Bladder Cancer Biomarker Discovery Using Global Metabolomic Profiling of Urine

Bladder cancer (BCa) is a common malignancy worldwide and has a high probability of recurrence after initial diagnosis and treatment. As a result, recurrent surveillance, primarily involving repeated cystoscopies, is a critical component of post diagnosis patient management. Since cystoscopy is invasive, expensive and a possible deterrent to patient compliance with regular follow-up screening, new non-invasive technologies to aid in the detection of recurrent and/or primary bladder cancer are strongly needed. In this study, mass spectrometry based metabolomics was employed to identify biochemical signatures in human urine that differentiate bladder cancer from non-cancer controls. Over 1000 distinct compounds were measured including 587 named compounds of known chemical identity. Initial biomarker identification was conducted using a 332 subject sample set of retrospective urine samples (cohort 1), which included 66 BCa positive samples. A set of 25 candidate biomarkers was selected based on statistical significance, fold difference and metabolic pathway coverage. The 25 candidate biomarkers were tested against an independent urine sample set (cohort 2) using random forest analysis, with palmitoyl sphingomyelin, lactate, adenosine and succinate providing the strongest predictive power for differentiating cohort 2 cancer from non-cancer urines. Cohort 2 metabolite profiling revealed additional metabolites, including arachidonate, that were higher in cohort 2 cancer vs. non-cancer controls, but were below quantitation limits in the cohort 1 profiling. Metabolites related to lipid metabolism may be especially interesting biomarkers. The results suggest that urine metabolites may provide a much needed non-invasive adjunct diagnostic to cystoscopy for detection of bladder cancer and recurrent disease management.     

Proteomic analysis of urinary biomarker candidates for nonmuscle invasive bladder cancer

Nonmuscle invasive tumors of the bladder often recur and thereby bladder cancer patients need regular re-examinations which are invasive, unpleasant, and expensive. A noninvasive and less expensive method, e.g. a urine dipstick test, for monitoring recurrence would thus be advantageous. In this study, the complementary techniques mass spectrometry (MS) and Western blotting (WB)/dot blot (DB) were used to screen the urine samples from bladder cancer patients. High resolving MS was used to analyze and quantify the urinary proteome and 29 proteins had a significantly higher abundance (p<0.05) in bladder cancer samples compared with control urine samples. The increased abundance found in urine from bladder cancer patients compared with controls was confirmed with Western blot for four selected proteins; fibrinogen β chain precursor, apolipoprotein E, α-1-antitrypsin, and leucine-rich α-2-glycoprotein 1. Dot blot analysis of an independent urine sample set pointed out fibrinogen β chain and α-1-antitrypsin as most interesting biomarkers having sensitivity and specificity values in the range of 66-85%. Exploring the Human Protein Atlas (HPA) also revealed that bladder cancer tumors are the likely source of these proteins. They have the potential of being useful in diagnosis, monitoring of recurrence and thus may improve the treatment of bladder tumors, especially nonmuscle invasive tumors.     

Genetic and epigenetic aspects of bladder cancer

Transitional cell carcinoma of the urinary bladder has a diverse collection of biologic and functional characteristics. This is reflected in differing clinical courses. The diagnosis of bladder cancer is based on the information provided by cystoscopy, the gold standard in combination with urinary cytology findings. Many tumor markers have been evaluated for detecting and monitoring the disease in serum, bladder washes, and urinary specimens. However, none of these biomarkers reported to date has shown sufficient sensitivity and specificity for the detection of the whole spectrum of bladder cancer diseases in routine clinical practice. The limited value of established prognostic markers requires the analysis of new molecular parameters of interest in predicting the prognosis of bladder cancer patients; in particular, the high-risk patient groups at risk of progression and recurrence. Over the past decade, there has been major progress elucidating of the molecular genetic and epigenetic changes leading to the development of transitional cell carcinoma. This review focuses on the recent advances of genetic and epigenetic aspects in bladder cancer, and emphasizes how molecular biology would be likely to affect the future therapies.     

