Automation and standardisation of a quantitative multiplex PCR assay using PCR.Ai.

BACKGROUND: We previously undertook a prospective clinical study to evaluate PCR.Ai's (www.pcr.ai) accuracy and impact when automating the manual data-analysis and quality control steps associated with routine clinical pathogen testing using a non-quantitative multiplex quantitative real-time PCR (qPCR). In this study we demonstrated 100 % concurrence between our manual routine analysis method and PCR.Ai. This paper expands the evaluation of PCR.Ai's (www.pcr.ai) accuracy and impact using a multiplex quantitative real-time PCR (qPCR). OBJECTIVES: We evaluated the impact of PCR.Ai when used as the final interpretation/verification step for routine in-house multiplex quantitative qPCR tests for CMV, EBV and adenovirus from blood samples for a total of 1350 interpretations. STUDY DESIGN: We compared PCR.Ai to our existing manual interpretation, to determine accuracy and hands on time savings. RESULTS AND CONCLUSIONS: There was 100 % concurrence between validated CMV, EBV and adenovirus detection and quantitation by our manual routine analysis method and PCR.Ai. Furthermore, there were significant routine savings with PCR.Ai of 63 minutes/ run. Our conclusion is that for quantitative tests PCR.Ai is a highly accurate time-saving tool that reduces complexity of qPCR analysis and hence the need for specialists and hands-on time. It demonstrated capabilities to enable us to get results out more quickly with lower costs and less risk of errors.

The effect of pre-analytical variables on downstream application and data analysis of human endometrial biopsies.

STUDY QUESTION: What are the effects of pre-analytical variables on the downstream analysis of patient-derived endometrial biopsies? SUMMARY ANSWER: There are distinct differences in the protein levels of the master regulator of oxygen homeostasis, hypoxia-inducible factor-1-alpha (HIF1α), and the protein and mRNA levels of three related genes, carbonic anhydrase 9 (CA9), vascular endothelial growth factor A (VEGFA) and progesterone receptor (PR) in human endometrial biopsies, depending on the pre-analytical variables: disease status (cancer vs benign), timing of biopsy (pre- vs post-hysterectomy) and type of biopsy (pipelle vs full-thickness). WHAT IS KNOWN ALREADY: Patient-derived biopsies are vital to endometrial research, but pre-analytical variables relating to their collection may affect downstream analysis, as is evident in other tissues. STUDY DESIGN SIZE DURATION: A prospective observational study including patients undergoing hysterectomy for endometrial cancer (EC) or benign indications was conducted at a large tertiary gynaecological unit in the UK. Endometrial biopsies were obtained at different time points (pre- or post-hysterectomy) using either a pipelle endometrial sampler or as a full-thickness wedge biopsy. PARTICIPANTS/MATERIALS SETTING METHODS: The changes in HIF1α, CA9, VEGFA and PR protein levels were measured by semi-quantitative analysis of immunostaining, and the expression levels of three genes (CA9, VEGFA and PR) were investigated by quantitative real-time PCR, in endometrial biopsies from 43 patients undergoing hysterectomy for EC (n = 22) or benign gynaecological indications (n = 21). MAIN RESULTS AND THE ROLE OF CHANCE: An increase in HIF1α immunostaining was observed in EC versus benign endometrium (functionalis glands) obtained pre-hysterectomy (P < 0.001). An increase in CA9 immunostaining was observed in EC versus benign endometrial functionalis glands at both pre- and post-hysterectomy time points (P = 0.03 and P = 0.003, respectively). Compared with benign endometrial pipelle samples, EC samples demonstrated increased mRNA expression of CA9 (pre-hysterectomy P < 0.001, post-hysterectomy P = 0.008) and VEGFA (pre-hysterectomy P = 0.004, post-hysterectomy P = 0.002). In benign uteri, HIF1α immunoscores (functionalis glands, P = 0.03 and stroma, P = 0.009), VEGFA immunoscores (functionalis glands, P = 0.03 and stroma, P = 0.01) and VEGFA mRNA levels (P = 0.008) were increased in matched post-hysterectomy versus pre-hysterectomy samples. Similarly, in EC, an increase in VEGFA immunoscores (epithelial and stromal) and VEGFA mRNA expression was observed in the matched post-hysterectomy versus pre-hysterectomy biopsies (P = 0.008, P = 0.004 and P = 0.018, respectively). Full-thickness benign post-hysterectomy endometrial biopsies displayed increased VEGFA (P = 0.011) and PR (P = 0.006) mRNA expression compared with time-matched pipelle biopsies. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: This descriptive study explores the effect of pre-analytical variables on the expression of four proteins and three hypoxia-related genes in a limited number of endometrial biopsies from patients with EC and benign controls. Due to the small number, it was not possible to investigate other potential variables such as menstrual cycle phase, region-specific differences within the endometrium, grade and stage of cancer, and surgical technicalities. WIDER IMPLICATIONS OF THE FINDINGS: Careful consideration of the effects of these pre-analytical variables is essential when interpreting data relating to human endometrial biopsies. A standardized approach to endometrial tissue collection is essential to ensure accurate and clinically transferrable data. STUDY FUNDING/COMPETING INTERESTS: The authors have no conflicts of interest to declare. The work included in this manuscript was funded by Wellbeing of Women project grants RG1073 and RG2137 (D.K.H.), Wellbeing of Women Entry-Level Scholarship ELS706 and Medical Research Council MR/V007238/1 (A.M./D.K.H.), Liverpool Women's Hospital Cancer Charity (M.A.) and University of Liverpool (L.B., L.R. and E.N.).

