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bladder pain syndrome
Urological Science xxx (2015) 1e5
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Review article
Biomarkers for patients with interstitial cystitis/bladder pain syndrome Hsin-Tzu Liu, Hann-Chorng Kuo* Department of Urology, Buddhist Tzu Chi General Hospital and Tzu Chi University, Hualien, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 December 2014 Received in revised form 15 February 2015 Accepted 24 February 2015 Available online xxx
Interstitial cystitis or bladder pain syndrome (IC/BPS) is a disease of unknown etiology manifested with bladder pain and frequency urgency symptoms. Although several pathophysiologic mechanisms have been proposed, the underlying mechanism of IC/BPS is still unclear. Accumulated evidence supports that IC/BPS is actually a spectrum of clinical phenomena that involves several different genes and environmental factors. Heterogeneous syndromes are seen in patients with IC/PBS, which suggests that the disease should be classified into different subtypes. Abnormal expressions of several bladder epithelial markers, including mast cells, epithelial differentiation proteins, cell membrane proteins, neurotransmitters, and cytokines, are present in IC/BPS. This review discusses the possible biomarkers that may play crucial roles in IC/BPS, and especially focuses on those that have the potential to be used as biomarkers for prognosis and for the determination of the best treatment for patients. Copyright © 2015, Taiwan Urological Association. Published by Elsevier Taiwan LLC. All rights reserved.
Keywords: biomarker bladder diagnosis pathophysiology
1. Introduction Interstitial cystitis or bladder pain syndrome (IC/BPS) is a chronic condition of unknown etiology that is characterized by clinical symptoms of urinary urgency/frequency and results in recurring discomfort or pain in the bladder and pelvic region. Although many theories have been proposed, there is still a lack of consensus regarding the pathophysiology of IC/BPS. No single test can be used to diagnose IC/BPS, and therefore a combination of several diagnostic tests is required to rule out other diseases and confirm the presence of IC/BPS. Currently, the diagnosis of IC/BPS is based on the presence of pain related to the bladder that is normally accompanied by frequency and urgency of urination, and the patient should be absent of other diseases that could cause the same symptoms. Although several pathophysiologic mechanisms have been proposed, the underlying mechanism of IC/BPS is still unclear, and universally accepted diagnostic criteria for the disease are still under development. As many different disorders may cause the same symptoms as those present in IC/BPS, we need to discover reliable markers that can not only help to correctly diagnose the disease,
* Corresponding author. Department of Urology, Buddhist Tzu Chi General Hospital, 707, Section 3, Chung-Yang Road, Hualien, Taiwan. E-mail address: [email protected] (H.-C. Kuo).
but more importantly, to identify a specific treatment that is best for patients. 2. Discovery of biomarkers for IC/BPS to improve treatment Biomarkers provide a powerful approach to understanding the progress of a disease. For the best biomarkers, it should be easy to perform tests that are accurate tools for diagnosis and prognosis. Accumulated evidence supports that IC/BPS is actually a spectrum of clinical phenomena that involves several different genes and environmental factors. In order to validate the best biomarker(s), it is necessary to determine diagnostic criteria that can identify different subsets of patients, and certain biomarkers can be used to screen patients who will respond to a specific treatment or to measure the outcome of a treatment. Heterogeneous syndromes are seen in patients with IC/PBS, which suggests that the disease should be classified into different subtypes. For example, Bouchelouche and Nordling1 concluded that the disease can be classified into a classic (ulcer) type and a non-nuclear type, and different approaches may be required to treat these two types of patient. In 2008, the European Society for the Study of Interstitial Cystitis (ESSIC) proposed a new classification system,2 which classifies the disease into more different subtypes/stages. When a widely acceptable classification system is developed, identifying markers for research is required to help increase our understanding of the
http://dx.doi.org/10.1016/j.urols.2015.02.002 1879-5226/Copyright © 2015, Taiwan Urological Association. Published by Elsevier Taiwan LLC. All rights reserved.
Please cite this article in press as: Liu H-T, Kuo H-C, Biomarkers for patients with interstitial cystitis/bladder pain syndrome, Urological Science (2015), http://dx.doi.org/10.1016/j.urols.2015.02.002
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molecular mechanism of the disease and further improve practical clinical treatment. 3. Pathogenesis of IC/BPS It is now generally agreed that infection is not a direct etiology of IC/BPS, as several studies have failed to detect bacterial DNA in patients with IC/BPS.3,4 It is known that a defect in bladder urothelial permeability is associated with the pathogenesis of IC/BPS, but the mechanism that causes urothelial disruption is still unknown. It is possible that a bacterial endotoxin, such as lipopolysaccharide, might cause urothelial damage and indirectly contribute to the development of the disease. Recent studies in humans with IC/BPS and in animal models of IC have highlighted directions for better treatment of the disease. Activation and increased numbers of mast cells in the bladder urothelium are seen in patients with IC/BPS.5 Mast cells secrete several inflammatory mediators, including substance P and nerve growth factor (NGF), which are associated with the proliferation of nerve fibers.6,7 In addition, mast cells also release cytokines and other proinflammatory and nociceptive mediators.8 Study has also shown that stress increases IC/BPS symptoms in patients,9 which may be due to stress-induced neurogenic chronic inflammation that causes overactivation of the sympathetic nervous system. This review discusses the possible biomarkers that may play crucial roles in IC/BPS, and especially focuses on those that have the potential to be used as biomarkers for prognosis and for the determination of the best treatment for patients. 