Nerve growth factor (NGF): a potential urinary biomarker for overactive bladder syndrome (OAB)?

Authors


Correspondence: Jai Seth, Department of Uro-Neurology, National Hospital for Neurology and Neurosurgery, UCL Hospitals Foundation Trust, London WC1N 3BG, UK.

e-mail: seth.jai@gmail.com

Abstract

What's known on the subject? and What does the study add?

The search for a biomarker in overactive bladder syndrome (OAB) is an emerging field of interest, as bladder dysfunction is a common complaint that causes significant morbidity. A biomarker may give us insight as a diagnostic tool, and also inform us about how severe the condition is, how it may progress and how it may best be treated. The protein of interest here is nerve growth factor (NGF) and it has been shown to be a dynamic molecule in the bladder of patients with OAB. Urinary levels have been seen to rise in patients with OAB and fall in those who respond to treatment. However, there have also been many studies that examine this trend in numerous other conditions, e.g. interstitial cystitis, bladder outflow obstruction, renal stone disease and patients with neurological impairment after stroke. As a result the specificity of this as a potential urinary biomarker for OAB is questioned.

This is a review of published studies, which discusses the pros and cons of NGF as a potential urinary biomarker. The evidence is examined and the studies are summarised together in a Table. Questions remain about the reliability, practicality and specificity of NGF as a biomarker for OAB. These questions need to be addressed by further studies that could clarify the points raised.

Objective

  • To review the current literature on the use of urinary nerve growth factor (NGF) as a potential biomarker for overactive bladder syndrome (OAB).

Method

  • A comprehensive electronic literature search was conducted using the PubMed database to identify publications relating to urinary NGF.

Results

  • There are a growing number of publications that have measured urinary NGF levels in different types of bladder dysfunction. These range from OAB, bladder pain syndrome, idiopathic and neurogenic detrusor overactivity, bladder oversensitivity and bladder outflow obstruction. Urinary NGF levels do appear to be raised in these pathological states when compared with healthy control samples.
  • In patients with OAB, these raised urinary NGF levels appear to also reduce after successful treatment with antimuscarinics and botulinum toxin A, which indicates a potential use in monitoring responses to treatment.
  • However, raised levels are not limited to OAB, which questions its specificity.
  • Urinary NGF measurements are performed with an enzyme-linked immunosorbent assay using polyclonal antibodies to NGF. The technique requires standardisation, and the different antibodies to NGF require validating.
  • Also a definition of what is the ‘normal’ range of NGF in urine is still required before it can be used as a diagnostic and prognostic tool.

Conclusions

  • Whilst the evidence for an increased urinary NGF in OAB appears convincing, many questions about its validity remain including: specificity, sensitivity, cost- and time-effectiveness.
  • Many criteria for what constitutes a biomarker still need to be evaluated and met before this molecule can be considered for this role.
Abbreviations
BTX-A

botulinum toxin-A

Cr

creatinine

(I)(N)DO

(idiopathic) (neurogenic) detrusor overactivity

ELISA

enzyme-linked immunosorbent assay DRG, dorsal root ganglia

IC/PBS

interstitial cystitis/painful bladder syndrome

LUT(D)

lower urinary tract (dysfunction)

NGF

nerve growth factor

OAB

overactive bladder syndrome

UI

urinary incontinence

Introduction

In 1950, Nobel prize winners Stanley Cohen and Rita Levi-Montalcini, first discovered and explored the role of nerve growth factor (NGF), in the regulation of the developing neurological system [1]. Their early work showed that the transfer of mice tumour cells into chick embryos, promoted the development of the nervous system, particularly the sensory and sympathetic neurones. It was concluded that a growth-promoting substance was being released from the tumour cells, which had a selective influence on certain nerves. This substance was identified as NGF, which is a small signalling protein, which in the bladder, is produced by detrusor smooth muscle cells and by urothelial cells. NGF, from the family of neurotrophins, is now known to be necessary to promote growth and survival of sympathetic fibres and sensory nerves, which are both vital for normal bladder functioning [2].

The overactive bladder (OAB) is a clinical syndrome consisting of urinary urgency, frequency with or without urinary incontinence (UI) [3]. The diagnosis of OAB is a clinical one, which can be assessed, somewhat subjectively by patient reported symptoms. The symptoms can be severely bothersome and negatively affect health-related quality of life. Symptoms of OAB, and in particular those with urgency UI, are also associated with detrusor overactivity (DO), a urodynamic diagnosis, which may also be related to raised NGF levels in the lower urinary tract [4].