Phosphate stress triggers the conversion of glycerol into l-carnitine in Pseudomonas fluorescens.

Glycerol, a by-product of the biofuel industry is transformed into l-carnitine when the soil microbe Pseudomonas fluorescens is cultured in a phosphate-limited mineral medium (LP). Although the biomass yield was similar to that recorded in phosphate-sufficient cultures (HP), the rate of growth was slower. Phosphate was completely consumed in the LP cultures while in the HP media, approximately 35 % of the initial phosphate was detected at stationary phase of growth. The enhanced production of α-ketoglutarate (KG) in HP cultures supplemented with manganese was recently reported (Alhasawi et al., 2017). l-carnitine appeared to be a prominent metabolite in the spent fluid while the soluble cellular-free extract was characterized with peaks attributable to lysine, γ-butyrobetaine (GB), acetate and succinate in the LP cultures. Upon incubation with glycerol and NH4Cl, the resting cells readily secreted l-carnitine and revealed the presence of such precursors like GB, lysine and methionine involved in the synthesis of this trimethylated moiety. Functional proteomic studies of select enzymes participating in tricarboxylic acid cycle (TCA), oxidative phosphorylation (OP), glyoxylate cycle and l-carnitine synthesis revealed a major metabolic reconfiguration evoked by phosphate stress. While isocitrate dehydrogenase-NAD+ dependent (ICDH-NAD+) and Complex I were markedly diminished, the activities of γ-butyrobetaine aldehyde dehydrogenase (GBADH) and l-carnitine dehydrogenase (CDH) were enhanced. Real-time quantitative polymerase chain reaction (RT-qPCR) analyses pointed to an increase in transcripts of the enzymes γ-butyrobetaine dioxygenase (bbox1), S-adenosylmethionine synthase (metK) and l-carnitine dehydrogenase (lcdH). The l-carnitine/γ-butyrobetaine antiporter (caiT) was enhanced more than 400-fold in the LP cultures compared to the HP controls. This metabolic reprogramming modulated by phosphate deprivation may provide an effective technology to transform glycerol, an industrial waste into valuable l-carnitine.

Fallopian tube epithelial cells express androgen receptor and have a distinct hormonal responsiveness when compared with endometrial epithelium.

