Enhanced Detection of Genitourinary Cancers Using Fragmentation and Copy Number Profiles Obtained from Urinary Cell-Free DNA.

BACKGROUND: Recent studies have reported that examining the fragmentation profiles (FP) of plasma cell-free DNA (cfDNA) further improves the clinical sensitivity of tumor detection. We hypothesized that considering the differences of the FP of urinary cfDNA would increase the clinical sensitivity of genitourinary (GU) cancer detection. METHODS: 177 patients with GU cancer and 94 individuals without tumors were enrolled in the discovery cohort. An independent validation dataset comprising 30 patients without tumors and 66 patients with GU cancer was also collected. We constructed an ensemble classifier, GUIDER, to detect and localize GU cancers using fragmentation and copy number profiles obtained from shallow whole-genome sequencing of urinary cfDNA. RESULTS: Urinary cfDNA of patients with GU cancer had a higher proportion of long fragments (209-280 bp) and a lower proportion of short fragments (140-208 bp) compared to controls. The overall mean classification accuracy of the FP was 74.62%-85.39% for different algorithms, and integration of the FP and copy number alteration (CNA) features further enhanced the classification of samples from patients with GU cancer. The mean diagnostic accuracy was further improved by the ensemble classifier GUIDER, which integrated the FP and CNA profiles and resulted in a higher mean accuracy (87.52%) compared to the analysis performed without FP features (74.62%). GUIDER performed well in an independent validation dataset. CONCLUSIONS: The lengthening and shortening of urinary cfDNA within specific size ranges were identified in patients with GU cancer. Integration of the FP should further enhance the ability to use urinary cfDNA as a molecular diagnostic tool.

Metabolomic Heterogeneity of Urogenital Tract Cancers Analyzed by Complementary Chromatographic Techniques Coupled with Mass Spectrometry.

BACKGROUND: In regard to urogenital tract cancer studies, an estimated 340,650 new cases and 58,360 deaths from genital system cancer and about 141,140 new cases and 29330 deaths from urinary system were projected to occur in the United States in 2012. The main drawbacks of currently available diagnostic tests constitute the low specificity, costliness and quite high invasiveness. OBJECTIVE: The main goal of this pilot study was to determine and compare urine metabolic fingerprints in urogenital tract cancer patients and healthy controls. METHOD: A comparative analysis of the metabolic profile of urine from 30 patients with cancer of the genitourinary system (bladder (n=10), kidney (n=10) and prostate (n=10)) and 30 healthy volunteers as a control group was provided by LC-TOF/MS and GCQqQ/ MS. The data analysis was performed by the use of U-Mann Whitney test or Student's t-test, principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA). RESULTS: As a result, 33, 43, and 22 compounds were identified as statistically significant in bladder, prostate and kidney cancer, respectively, compared to healthy groups. CONCLUSION: Diverse compounds such as purine, sugars, amino acids, nucleosides, organic acids which play a role in purine metabolism, in tricarboxylic acid cycle, in amino acid metabolism or in gut microbiota metabolism were identified. Only two metabolites namely glucocaffeic acid and lactic acid were found to be in common in studied three types of cancer.

Diagnostic performance of the urinary canine calgranulins in dogs with lower urinary or urogenital tract carcinoma.

BACKGROUND: Onset of canine transitional cell carcinoma (TCC) and prostatic carcinoma (PCA) is usually insidious with dogs presenting at an advanced stage of the disease. A biomarker that can facilitate early detection of TCC/PCA and improve patient survival would be useful. S100A8/A9 (calgranulin A/B or calprotectin) and S100A12 (calgranulin C) are expressed by cells of the innate immune system and are associated with several inflammatory disorders. S100A8/A9 is also expressed by epithelial cells after malignant transformation and is involved in the regulation of cell proliferation and metastasis. S100A8/A9 is up-regulated in human PCA and TCC, whereas the results for S100A12 have been ambiguous. Also, the urine S100A8/A9-to-S100A12 ratio (uCalR) may have potential as a marker for canine TCC/PCA. Aim of the study was to evaluate the diagnostic accuracy of the urinary S100/calgranulins to detect TCC/PCA in dogs by using data and urine samples from 164 dogs with TCC/PCA, non-neoplastic urinary tract disease, other neoplasms, or urinary tract infections, and 75 healthy controls (nested case-control study). Urine S100A8/A9 and S100A12 (measured by species-specific radioimmunoassays and normalized against urine specific gravity [S100A8/A9USG; S100A12USG], urine creatinine concentration, and urine protein concentration and the uCalR were compared among the groups of dogs. RESULTS: S100A8/A9USG had the highest sensitivity (96%) and specificity (66%) to detect TCC/PCA, with specificity reaching 75% after excluding dogs with a urinary tract infection. The uCalR best distinguished dogs with TCC/PCA from dogs with a urinary tract infection (sensitivity: 91%, specificity: 60%). Using a S100A8/A9USG ≥ 109.9 to screen dogs ≥6 years of age for TCC/PCA yielded a negative predictive value of 100%. CONCLUSIONS: S100A8/A9USG and uCalR may have utility for diagnosing TCC/PCA in dogs, and S100A8/A9USG may be a good screening test for canine TCC/PCA.