4. Markers in bladder tissue 4.1. Mast cells Several reports have indicated that bladder specimens of patients with IC/BPS show increased numbers of infiltrated mast cells.5,10e13 Other studies have also shown that mast cells are found both in the epithelium and in bladder washings of patients with IC/ BPS, but not in normal individuals.12,14 This evidence suggests that the disease is mediated by the immune system, and the abnormalities are possibly caused by dysregulation of the inflammatory response. In addition to the increased number of mast cells in the bladder in IC/BPS, the mast cells are mainly activated, as they are partially or completely degranulated.11 Thus, the increased cell number and activation of mast cells in the bladder suggests that mucosal mast cells are one of the main events involved in the etiology of IC/BPS. Although it is now known that there are increased numbers and increased activation of mast cells in the bladder of patients with IC/ BPS, the cell count in tissue and the percentage of activated mast cells are significantly influenced by sample processing. Therefore, a standardized method and a certain number of control individuals are required to validate the use of mast cells as a biomarker of IC/ BPS. 4.2. Bladder urothelial defects Primary urothelial lining defects are thought to play an important role in IC/BPS.15 Urothelial defects are significantly associated with chronic inflammation, and increased urothelial apoptosis may be the causative factor of the disease.16 Several studies have also shown that decreased tight junction protein zonula occludens-1 expression occurs in IC/BPS, suggesting that urothelial barrier defects may lead to increased urothelial leakage and painful bladder symptoms.17e19 Several studies have linked overactive bladder (OAB) and IC/BPS to chronic
inflammation, showing that the levels of bladder and urinary NGF, cytokines, and serum C-reactive protein are elevated in both patients with OAB and those with IC/BPS.20e25 Although it has been suggested that OAB and IC/BPS might share a common pathway, our recent study demonstrated that although mast cell infiltration was found in both diseases, abnormal urothelial barrier function only occurred in patients with IC/BPS, and not in those with OAB.26 Proper function of the urothelium requires normal epithelial integrity, which relies on intercellular adhesion molecules and a layer of molecular components on the apical surface of the urothelium. E-cadherin is one of the intercellular junction proteins that have been suggested to be involved in the barrier function of the urothelium. The role of E-cadherin in the pathophysiology of IC/ BPSS remains controversial. Earlier studies demonstrated elevated E-cadherin in the bladder of patients with IC/BPS, and it has been suggested that this is due to adaptation to increased bladder permeability.27,28 However, recent studies from our group and others have shown decreased or abnormal expression of E-cadherin in IC/BPS.16,19,26 The apical surface of the urothelium is coated with a layer of glycoproteins, proteoglycans, and glycolipids, with a superficial proportion of glycosaminoglycans (GAGs) on the top of the layer.29 The GAG or mucin layer is thought to act as a nonspecific anti-adherence factor and as a defense mechanism against infection,30,31 and several lines of evidence support that surface mucosal and GAG dysfunction contribute to IC/PBS.32 Since these studies, GAG replenishment therapy has been under development.33 Although GAGs in the bladder urothelium have an important role, the mucin GAG-layer lining is constituted by a group of different glycoproteins, proteoglycans, and other molecules, and further studies to identify the key molecules in IC/BPS will help to improve the efficacy of treatment and identify biomarkers of the disease. 5. Serum and urinary biomarkers for OAB and IC/BPS Serum and urinary cytokines, chemokines, and other informatory mediators are potential molecules that could be developed as useful markers for IC/BPS as they are noninvasive predictors. Cytokines and chemokines have crucial roles in the pathogenesis of several chronic inflammatory diseases. As bladder inflammation is one of the major causes of IC/BPS, it is likely that the biomarkers from serum and urine have a prognostic role and can serve as tools for the selection of suitable therapeutic agents for treatment. 5.1. Cytokines Increased levels of cytokines and chemokines have been found in the urine of patients with IC/BPS. Studies by Erickson et al34 and Sakthivel et al35 found that several proinflammatory mediators, such as interleukin-6 (IL-6) and CXC chemokines, were increased in urinary and serum samples of patients with IC/BPS. Lamale and coworkers36 proposed the use of a combination of methylhistamine and IL-6 as a sensitive and specific marker for IC/BPS. A study by Ogawa and colleagues37 also confirmed upregulation of several CXCR3 binding chemokines in patients with ulcerative IC/BPS. Controversial results have been reported for other cytokines, including IL-2, IL-8, interferon-g, and tumor necrosis factor-a, which were only found to be increased in patients in some earlier studies.34,38,39 Our recent study revealed that the serum IL-1b, IL-6, tumor necrosis factor-a, and IL-8 levels were significantly higher in the serum of patients with IC/BPS than in control patients (Table 1).40
Please cite this article in press as: Liu H-T, Kuo H-C, Biomarkers for patients with interstitial cystitis/bladder pain syndrome, Urological Science (2015), http://dx.doi.org/10.1016/j.urols.2015.02.002
H.-T. Liu, H.-C. Kuo / Urological Science xxx (2015) 1e5 Table 1 Expression of serum interleukin (IL)-1b, IL-6, tumor necrosis factor (TNF-a) and IL-8 in interstitial cystitis or bladder pain syndrome (IC/PBS) patients and controls.
Gender Age (y) IL-1b (pg/mL) IL-6 (pg/mL) TNF-a (pg/mL) IL-8 (pg/mL)
Controls (n ¼ 26)
IC/PBS (n ¼ 30)
p
F:16 M:10 32.36 ± 1.56 (22e55) 1.64 ± 0.47 (0.00e6.08) 0.79 ± 0.21 (0.00e3.67) 0.91 ± 0.17 (0.00e4.64) 1.45 ± 0.21 (0.00e4.09)
F: 26 M: 4 50.57 ± 2.68 (24e86) 6.45 ± 0.71 (2.77e23.96) 1.52 ± 0.24 (0.00e6.14) 2.63 ± 0.60 (0.62e13.70) 3.23 ± 0.48 (0.00e15.08)
<0.001 <0.001 <0.001 <0.001 <0.001
Data are presented as mean ± standard error (minemax). F ¼ female; M ¼ male.