STUDY QUESTION: How does steroid receptor expression, proliferative activity and hormone responsiveness of the fallopian tube (FT) epithelium compare to that of the endometrial epithelium? SUMMARY ANSWER: Proliferative indices, hormone receptor expression-scores and in vitro response to oestrogen and androgens of the human FT demonstrate a distinct pattern from the matched endometrium. WHAT IS KNOWN ALREADY: The FT epithelium exists as a continuum of the endometrium, and both express steroid hormone receptors. The ovarian steroid hormones regulate cyclical proliferation and regeneration of the endometrium, but their effects on steroid hormone receptor expression and proliferation in the FT have not yet been fully elucidated. STUDY DESIGN, SIZE, DURATION: We included women with proven fertility, undergoing hysterectomy and bilateral salpingo-oophorectomy for benign, gynaecological conditions at Liverpool Women's NHS Foundation Trust. They had no known endometrial or tubal pathology and were not on hormonal treatments for at least 3 months preceding sample collection in this prospective observational study (conducted between 2010 and 2018). A full-thickness sample of the endometrium and a sample from the FT were collected from each woman. PARTICIPANTS/MATERIALS, SETTING, METHODS: The differential protein and mRNA levels of steroid hormone receptors, oestrogen receptors α and ß, androgen receptor (AR) and progesterone receptor (PR), and the proliferative marker (Ki67) of the endometrium and the FT tissue samples from 47 healthy women undergoing surgery (37 premenopausal and 10 postmenopausal) were investigated using immunohistochemistry and quantitative real-time PCR. The comparative responsiveness to oestrogen and androgen of the endometrium and the fimbrial end of the FT was analysed using an in vitro short-term explant culture model. The endpoints assessed in the explants were the changes in mRNA and protein levels for AR, PR and the epithelial proliferative index after 24 h treatment with oestradiol (E2) or dihydrotestosterone (DHT). MAIN RESULTS AND THE ROLE OF CHANCE: The premenopausal endometrial functionalis glands (FG) displayed the well-known cyclic variation in cellular proliferation and steroid receptor scores. Compared with the endometrial FG, the matched FT epithelium (both fimbrial or isthmic ends) displayed a significantly lower proportion of cells expressing Ki67 (2.8% ± 2.2%, n = 18 vs 30.0% ± 26.3%, n = 16, P = 0.0018, respectively) accompanied with a significantly higher AR immunoscores (6.7 ± 2.7, n = 16 vs 0.3 ± 1.0, n = 10, P = 0.0136). The proportion of cells expressing Ki67 and the AR immunoscores of the FT epithelium correlated positively with endometrial luminal epithelium (r = 0.62, P = 0.005, and r = 0.68, P = 0.003, respectively). In vitro experiments suggested the tubal explants to be apparently less responsive to E2 yet more sensitive to DHT compared with the matched endometrium explants. LIMITATIONS, REASONS FOR CAUTION: The short-term in vitro nature of the tissue explant cultures used in the study may not be representative of how different anatomical regions of the endometrium and FT behave in vivo. Our study included a high proportion of older premenopausal women with a regular menstrual cycle, which may therefore affect extrapolation of findings to a younger group. WIDER IMPLICATIONS OF THE FINDINGS: Advancing our understanding of tubal and endometrial epithelial cell function has important implications for the diagnosis and treatment of diseases such as infertility, ectopic pregnancy, endometriosis and cancer. STUDY FUNDING/COMPETING INTEREST(S): The work included in this article was funded by Wellbeing of Women project grants RG1073 and RG2137 (D.K.H.) and Wellbeing of Women Entry-Level Scholarship ELS706 (A.M). A.M. was also supported by an NIHR ACF fellowship grant. Further support received from Liverpool Women's Hospital NHS Trust (S.M.), University of Liverpool (E.B. and A.W.). All authors declare there are no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Identification of a Novel Enhancer/Chromatin Opening Element Associated with High-Level γ-Globin Gene Expression.