In the healthy bladder, sensory C-fibre activity, maintains a quiescent role in the sensation of physiological bladder filling, except nearing functional bladder capacity, only being activated by distension that is greater than that required to stimulate Aδ fibres [5]. However, in an OAB, neuronal and non-neuronal release of neurotransmitter substances, from the urothelium and suburothelium, have a stimulatory effect on C-fibre activity. It is thought that NGF, which is expressed in excess in pathological bladder states, is one of these stimulatory substances, and has a knock on affect to sensitise afferent C-fibres, leading to sensory urgency, and ultimately reflex bladder hyperactivity, causing urinary frequency and urgency UI. The overexpression of NGF has led to the interest in this secretory protein as a potential urinary biomarker for the OAB, which represents a considerable health and economic burden. This article reviews the evidence, and discusses the prospect of NGF as a biomarker for OAB.

Tissue NGF Levels: Animal and Human Studies

Evidence has been accumulated from numerous animals studies, discussed below, to show the importance of NGF in the pathological bladder [6]. In a study in cats, examining a naturally occurring model of feline interstitial cystitis (IC), investigators noticed an increase in the levels of NGF and Substance P in the urothelium, along with an increase in the cell body size of the dorsal root ganglia (DRG) when compared with controls. This suggests that the inflamed urothelium may be an important source of increased NGF, which may act as a transducer of physical and chemical stimuli, by undergoing retrograde transport to DRGs [7].

In rat models of spinal cord injury causing neurogenic DO (NDO) or cyclophosphamide-induced cystitis and overactivity, levels of NGF have also been found to be increased in the bladder, the DRG and the lumbosacral spinal cord [8]. Systemic treatment of rats with this experimental cystitis model, with an NGF sequestering molecule, has been shown to reduce the bladder overactivity that develops [9]. Another study that reported a significant increase in NGF production, after cyclophosphamide -induced cystitis in female rats, showed this was also associated with a decreased intercontraction interval [10]. Once hyaluronic acid was administered intravesically, this significantly reduced the urinary NGF levels and increased the intercontraction interval. Furthermore, the application of hyaluronidase, which counteracts the action of hyaluronic acid, eliminated the suppressive effect of hyaluronic acid on NGF levels [10].

Furthermore, chronic administration of NGF into the bladder or spinal cord of rats has been shown to induce bladder hyperactivity [11]. One study explored the role of NGF by generating transgenic mice, to cause chronic overexpression of NGF in the urothelium, and examined certain characteristics in comparison to wild-type littermate controls [12]. In these mice, the overexpression of NGF led to a reduced bladder capacity that exhibited an increase in the number and amplitude of non-voiding reflex bladder contractions, when compared with the controls. There was also marked nerve fibre hyperplasia in the lamina propria and detrusor smooth muscle, along with causing elevated numbers of inflammatory cells, e.g. tissue mast cells. Sensory afferent nerves also showed increased density of calcitonin gene-related peptide and Substance P containing nerve fibres [12]. These neuropeptides are released in inflammatory states, and through their release, NGF may also be implicated in visceral nociception by producing hyperalgesia. It is thought that NGF can also modulate the expression of membrane ion channels, e.g. P2X3 (purine receptor), TRPV1 (transient receptor potential vanilloid 1), and voltage-gated sodium channels in the bladder, which are thought to play a major role in inflammation, tissue-induced pain and hypersensitivity [12].

Another study showed that raised NGF levels can also be related to DO secondary to BOO. In this study, 40 rats had a BOO created by partial ligation of the urethral outlet, and were shown to develop DO by cystometry. Urine was collected before ligation, during the obstructed period and after relief of the obstruction. NGF levels were reported to be significantly raised from baseline levels during the period with DO, which then decreased back to baseline levels after the DO disappeared after the relief of the BOO. In the rats with persistent DO, despite removal of the urethral obstruction, the urinary NGF levels remained significantly raised from baseline and compared with the control rats [13].

