Adam J. Devall

Hyperalgesia in the setting of anxiety: sex differences and effects of the oestrous cycle in Wistar rats.

Sex differences to noxious thermal cutaneous stimulation were compared in Wistar rats. Male and female rats showed similar baseline tail flick latencies. However, sex differences emerged when nociceptive testing was carried out in the setting of mild non-noxious anxiogenic stress (4Hz vibration for 5min). On cessation of vibration stress 16/35 (46%) of male rats showed hyperalgesia (decrease in tail flick latency lasting >20min) whist the reminder showed a brief (<2min) hypoalgesia. In 15 animals re-tested the next day, stress-induced hyperalgesia was reproducible (n=7) but the hypoalgesia initially present in 8 rats was less stable, being reduced (n=2) or replaced by weak hyperalgesia (n=3) in some cases. The response of females was oestrous cycle dependent. On cessation of the vibration stress, females in late dioestrus displayed rapid onset hyperalgesia lasting 10min (n=12) whilst others showed either brief (<2min) hypoalgesia (proestrus, n=13 and early dioestrus, n=9) or brief (<2min) delayed hyperalgesia (oestrus, n=16). On re-testing the next day, when most rats were in a different stage of their cycle, the responsiveness of individual female rats changed according to cycle stage. Thus in females, stage of the oestrous cycle rather than trait differences between individuals appears to be the important determinant of responsiveness to stress. Hyperalgesia in females in late dioestrus correlated with increased anxiety behaviour in a novel environment: rats in late dioestrus showed longer latencies to re-enter the inner zone of an open field compared to rats in other cycle stages. Rats undergoing withdrawal from a progesterone dosing regimen (5mgkg(-1) IP twice daily for 6 days) to mimic the fall in progesterone that occurs naturally during late dioestrus, exhibited a stress-induced hyperalgesia similar to animals in late dioestrus. Falling levels of progesterone during late dioestrus may therefore be a pre-disposing factor for the development of stress-induced hyperalgesia in females.

Genomic complexity of urothelial bladder cancer revealed in urinary cfDNA

Urothelial bladder cancers (UBCs) have heterogeneous clinical characteristics that are mirrored in their diverse genomic profiles. Genomic profiling of UBCs has the potential to benefit routine clinical practice by providing prognostic utility above and beyond conventional clinicopathological factors, and allowing for prediction and surveillance of treatment responses. Urinary DNAs representative of the tumour genome provide a promising resource as a liquid biopsy for non-invasive genomic profiling of UBCs. We compared the genomic profiles of urinary cellular DNA and cell-free DNA (cfDNA) from the urine with matched diagnostic formalin-fixed paraffin-embedded tumour DNAs for 23 well-characterised UBC patients. Our data show urinary DNAs to be highly representative of patient tumours, allowing for detection of recurrent clinically actionable genomic aberrations. Furthermore, a greater aberrant load (indicative of tumour genome) was observed in cfDNA over cellular DNA (P<0.001), resulting in a higher analytical sensitivity for detection of clinically actionable genomic aberrations (P<0.04) when using cfDNA. Thus, cfDNA extracted from the urine of UBC patients has a higher tumour genome burden and allows greater detection of key genomic biomarkers (90%) than cellular DNA from urine (61%) and provides a promising resource for robust whole-genome tumour profiling of UBC with potential to influence clinical decisions without invasive patient interventions.

Urinary EpCAM in urothelial bladder cancer patients: characterisation and evaluation of biomarker potential