The organization of the five ß-type globin genes on chromosome 11 reflects the timing of expression during erythroid cell development, with the embryonic ε-globin gene being located at the 5' end, followed by the two fetal γ-globin genes, and with the adult ß- and δ-globin genes being located at the 3' end. Here, we functionally characterized a DNase I-hypersensitive site (HS) located 4 kb upstream of the Gγ-globin gene (HBG-4kb HS). This site is occupied by transcription factors USF1, USF2, EGR1, MafK, and NF-E2 in the human erythroleukemia cell line K562 and exhibits histone modifications typical for enhancers. We generated a synthetic zinc finger (ZF) DNA-binding domain targeting the HBG-4kb HS (HBG-4kb ZF). The HBG-4kb ZF interacted with the target site in vitro and in the context of cells with a high affinity and specificity. Direct delivery of the HBG-4kb ZF to K562 and primary human erythroid cells caused a reduction in γ-globin gene expression which was associated with decreased binding of transcription factors and active histone marks at and downstream of the HS. The data demonstrate that the HBG-4kb HS is important for fetal globin production and suggest that it may act by opening chromatin in a directional manner.

Robotic arm-assisted versus conventional unicompartmental knee arthroplasty: Exploratory secondary analysis of a randomised controlled trial.

OBJECTIVES: This study reports on a secondary exploratory analysis of the early clinical outcomes of a randomised clinical trial comparing robotic arm-assisted unicompartmental knee arthroplasty (UKA) for medial compartment osteoarthritis of the knee with manual UKA performed using traditional surgical jigs. This follows reporting of the primary outcomes of implant accuracy and gait analysis that showed significant advantages in the robotic arm-assisted group. METHODS: A total of 139 patients were recruited from a single centre. Patients were randomised to receive either a manual UKA implanted with the aid of traditional surgical jigs, or a UKA implanted with the aid of a tactile guided robotic arm-assisted system. Outcome measures included the American Knee Society Score (AKSS), Oxford Knee Score (OKS), Forgotten Joint Score, Hospital Anxiety Depression Scale, University of California at Los Angeles (UCLA) activity scale, Short Form-12, Pain Catastrophising Scale, somatic disease (Primary Care Evaluation of Mental Disorders Score), Pain visual analogue scale, analgesic use, patient satisfaction, complications relating to surgery, 90-day pain diaries and the requirement for revision surgery. RESULTS: From the first post-operative day through to week 8 post-operatively, the median pain scores for the robotic arm-assisted group were 55.4% lower than those observed in the manual surgery group (p = 0.040).At three months post-operatively, the robotic arm-assisted group had better AKSS (robotic median 164, interquartile range (IQR) 131 to 178, manual median 143, IQR 132 to 166), although no difference was noted with the OKS.At one year post-operatively, the observed differences with the AKSS had narrowed from a median of 21 points to a median of seven points (p = 0.106) (robotic median 171, IQR 153 to 179; manual median 164, IQR 144 to 182). No difference was observed with the OKS, and almost half of each group reached the ceiling limit of the score (OKS > 43). A greater proportion of patients receiving robotic arm-assisted surgery improved their UCLA activity score.Binary logistic regression modelling for dichotomised outcome scores predicted the key factors associated with achieving excellent outcome on the AKSS: a pre-operative activity level > 5 on the UCLA activity score and use of robotic-arm surgery. For the same regression modelling, factors associated with a poor outcome were manual surgery and pre-operative depression. CONCLUSION: Robotic arm-assisted surgery results in improved early pain scores and early function scores in some patient-reported outcomes measures, but no difference was observed at one year post-operatively. Although improved results favoured the robotic arm-assisted group in active patients (i.e. UCLA ⩾ 5), these do not withstand adjustment for multiple comparisons.Cite this article: M. J. G. Blyth, I. Anthony, P. Rowe, M. S. Banger, A. MacLean, B. Jones. Robotic arm-assisted versus conventional unicompartmental knee arthroplasty: Exploratory secondary analysis of a randomised controlled trial. Bone Joint Res 2017;6:631-639. DOI: 10.1302/2046-3758.611.BJR-2017-0060.R1.

Contributions of Nonhuman Primates to Research on Aging.

