation pneumonitis for non-conventional fraction sizes used in the treatment of lung cancer. MATERIALS AND METHODS: Patients requiring computed tomography planning for thoracic radiotherapy between January 1999 and January 2002 were identified. The patients receiving radical or high-dose palliative radiotherapy had DVH produced routinely during planning. These were retrospectively reviewed and the case notes accessed for additional pre-treatment parameters, demographics and evidence of radiation pneumonitis. The severity of the pneumonitis was then scored using Radiation Therapy Oncology Group criteria. Data were analysed using the SPSS computer program. RESULTS: One hundred and sixty consecutive patients were reviewed. Ninety patients received hypofractionated treatment (fraction size > 2.5 Gy) and 57 continuous hyperfractionated accelerated radiation therapy (CHART) (fraction size 1.5 Gy). Lung V20Gy values ranged from 3% to 53%, with a median value of 24%. Only six patients reported grade 2, and 16 patients grade 3 pneumonitis. Two patients developed fatal, grade 5 pneumonitis. No correlation between pneumonitis score and V20Gy or other possible predictive factors was found. CONCLUSION: The 15% grade 2-5 pneumonitis rate we document is at the lower end of the spectrum reported in other studies. This suggests that using published data on limiting V20Gy values to reduce the risk of radiation pneumonitis can be extrapolated to planning treatment with non-conventionally fractionated radiotherapy.

The impact of virtual simulation in palliative radiotherapy for non-small-cell lung cancer.

BACKGROUND AND PURPOSE: Radiotherapy is widely used to palliate local symptoms in non-small-cell lung cancer. Using conventional X-ray simulation, it is often difficult to accurately localize the extent of the tumour. We report a randomized, double blind trial comparing target localization with conventional and virtual simulation. METHODS: Eighty-six patients underwent both conventional and virtual simulation. The conventional simulator films were compared with digitally reconstructed radiographs (DRRs) produced from the computed tomography (CT) data. The treatment fields defined by the clinicians using each modality were compared in terms of field area, position and the implications for target coverage. RESULTS: Comparing fields defined by each study arm, there was a major mis-match in coverage between fields in 66.2% of cases, and a complete match in only 5.2% of cases. In 82.4% of cases, conventional simulator fields were larger (mean 24.5+/-5.1% (95% confidence interval)) than CT-localized fields, potentially contributing to a mean target under-coverage of 16.4+/-3.5% and normal tissue over-coverage of 25.4+/-4.2%. CONCLUSIONS: CT localization and virtual simulation allow more accurate definition of the target volume. This could enable a reduction in geographical misses, while also reducing treatment-related toxicity.

A study of waiting times and waiting lists for radiation therapy patients.

A simple mathematical model based on queueing theory is introduced, and used to obtain a sensitive measure of the capacity of a radiation therapy centre to provide service. The model illustrates the relationships among the relevant variables: the patient waiting time, the number on the waiting list, the rate at which requests for radiation therapy are received and the rate at which courses of treatment are commenced. In particular, the rate at which the waiting time increases is equal to the difference between the request rate and the start rate, expressed as a fraction of the start rate, and is therefore a measure of the deficiency in service capacity of a facility. A study of the records of patients treated on the linear accelerators of the Tom Baker Cancer Centre from January 1991 to June 1994 shows that this relationship holds to a high degree of accuracy for average values of the various parameters in spite of the considerable variation in these values, and particularly variation in the individual waiting times. This finding suggests that such an approach would be useful in assessing and comparing the performance of radiation therapy facilities.

Malignant schwannoma following treatment for Hodgkin's disease.

Two cases of malignant schwannoma are reported 13 and 15 years following combined modality treatment for Hodgkin's disease. Both patients survived for only 15 months. The aetiology of this rare condition is discussed.

Mild and moderate dyskaryosis: can women be selected for colposcopy on the basis of social criteria?

OBJECTIVE: To describe the distribution of cervical intraepithelial neoplasia grades among women with mild and moderate dyskaryosis after a single cervical smear and to determine whether social criteria could help identify women who are at increased risk of grade II or III disease. DESIGN: Cross sectional analysis within a randomised prospective study. Subjects had a repeat smear, a colposcopic examination, and an excision biopsy of the transformation zone. In addition, women were asked to complete a social questionnaire. SETTING: Colposcopy clinic, Aberdeen. SUBJECTS: 228 women with a single smear test showing mild or moderate dyskaryosis. MAIN OUTCOME MEASURES: Histology, age, sexual and contraceptive history, cigarette smoking. RESULTS: 159 (70%) women had cervical intraepithelial neoplasia grades II or III. Among current smokers the prevalence of grade II and III disease was higher in women who smoked greater than or equal to 20 cigarettes a day (84%) than among those who smoked less (66%; p less than 0.04). Women with more than one sexual partner also had a higher prevalence (75%) than women with only one partner (50%; p = 0.0028). Use of oral contraceptives and younger age were not significantly associated. The prevalence of grade II or III disease was up to 66% in the lower risk groups. CONCLUSIONS: Because of the high prevalence of cervical intraepithelial neoplasia grades II and III in both the high and the low risk groups social factors are not useful for selecting women with mild or moderate dyskaryosis for either early referral to colposcopy or cytological surveillance.

A population-based study of microinvasive disease of the cervix--a colposcopic and cytologic analysis.

A study of 61 cases of microinvasion of the cervix occurring in our population between 1980 and 1989 is reported. The mean age of the women was 39 years, compared with 30 years for cervical intraepithelial grade 3 (CIN III) and 47 years for frank invasion, respectively. Colposcopic suspicion of microinvasion was present in 31 cases, giving a sensitivity of colposcopic diagnosis of 50% and a specificity of 91%. In 21 cases (34%) there was no suspicion either cytologically or colposcopically of microinvasion. Colposcopy predicted microinvasion more accurately with increasing depth of invasion. In 28 women there had been previous smears within 10 years available for review. The time interval between the first abnormal smear and the histological diagnosis ranged from 1 month to 9.8 years (mean, 4 years).