Optical sensory arrays for the detection of urinary bladder cancer‐related volatile organic compounds

Non‐invasive detection of urinary bladder cancer remains a significant challenge. Urinary volatile organic compounds (VOCs) are a promising alternative to cell‐based biomarkers. Herein, we demonstrate a novel diagnosis system based on an optic fluorescence sensor array for detecting urinary bladder cancer VOCs biomarkers. This study describes a fluorescence‐based VOCs sensor array detecting system in detail. The choice of VOCs for the initial part was based on an extensive systematic search of the literature and then followed up using urinary samples from patients with urinary bladder transitional cell carcinoma. Canonical discriminant analysis and partial least squares discriminant analysis (PLS‐DA) were employed and correctly detected 31/48 urinary bladder cancer VOC biomarkers and achieved an overall 77.75% sensitivity and 93.25% specificity by PLS‐DA modelling. All five urine samples from bladder cancer patients, and five healthy controls were successfully identified with the same sensor arrays. Overall, the experiments in this study describe a real‐time platform for non‐invasive bladder cancer diagnosis using fluorescence‐based gas‐sensor arrays. Pure VOCs and urine samples from the patients proved such a system to be promising; however, further research is required using a larger population sample.      

Prostaglandin E2 concentrations in naturally occurring canine cancer

The purpose of this study was to determine the PGE2 concentration in naturally-occurring cancer in pet dogs and in canine cancer cell lines in order to identify specific types of canine cancer with high PGE2 production which could serve as preclinical models to evaluate anticancer strategies targeting PGE2. PGE2 concentrations were measured by enzyme immunoassay in canine melanoma, soft tissue sarcoma, transitional cell carcinoma, osteosarcoma, and prostatic carcinoma cell lines; in 80 canine tumor tissue samples including oral melanoma (MEL), oral squamous cell carcinoma (SCC), transitional cell carcinoma of the urinary bladder (TCC), lymphoma (LSA), mammary carcinoma (MCA), osteosarcoma (OSA), prostatic carcinoma (PCA); and in corresponding normal organ tissues. High concentrations of PGE(2)(range 400-3300 pg/10(4)cells) were present in cell culture medium from the transitional cell carcinoma, prostatic carcinoma, and osteosarcoma cell lines. PGE2 concentrations in tumor tissues were elevated (tumor PGE2 concentration>mean+2X sd PGE(2)concentration of normal organ tissue) in 21/22 TCC, 5/6 PCA, 7/10 SCC, 5/10 MEL, 3/8 MCA, 4/15 OSA, and 0/9 LSA. Results of this study will help guide future investigations of anticancer therapies that target cyclooxygenase and PGE2.     

Urine cytology evaluation in cases of uretero-ileal cutaneous diversion

Cytological examination of urine from the ileal conduit in cases of bladder cancer treated by radical surgery can be an important and effective follow-up procedure. A total of 19 patients (18 males and one female) on whom radical cystectomy for cancer was performed were studied. Three urine specimens were examined in each case using routine cytological methods. Three cases of recurrent carcinoma (mainly of papillary type) were diagnosed cytologically before any clinical evidence of disease. the cytological examination of urine at 3-6 month intervals after cystectomy for bladder carcinoma is considered advisable in all cases, since the recurrence rate of transitional cell neoplasms in the upper urinary tract after cystectomy for transitional carcinoma is quite high.     