Another study reported increased mRNA expression of NGF in the bladder in BOO rat models associated with DO, and in continued DO after BOO relief [14]. More recently, a study reported raised tissue NGF levels, quantified by immunofluorescence staining and Western blotting in an unstable BOO rat model, which were then significantly reduced after detrusor botulinum toxin A (BTX-A) injection [15]. It has been shown that once this excess quantity of expressed NGF is sequestered, it can reduce the observed bladder dysfunction. After spinal cord injury in rats, the resulting detrusor hyperreflexia and detrusor-sphincter dyssynergia can be suppressed by intrathecal application of NGF antibodies, which neutralises the NGF in the spinal cord [16, 17]. This sequestration of NGF produces effects similar to treatments, e.g. resiniferatoxin or capsaicin, which desensitise C-fibre afferents.

Similarly to the studies in animals, which prove that NGF plays an important role in bladder dysfunction, human studies have also corroborated this. One study that quantified human bladder tissue levels of NGF using tissue enzyme-linked immunosorbent assay (ELISA) techniques, collected bladder biopsies before and at 1 and 3 months after detrusor BTX-A injection in patients with NDO [18]. As the urodynamic parameters improved over this time frame, the tissue NGF levels were also noted to decrease significantly. This suggests that BTX-A may decrease the levels of neurotransmitters, which may otherwise stimulate NGF production and release. In bladder biopsies from patients with IC/painful bladder syndrome (IC/PBS), an increased expression of NGF mRNA has been reported, which is reduced 2 weeks after successful intravesical injection with BTX-A, which corresponded with a decrease in pain scores [19, 20]. However, NGF mRNA levels were not reduced in non-responders with persistent pain after BTX-A. Interestingly, decreased NGF mRNA levels correlated with decreases in pain scores, but not with changes in maximum bladder capacity, which suggests NGF may contribute more to pain perception, and increased afferent excitability. This is supported by the action of NGF on TrkA (neurotrophic tyrosine kinase receptor type 1) receptors, via which it further mediates the release of Substance P, which is implicated in inflammatory responses and nociception [21].

Urinary NGF a Potential Biomarker for OAB/DO?

The accumulation of the above findings, have led to the evaluation of urinary levels of NGF as a potential biomarker and as a simple, cost-effective method to objectively assess therapeutic outcomes in patients with OAB [22]. Such a tool may also be of use as a prognostic indicator and provide information about which patients may respond better to certain treatments.

A biomarker should be ‘a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention’. A suitable biomarker needs to be easily accessible, reliable, and as a test repeatable, whilst offering high specificity and sensitivity to diagnose and monitor OAB. There should be a relationship with the severity of OAB. Furthermore it must provide information that is not already available from the clinical assessment, improve the outcome of the disease, and predict responses to specific therapies. The cost and time aspect of the test should be acceptable, and ultimately save costs by avoiding unnecessary therapies [23].

The levels of NGF in the urine can be quantified using an ELISA system, with most studies reporting results from the same kit (Emax, Promega, Madison, WI, USA) for reproducibility. The antibodies that come within this kit have not specifically been developed or marketed for measurement in the urine, and are only being used within a research domain. The technique uses a sandwich ELISA assay, using a polyclonal antibody to NGF to initially coat a well, and after incubation with the patient sample, a second monoclonal antibody is then used to sandwich the molecule, between the two antibodies. A tertiary species-specific antibody is then used, which is conjugated with horseradish peroxidase, which is then incubated with a chromogen, to induce a colour change, the intensity of which is proportional to the concentration of the substrate within the well. The total urinary NGF level is normalised to the concentration of urinary creatinine (Cr), to overcome the differing dilutions of urine between patients. Other NGF ELISA kits are available and a validation and standardisation between these different kits needs to be performed to check reproducibility.

Table 1 summarises published studies that have measured urinary NGF levels in patients with various bladder dysfunctions. Baseline urinary NGF levels have repeatedly been shown to be highly expressed, in patients with OAB when compared with controls. This increase is even more significant in patients with OAB and urgency UI (termed OAB-wet), compared with those without (OAB-dry) [22]. It has been postulated that the difference between the OAB-dry and OAB-wet groups, may be due to the higher percentage of DO in the OAB-wet patients, which may suggest that higher levels of urinary NGF may predispose the occurrence of DO [6]. However, urinary NGF levels were not raised in patients with bladder oversensitivity alone [24]. Patients with mixed UI, with co-existing DO, were also shown to have higher NGF levels than patients with pure stress UI alone. However, in the latter group, urinary levels were raised in those patients who developed de novo DO after anti-UI surgery, which resulted from BOO [25]. In antimuscarinic-refractory patients with OAB, serum levels of NGF were also assayed, and correlated with urinary findings, by being raised compared with controls. These patients were treated with 3 months solifenacin, after which serum and urinary levels remained raised. The complex pathophysiology of OAB, may therefore be multimodal, involving several neurotransmitter and inflammatory pathways [26].