Background:Epithelial cell adhesion molecule is overexpressed in bladder tumours and released from bladder cancer cells in vitro. We test the hypotheses that urinary EpCAM could act as a biomarker for primary bladder cancer detection and risk stratification.Methods:Epithelial cell adhesion molecule was measured by ELISA in urine from 607 patients with primary bladder tumours and in urine from 53 non-cancer controls. Mann–Whitney tests and ROC analyses were used to determine statistical significance and discrimination between non-cancer controls and different stages and grades of disease. Multivariable modelling and Kaplan–Meier analyses were used to determine prognostic significance. The structure of urinary EpCAM was investigated by western blotting and mass spectrometry.Results:Urinary EpCAM levels increase with stage and grade of bladder cancer. Alongside grade and stage, elevated urinary EpCAM is an independent indicator of poor prognosis with a hazard ratio of 1.76 for bladder cancer-specific mortality. The soluble form of EpCAM in urine is the extracellular domain generated by cleavage between ala243 and gly244. Further studies are required to define the influence of other urinary tract malignancies and benign urological conditions on urinary EpCAM.Conclusion:The extracellular domain of EpCAM is shed into urine by bladder tumours. Urinary EpCAM is a strong indicator of bladder cancer-specific survival, and may be useful within a multi-marker panel for disease detection or as a stand-alone marker to prioritise the investigation and treatment of patients. The mechanisms and effects of EpCAM cleavage in bladder cancer are worthy of further investigation, and may identify novel therapeutic targets.

Protein shedding in urothelial bladder cancer: prognostic implications of soluble urinary EGFR and EpCAM

Background:Better biomarkers must be found to develop clinically useful urine tests for bladder cancer. Proteomics can be used to identify the proteins released by cancer cell lines and generate candidate markers for developing such tests.Methods:We used shotgun proteomics to identify proteins released into culture media by eight bladder cancer cell lines. These data were compared with protein expression data from the Human Protein Atlas. Epidermal growth factor receptor (EGFR) was identified as a candidate biomarker and measured by ELISA in urine from 60 noncancer control subjects and from 436 patients with bladder cancer and long-term clinical follow-up.Results:Bladder cancer cell lines shed soluble EGFR ectodomain. Soluble EGFR is also detectable in urine and is highly elevated in some patients with high-grade bladder cancer. Urinary EGFR is an independent indicator of poor bladder cancer-specific survival with a hazard ratio of 2.89 (95% CI 1.81–4.62, P<0.001). In multivariable models including both urinary EGFR and EpCAM, both biomarkers are predictive of bladder cancer-specific survival and have prognostic value over and above that provided by standard clinical observations.Conclusions:Measuring urinary EGFR and EpCAM may represent a simple and useful approach for fast-tracking the investigation and treatment of patients with the most aggressive bladder cancers.

Quantitative genome-wide methylation analysis of high-grade non-muscle invasive bladder cancer.

ABSTRACT High-grade non-muscle invasive bladder cancer (HG-NMIBC) is a clinically unpredictable disease with greater risks of recurrence and progression relative to their low-intermediate-grade counterparts. The molecular events, including those affecting the epigenome, that characterize this disease entity in the context of tumor development, recurrence, and progression, are incompletely understood. We therefore interrogated genome-wide DNA methylation using HumanMethylation450 BeadChip arrays in 21 primary HG-NMIBC tumors relative to normal bladder controls. Using strict inclusion-exclusion criteria we identified 1,057 hypermethylated CpGs within gene promoter-associated CpG islands, representing 256 genes. We validated the array data by bisulphite pyrosequencing and examined 25 array-identified candidate genes in an independent cohort of 30 HG-NMIBC and 18 low-intermediate-grade NMIBC. These analyses revealed significantly higher methylation frequencies in high-grade tumors relative to low-intermediate-grade tumors for the ATP5G2, IRX1 and VAX2 genes (P<0.05), and similarly significant increases in mean levels of methylation in high-grade tumors for the ATP5G2, VAX2, INSRR, PRDM14, VSX1, TFAP2b, PRRX1, and HIST1H4F genes (P<0.05). Although inappropriate promoter methylation was not invariantly associated with reduced transcript expression, a significant association was apparent for the ARHGEF4, PON3, STAT5a, and VAX2 gene transcripts (P<0.05). Herein, we present the first genome-wide DNA methylation analysis in a unique HG-NMIBC cohort, showing extensive and discrete methylation changes relative to normal bladder and low-intermediate-grade tumors. The genes we identified hold significant potential as targets for novel therapeutic intervention either alone, or in combination, with more conventional therapeutic options in the treatment of this clinically unpredictable disease.