Urine metabolite profiling offers potential early diagnosis of oral cancer

Oral cancer is the sixth most common human cancer, with a high morbidity rate and an overall 5-year survival rate of less than 50%. It is often not diagnosed until it has reached an advanced stage. Therefore, an early diagnostic and stratification strategy is of great importance for oral cancer. In the current study, urine samples of patients with oral squamous cell carcinoma (OSCC, n = 37), oral leukoplakia (OLK, n = 32) and healthy subjects (n = 34) were analyzed by gas chromatography-mass spectrometry (GC–MS). Using multivariate statistical analysis, the urinary metabolite profiles of OSCC, OLK and healthy control samples can be clearly discriminated and a panel of differentially expressed metabolites was obtained. Metabolites, valine and 6-hydroxynicotic acid, in combination yielded an accuracy of 98.9%, sensitivity of 94.4%, specificity of 91.4%, and positive predictive value of 91.9% in distinguishing OSCC from the controls. The combination of three differential metabolites, 6-hydroxynicotic acid, cysteine, and tyrosine, was able to discriminate between OSCC and OLK with an accuracy of 92.7%, sensitivity of 85.0%, specificity of 89.7%, and positive predictive value of 91.9%. This study demonstrated that the metabolite markers derived from this urinary metabolite profiling approach may hold promise as a diagnostic tool for early stage OSCC and its differentiation from other oral conditions.     

细胞角蛋白20在膀胱癌中的研究进展

膀胱肿瘤是最常见的泌尿系统肿瘤,其中上皮性肿瘤占95％以上,绝大多数为尿路移行上皮细胞癌。膀胱癌的早期症状不明显,复发率较高,早期诊断和治疗对提高其疗效非常重要。近年来,诊断膀胱肿瘤的新方法不断出现,显著提高了膀胱肿瘤诊断及预后预测水平。其中,膀胱肿瘤标记物检测已成为膀胱肿瘤的诊断新方法,具有十分重要的临床意义。研究发现,细胞角蛋白20fcytokeratin20,CK20）是中间纤维家族成员之一,在正常膀胱组织中特异性表达于伞细胞,在膀胱癌中特异性表达于膀胱移行细胞癌,其诊断膀胱肿瘤的特异性和灵敏性均较高,且与膀胱肿瘤的临床分级、病理分期和转移均密切相关,因此可作为辅助诊断膀胱肿瘤的检测标志物及治疗和预后评估指标。本文将就其在膀胱癌中的研究进展综述如下。     

细胞角蛋白20 在膀胱癌中的研究进展

膀胱肿瘤是最常见的泌尿系统肿瘤,其中上皮性肿瘤占95%以上,绝大多数为尿路移行上皮细胞癌。膀胱癌的早期症状不 明显,复发率较高,早期诊断和治疗对提高其疗效非常重要。近年来,诊断膀胱肿瘤的新方法不断出现,显著提高了膀胱肿瘤诊断 及预后预测水平。其中,膀胱肿瘤标记物检测已成为膀胱肿瘤的诊断新方法,具有十分重要的临床意义。研究发现,细胞角蛋白20 (cytokeratin 20,CK20)是中间纤维家族成员之一,在正常膀胱组织中特异性表达于伞细胞,在膀胱癌中特异性表达于膀胱移行细 胞癌,其诊断膀胱肿瘤的特异性和灵敏性均较高,且与膀胱肿瘤的临床分级、病理分期和转移均密切相关,因此可作为辅助诊断 膀胱肿瘤的检测标志物及治疗和预后评估指标。本文将就其在膀胱癌中的研究进展综述如下。     

Bladder cancer determination via two urinary metabolites: a biomarker pattern approach

The purpose of this study was to use metabonomic profiling to identify a potential specific biomarker pattern in urine as a noninvasive bladder cancer (BC) detection strategy. A liquid chromatography-mass spectrometry based method, which utilized both reversed phase liquid chromatography and hydrophilic interaction chromatography separations, was performed, followed by multivariate data analysis to discriminate the global urine profiles of 27 BC patients and 32 healthy controls. Data from both columns were combined, and this combination proved to be effective and reliable for partial least squares-discriminant analysis. Following a critical selection criterion, several metabolites showing significant differences in expression levels were detected. Receiver operating characteristic analysis was used for the evaluation of potential biomarkers. Carnitine C9:1 and component I, were combined as a biomarker pattern, with a sensitivity and specificity up to 92.6% and 96.9%, respectively, for all patients and 90.5% and 96.9%, respectively for low-grade BC patients. Metabolic pathways of component I and carnitine C9:1 are discussed. These results indicate that metabonomics is a practicable tool for BC diagnosis given its high efficacy and economization. The combined biomarker pattern showed better performance than single metabolite in discriminating bladder cancer patients, especially low-grade BC patients, from healthy controls.     

