Biomarkers for Interstitial Cystitis/Painful Bladder Syndrome

“Urinary elevation of proteins associated with interstitial cystitis/painful bladder syndrome may be due to lesser dilution of proteins shed into urine or due to lower levels of proteases in urine compared to plasma.”

Interstitial cystitis/painful bladder syndrome (IC/PBS) is a chronic bladder disorder with an estimated US prevalence of 3–8 million women and 1–4 million men [1]. Prominent symptoms of IC/PBS include urinary urgency, frequency, pain, pressure and/or discomfort, perceived to be related to the urinary bladder in the absence of urinary tract infection [2]. These symptoms often overlap with a host of other urinary disorders including chronic urethral syndrome, overactive bladder, hypersensitive bladder, vulvodynia, endometriosis in women and prostatitis in men. These overlapping symptoms make it difficult for experts to arrive at a consensus on the etiology and pathology of IC/PBS. Although National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) diagnostic guidelines are available, diagnosis historically has been largely dependent on the physician's judgement and exclusion of other disorders [3].

Current uncertainty regarding the etiology and pathology of IC/PBS ultimately impacts its treatment and hampers future drug development. Similar scenarios involving other diseases with overlapping symptoms have been resolved with biomarkers. A biomarker is a simple and elegant concept of objectively measuring a constituent or characteristic that is indicative of health, disease or treatment response. Biomarkers can be gene related [4], which tends to identify predisposing risk factors for a disease, or they can be protein related [5], which have the potential to identify the point in time when predisposition is developing into a disease. It is needless to say that there exists an unmet need for noninvasive biomarkers of IC/PBS, which can enable the expedited diagnosis and advance the mechanistic understanding of IC/PBS. Here, we summarize the current understanding of emerging biomarkers.

There are several considerations in the biomarker discovery for IC/PBS as it is a complex and heterogeneous disease with multiple possible etiologies. Prominent etiologic theories for IC/PBS include bladder epithelial dysfunction [6], mast cell activation [7], immune system abnormalities and pelvic floor hypertonicity [8]. It is highly controversial among experts whether IC/PBS symptoms primarily emanate from a localized bladder pathology or are due to pelvic floor hypertonicity. A number of single protein biomarkers in different biofluids associated with IC/PBS have been detected using a variety of techniques that can potentially classify the heterogeneity of IC/PBS into different etiology based subsets. However, none of them have sufficient demonstration of analytical and clinical validity to meet the US FDA standards for clinical use. Hence the search for biomarkers for IC/PBS continues.

Cystoscopic examination is a standard routine for IC/PBS patients, which generally classifies them as either having the ulcerative or nonulcerative type of IC/PBS [5]. Ulcerative IC/PBS accounts for 10% of cases and refers to Hunner's lesions visible on the bladder epithelium during cystoscope exam. Patients not having Hunner's lesions are classified as nonulcerative IC/PBS. However, this gross morphological discrimination with cystoscopy is unable to distinguish patients having abnormalities in the bladder from those having abnormalities in the pelvic floor. As alluded to earlier, bladder tissue and the urine proteome is inherently dynamic and the complete understanding of the IC/PBS associated proteomic changes has the potential to advance the understanding of molecular mechanisms underlying IC/PBS symptoms. Evidence of Hunner's lesions in IC/PBS patients is associated with high expression of T- and B-cell markers [9], low expression of urothelial markers, focal lymphoid aggregates in bladder submucosa and high immunoglobulin concentration in urine compared to patients without Hunner's lesions. Other studies compared IC/PBS patients with control subjects and reported elevation of cytokines [10], chemokines and enhanced immunoreactivity for muscarinic M2, purinergic P2X1, P2X2 and histamine H1 receptors [11].

“Latest advances in technology have the potential to make urine a nonsurgical biopsy of urinary tract.”

There is an obvious preference for noninvasive biomarkers such as proteins found in urine, which is an easy-to-collect biofluid. In addition, the urine proteome shows a better association with IC/PBS symptoms than the plasma proteome for several reasons. IC/PBS is a bladder-related disease and urine resides in the bladder for a long time to capture proteins and peptides shed from the bladder. One such peptide is a glycosylated nonapeptide, also known as antiproliferative factor (APF). APF was detected in the urine of IC/PBS patients based on the inhibitory effect of urine on the proliferation of cultured urothelial cells measured by thymidine incorporation assay [12]. Lower urinary APF activity after hydrodistension in 33 IC/PBS patients [13] showed the promise of this peptide in IC/PBS, which were tempered by the lack of significant difference in the APF activity for the urine specimens of ulcerative and nonulcerative IC/PBS patients [12].

By contrast, urine levels of IL-8 (CXCL-8) were able to discriminate ulcerative and nonulcerative IC/PBS. Using antibody-based multiplex assays, our group reported that CXCL-8 is elevated along with other members of CXC family of chemokines namely CXCL-1 and CXCL-10 in urine of ulcerative IC/PBS patients [5]. A earlier study by Erickson et al. reported a positive association between urinary CXCL-8 and bladder mast cell counts of IC/PBS patients [12]. A subsequent study from her group also reproduced the elevation of CXCL-10 in urine of ulcerative IC/PBS [14]. Longitudinal analysis of urine samples from IC/PBS patients at baseline and follow-up at 4 and 24 weeks demonstrated a treatment-associated reduction in chemokine levels following hydrodistension [10] and sacral neuromodulation [15], respectively.