Aging is the biological process of declining physiologic function associated with increasing mortality rate during advancing age. Humans and higher nonhuman primates exhibit unusually longer average life spans as compared with mammals of similar body mass. Furthermore, the population of humans worldwide is growing older as a result of improvements in public health, social services, and health care systems. Comparative studies among a wide range of organisms that include nonhuman primates contribute greatly to our understanding about the basic mechanisms of aging. Based on their genetic and physiologic relatedness to humans, nonhuman primates are especially important for better understanding processes of aging unique to primates, as well as for testing intervention strategies to improve healthy aging and to treat diseases and disabilities in older people. Rhesus and cynomolgus macaques are the predominant monkeys used in studies on aging, but research with lower nonhuman primate species is increasing. One of the priority topics of research about aging in nonhuman primates involves neurologic changes associated with cognitive decline and neurodegenerative diseases. Additional areas of research include osteoporosis, reproductive decline, caloric restriction, and their mimetics, as well as immune senescence and chronic inflammation that affect vaccine efficacy and resistance to infections and cancer. The purpose of this review is to highlight the findings from nonhuman primate research that contribute to our understanding about aging and health span in humans.

Clinical outcomes of elderly patients receiving neoadjuvant chemoradiation for locally advanced rectal cancer.

BACKGROUND: Studies of clinical outcomes of elderly patients treated with neoadjuvant chemoradiation (nCRT) for locally advanced rectal cancer (LARC) are limited. Our aim was to assess the impact of age on clinical outcomes in a large multi-institutional database. PATIENTS AND METHODS: Data for patients diagnosed with LARC who received nCRT and curative-intent surgery between 2005 and 2012 were collected from five major Canadian cancer centers. Age was analyzed as a continuous and dichotomous variable (< 70 versus ≥ 70 years) and correlated with disease-free survival (DFS), cancer-specific survival (CSS) and overall survival (OS). Cox regression models were used to adjust for important prognostic factors. RESULTS: Of 1172 patients included, 295 (25%) were ≥ 70 years, and they were less likely to receive adjuvant chemotherapy (ACT; 60% versus 79%, P < 0.0001), oxaliplatin-based ACT (12% versus 31%, P < 0.0001), less likely to complete nCT (76% versus 86%, P < 0.001), and more likely to be anemic at initiation of nCRT (42% versus 30%, P = 0.0004). In multivariate analyses, age ≥ 70 years was associated with similar DFS [hazard ratio (HR) 0.93, 95% confidence interval (CI) 0.68-1.26, P = 0.63], similar CSS (HR 0.81, 95% CI 0.46-1.41, P = 0.45), and similar OS (HR 1.28, 95% CI 0.88-1.86, P = 0.20), compared with the younger age group. As a continuous variable, increasing age was not predictive of DFS (HR 1.00, 95% CI 0.99-1.02, P = 0.49) or CSS (HR 1.002, 95% CI 0.98-1.02, P = 0.88); however, it correlated with an inferior OS (HR 1.02, 95% CI 1.00-1.03, P = 0.04). CONCLUSIONS: Elderly patients (≥ 70 years) who receive nCRT followed by surgery appear to have similar outcomes compared with younger patients. Decisions regarding eligibility for nCRT and surgery should not be based on age alone.

Ileocaecal junction carcinoma: a clinicopathological study of 199 cases.