N-methylpyridoxamine: novel canine vitamin B6 urine metabolite

Cation-exchange HPLC analysis of urine from dogs given large daily doses of pyridoxamine revealed an unidentified metabolite hypothesized to be N-methylpyridoxamine. Identity was established by N-methylpyridoxamine synthesis and HPLC comparison to the canine metabolite. Compound synthesis was confirmed by IR, NMR, UV-vis and emission spectroscopy. It seems to have less fluorescent character than other routinely-measured vitamin B(6) metabolites. Upon administration of substantial pyridoxamine doses, N-methylpyridoxamine appears to be a quantifiable canine urine metabolite, although, at either pharmacological or dietary pyridoxamine intakes, its relevance to vitamin B(6) metabolism in other species, including humans, is not yet determined.     

A multiplex biomarker approach for the diagnosis of transitional cell carcinoma from canine urine

Transitional cell carcinoma (TCC), the most common cancer of the urinary bladder in dogs, is usually diagnosed at an advanced disease stage with limited response to chemotherapy. Commercial screening tests lack specificity and current diagnostic procedures are invasive. A proof of concept pilot project for analyzing the canine urinary proteome as a noninvasive diagnostic tool for TCC identification was conducted. Urine was collected from 12 dogs in three cohorts (healthy, urinary tract infection, TCC) and analyzed using liquid chromatography tandem mass spectrometry. The presence of four proteins (macrophage capping protein, peroxiredoxin 5, heterogeneous nuclear ribonucleoproteins A2/B, and apolipoprotein A1) was confirmed via immunoblot. Of the total 379 proteins identified, 96 were unique to the TCC group. A statistical model, designed to evaluate the accuracy of this multiplex biomarker approach for diagnosis of TCC, predicted the presence of disease with 90% accuracy.     

Proteomic analysis of urinary fibrinogen degradation products in patients with urothelial carcinomas

Despite many years of research efforts and continued progress in the identification of urine markers for detection of bladder cancer, none of the markers described to date has been able to replace cystoscopy and urine cytology, the current gold standards for diagnosis. Here, we present a comprehensive gel-based proteomic study in which we have analyzed the presence and origin of fibrinogen (FG) and its degradation products (FDPs) in the urine of patients with and without urothelial carcinoma (UCs), with the aim of evaluating the potential of two-dimensional (2D) gel FDP profiling for detecting bladder cancer. A total of 151 urine samples collected from patients with Ucs of varying degrees of atypia and stages of invasion were compared with a control group consisting of 34 healthy volunteers with no clinical history of bladder cancer. The level and degree of degradation of FG in the urine were determined by 2D gel Western blotting in combination with enhanced chemilumenscence detection. Elevated levels of urine FG/FDPs were found in 99% of patients bearing superficial tumors, in 97% of the cases with early invasive disease, and in 96% of patients with highly invasive tumors. 2D gel profiling of urine FG/FDPs showed that the FG chains exhibited differential susceptibility to in situ proteolysis, with the α-chain being the most susceptible and the γ-chain the most resistant. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identified peptide sequence regions in several of the most representative and common FDPs, which can be of value for producing novel specific antibodies to detect FG/FDPs in the urine. In addition, we present evidence indicating that FG is not produced by normal or malignant urothelium, although it is present both in the stroma of malignant tissue and tumor lesions. Taken together, the data indicate that increased levels of FG/FDPs amounts in the urine are a characteristic feature of bladder cancer, and emphasize the value of 2D gel profiling of urine FG/FDPs for detecting low-grade, noninvasive UCs.     