5.2. Nerve system inflammation In patients with IC/PBS, neurotrophins, including NGF, neurotrophin-3, and glial-cell-derived neurotrophic factor, have been detected in the urine.41 NGF is a small secreted protein that induces differentiation and survival of target neurons. NGF has been shown to play a role in altered lower urinary tract function. Here, NGF acts as a chemical mediator in C-fiber afferents that may regulate the change in urinary bladder function.42,43 The levels of several neurotrophic factors, including NGF, increase in the bladder after spinal cord injury,42,44 and NGF has been found to increase in the lumbosacral spinal cord and dorsal root ganglia after spinal cord injury in a rat model.45 Patients with lower urinary tract diseases, including IC/BPS23e25,41,46 and bladder outlet obstruction,47 have been found to have increased NGF levels in the urine and/or bladder tissue. In the bladder, NGF is expressed in the urothelial cells and smooth muscle.48 Normally, NGF exists in the afferent nerves and ganglia of the bladder wall and is responsible for the normal sensation of bladder distention and conduction of abnormal sensory input.48 NGF is also released by mast cells.49 In a transgenic mouse model, chronic NGF overexpression in the bladder led to neuronal hyperinnervation, pelvic sensitivity, and changes in urinary bladder function.50 Serum NGF levels have been found to increase in several systemic diseases, including allergic diseases, autoimmune diseases, and psychosocial stress,51e53 and therefore analysis of both urinary and serum NGF in patients with IC/BPS provides insight into the development of the disease. In samples of patients with IC/BPS, increased levels of NGF have been noted in the urine23,24,41,46 and bladder tissue.25 Our recent study showed that the serum NGF level of IC/BPS patients was elevated, while the increased serum NGF level was not correlated with the urinary NGF level and was not related to the severity of IC/BPS (Fig. 1).54 These findings suggest that NGF is associated with bladder function, and elevated urinary NGF levels reflect that chronic inflammation is occurring in the urinary bladder of IC/BPS patients.
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NGF has been suggested to act as a local stress mediator in perceived stress and allergy, and increased NGF leads to deteriorating allergic inflammation in stress-induced worsening of allergic inflammation.55 Stress has been shown to be involved in the pathogenesis of IC/BPS,9 and has also been found to induce NGF expression in the urinary bladder in an animal model.56 The urinary NGF level has been shown to be closely related to the visual analog scale score for pain and response to conventional treatment for IC/ BPS. Several studies have shown that injections of botulinum toxin A reduce bladder pain in patients with refractory IC/BPS,57,58 and the increased NGF levels in the bladder tissue of patients with IC/ BPS can decrease to within the normal range after botulinum toxin A treatment.59 However, the role of NGF may be due to its association with bladder function, as NGF also increases in patients with other bladder diseases, such as OAB and urinary tract infection. Although NGF might be able to be developed as an indicator for treatment, in order to be a sensitive molecular diagnostic tool for IC/BPS, it is likely that a combination of other urinary or tissue biomarkers is required. 6. Recent development 6.1. Genetic biomarkers Several pilot studies have attempted to use microarray technology to reveal genes involved in the pathobiology underlying IC/ BPS.60,61 The results of these studies are promising, and have demonstrated through biopsy of bladder tissue that a number of genes with differential expressions exist between IC/BPS patients and healthy controls. However, further studies are needed in order to validate the target genes to enable identification of genes that can be used as new biomarkers of IC/BPS. This also indicates that the fundamental disease processes of IC/BPS are associated with a heterogeneous and complicated transcriptional network. In addition to microarray, high-throughput sequencing technologies provide new methods for the analysis of gene expression. With the recent increases in specificity and sensitivity and a decrease in cost, RNA sequencing technology has been shown to improve the accuracy of analysis of differential gene expressions, especially for low-abundance transcripts.62 Therefore, RNA sequencing technology may be a better tool for enhanced detection of differential expression in future studies of IC/BPS. These gene expression-based predictive models can narrow down the number of potential biomarkers to be studied and their roles of importance in the disease. 6.2. Reclassification of IC/BPS Recently, researchers have suggested that IC/BPS should be reclassified. ESSIC is classified into several subtypes.63 Elucidating
Fig. 1. Nerve growth factor (NGF)-immunoreactivity in the bladder of patients with interstitial cystitis or bladder pain syndrome (IC/BPS) shows significantly increased density in submucosal and urothelial cells at baseline (A) 200 and (B) 400. NGF staining in the control was localized to apical urothelial cells (C) 200.
Please cite this article in press as: Liu H-T, Kuo H-C, Biomarkers for patients with interstitial cystitis/bladder pain syndrome, Urological Science (2015), http://dx.doi.org/10.1016/j.urols.2015.02.002