Table 1. Summary of studies examining urinary NGF levels
Author and YearConditions investigated and sample sizesConclusions
Antunes-Lopes et al. [42]Females with OAB (37) and healthy controls (40)Urine NGF/Cr and BDNF/Cr (Brain-derived Neurotrophic Factor) were significantly higher in OAB than healthy controls. These levels decreased after anti-muscarinic treatment. No significantly raised urinary glial cell-line derived neurotrophic factor/Cr in OAB compared with controls. Reduction in number of urgency episodes per week correlated with BDNF/Cr but not with NGF/Cr. There was no circadian variation to urinary NGF/Cr levels.
Liu et al. (2012) [31]Interstitial cystitis (IC) (30) and heathy controls (28)Increased mean urine NGF/Cr and serum NGF levels in patients with IC compared with controls.
Liu et al. (2011) [26]Serum and urinary NGF levels in OAB (34), wet (17) and dry (17)Serum NGF and urinary NGF/Cr levels were significantly raised in patients with OAB when compared with healthy asymptomatic controls. Serum NGF correlated with urinary NGF levels. No significant differences in serum NGF were detected between the OAB dry and wet groups. Serum and urinary levels remain unchanged before and after solifenacin treatment.
Liu et al. (2011) [44]OAB dry (113), OAB wet (104), controls (84)Urinary NGF levels were significantly higher in OAB dry and even higher in OAB wet patients. Urinary NGF levels were not associated with menopause, ageing or higher body mass index.
Pinto et al. (2010) [43]Bladder pain syndrome/Interstitial cystitis (10) treated with intra-trigonal botulinum toxin AUrinary NGF and BDNF (brain derived neurotrophic factor) levels were measured at baseline, 1, 3 and 6 months after injection. Urinary NGF and BDNF levels were significantly reduced after the injection at one month, which corresponded with the improvement in pain intensity on a visual analogue scale. At three months, levels remain reduced however not as low as at one month. At six months levels have risen back up to baseline levels.
Liu et al. (2009) [45]Interstitial cystitis/painful bladder syndrome (IC/PBS) (122) and normal controls (28)Urinary NGF /Cr levels were significantly greater in patients with IC/PBS than in controls. NGF/Cr levels were very significantly higher when the bladder was distended in patients with IC/PBS. Patients who responded to treatment with an improvement in visual analogue scores of >2 had significantly lower NGF/Cr levels than non-responders who had improvements of <2.
Kuo et al. (2010) [24]Urinary NGF versus Bladder wall thickness (DWT) as a biomarker for OAB (81)DWT was not significantly greater in OAB patients compared to controls. Urinary NGF/Cr levels were significantly increased in patients with incontinence and OAB and those with DO.
Liu et al. (2010) [46]Interstitial cystitis IC/bladder pain syndrome (40), OAB (54) mixture of DO or increased bladder sensation (IBS), controls (27)Urinary NGF/Cr levels were raised in the 40 patients with IC, and 23 with DO, but not in the 31 with IBS or controls. Urinary NGF/Cr levels were not significantly different between the IC or DO groups.
Liu et al. (2009) [27]OAB (70) all treated with antimuscarinics, 50 responders, 20 non-responders, controls (38)Urinary NGF/Cr levels were significantly higher in patients with OAB taking and responding to antimuscarinics compared with non-responders and controls.
Liu et al. (2009) [37]Cerebrovascular accident (CVA) (93)Urinary NGF/Cr levels correlated with severity of neurological impairment after CVA.
Liu et al. (2009) [34]Urine collection at OAB (39) at first sensation of bladder filling (FSF), urge sensation (US) and controls at FSF and US (35)Urinary NGF/Cr levels were significantly higher in OAB patients than controls. In controls urinary NGF/Cr levels were very low at first sensation of bladder filling (FSF), but significantly higher at urge sensation (US). In OAB patients there was no significant difference in urinary NGF/Cr levels at FSF and US.
Liu et al. (2008) [25]Mixed urinary incontinence (urinary stress incontinence (USI) and OAB) (38), DO alone (26), persistent USI after anti-incontinence surgery (21), de novo DO (15), control (31).Urinary NGF/Cr levels were significantly higher in women with mixed urinary stress incontinence and DO than those with pure stress incontinence or controls. Urinary NGF/Cr levels were undetectable in women with persistent USI, but significantly higher in those with de novo DO after anti-incontinence surgery.
Liu et al. (2009) [4]IDO (143), NDO (100). Mixture of untreated, well treated and failed treatment with antimuscarinics. BoNT/A was given to IDO (24) and NDO (19), Controls (38).Urinary NGF/Cr levels were significantly higher in 66 patients with IDO and 59 with NDO compared to controls. Patients with well-treated IDO or NDO had reduced NGF/Cr levels, whereas those with failed-treated IDO or NDO did not. Patients who responded to BoNT-A treatment had significantly reduced urinary NGF/Cr levels in both the IDO and NDO groups compared to baseline levels. However, the NGF levels remained significantly higher at 3 months in 7 IDO and 5 NDO patients who failed BoNT-A treatment.
Yokoyama et al. (2008) [47]OAB total (51) due to IDO (13), sensory urgency (6), bladder outflow obstruction (BOO) (16), and NDO (16) due to spinal cord injury (SCI) or CVAThe urinary NGF levels were significantly raised in patients with NDO due to SCI, BOO and sensory urgency when compared with controls. There was no significant difference in urinary levels between those with IDO, NDO due to CVA and controls.
Jacobs et al. (2010) [29]Neurogenic overactive bladder (NOAB) (13), idiopathic overactive bladder (17), interstitial cystitis/painful bladder syndrome (IC/PBS) (8), prostate cancer (7), active bladder cancer (4), and nephrolithiasis (4)Urinary NGF/Cr levels were significantly raised in NOAB and IC/PBS groups when compared with controls. Raised levels approached significance in the patients with nephrolithiasis.
Liu et al. (2008) [35]Bladder outflow obstruction (BOO) (153): BOO non OAB (21), BOO and OAB (25), BOO and DO (47), BOO on successful medical treatment (60), Controls (38).The urinary NGF/Cr levels were very low in the control group and in patients with BOO/non-OAB and significantly greater in patients with BOO/OAB and BOO/DO. The urinary NGF/Cr levels returned to normal levels after successful relief of OAB symptoms with medical treatment.
Liu et al. (2008) [48]Normal controls (40), patients with increased bladder sensation (23), OAB dry (54), OAB wet (80)Urinary NGF/Cr levels were low in controls and patients with increased bladder sensation. Levels in patients with OAB dry were significantly higher than controls. Patients with OAB wet had significantly higher levels than those with OAB dry.
Kim et al. (2006) [49]OAB (65) controls (20)Urinary NGF levels were significantly higher in OAB patients than controls
Kim et al. (2005) [50]OAB (75) and controls (20)Urinary NGF levels and PGE2 (prostaglandin E2) were significantly higher in OAB patients than controls