AIM: The ileocaecal junction (ICJ) region is an epithelial transition zone in which carcinomas are frequently diagnosed. However, it is currently unknown whether ICJ carcinomas (ICJ-CAs) have distinctive features. This study aimed to characterize the clinicopathological features of ICJ-CAs. METHOD: All ileal and colorectal resections for carcinoma, performed in Calgary, Canada between January 2009 and June 2012, were reviewed. Carcinomas in which the epicentre was within 5 cm of the ileocaecal valve (ICV) were defined as ICJ-CAs. Of 1003 carcinomas studied, 199 (19.8%) were ICJ-CAs, including 93 (9.3%) that crossed the ICV. Comparison of clinicopathological features with carcinomas of the other ileo-colorectal regions was made. Survival was also assessed. RESULTS: Clinically, ICJ-CAs were more common in female than male patients (56.3% female) compared with left-colonic (42.9% female) and rectal (37.9% female) carcinomas, and were more common in older age-groups of patients (71.8 ± 12.7 years) compared with appendiceal (62.6 ± 11.3 years), left-colonic (69.4 ± 12.3 years) and rectal (67.1 ± 11.9 years) carcinomas. Macroscopically, ICJ-CAs were similar to other colorectal carcinomas and were mostly described as ulcerated (63.3%). Histologically, ICJ-CAs had more mucinous, signet-ring cell and/or neuroendocrine features (39.7%, 8.0% and 7.5%, respectively) than did carcinomas of the left colon (16.8%, 1.6% and 1.1%, respectively) and the rectum (14.1%, 1.0% and 0.0%, respectively). They were higher grade (20.1% were high grade) than those of the left-colon (10.3%) and the rectum (9.8%). ICJ-CAs presented at a higher T-stage (25.6% were T4) compared with rectal carcinomas (11.6%). Most significantly, ICJ-CAs presented at a higher N-stage (25.6% were N2) than did right-colonic (14.1%) and rectal (16.2%) carcinomas. Although survival of patients with ICJ-CAs did not differ from those with right-colonic carcinomas, those with carcinomas directly involving the ICV did show a significantly decreased survival. CONCLUSION: ICJ-CAs display several distinct clinicopathological features that may require special diagnostic, prognostic and management attention.

One compared with two cycles of mitomycin C in chemoradiotherapy for anal cancer: analysis of outcomes and toxicity.

BACKGROUND: Concurrent chemoradiation with fluorouracil (5fu) and mitomycin C (mmc) is standard treatment for anal canal carcinoma (acc). The current protocol in Alberta is administration of 5fu and mmc during weeks 1 and 5 of radiation. However, administration of the second bolus of mmc has been based largely on centre preference. Given limited published data on outcomes with different mmc regimens, our objective was to compare the efficacy and toxicity of 1 compared with 2 cycles of mmc in acc treatment. METHODS: Our retrospective study evaluated 169 acc patients treated with radical chemoradiotherapy between 2000 and 2010 at two tertiary cancer centres. All patients were treated with 2 cycles of 5fu and with 1 cycle (mmc1) or 2 cycles (mmc2) of mmc. Acute toxicities, disease-free (dfs) and overall survival (os) were analyzed. RESULTS: Baseline demographics, performance status, and stage were similar in the groups of patients who received mmc1 (52%) and mmc2 (48%). Before treatment, median hematologic parameters were comparable, except for white blood cell count, which was higher in the mmc2 group, but within normal range. The 5-year os and dfs were similar (75.1% and 54.2% for mmc1 vs. 70.7% and 44.2% for mmc2, p = 0.98 and p = 0.63 respectively). On multivariate analysis, mmc2 was the factor most strongly associated with specific acute toxicities: grade 3+ leukopenia (hazard ratio: 4.82; p < 0.01), grade 3+ skin toxicity (hazard ratio: 4.76; p < 0.001), and hospitalizations secondary to febrile neutropenia (hazard ratio: 9.91; p = 0.001). CONCLUSIONS: In definitive chemoradiotherapy for acc, 1 cycle of mmc appears to offer outcomes similar to those achieved with 2 cycles, with significantly less acute toxicity.

Ovarian tumours, their characterisation and origins and ovarian screening.

Ultrasound assessment of ovarian tumours cannot accurately predict pathology. Recent reviews suggest that the source of many epithelial ovarian carcinomas may be the distal fallopian tube. Further study of the natural history of these tubal lesions is required before resuming ovarian screening.

S100ß as a novel and accessible indicator for the presence of monocyte-driven encephalitis in AIDS.