Cost-effectiveness of Common Diagnostic Approaches for Evaluation of Asymptomatic Microscopic Hematuria.

Importance: Asymptomatic microscopic hematuria (AMH) is highly prevalent and may signal occult genitourinary (GU) malignant abnormality. Common diagnostic approaches differ in their costs and effectiveness in detecting cancer. Given the low prevalence of GU malignant abnormality among patients with AMH, it is important to quantify the cost implications of detecting cancer for each approach. Objective: To estimate the effectiveness, costs, and incremental cost per cancer detected (ICCD) for 4 common diagnostic approaches evaluating AMH. Design, Setting, and Participants: A decision-analytic model-based cost-effectiveness analysis using inputs from the medical literature. PubMed searches were performed to identify relevant literature for all key model inputs, each of which was derived from the clinical study with the most robust data and greatest applicability. Analysis included adult patients with AMH on routine urinalysis with subgroups of high-risk patients (males, smokers, age ≥50 years) seen in the primary care or urologic referral setting. Interventions: Four diagnostic approaches were evaluated relative to the reference case of no evaluation: (1) computed tomography (CT) alone; (2) cystoscopy alone; (3) CT and cystoscopy combined; and (4) renal ultrasound and cystoscopy combined. Main Outcomes and Measures: At termination of the diagnostic period, cancers detected, costs (payer perspective), and ICCD per 10 000 patients evaluated for AMH. Results: Of the 4 diagnostic approaches analyzed, CT alone was dominated by all other strategies, detecting 221 cancers at a cost of $9 300 000. Ultrasound and cystoscopy detected 245 cancers and was most cost-effective with an ICCD of $53 810. Replacing ultrasound with CT detected just 1 additional cancer at an ICCD of $6 480 484. Ultrasound and cystoscopy remained the most cost-effective approach in subgroup analysis. The model was not sensitive to any inputs within the proposed ranges. Using probabilistic sensitivity analysis, ultrasound and cystoscopy was the dominant strategy in 100% of simulations. Conclusions and Relevance: The combination of renal ultrasound and cystoscopy is the most cost-effective among 4 diagnostic approaches for the initial evaluation of AMH. The use of ultrasound in lieu of CT as the first-line diagnostic strategy will optimize cancer detection and reduce costs associated with evaluation of AMH. Given our findings, we need to critically evaluate the appropriateness of our current clinical practices, and potentially alter our guidelines to reflect the most effective screening strategies for patients with AMH.

Increased risk of malignancy for non-atypical urothelial cell groups compared to negative cytology in voided urine. Morphological changes with LBC.

Liquid-based cytology (LBC) has recently become the preferred method for urine cytology analysis, but differences with conventional cytology (CC) have been observed. The purpose of this study is to analyze these differences and the clinical relevance of non-atypical urothelial cell groups (UCG) in voided urine specimens. Reporting terminology is discussed. Initially, diagnostic categories from 619 LBC and 474 CC samples, reviewed by five different pathologists, were compared (phase 1). Five years after LBC was implemented and applying strict cytologic criteria for UCG diagnosis, 760 samples were analyzed (phase 2) and compared to previous LBC specimens. Diagnostic differences, interobserver variability and clinicopathological correlation with a 6-month follow-up, were analyzed. UCG increased from 6.5% with CC to 20.7% (218%, 3.2 fold, P < 0.0001) with LBC. This difference was not related to interobserver variability. Five years later, the rate of UCG had decreased to 13 2%. While 6% of cases with a negative cytology had urothelial carcinoma (UC) within 6 months of diagnosis, this percentage increased to 15.7% with UCG. The sensitivity of the UCG category for UC was low (30.4%), but the specificity and the negative predictive value (NPV) were high (87.1% and 94%, respectively). LBC increases UCG when compared to CC. This can be corrected with observers experience and using set cytological criteria. Due to its association with carcinoma, the presence of UCG in voided urine should be framed in a diagnostic category other than "negative for malignancy." Diagn. Cytopathol. 2016;44:582-590. © 2016 Wiley Periodicals, Inc.

Statistical-based approach in potential diagnostic application of urinary nucleosides in urogenital tract cancer.

AIM: We aimed at evaluation the potential diagnostic role of urinary nucleosides in urogenital tract cancer. MATERIALS & METHODS: Concentrations of 12 nucleosides determined by LC-MS/MS were subjected to correlation, association and interaction analyses. RESULTS: We identified six pairs of nucleosides differently correlated in the group of patients and controls (p < 0.05). N-2-methylguanosine (odds ratio: 4.82; 95% CI: 1.78-12.93; p = 0.002) and N,N-dimethylguanosine (odds ratio: 5.45; 95% CI: 1.78-16.44; p = 0.003), were significantly associated with the disease risk (p-corrected = 0.004). Interaction between N-2-methylguanosine and adenosine (p-interaction = 0.019) suggested their multiplicative effect on the outcome. CONCLUSION: Urinary nucleosides, namely N,N-dimethylguanosine and N-2-methylguanosine may have the potential to serve as prognostic biomarkers. Gender-specific differences in urogenital tract cancer are likely to occur.