NGF urine levels also improve in those patients who have responded to medical therapy, with anti-muscarinics and BTX-A [22, 27]. However, these levels do not reduce in patients who have not responded to treatment with either of these methods. It was also evident that once patients discontinue antimuscarinic treatment for 1 month, the levels re-elevated in almost half of the responders. This suggests that urinary NGF levels could have potential use as an objective tool for assessing the response of DO to therapy, as the levels appear to fall as DO is treated. As both of these treatment methods either inhibit the release or action of acetylcholine, it may be inferred that acetylcholine, which also acts at muscarinic receptors on urothelial and suburothelial cells [28], is a required substance for the release of NGF.

An issue which presents itself for the potential use of this protein as an OAB marker is its specificity, as raised levels are not limited to OAB. Links have also been reported for raised urinary NGF levels and painful inflammatory conditions of the urinary tract, e.g. IC/PBS, BOO, chronic prostatitis, renal stone disease, UTI, and bladder cancer [6, 29, 30]. In a group of patients with interstitial cystitis, both mean urine and serum NGF have been found to be significantly raised compared to healthy controls [31]. A common trend for patients with raised urinary NGF levels appears to be the presence of inflammation, which is an increasingly recognised process in the development of OAB. It has been shown that at least half of biopsy samples from patients with NDO and idiopathic DO (IDO) have histological evidence of chronic inflammation [32]. This would be consistent with the finding that urinary NGF levels are raised with bacterial cystitis, when compared with controls, and that once the infection has been treated with antibiotics for a week, these levels significantly decrease [6]. Furthermore, in patients with concurrent UTI and OAB, despite treatment with antibiotics, urinary NGF levels remain significantly raised. Patients with IC/PBS, who responded to treatment with hydrodistension with an improved visual analogue scale pain score, also had a corresponding reduction in urinary NGF/Cr level. This reduction in levels was not seen those who did not respond to treatment, which is a further suggestion of potential use of NGF in monitoring the response to treatment.