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subtype-specific biomarkers may be more clinically relevant. For instance, Logadottir et al64 studied biopsies from patients with ESSIC Type 3C, and found that the expressions of IL-6, IL-10, and IL17A, and inducible nitric oxide synthase mRNA, were significantly higher in the patients than in healthy controls. Another study took clinical presentation into consideration, and found that several genes are correlated with clinical factors.60 Nickel et al65 also proposed that a phenotype-directed therapeutic approach can significantly improve the treatment outcome. Homma and colleagues66 showed that there are different gene expression patterns for classic and nonclassic IC. Tripp and coworkers67 developed a system to classify IC/BPS into several pain-location phenotypes, and suggested that the system can enable patients to receive better treatment as it can provide improved pain management. Therefore, a larger patient sample that can be subclassified into different types will help to identify reliable biomarkers for IC/BPS. In combination with consideration of disease progress and clinical factors, a noninvasive method can be developed to analyze the efficiency of treatment. 7. Conclusion Abnormal expressions of several bladder epithelial markers, including mast cells, epithelial differentiation proteins, cell membrane proteins, neurotransmitters, and cytokines, are present in IC/ BPS. Clinical and experimental data have indicated a direct link between increased levels of NGF in the bladder tissue and urine and painful inflammatory conditions in IC/BPS. However, NGF is not the only protein that contributes to IC/BPS; therefore, a combination test with measurement of other inflammatory proteins in the urine, or other marker(s) in tissue specimens, is required. Because the urinary NGF level can also be increased in many lower urinary tract diseases, such as stones, tumors, acute bacterial infection, as well as in IC/PBS, it is important to exclude these diseases. Current findings suggest that the urinary NGF level can be monitored as a biomarker for IC/PBS severity and for the response of patients to treatment. Several recently identified cytokines and mediators identified to be associated with the disease can be useful tools to assess treatment outcome. Further studies and subclassification of IC/PBS are required to identify new biomarkers for diagnosis, as well as for treatment efficiency in patients with IC/PBS. Conflicts of interest All contributing authors declare no conflicts of interest. References 1. Bouchelouche K, Nordling J. Recent developments in the management of interstitial cystitis. Curr Opin Urol 2003;13:309e13. 2. van de Merwe JP, Nordling J, Bouchelouche P, Bouchelouche K, Cervigni M, Daha LK, et al. Diagnostic criteria, classification, and nomenclature for painful bladder syndrome/interstitial cystitis: an ESSIC proposal. Eur Urol 2008;53: 60e7. 3. Haarala M, Jalava J, Laato M, Kiilholma P, Nurmi M, Alanen A. Absence of bacterial DNA in the bladder of patients with interstitial cystitis. J Urol 1996;156:1843e5. 4. Keay S, Zhang CO, Baldwin BR, Jacobs SC, Warren JW. Polymerase chain reaction amplification of bacterial 16S rRNA genes in interstitial cystitis and control patient bladder biopsies. J Urol 1998;159:280e3. 5. Theoharides TC, Kempuraj D, Sant GR. Mast cell involvement in interstitial cystitis: a review of human and experimental evidence. Urology 2001;57: 47e55. 6. Theoharides TC, Sant GR, el-Mansoury M, Letourneau R, Ucci Jr AA, Meares Jr EM. Activation of bladder mast cells in interstitial cystitis: a light and electron microscopic study. J Urol 1995;153:629e36. 7. Lundeberg T, Liedberg H, Nordling L, Theodorsson E, Owzarski A, Ekman P. Interstitial cystitis: correlation with nerve fibres, mast cells and histamine. Br J Urol 1993;71:427e9.
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H.-T. Liu, H.-C. Kuo / Urological Science xxx (2015) 1e5 37. Ogawa T, Homma T, Igawa Y, Seki S, Ishizuka O, Imamura T, et al. CXCR3 binding chemokine and TNFSF14 over expression in bladder urothelium of patients with ulcerative interstitial cystitis. J Urol 2010;183:1206e12. 38. Felsen D, Frye S, Trimble LA, Bavendam TG, Parsons CL, Sim Y, et al. Inflammatory mediator profile in urine and bladder wash fluid of patients with interstitial cystitis. J Urol 1994;152:355e61. 39. Peters KM, Diokno AC, Steinert BW. Preliminary study on urinary cytokine rin treat levels in interstitial cystitis: does intravesical bacille Calmette-Gue interstitial cystitis by altering the immune profile in the bladder? Urology 1999;54:450e3. 40. Liu HT, Kuo HC. Increased pro-inflammatory cytokine and chemokine expressions in serum of patients with interstitial cystitis/bladder pain syndrome. ICS 2012-The 42nd Annual Meeting of the International Continence Society (ICS). Beijing, China. 2012. p. 480. 41. Okragly AJ, Niles AL, Saban R, Schmidt D, Hoffman RL, Warner TF, et al. Elevated tryptase, nerve growth factor, neurotrophin-3 and glial cell line-derived neurotrophic factor levels in the urine of interstitial cystitis and bladder cancer patients. J Urol 1999;161:438e41. 42. Vizzard MA. Changes in urinary bladder neurotrophic factor mRNA and NGF protein following urinary bladder dysfunction. Exp Neurol 2000;161:273e84. 43. Yoshimura N. Bladder afferent pathway and spinal cord injury: possible mechanisms inducing hyperreflexia of the urinary bladder. Prog Neurobiol 1999;57:583e606. 44. Vizzard MA. Neurochemical plasticity and the role of neurotrophic factors in bladder reflex pathways after spinal cord injury. Prog Brain Res 2006;152: 97e115. 45. Satoshi S, Sasaki K, Igawa Y, Nishizawa O, Chancellor M, de Groat W, et al. Detrusor overactivity induced by increased levels of nerve growth factor in bladder afferent pathways in rats. Neurourol Urodyn 2002;22:375e7. 46. Jacobs BL, Smaldone MC, Tyagi V, Philips BJ, Jackman SV, Leng WW, et al. Increased nerve growth factor in neurogenic overactive bladder and interstitial cystitis patients. Can J Urol 2010;17:4989e94. 47. Liu HT, Kuo HC. Urinary nerve growth factor levels are increased in patients with bladder outlet obstruction with overactive bladder symptoms and reduced after successful medical treatment. Urology 2008;72:104e8. discussion 8. 48. Steers WD, Tuttle JB. Mechanisms of disease: the role of nerve growth factor in the pathophysiology of bladder disorders. Nat Clin Pract Urol 2006;3:101e10. 49. Molderings GJ. Mast cell function in physiology and pathophysiology. Biotrend Rev 2010;5:1e9. 50. Schnegelsberg B, Sun TT, Cain G, Bhattacharya A, Nunn PA, Ford AP, et al. Overexpression of NGF in mouse urothelium leads to neuronal hyperinnervation, pelvic sensitivity, and changes in urinary bladder function. Am J Physiol Regul Integr Comp Physiol 2010;298:R534e47. 51. Alleva E, Santucci D. Psychosocial vs. “physical” stress situations in rodents and humans: role of neurotrophins. Physiol Behav 2001;73:313e20. 52. Aloe L, Skaper SD, Leon A, Levi-Montalcini R. Nerve growth factor and autoimmune diseases. Autoimmunity 1994;19:141e50.