It is generally understood that a majority of IC/PBS patients in the population do not show Hunner's lesions and therefore belong to the nonulcerative phenotype. Therefore, a biomarker that can further classify such patients as either having a bladder pathology [16] or pelvic floor hypertonicity [8] can help in clinical decision-making. IC/PBS patients identified as having bladder pathology will be good candidates for glycosaminoglycan replacement therapy by pentosan polysulfate or other intravesical treatments such as hyaluronic acid. On the other hand, IC/PBS patients diagnosed as having pelvic floor hypertonicity are more likely to benefit from physical therapy instead of pentosan polysulfate. Biomarkers can not only allow objective assessment of therapeutic response, but also foretell the therapeutic outcome from drugs like pentosan polysulfate, which has a late onset of action [17] and provide the biological basis for lack of response in non-responders. Potential therapeutic biomarkers [15] unique to each etiology of IC/PBS [18] can suitably identify patients for targeted therapeutic intervention and allow clinicians to propose a specific and tailored treatment to each patient with an improved safety profile.

Overall, studies have demonstrated that a decrease of APF [13] and chemokines in urine [10,15] was in concert with treatment mediated improvement in standardized measures for clinical symptoms of IC/PBS. In another study, urine levels of monocyte chemoattractant protein-1 positively correlated with lower bladder capacity and higher symptom scores of IC/PBS patients enrolled in the BCG trial [19]. Promising preclinical data are available to support the potential of urinary cytokines and chemokines as biomarkers for IC/PBS. The concentration of cytokines and chemokines associated with IC/PBS is usually higher in spot urine specimens than in corresponding plasma samples. Urinary elevation of proteins associated with IC/PBS may be due to lesser dilution of proteins shed into urine or due to lower levels of proteases in urine compared to plasma [20]. Besides, it is the innate nature of systemic circulation to maintain homeostasis in the concentrations of blood constituents including cytokines so any excess contributed from bladder into the plasma is likely to be filtered by kidney into urine.

Another consideration for biomarker discovery is that IC/PBS is not a static disease, but can present itself along the continuum of mild to moderate to severe symptoms. In recent years, a new technique of metabolomics is gaining interest for the understanding of disease mechanisms and biomarker discovery [21]. Using metabolomics profiling, urine levels of tyramine and 2-oxoglutarate were found to be significantly elevated in IC/PBS subjects compared to controls. Interestingly, urine levels of 2-oxoglutarate also retarded the growth of normal bladder epithelial cells. Levels of phenylacetylglutamine were found to be elevated in urine of mild to moderate IC/PBS patients [22] and creatinine corrected phenylacetylglutamine levels descended in the following order: mild to moderate IC/PBS > controls > severe IC/PBS. Drastically reduced bladder capacity of severe IC/PBS patients reported by several groups [4,19] may suggest the need for correcting the urine levels of proteins and metabolites associated with severe IC/PBS for the lower bladder capacity.

Presumably out of simplicity, several studies on biomarkers for IC/PBS look for the differences between IC/PBS patients and asymptomatic controls. IC/PBS patients are considered to have a deficient epithelial barrier formed by the layer of glycosaminoglycan and were therefore expected to have increased levels of glycosaminoglycan or its degradation products in urine. Incidentally, total content of urinary hexosamines was found to be significantly increased in IC/PBS patients relative to controls and the increase in glucosamine was greater than that for galactosamine [16]. While such studies are valuable in elucidating the molecular basis of IC/PBS, they may not help in differentiating the IC/PBS from other overlapping diseases.

A useful biomarker should have high sensitivity, specificity and reduce the number of false negatives during the testing process. Available knowledge about the clinical presentation of IC/PBS is relevant in assessing the value of statistical association seen between a clinical characteristic of patients and a putative biomarker. A recent study reported that YKL-40 antigenic expression in detrusor mast cell granules and submucosal macrophages was associated with detrusor fibrosis [23], which need to be considered in light of the fact that detrusor fibrosis is not noticeable in all IC/PBS patients. The likelihood of finding a single molecule that fits the criteria of a good biomarker, which can also distinguish IC/PBS from other lower urinary tract conditions and asymptomatic controls at all symptomatic stages of the IC/PBS, is extremely low. However, multimarker panels may be able to encompass the molecular heterogeneity of IC/PBS seen within the population and are likely to be clinically effective. Latest advances in technology have the potential to make urine a nonsurgical biopsy of urinary tract. The richness of urine as a biofluid for biomarker discovery is highlighted by the discovery of around 1543 proteins upon mass spectrometric analysis [24]. However, most techniques for urine proteomics are not that efficient at detecting proteins downregulated due to IC/PBS. In contrast, tissue or urine sediment [25] based genomic or miRNA analysis are better suited to pick genomic or proteomic biomarkers that are downregulated in bladder of IC/PBS patients [4].

The final characteristic of a biomarker is that it should be analyzed quickly and without very specialized equipment in a normal hospital laboratory setting. This is not an initial requirement for the biomarker since analytic procedures can be developed once a molecule of interest has been identified. However, methods of analysis generally have specific conceptual and technical considerations. Compared to bioassays based on thymidine incorporation for APF, assays based on immunological, chromatographic or spectrometric principles are easier to validate in order to satisfy FDA requirements. Some groups have published NMR-based metabolomics analysis or miRNA analysis [26] for identifying potential biomarker candidates, but these methods may not be feasible as a point of care diagnostic test.

There is still a lot of work to be done in identifying a biomarker for IC/PBS, but significant research is underway. There is a need for well-structured clinical studies to satisfy the rigorous criteria that must be met to qualify biomarkers for specific contexts of use in clinic. Currently, biomarker research in IC/PBS is lagging behind because of the long interval required for validation, testing and approval of the assays, and the lack of standardization for urine or tissue biopsy collection. To enhance the development of new biomarkers, a more structured approach is required.