Some investigations have focused on the relationship between bladder stretch and NGF release, as it is thought that the secretion of NGF from the bladder depends on stretch and duration of distension [24]. Detrusor smooth muscle stretch stimulates increased NGF production and hyperactive voiding in a rat model [33]. Urinary urgency occurs with full distension of the bladder, and fast filling and distension during a cystometric investigation can also induce sensory urgency or provoke DO. Urinary NGF/Cr levels in OAB-wet patients are significantly raised, and less so with OAB-dry, with natural filling (by drinking water), when compared with levels from artificial stretch obtained with catheter filling [24]. This could be because natural filling allows more time for NGF to be released from the bladder. In control patients it has been shown that NGF levels are higher when volunteers wait to void at strong urge sensation, rather than sooner at the first sensation of bladder filling. However, as NGF levels are pathologically raised in OAB at small bladder volumes, it does not significantly elevate at sensory urgency sensation [34].

OAB is associated with BOO, and in men with BOO and OAB, and BOO and DO, urinary NGF/Cr levels are significantly higher than in those with BOO and no OAB [35]. These levels are lower in treated patients after relief of the OAB associated with BOO [6]. Chronic stretching of the urothelium, as with BPH, can stimulate NGF production, among other transmitters, and chronically sensitise afferent pathways and DRG constituency, which causes increased excitability. In rat BOO models, immunity to NGF prevented obstruction-induced hypertrophy of DRG neurones, less retrograde sacral afferent activity, and eliminated the spinal micturition reflex [36].

Not all patients with OAB have raised urinary NGF values at baseline, which was up to 30% in some cohorts [27]. In a cross-sectional study in 143 patients with IDO, urinary NGF levels were seen to be raised in 66 of these patients. These findings suggest that there may be other causes of OAB pathophysiology, some which do not involve inflammatory or NGF pathways. Thus, as a potential biomarker, there will therefore be significant proportions of patients with OAB or DO, who do not have raised urinary NGF levels, which lowers its sensitivity.

Also, it was noticed that in responders to antimuscarinics, who had significantly improved urinary symptom scores, and a corresponding fall in urinary NGF levels after 3 months of therapy, that these levels although reduced were still significantly higher than those of controls. This may be due to incomplete resolution of the underlying OAB pathophysiology, and also that overactivity of the cholinergic system is not the sole cause of OAB.

The presence of NGF in the urine has been affiliated with the raised NGF level expression identified in the urothelium. However, it is not entirely clear if this is the sole source of urinary NGF. The circulating serum NGF levels may also represent some of the sourcing of urinary NGF, via renal excretion, the proportion to which is unknown. Urinary NGF levels have also been identified to correlate with the severity of neurological impairment after cerebrovascular accident [37]. It is thought that after the brain injury in an acute phase of a stroke, levels of circulating neurotrophins are raised, which may have a knock-on effect on urinary levels [6]. Furthermore increased serum NGF levels have been identified in numerous other medical conditions, e.g. asthma, allergy and Alzheimer's [38, 39]. Therefore, as a potential biomarker for OAB, this raises the possibility that in certain neurological conditions, or other medical conditions, when patients do not have any lower urinary tract (LUT) complaints, urinary NGF levels could be silently raised, causing false positives.

Pitfalls with NGF as a Biomarker

Many proteins are involved in the complex pathophysiology of the OAB, of which NGF is only one. It seems to play a role in LUT dysfunction (LUTD), and is closely associated with inflammation in the bladder. However, due to the numerous sensory neurotransmitters and inflammatory substances that are likely to also play an important role, e.g. Substance P, ATP, calcitonin gene-related peptide, other neurotrophins and inflammatory markers, NGF alone may be insufficient to act as a urinary marker for OAB.