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53. Bonini S, Lambiase A, Bonini S, Levi-Schaffer F, Aloe L. Nerve growth factor: an important molecule in allergic inflammation and tissue remodelling. Int Arch Allergy Immunol 1999;118:159e62. 54. Liu HT, Kuo HC. Increased urine and serum nerve growth factor levels in interstitial cystitis suggest chronic inflammation is involved in the pathogenesis of disease. PLoS One 2012;7:e44687. 55. Peters EM, Liezmann C, Spatz K, Daniltchenko M, Joachim R, Gimenez-Rivera A, et al. Nerve growth factor partially recovers inflamed skin from stress-induced worsening in allergic inflammation. J Invest Dermatol 2011;131:735e43. 56. Merrill L, Malley S, Vizzard MA. Repeated variate stress in male rats induces increased voiding frequency, somatic sensitivity, and urinary bladder nerve growth factor expression. Am J Physiol Regul Integr Comp Physiol 2013;305: R147e56. 57. Kuo HC, Chancellor MB. Comparison of intravesical botulinum toxin type A injections plus hydrodistention with hydrodistention alone for the treatment of refractory interstitial cystitis/painful bladder syndrome. BJU Int 2009;104: 657e61. 58. Pinto R, Lopes T, Silva J, Silva C, Dinis P, Cruz F. Persistent therapeutic effect of repeated injections of onabotulinum toxin a in refractory bladder pain syndrome/interstitial cystitis. J Urol 2013;189:548e53. 59. Liu HT, Kuo HC. Intravesical botulinum toxin A injections plus hydrodistension can reduce nerve growth factor production and control bladder pain in interstitial cystitis. Urology 2007;70:463e8. 60. Colaco M, Koslov DS, Keys T, Evans RJ, Badlani GH, Andersson KE, et al. Correlation of gene expression with bladder capacity in interstitial cystitis/bladder pain syndrome. J Urol 2014;192:1123e9. 61. Tseng LH, Chen I, Chen MY, Lee CL, Lin YH, Lloyd LK. Genome-based expression profiles as a single standardized microarray platform for the diagnosis of bladder pain syndrome/interstitial cystitis: an array of 139 genes model. Int Urogynecol J Pelvic Floor Dysfunct 2009;20:515e22. 62. Wang C, Gong B, Bushel PR, Thierry-Mieg J, Thierry-Mieg D, Xu J, et al. The concordance between RNA-seq and microarray data depends on chemical treatment and transcript abundance. Nat Biotechnol 2014;32:926e32. 63. ESSIC. Bladder pain syndrome. 2007. http://www.essic.eu/pdf/ESSICconsensus2007. pdf [accessed 13.02.15]. 64. Logadottir Y, Delbro D, Fall M, Gjertsson I, Jirholt P, Lindholm C, et al. Cytokine expression in patients with bladder pain syndrome/interstitial cystitis ESSIC type 3C. J Urol 2014;192:1564e8. 65. Nickel JC, Irvine-Bird K, Jianbo L, Shoskes DA. Phenotype-directed management of interstitial cystitis/bladder pain syndrome. Urology 2014;84:175e9. 66. Homma Y, Nomiya A, Tagaya M, Oyama T, Takagaki K, Nishimatsu H, et al. Increased mRNA expression of genes involved in pronociceptive inflammatory reactions in bladder tissue of interstitial cystitis. J Urol 2013;190:1925e31. 67. Tripp DA, Nickel JC, Wong J, Pontari M, Moldwin R, Mayer R, et al. Mapping of pain phenotypes in female patients with bladder pain syndrome/interstitial cystitis and controls. Eur Urol 2012;62:1188e94.
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Review article
Biomarkers for patients with interstitial cystitis/bladder pain syndrome Hsin-Tzu Liu, Hann-Chorng Kuo* Department of Urology, Buddhist Tzu Chi General Hospital and Tzu Chi University, Hualien, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 December 2014 Received in revised form 15 February 2015 Accepted 24 February 2015 Available online xxx
Interstitial cystitis or bladder pain syndrome (IC/BPS) is a disease of unknown etiology manifested with bladder pain and frequency urgency symptoms. Although several pathophysiologic mechanisms have been proposed, the underlying mechanism of IC/BPS is still unclear. Accumulated evidence supports that IC/BPS is actually a spectrum of clinical phenomena that involves several different genes and environmental factors. Heterogeneous syndromes are seen in patients with IC/PBS, which suggests that the disease should be classified into different subtypes. Abnormal expressions of several bladder epithelial markers, including mast cells, epithelial differentiation proteins, cell membrane proteins, neurotransmitters, and cytokines, are present in IC/BPS. This review discusses the possible biomarkers that may play crucial roles in IC/BPS, and especially focuses on those that have the potential to be used as biomarkers for prognosis and for the determination of the best treatment for patients. Copyright © 2015, Taiwan Urological Association. Published by Elsevier Taiwan LLC. All rights reserved.
Keywords: biomarker bladder diagnosis pathophysiology
1. Introduction Interstitial cystitis or bladder pain syndrome (IC/BPS) is a chronic condition of unknown etiology that is characterized by clinical symptoms of urinary urgency/frequency and results in recurring discomfort or pain in the bladder and pelvic region. Although many theories have been proposed, there is still a lack of consensus regarding the pathophysiology of IC/BPS. No single test can be used to diagnose IC/BPS, and therefore a combination of several diagnostic tests is required to rule out other diseases and confirm the presence of IC/BPS. Currently, the diagnosis of IC/BPS is based on the presence of pain related to the bladder that is normally accompanied by frequency and urgency of urination, and the patient should be absent of other diseases that could cause the same symptoms. Although several pathophysiologic mechanisms have been proposed, the underlying mechanism of IC/BPS is still unclear, and universally accepted diagnostic criteria for the disease are still under development. As many different disorders may cause the same symptoms as those present in IC/BPS, we need to discover reliable markers that can not only help to correctly diagnose the disease,
* Corresponding author. Department of Urology, Buddhist Tzu Chi General Hospital, 707, Section 3, Chung-Yang Road, Hualien, Taiwan. E-mail address: [email protected] (H.-C. Kuo).