AIMS: The pathogenesis of human/simian immunodeficiency virus encephalitis (HIVE/SIVE) remains incompletely understood, but is associated with alterations in the blood-brain barrier. At present, it is not possible to easily determine if an individual has HIVE/SIVE before post mortem examination. METHODS: We have examined serum levels of the astroglial protein S100ß in SIV-infected macaques and show that it can be used to determine which animals have SIVE. We also checked for correlations with inflammatory markers such as CCL2/MCP-1, IL-6 and C-reactive protein. RESULTS: We found that increased S100ß protein in serum correlated with decreased expression of the tight junction protein zonula occludens-1 on brain microvessels. Furthermore, the decrease in zonula occludens-1 expression was spatially related to SIVE lesions and perivascular deposition of plasma fibrinogen. There was no correlation between encephalitis and plasma levels of IL-6, MCP-1/CCL2 or C-reactive protein. CONCLUSIONS: Together, these data indicate that SIVE lesions are associated with vascular leakage that can be determined by S100ß protein in the periphery. The ability to simply monitor the presence of SIVE will greatly facilitate studies of the neuropathogenesis of AIDS.

Immunohistochemical expression of VEGF predicts response to platinum based chemotherapy in patients with epithelial ovarian cancer.

PURPOSE: For patients with epithelial ovarian cancer (EOC) cytoreduction, with a combination of taxane and platinum, is the standard of care. Despite this, approximately 50% of patients with advanced disease will relapse and moreover 15-20% of cases of EOC are resistant to platinum based chemotherapy. Vascular Endothelial Growth Factor (VEGF), an angiogenic factor, is associated with poor prognosis. This study was undertaken to examine whether there is an association between VEGF-A expression in the tumour of EOC patients and their response to platinum based chemotherapy. METHODS: The study cohort consisted of 66 patients with advanced stage EOC (FIGO III-IV). Ovarian cancer tissue was analysed for VEGF-A expression immunohistochemically. Protein expression was measured and correlated, with platinum sensitivity and overall patient survival. RESULTS: Median age of patients was 53 years, 45 patients had platinum sensitive disease (68%), the remaining patients being platinum resistant (32%). Of the platinum resistant group, 18 (86%) patients had high VEGF score compared to only 1 (2%) with high VEGF score in the platinum sensitive group. Median survival was 11 months in the patient group with high VEGF score versus 32 months in that cohort with low VEGF score. VEGF expression was significantly inversely correlated with overall survival (P < 0.0001). CONCLUSION: We demonstrated that tumours of patients with platinum resistant EOC exhibit higher levels of VEGF expression compared to the platinum sensitive group. VEGF in EOC, may be of clinical and therapeutic relevance and suggests a role for first line anti-angiogenic therapy.

Altered expression and coregulation of dopamine signalling genes in schizophrenia and bipolar disorder.

INTRODUCTION: signalling through dopamine receptors is of critical importance in the brain and is implicated in schizophrenia and bipolar disorder, but its underlying molecular mechanisms remain poorly understood. MATERIALS AND METHODS: using a yeast two-hybrid approach, we previously identified 11 novel dopamine receptor-interacting proteins. Here we compare gene expression levels for 17 genes [including all 11 dopamine receptor interacting proteins, all 5 dopamine receptors (DRD1-DRD5) and DARPP-32] by real-time polymerase chain reaction, using prefrontal cortex post mortem brain samples from 33 schizophrenic, 32 bipolar disorder and 34 control subjects. RESULTS: the expression of C14ORF28, GNB2L1, MLLT3, DRD2 and DARPP-32 genes was altered in schizophrenia and/or bipolar disorder samples relative to controls (P < 0.05). Hierarchical clustering analysis revealed the expression of these five genes (C14ORF28, GNB2L1, MLLT3, DARPP-32, DRD2) is closely correlated in patients. However, in controls, DRD2 expression in relation to the other genes appears to be very different, suggesting abnormal DRD2 activity is an important trigger in the pathophysiology of schizophrenia and bipolar disorder. CONCLUSIONS: our data suggest: (i) C14ORF28, GNB2L1, MLLT3, DRD2 and DARPP-32 are important in the pathogenesis of schizophrenia and bipolar disorder; (ii) these two disorders share common disease-related mechanisms linked to dopamine signalling; (iii) the expression of these genes is closely correlated; and (iv) DRD2 provides the initial trigger in the pathogenesis of these disorders.