Diagnostic accuracy has been a subject of much attention, and applies to all medical tests. This refers to the ability of a test to identify the condition of interest. This involves comparing a test with the reference or ‘gold’ standard in a group of patients suspected of having the condition of interest. Accuracy refers to the degree of agreement between the new test and the gold standard [40]. Recent development of Standards for Reporting Diagnostic Accuracy (STARD) guidelines, which was a multidisciplinary effort of clinicians and scientists, have been created to provide a checklist for reporting the accuracy of a diagnostic test [41]. This checklist addresses several quality related issues, and an official assessment of this proposed biomarker would need to be performed in accordance with STARD guidance.

As a potential biomarker it is a non-invasive investigation, as urine is relatively easy to collect from the patient. The expression of this abundant protein can change with underlying pathological processes. As patients subjectively respond to successful pharmacological intervention, NGF levels also reduce, which may be of use as an objective tool to monitor the response to a treatment. However, a biomarker should also have a high level of specificity and sensitivity, add new information to the clinical assessment, and have a clear association with the severity of OAB and improve the outcome of the disease, which urinary NGF does not do. For the latter, not all of patients with symptomatic OAB will have significantly raised urinary NGF levels, and this suggests the possible variation in pathophysiology that exists in this condition. As a result, the consideration of NGF as a sole biomarker for this condition may be limited.

Clearly, as NGF levels may be raised in numerous conditions, an exclusion criteria needs to be established to highlight patient suitability, and avoid performing measurements in patients who may have false positive results for OAB. Other groups that may also require exclusion are those with active or recent UTIs, and those with indwelling catheters or performing clean intermittent self-catheterisation, which may be a significant proportion of the neurogenic population, as the minor trauma may theoretically exacerbate urothelial release; however, this is yet to be investigated. The exact source of the NGF in the urine is also unclear, with regards to the proportion that is attributed to the urothelium, and the proportion that may be from systemically circulating NGF, which may undergo renal excretion. More study is also required within a control population, to assess within-person biological variation; levels may change throughout the day. So variables such as the time of sample collection, whether a first morning void, or a later sample and bladder volume or degree of bladder stretch when providing a sample, may also influence the urinary levels. This question of the variability of urinary NGF/Cr levels during the day has been explored in a recent study, which confirmed no circadian variation [42]. These variables may have an impact on the end result and also need to be standardised for collection. The methods currently reported could be addressed by collecting samples and immediately placing on ice, followed by rapid transfer to the laboratory and processing. If processing is delayed this may risk NGF degradation, and hence levels may be affected. Further studies are required on assessing the stability of this protein in urine, to help standardise methods of collection in an outpatient setting. The costs of the detection techniques may also need to be considered, to gather information for cost-effectiveness calculations.

Conclusions

Whilst the evidence for an increased urinary NGF in OAB appears convincing, many questions about its validity remain including specificity, sensitivity, cost- and time-effectiveness. Many criteria for what constitutes a biomarker still need to be evaluated and met before this molecule can be considered for this role. However, exploring the role of NGF in these conditions has elucidated more information about the complex urothelial signalling mechanisms that exist, the pathophysiology of OAB, and the role of the neurotrophins in particular. Other promising biomarkers are also being explored such as inflammatory molecules and brain-derived neurotrophic factor [43], also within the neurotrophin family, which may identify some more reliable biomarker candidates. It may well be that the ideal urinary biomarker for any particular type of LUTD may be the presence of a combination of proteins, the levels of which may differ between conditions and patients. Used together, their sensitivities and specificities may be combined, to make it a stronger investigation.

Conflict of Interest/Financial Declaration

Mohammad S. Khan, Frank van der Aa, Dirk de Ridder and Arun Sahai have all been Investigators for Allergan, Ltd. Mohammad S. Khan and Arun Sahai have received unrestricted educational grants from Allergan, Ltd. Arun Sahai has spoken on behalf of Allergan, Ltd at educational meetings.

Jai H. Seth, Arun Sahai, Jalesh N. Panicker and Clare J. Fowler acknowledge financial support from the InComb project. Mohammad S. Khan, Prokar Dasgupta and Arun Sahai acknowledge financial support from the Department of Health via the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre award to Guy's and St Thomas' NHS Foundation Trust in partnership with King's College London and King's College Hospital NHS Foundation Trust. They also acknowledge the support of the MRC Centre for Transplantation.

Jai H. Seth, Jalesh N. Panicker and Clare J. Fowler would like to acknowledge UCLH/UCL Institute of Neurology and received a proportion of funding from the Department of Health's NIHR Biomedical Research Centres funding scheme.

Ancillary