but more importantly, to identify a specific treatment that is best for patients. 2. Discovery of biomarkers for IC/BPS to improve treatment Biomarkers provide a powerful approach to understanding the progress of a disease. For the best biomarkers, it should be easy to perform tests that are accurate tools for diagnosis and prognosis. Accumulated evidence supports that IC/BPS is actually a spectrum of clinical phenomena that involves several different genes and environmental factors. In order to validate the best biomarker(s), it is necessary to determine diagnostic criteria that can identify different subsets of patients, and certain biomarkers can be used to screen patients who will respond to a specific treatment or to measure the outcome of a treatment. Heterogeneous syndromes are seen in patients with IC/PBS, which suggests that the disease should be classified into different subtypes. For example, Bouchelouche and Nordling1 concluded that the disease can be classified into a classic (ulcer) type and a non-nuclear type, and different approaches may be required to treat these two types of patient. In 2008, the European Society for the Study of Interstitial Cystitis (ESSIC) proposed a new classification system,2 which classifies the disease into more different subtypes/stages. When a widely acceptable classification system is developed, identifying markers for research is required to help increase our understanding of the
http://dx.doi.org/10.1016/j.urols.2015.02.002 1879-5226/Copyright © 2015, Taiwan Urological Association. Published by Elsevier Taiwan LLC. All rights reserved.
Please cite this article in press as: Liu H-T, Kuo H-C, Biomarkers for patients with interstitial cystitis/bladder pain syndrome, Urological Science (2015), http://dx.doi.org/10.1016/j.urols.2015.02.002
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molecular mechanism of the disease and further improve practical clinical treatment. 3. Pathogenesis of IC/BPS It is now generally agreed that infection is not a direct etiology of IC/BPS, as several studies have failed to detect bacterial DNA in patients with IC/BPS.3,4 It is known that a defect in bladder urothelial permeability is associated with the pathogenesis of IC/BPS, but the mechanism that causes urothelial disruption is still unknown. It is possible that a bacterial endotoxin, such as lipopolysaccharide, might cause urothelial damage and indirectly contribute to the development of the disease. Recent studies in humans with IC/BPS and in animal models of IC have highlighted directions for better treatment of the disease. Activation and increased numbers of mast cells in the bladder urothelium are seen in patients with IC/BPS.5 Mast cells secrete several inflammatory mediators, including substance P and nerve growth factor (NGF), which are associated with the proliferation of nerve fibers.6,7 In addition, mast cells also release cytokines and other proinflammatory and nociceptive mediators.8 Study has also shown that stress increases IC/BPS symptoms in patients,9 which may be due to stress-induced neurogenic chronic inflammation that causes overactivation of the sympathetic nervous system. This review discusses the possible biomarkers that may play crucial roles in IC/BPS, and especially focuses on those that have the potential to be used as biomarkers for prognosis and for the determination of the best treatment for patients. 4. Markers in bladder tissue 4.1. Mast cells Several reports have indicated that bladder specimens of patients with IC/BPS show increased numbers of infiltrated mast cells.5,10e13 Other studies have also shown that mast cells are found both in the epithelium and in bladder washings of patients with IC/ BPS, but not in normal individuals.12,14 This evidence suggests that the disease is mediated by the immune system, and the abnormalities are possibly caused by dysregulation of the inflammatory response. In addition to the increased number of mast cells in the bladder in IC/BPS, the mast cells are mainly activated, as they are partially or completely degranulated.11 Thus, the increased cell number and activation of mast cells in the bladder suggests that mucosal mast cells are one of the main events involved in the etiology of IC/BPS. Although it is now known that there are increased numbers and increased activation of mast cells in the bladder of patients with IC/ BPS, the cell count in tissue and the percentage of activated mast cells are significantly influenced by sample processing. Therefore, a standardized method and a certain number of control individuals are required to validate the use of mast cells as a biomarker of IC/ BPS. 4.2. Bladder urothelial defects Primary urothelial lining defects are thought to play an important role in IC/BPS.15 Urothelial defects are significantly associated with chronic inflammation, and increased urothelial apoptosis may be the causative factor of the disease.16 Several studies have also shown that decreased tight junction protein zonula occludens-1 expression occurs in IC/BPS, suggesting that urothelial barrier defects may lead to increased urothelial leakage and painful bladder symptoms.17e19 Several studies have linked overactive bladder (OAB) and IC/BPS to chronic
inflammation, showing that the levels of bladder and urinary NGF, cytokines, and serum C-reactive protein are elevated in both patients with OAB and those with IC/BPS.20e25 Although it has been suggested that OAB and IC/BPS might share a common pathway, our recent study demonstrated that although mast cell infiltration was found in both diseases, abnormal urothelial barrier function only occurred in patients with IC/BPS, and not in those with OAB.26 Proper function of the urothelium requires normal epithelial integrity, which relies on intercellular adhesion molecules and a layer of molecular components on the apical surface of the urothelium. E-cadherin is one of the intercellular junction proteins that have been suggested to be involved in the barrier function of the urothelium. The role of E-cadherin in the pathophysiology of IC/ BPSS remains controversial. Earlier studies demonstrated elevated E-cadherin in the bladder of patients with IC/BPS, and it has been suggested that this is due to adaptation to increased bladder permeability.27,28 However, recent studies from our group and others have shown decreased or abnormal expression of E-cadherin in IC/BPS.16,19,26 The apical surface of the urothelium is coated with a layer of glycoproteins, proteoglycans, and glycolipids, with a superficial proportion of glycosaminoglycans (GAGs) on the top of the layer.29 The GAG or mucin layer is thought to act as a nonspecific anti-adherence factor and as a defense mechanism against infection,30,31 and several lines of evidence support that surface mucosal and GAG dysfunction contribute to IC/PBS.32 Since these studies, GAG replenishment therapy has been under development.33 Although GAGs in the bladder urothelium have an important role, the mucin GAG-layer lining is constituted by a group of different glycoproteins, proteoglycans, and other molecules, and further studies to identify the key molecules in IC/BPS will help to improve the efficacy of treatment and identify biomarkers of the disease. 5. Serum and urinary biomarkers for OAB and IC/BPS Serum and urinary cytokines, chemokines, and other informatory mediators are potential molecules that could be developed as useful markers for IC/BPS as they are noninvasive predictors. Cytokines and chemokines have crucial roles in the pathogenesis of several chronic inflammatory diseases. As bladder inflammation is one of the major causes of IC/BPS, it is likely that the biomarkers from serum and urine have a prognostic role and can serve as tools for the selection of suitable therapeutic agents for treatment. 5.1. Cytokines Increased levels of cytokines and chemokines have been found in the urine of patients with IC/BPS. Studies by Erickson et al34 and Sakthivel et al35 found that several proinflammatory mediators, such as interleukin-6 (IL-6) and CXC chemokines, were increased in urinary and serum samples of patients with IC/BPS. Lamale and coworkers36 proposed the use of a combination of methylhistamine and IL-6 as a sensitive and specific marker for IC/BPS. A study by Ogawa and colleagues37 also confirmed upregulation of several CXCR3 binding chemokines in patients with ulcerative IC/BPS. Controversial results have been reported for other cytokines, including IL-2, IL-8, interferon-g, and tumor necrosis factor-a, which were only found to be increased in patients in some earlier studies.34,38,39 Our recent study revealed that the serum IL-1b, IL-6, tumor necrosis factor-a, and IL-8 levels were significantly higher in the serum of patients with IC/BPS than in control patients (Table 1).40
Please cite this article in press as: Liu H-T, Kuo H-C, Biomarkers for patients with interstitial cystitis/bladder pain syndrome, Urological Science (2015), http://dx.doi.org/10.1016/j.urols.2015.02.002
H.-T. Liu, H.-C. Kuo / Urological Science xxx (2015) 1e5 Table 1 Expression of serum interleukin (IL)-1b, IL-6, tumor necrosis factor (TNF-a) and IL-8 in interstitial cystitis or bladder pain syndrome (IC/PBS) patients and controls.