Comparative Gene Expression Analyses Identify Luminal and Basal Subtypes of Canine Invasive Urothelial Carcinoma That Mimic Patterns in Human Invasive Bladder Cancer

More than 160,000 people are expected to die from invasive urothelial carcinoma (iUC) this year worldwide. Research in relevant animal models is essential to improving iUC management. Naturally-occurring canine iUC closely resembles human iUC in histopathology, metastatic behavior, and treatment response, and could provide a relevant model for human iUC. The molecular characterization of canine iUC, however, has been limited. Work was conducted to compare gene expression array results between tissue samples from iUC and normal bladder in dogs, with comparison to similar expression array data from human iUC and normal bladder in the literature. Considerable similarities between enrichment patterns of genes in canine and human iUC were observed. These included patterns mirroring basal and luminal subtypes initially observed in human breast cancer and more recently noted in human iUC. Canine iUC samples also exhibited enrichment for genes involved in P53 pathways, as has been reported in human iUC. This is particularly relevant as drugs targeting these genes/pathways in other cancers could be repurposed to treat iUC, with dogs providing a model to optimize therapy. As part of the validation of the results and proof of principal for evaluating individualized targeted therapy, the overexpression of EGFR in canine bladder iUC was confirmed. The similarities in gene expression patterns between dogs and humans add considerably to the value of naturally-occurring canine iUC as a relevant and much needed animal model for human iUC. Furthermore, the finding of expression patterns that cross different pathologically-defined cancers could allow studies of dogs with iUC to help optimize cancer management across multiple cancer types. The work is also expected to lead to a better understanding of the biological importance of the gene expression patterns, and the potential application of the cross-species comparisons approach to other cancer types as well.     

Urinary proteomic profiling for diagnostic bladder cancer biomarkers

The ability to detect and monitor bladder cancer in noninvasively obtained urine samples is a major goal. While a number of protein biomarkers have been identified and commercially developed, none have greatly improved the accuracy of sample evaluation over invasive cystoscopy. The ongoing development of high-throughput proteomic profiling technologies will facilitate the identification of molecular signatures that are associated with bladder disease. The appropriate use of these approaches has the potential to provide efficient biomarkers for the early detection and monitoring of recurrent bladder cancer. Identification of disease-associated proteins will also advance our knowledge of tumor biology, which, in turn, will enable development of targeted therapeutics aimed at reducing morbidity from bladder cancer. In this article, we focus on the accumulating proteomic signatures of urine in health and disease, and discuss expected future developments in this field of research.     

荧光原位杂交应用于尿路上皮癌尿液早期诊断的研究进展

尿路上皮癌（urothelial carcinoma,uc）是泌尿系统最常见恶性肿瘤之一,早期诊断是提高该类疾病疗效的关键所在,荧光原位杂交（fluorescencein situ hybridization,FISH）通过尿液来检测UC,具有快速、无创伤性、敏感度高和特异性强等优点。FISH提高了尿细胞学在低级别或浅表性膀胱UC诊断的敏感性,且减少了血尿、尿路感染及膀胱内灌注治疗等对细胞形态的影响而引起的假阳性,提高检测的特异性。对于诊断上尿路UC,FISH的敏感性与特异性更高。膀胱UC患者9号染色体p16抑癌基因丢失与复发明显相关。FISH既能预测膀胱UC的复发性,更能监测UC的复发,但仍需大样本、多中心的前瞻性研究。本文将FISH在膀胱UC、上尿路UC早期诊断以及膀胱UC术后监测等方面的临床应用研究报道进行综述。     

Micropapillary variant of transitional cell carcinoma of the urinary bladder: a report of three cases with cytologic diagnosis in urine specimens

BACKGROUND: Micropapillary transitional cell carcinoma is a recently described, aggressive variant of bladder cancer. Its cytologic features in urine have not been previously characterized. CASES: Three cases illustrate the urinary cytologic features of this high grade urothelial carcinoma and its concurrent biopsy findings. This tumor is similar to low. grade urothelial lesions of the bladder, tends to present as micropapillary clusters in urine and yet has high grade nuclear features within these clusters that help with the differential diagnosis of a flat, high grade urothelial carcinoma. CONCLUSION: The micropapillary type of transitional cell carcinoma is a distinct morphologic entity with an aggressive clinical course. Recognizing its presence in urinary cytology, albeit a rare occurrence, is important in distinguishing this lesion from the more indolent, low grade papillary lesions and high grade urothelial carcinomas, which continuously shed single malignant urothelial cells.     