Differential Activation of the Periaqueductal Gray by Mild Anxiogenic Stress at Different Stages of the Estrous Cycle in Female Rats

The effect of acute exposure to mild anxiogenic stress on cutaneous nociceptive threshold was investigated in female Wistar rats at different stages of the estrous cycle. Baseline tail flick latencies did not change significantly during the cycle. However after brief exposure to vibration stress (4 Hz for 5 min), rats in late diestrus, but not at other cycle stages, developed a hyperalgesia (decrease in tail flick latency). Animals in late diestrus revealed a more than fivefold increase in the density of Fos-like immunoreactive nuclei in the dorsolateral, lateral, and ventrolateral columns in the caudal half of the periaqueductal gray matter (PAG). There was no change in the density of Fos-like immunoreactive nuclei in the PAG in rats in estrus and early diestrus, although rats in proestrus showed a smaller (50%) but significant increase. Rats undergoing withdrawal from a progesterone dosing regimen (5 mg/kg i.p. twice daily for 6 days) designed to mimic the fall in progesterone that occurs naturally during late diestrus, exhibited a stress-induced hyperalgesia that was similar to animals in late diestrus and a significant increase in Fos-positive cells in the PAG. We suggest that falling levels of progesterone during late diestrus may be a predisposing factor for the development of stress-induced hyperalgesia, which is linked to differential activation of descending pain control circuits in the PAG. Similar changes in women, when progesterone levels fall during the late luteal phase of the menstrual cycle, may contribute to the development of premenstrual symptoms that include increased anxiety and hyperalgesia.

Methylation of HOXA9 and ISL1 Predicts Patient Outcome in High-Grade Non-Invasive Bladder Cancer

Introduction Inappropriate DNA methylation is frequently associated with human tumour development, and in specific cases, is associated with clinical outcomes. Previous reports of DNA methylation in low/intermediate grade non-muscle invasive bladder cancer (NMIBC) have suggested that specific patterns of DNA methylation may have a role as diagnostic or prognostic biomarkers. In view of the aggressive and clinically unpredictable nature of high-grade (HG) NMIBC, and the current shortage of the preferred treatment option (Bacillus:Calmette-Guerin), novel methylation analyses may similarly reveal biomarkers of disease outcome that could risk-stratify patients and guide clinical management at initial diagnosis. Methods Promoter-associated CpG island methylation was determined in primary tumour tissue of 36 initial presentation high-grade NMIBCs, 12 low/intermediate-grade NMIBCs and 3 normal bladder controls. The genes HOXA9, ISL1, NKX6-2, SPAG6, ZIC1 and ZNF154 were selected for investigation on the basis of previous reports and/or prognostic utility in low/intermediate-grade NMIBC. Methylation was determined by Pyrosequencing of sodium-bisulphite converted DNA, and then correlated with gene expression using RT-qPCR. Methylation was additionally correlated with tumour behaviour, including tumour recurrence and progression to muscle invasive bladder cancer or metastases. Results The ISL1 genes’ promoter-associated island was more frequently methylated in recurrent and progressive high-grade tumours than their non-recurrent counterparts (60.0% vs. 18.2%, p = 0.008). ISL1 and HOXA9 showed significantly higher mean methylation in recurrent and progressive tumours compared to non-recurrent tumours (43.3% vs. 20.9%, p = 0.016 and 34.5% vs 17.6%, p = 0.017, respectively). Concurrent ISL1/HOXA9 methylation in HG-NMIBC reliably predicted tumour recurrence and progression within one year (Positive Predictive Value 91.7%), and was associated with disease-specific mortality (DSM). Conclusions In this study we report methylation differences and similarities between clinical sub-types of high-grade NMIBC. We report the potential ability of methylation biomarkers, at initial diagnosis, to predict tumour recurrence and progression within one year of diagnosis. We found that specific biomarkers reliably predict disease outcome and therefore may help guide patient treatment despite the unpredictable clinical course and heterogeneity of high-grade NMIBC. Further investigation is required, including validation in a larger patient cohort, to confirm the clinical utility of methylation biomarkers in high-grade NMIBC.