Gender Age (y) IL-1b (pg/mL) IL-6 (pg/mL) TNF-a (pg/mL) IL-8 (pg/mL)
Controls (n ¼ 26)
IC/PBS (n ¼ 30)
p
F:16 M:10 32.36 ± 1.56 (22e55) 1.64 ± 0.47 (0.00e6.08) 0.79 ± 0.21 (0.00e3.67) 0.91 ± 0.17 (0.00e4.64) 1.45 ± 0.21 (0.00e4.09)
F: 26 M: 4 50.57 ± 2.68 (24e86) 6.45 ± 0.71 (2.77e23.96) 1.52 ± 0.24 (0.00e6.14) 2.63 ± 0.60 (0.62e13.70) 3.23 ± 0.48 (0.00e15.08)
<0.001 <0.001 <0.001 <0.001 <0.001
Data are presented as mean ± standard error (minemax). F ¼ female; M ¼ male.
5.2. Nerve system inflammation In patients with IC/PBS, neurotrophins, including NGF, neurotrophin-3, and glial-cell-derived neurotrophic factor, have been detected in the urine.41 NGF is a small secreted protein that induces differentiation and survival of target neurons. NGF has been shown to play a role in altered lower urinary tract function. Here, NGF acts as a chemical mediator in C-fiber afferents that may regulate the change in urinary bladder function.42,43 The levels of several neurotrophic factors, including NGF, increase in the bladder after spinal cord injury,42,44 and NGF has been found to increase in the lumbosacral spinal cord and dorsal root ganglia after spinal cord injury in a rat model.45 Patients with lower urinary tract diseases, including IC/BPS23e25,41,46 and bladder outlet obstruction,47 have been found to have increased NGF levels in the urine and/or bladder tissue. In the bladder, NGF is expressed in the urothelial cells and smooth muscle.48 Normally, NGF exists in the afferent nerves and ganglia of the bladder wall and is responsible for the normal sensation of bladder distention and conduction of abnormal sensory input.48 NGF is also released by mast cells.49 In a transgenic mouse model, chronic NGF overexpression in the bladder led to neuronal hyperinnervation, pelvic sensitivity, and changes in urinary bladder function.50 Serum NGF levels have been found to increase in several systemic diseases, including allergic diseases, autoimmune diseases, and psychosocial stress,51e53 and therefore analysis of both urinary and serum NGF in patients with IC/BPS provides insight into the development of the disease. In samples of patients with IC/BPS, increased levels of NGF have been noted in the urine23,24,41,46 and bladder tissue.25 Our recent study showed that the serum NGF level of IC/BPS patients was elevated, while the increased serum NGF level was not correlated with the urinary NGF level and was not related to the severity of IC/BPS (Fig. 1).54 These findings suggest that NGF is associated with bladder function, and elevated urinary NGF levels reflect that chronic inflammation is occurring in the urinary bladder of IC/BPS patients.
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NGF has been suggested to act as a local stress mediator in perceived stress and allergy, and increased NGF leads to deteriorating allergic inflammation in stress-induced worsening of allergic inflammation.55 Stress has been shown to be involved in the pathogenesis of IC/BPS,9 and has also been found to induce NGF expression in the urinary bladder in an animal model.56 The urinary NGF level has been shown to be closely related to the visual analog scale score for pain and response to conventional treatment for IC/ BPS. Several studies have shown that injections of botulinum toxin A reduce bladder pain in patients with refractory IC/BPS,57,58 and the increased NGF levels in the bladder tissue of patients with IC/ BPS can decrease to within the normal range after botulinum toxin A treatment.59 However, the role of NGF may be due to its association with bladder function, as NGF also increases in patients with other bladder diseases, such as OAB and urinary tract infection. Although NGF might be able to be developed as an indicator for treatment, in order to be a sensitive molecular diagnostic tool for IC/BPS, it is likely that a combination of other urinary or tissue biomarkers is required. 6. Recent development 6.1. Genetic biomarkers Several pilot studies have attempted to use microarray technology to reveal genes involved in the pathobiology underlying IC/ BPS.60,61 The results of these studies are promising, and have demonstrated through biopsy of bladder tissue that a number of genes with differential expressions exist between IC/BPS patients and healthy controls. However, further studies are needed in order to validate the target genes to enable identification of genes that can be used as new biomarkers of IC/BPS. This also indicates that the fundamental disease processes of IC/BPS are associated with a heterogeneous and complicated transcriptional network. In addition to microarray, high-throughput sequencing technologies provide new methods for the analysis of gene expression. With the recent increases in specificity and sensitivity and a decrease in cost, RNA sequencing technology has been shown to improve the accuracy of analysis of differential gene expressions, especially for low-abundance transcripts.62 Therefore, RNA sequencing technology may be a better tool for enhanced detection of differential expression in future studies of IC/BPS. These gene expression-based predictive models can narrow down the number of potential biomarkers to be studied and their roles of importance in the disease. 6.2. Reclassification of IC/BPS Recently, researchers have suggested that IC/BPS should be reclassified. ESSIC is classified into several subtypes.63 Elucidating
Fig. 1. Nerve growth factor (NGF)-immunoreactivity in the bladder of patients with interstitial cystitis or bladder pain syndrome (IC/BPS) shows significantly increased density in submucosal and urothelial cells at baseline (A) 200 and (B) 400. NGF staining in the control was localized to apical urothelial cells (C) 200.