Metabolomics as a diagnostic tool for hepatology: validation in a naturally occurring canine model

Human hepatopathies are a diagnostic challenge, with many distinct diseases having similar clinical signs and laboratory findings. Naturally occurring canine hepatic disease provides an excellent model for human diseases and similar diagnostic dilemmas exist; differentiating canine congenital portosystemic vascular anomalies (PVA) from acquired hepatopathies is difficult and traditionally requires invasive diagnostic procedures. The emerging post-genomic science of metabolomics is concerned with detecting global changes of populations of endogenous low molecular weight metabolites in biological samples and offers the possibility of identifying surrogate profiles of disease. Metabolomics couples sensitive metabolite analysis with sophisticated pattern recognition techniques. In this study, a metabolomic strategy has been employed to assess metabolite changes in the plasma of dogs with congenital PVA and acquired hepatic disease. Plasma samples were collected from 25 dogs, comprising 9 dogs with congenital PVA, 6 with acquired hepatopathy and 10 with non-hepatic disorders. Low molecular weight metabolites were analyzed by liquid chromatography-mass spectrometry (LC-MS). Following identification of metabolites, multivariate data analysis was used to compare profiles amongst groups. The analysis demonstrated significant disturbances in the plasma bile acid and phospholipid profiles of dogs with portovascular anomalies. In contrast to traditional laboratory parameters, the metabolomic strategy was able to produce a clear segregation between all three study groups. In conclusion, this study demonstrates the potential of metabolomics as a diagnostic tool for naturally occurring hepatic disease. With further validation, this approach will improve diagnostic capabilities, provide an insight into pathogenetic mechanisms, and ultimately inform therapeutic decision making in clinical hepatology. This work was supported in part by grants from The Royal Society, Petplan Charitable Trust and The Waltham Foundation.     

Diagnosis of bladder cancer with urinary cytology, immunocytology and DNA-image-cytometry.

DNA-image-cytometry and antibodies directed against the Lewis X- and the 486p 3/12 antigen were applied to improve diagnostic accuracy of urinary cytology for the detection of bladder cancer. Cytology, immunocytology and DNA-image-cytometry were performed in spontaneously voided urine samples and barbotage bladder washings from 71 patients. The DNA content was determined using the CM-1 Cytometer according to the recommendation of the ESCAP Consensus Report on Standardization of DNA-image-cytometry (1995). For immunocytological examination we used the monoclonal anti Lewis X antibody P-12 and antibody 486p 3/12. All patients underwent subsequent cystoscopy and for any suspicious lesion biopsy or transurethral resection was done. Histological findings revealed 31 patients with transitional cell carcinomas of different stages and grades of malignancy. 40 patients had various benign diseases of the urinary bladder. Cytology yielded a sensitivity of 68% and a specificity of 100%. DNA aneuploidy was detected in 81% of cancer patients with a specificity of 100%. By combination of these two methods the overall sensitivity increased to 87%. Immunocytology with Lewis X and 486p 3/12 antibodies showed reactivity in 84% and 87% in combination with a specificity of 80% and 70%, respectively. By combining urinary cytology, immunocytology and/or DNA-image-cytometry the overall sensitivity increased to 94% with no change in specificity. DNA-image-cytometry should be used to evaluate particularly urothelial cells suspicious for malignancy in urinary specimens. Because of low specificity the monoclonal antibodies against Lewis X- and 486p 3/12 antigens are not helpful in screening for bladder cancer. Nevertheless, their high sensitivity may justify their use in case DNA image cytometry is not available and in the follow up of patients with transitional cell carcinoma.