Elevation of brain allopregnanolone rather than 5-HT release by short term, low dose fluoxetine treatment prevents the estrous cycle-linked increase in stress sensitivity in female rats.

Withdrawal from long-term dosing with exogenous progesterone precipitates increased anxiety-linked changes in behavior in animal models due to the abrupt decrease in brain concentration of allopregnanolone (ALLO), a neuroactive metabolite of progesterone. We show that a withdrawal-like effect also occurs during the late diestrus phase (LD) of the natural ovarian cycle in rats, when plasma progesterone and ALLO are declining but estrogen secretion maintains a stable low level. This effect at LD was prevented by short-term treatment with low dose fluoxetine. During LD, but not at other stages of the estrous cycle, exposure to anxiogenic stress induced by whole body vibration at 4 Hz for 5 min evoked a significant decrease in tail flick latency (stress-induced hyperalgesia) and a decrease in the number of Fos-positive neurons present in the periaqueductal gray (PAG). The threshold to evoke fear-like behaviors in response to electrical stimulation of the dorsal PAG was lower in the LD phase, indicating an increase in the intrinsic excitability of the PAG circuitry. All these effects were blocked by short-term administration of fluoxetine (2 × 1.75 mg kg(-1) i.p.) during LD. This dosage increased the whole brain concentration of ALLO, as determined using gas chromatography-mass spectrometry, but was without effect on the extracellular concentration of 5-HT in the dorsal PAG, as measured by microdialysis. We suggest that fluoxetine-induced rise in brain ALLO concentration during LD offsets the sharp physiological decline, thus removing the trigger for the development of anxiogenic withdrawal effects.

Fluoxetine elevates allopregnanolone in female rat brain but inhibits a steroid microsomal dehydrogenase rather than activating an aldo‐keto reductase

Fluoxetine, a selective serotonin reuptake inhibitor, elevates brain concentrations of the neuroactive progesterone metabolite allopregnanolone, an effect suggested to underlie its use in the treatment of premenstrual dysphoria. One report showed fluoxetine to activate the aldo‐keto reductase (AKR) component of 3α‐hydroxysteroid dehydrogenase (3α‐HSD), which catalyses production of allopregnanolone from 5α‐dihydroprogesterone. However, this action was not observed by others. The present study sought to clarify the site of action for fluoxetine in elevating brain allopregnanolone.

Estrous cycle stage influences on neuronal responsiveness to repeated anxiogenic stress in female rats

Experiments were carried out to investigate (i) whether estrous cycle stage influences nociceptive responsiveness to anxiogenic stress and (ii) whether prior experience of the stressor modifies the response. Exposure to mild anxiogenic vibration stress evoked hyperalgesia, reflected by a decrease in tail flick latency, only in animals in the late diestrus phase. Stress evoked hyperalgesia in late diestrus regardless of whether the rat was experiencing the stress for the first time or had been exposed to the stress previously, when in another cycle stage. Whilst the behavioral response to stress appeared to be determined exclusively by estrous cycle stage, the stress-evoked pattern of Fos expression in the periaqueductal grey matter (PAG) depended not only on cycle stage but also on whether the rat had previous experience of the stress. First exposure to stress in late diestrus evoked a 50% decrease in Fos expression compared to non-stressed controls, particularly in the lateral and dorsolateral sectors of the rostral PAG. In contrast, in experienced rats in late diestrus the pattern of Fos expression increased up to 4-fold, particularly in the ventral half of the caudal PAG but also in the lateral and dorsolateral parts. At other cycle stages Fos expression was not changed except for an increase in rats in proestrus. The results suggest that in females, changes in gonadal hormone levels during the estrous cycle impact significantly on the processing of fear-inducing stimuli by the PAG. These hormonal influences may also influence how the PAG responds to a subsequent anxiogenic challenge.