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subtype-specific biomarkers may be more clinically relevant. For instance, Logadottir et al64 studied biopsies from patients with ESSIC Type 3C, and found that the expressions of IL-6, IL-10, and IL17A, and inducible nitric oxide synthase mRNA, were significantly higher in the patients than in healthy controls. Another study took clinical presentation into consideration, and found that several genes are correlated with clinical factors.60 Nickel et al65 also proposed that a phenotype-directed therapeutic approach can significantly improve the treatment outcome. Homma and colleagues66 showed that there are different gene expression patterns for classic and nonclassic IC. Tripp and coworkers67 developed a system to classify IC/BPS into several pain-location phenotypes, and suggested that the system can enable patients to receive better treatment as it can provide improved pain management. Therefore, a larger patient sample that can be subclassified into different types will help to identify reliable biomarkers for IC/BPS. In combination with consideration of disease progress and clinical factors, a noninvasive method can be developed to analyze the efficiency of treatment. 7. Conclusion Abnormal expressions of several bladder epithelial markers, including mast cells, epithelial differentiation proteins, cell membrane proteins, neurotransmitters, and cytokines, are present in IC/ BPS. Clinical and experimental data have indicated a direct link between increased levels of NGF in the bladder tissue and urine and painful inflammatory conditions in IC/BPS. However, NGF is not the only protein that contributes to IC/BPS; therefore, a combination test with measurement of other inflammatory proteins in the urine, or other marker(s) in tissue specimens, is required. Because the urinary NGF level can also be increased in many lower urinary tract diseases, such as stones, tumors, acute bacterial infection, as well as in IC/PBS, it is important to exclude these diseases. Current findings suggest that the urinary NGF level can be monitored as a biomarker for IC/PBS severity and for the response of patients to treatment. Several recently identified cytokines and mediators identified to be associated with the disease can be useful tools to assess treatment outcome. Further studies and subclassification of IC/PBS are required to identify new biomarkers for diagnosis, as well as for treatment efficiency in patients with IC/PBS. Conflicts of interest All contributing authors declare no conflicts of interest. References 1. Bouchelouche K, Nordling J. Recent developments in the management of interstitial cystitis. Curr Opin Urol 2003;13:309e13. 2. van de Merwe JP, Nordling J, Bouchelouche P, Bouchelouche K, Cervigni M, Daha LK, et al. Diagnostic criteria, classification, and nomenclature for painful bladder syndrome/interstitial cystitis: an ESSIC proposal. Eur Urol 2008;53: 60e7. 3. Haarala M, Jalava J, Laato M, Kiilholma P, Nurmi M, Alanen A. Absence of bacterial DNA in the bladder of patients with interstitial cystitis. J Urol 1996;156:1843e5. 4. Keay S, Zhang CO, Baldwin BR, Jacobs SC, Warren JW. Polymerase chain reaction amplification of bacterial 16S rRNA genes in interstitial cystitis and control patient bladder biopsies. J Urol 1998;159:280e3. 5. Theoharides TC, Kempuraj D, Sant GR. Mast cell involvement in interstitial cystitis: a review of human and experimental evidence. Urology 2001;57: 47e55. 6. Theoharides TC, Sant GR, el-Mansoury M, Letourneau R, Ucci Jr AA, Meares Jr EM. Activation of bladder mast cells in interstitial cystitis: a light and electron microscopic study. J Urol 1995;153:629e36. 7. Lundeberg T, Liedberg H, Nordling L, Theodorsson E, Owzarski A, Ekman P. Interstitial cystitis: correlation with nerve fibres, mast cells and histamine. Br J Urol 1993;71:427e9.
8. Cao J, Papadopoulou N, Kempuraj D, Boucher WS, Sugimoto K, Cetrulo CL, et al. Human mast cells express corticotropin-releasing hormone (CRH) receptors and CRH leads to selective secretion of vascular endothelial growth factor. J Immunol 2005;174:7665e75. 9. Rothrock NE, Lutgendorf SK, Kreder KJ, Ratliff T, Zimmerman B. Stress and symptoms in patients with interstitial cystitis: a life stress model. Urology 2001;57:422e7. 10. Feltis JT, Perez-Marrero R, Emerson LE. Increased mast cells of the bladder in suspected cases of interstitial cystitis: a possible disease marker. J Urol 1987;138:42e3. 11. Larsen S, Thompson SA, Hald T, Barnard RJ, Gilpin CJ, Dixon JS, et al. Mast cells in interstitial cystitis. Br J Urol 1982;54:283e6. €ck L, Fall M, Aldenborg F. Histamine and mucosal mast cells in inter12. Enerba stitial cystitis. Agents Actions 1989;27:113e6. 13. Kastrup J, Hald T, Larsen S, Nielsen VG. 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Please cite this article in press as: Liu H-T, Kuo H-C, Biomarkers for patients with interstitial cystitis/bladder pain syndrome, Urological Science (2015), http://dx.doi.org/10.1016/j.urols.2015.02.002