Prognostic significance of intratumoral vascular endothelial growth factor as a marker of tumour angiogenesis in epithelial ovarian cancer.

OBJECTIVES: The aim of this study was to examine the prognostic significance of vascular endothelial growth factor (VEGF) in epithelial ovarian cancer (EOC). METHODS: Surgical specimens of 105 patients with primary EOC FIGO Stages 1 to 4, who underwent surgical staging, were investigated. Expression of VEGF was evaluated by immunohistochemical staining using related monoclonal antibodies. The correlation of this data with survival and established prognostic factors such as histological grade, FIGO stage and residual tumour status was evaluated. Multivariate analysis and correlation tests were performed. RESULTS: The results of VEGF expression were correlated with clinicopathological variables and overall survival. No correlation between the VEGF expression and clinicopathological factors was identified. However, VEGF expression was found to be significantly correlated to survival, and a prognostic factor independent of the stage of disease and residual tumour status (p < 0.0001). CONCLUSION: High intratumoral VEGF expression, a marker of angiogenesis, appeared to be an independent prognostic factor for overall survival in women with EOC. Angiogenic evaluation of patients with EOC may play a role in predicting a subgroup of patients with aggressive disease. These patients could be the target of front-line molecular targeted therapy with anti-angiogenic agents.

Ovulation-stimulation drugs and cancer risks: a long-term follow-up of a British cohort.

To assess long-term health effects of ovarian-stimulation drugs we followed-up for over 20 years a British cohort of 7355 women with ovulatory disorders, 43% of whom were prescribed ovarian-stimulation drugs, and identified a total of 274 deaths and 367 incident cancers. Relative to the general population, the cohort experienced lower mortality from most causes, including from all neoplasms combined, and lower incidence of cervical cancer, but higher incidence of cancers of the breast (relative risk: 1.13; 95% CI 0.97, 1.30) and corpus uteri (2.02; 1.37, 2.87). There were, however, no significant differences in the risk of cancers of the breast, corpus uteri, ovary, or of any other site, between women who had been prescribed ovarian-stimulation drugs and those who had not. Further analyses by type of drug and dose revealed a dose-response gradient in the risk of cancer of the corpus uteri (P for linear trend=0.03), with women given >or=2250 mg of clomiphene having a 2.6-fold (2.62; 0.94, 6.82) increase in risk relative to those who were not treated. These findings do not support strong associations between ovulation-stimulation drugs and cancer risks, but they indicate the need for continued monitoring to establish whether risks are elevated in certain subgroups of users.

Displaced intracapuslar hip fractures in the working age alcohol-abusing patient.

Management of alcohol-abusing patients presenting with intracapsular hip fractures is controversial. The aim of this study was to compare the management and outcome of working-age alcohol-abusing patients with similar-aged controls. Patients were identified from a prospectively collected database of trauma admissions. Full case notes were available for 78 patients under 65 of age presenting with a displaced intacapsular fracture at a teaching hospital between 1998 and 2002. Thirty seven patients had evidence of alcohol abuse. Alcohol-abusing patients presented to hospital later (p = 0.05), underwent surgery a median of 18 hours later (p = 0.011) and required a longer post operative stay (p = 0.003) compared to non-abusers. Despite this, the results of internal fixation were comparable. There was no significant difference between alcohol-abusers and non-abusers in rates of avascular necrosis (6.9% vs 9.7%; odds ratio 0.69, 0.11-4.47) or revision surgery (0.21 vs 0.10 procedures/ patient; odds ratio 1.49, 0.30-7.33). The high rates of alcohol abuse in this low-velocity trauma population suggest such patients are at increased risk of osteoporosis. Routine screening for osteoporosis should be considered in working-age alcohol abusers. After subcapital fracture, reduction and internal fixation is an acceptable treatment in this sub-group of patients.