Advanced assessment of the endogenous hormone level as a potential biomarker of the urogenital tract cancer.

The evaluation of the relationships between the hormones involved in the urogenital tract cancer, including bladder, kidney, prostate, and testis, could prove important from diagnostic point of view. The determination of the steroid hormone profiles may likely provide a biomarker for discrimination of hormone-related diseases, as well as for differentiation of healthy volunteers from patients with cancer. The aim of the study was to demonstrate the changes in the steroid hormone profile (comprising corticosteroids, androgens and progesterone) in the urine of patients with the urogenital tract cancer versus urine from healthy subjects. A reliable analytical method based on liquid chromatography coupled with mass spectrometry was successfully applied to determine the urinary profiles of 6 endogenous steroids: cortisol, cortisone, corticosterone, testosterone, epitestosterone and progesterone for 92 urogenital tract cancer patients and 100 healthy controls. The obtained data was further evaluated by in-depth chemometric analysis, including the applied standardized Kennard-Stone's algorithm to pre-process the data. Mann-Whitney U test revealed statistically significant (p <0.05) differences in concentration of androgens and progesterone in the case of bladder cancer for male and female population, for male also cortisol and cortisone levels were significantly increased. PCA analysis proved a reasonable trend for differentiating healthy and cancer patients, and finally, applying PLS-DA model we were able to correctly classify 80.56%of cancer patients. Our results indicate that steroid hormone profile determination could be a promising approach for early diagnosis of urogenital tract cancer. However our preliminary results require an extension both in patient number and steroid profile.

Steroid profiles as potential biomarkers in patients with urogenital tract cancer for diagnostic investigations analyzed by liquid chromatography coupled to mass spectrometry.

Large discrepancy remains for the hormone-responsible cancers with regards to the conditions generating the optimal opportunity for cancerogenesis. In the research, altered steroid profiles were observed in patients with urogenital tract cancer diseases, namely bladder, kidney, prostate and testis ones. The presented steroid profiles from 154 subjects, including 77 urogenital tract cancer patient and 77 healthy controls were determined by liquid chromatography coupled to mass spectrometry method. Because the original experimental data obtained as a result of analytical experiment in order to interpret them in better way required the appropriate pre-treatment, the data were standardized by scaling and centering. In order to determine which samples form a collection for a high-capacity predictive model, Kennard-Stone's algorithm was used. A principal component analysis of preprocessed data provided better consistency of the steroid profiles with health status of subjects than PCA profiles without data preprocessing and showed a tendency to separate clusters of cancer patients from healthy subjects. The discriminant analysis was also performed and the percent of correct classification of cancer patients and control group was calculated. Finally, detailed studies examined the role of steroid profiles measured in urine, and considered as potential biomarkers related to urogenital cancer and associated renal dysfunctions.

Comparison of RP-HPLC columns used for determination of nucleoside metabolic patterns in urine of cancer patients.

BACKGROUND: Metabolic profiling allows the measurement of a large set of both known and unknown metabolites (such as nucleosides and nucleobases) present in a biological sample (e.g., urine). RESULTS: Separation of the isolated urinary nucleosides was performed on two connected Gemini C18 columns - 3 µm pore size (50 cm total length) - at 55°C using mobile-phase gradient elution. The Mann-Whitney U test was used to distinguish differences in the concentration of compounds in urine from urogenital cancer patients and healthy controls. Comparison of mean concentration values from the healthy and cancer groups revealed statistically significant differences (p < 0.01) for most of the metabolites studied (excluding m7G, m3C and A). Observed elevated levels of nucleosides mean concentrated values in urine in the case of cancer patients are between 1.5 and 2.0. CONCLUSION: These results verify the usefulness of the RP-HPLC method to investigate the urinary pattern of normal and modified nucleosides.

Development and validation of urinary nucleosides and creatinine assay by capillary electrophoresis with solid phase extraction.

For the analysis of metabolite nucleoside profiles, capillary electrophoretic (CE) methods preceded by appropriate solid phase extraction procedures have been developed. The approach has been proposed for the determination of 13 nucleosides and creatinine in human urine. A background solution composed of 100 mM borate-72 mM phosphate-160 mM SDS and a fused silica capillary of 70 cm length to detector and 50 microm i.d. were used. The methods developed were statistically validated for their linearity, trueness, precision and selectivity. Stability of the analyzed nucleoside profiles in urine during storage was checked. Validation parameters of solid phase extraction procedures for urinary nucleosides were evaluated. The developed analytical methods were employed for the analysis of 22 urine samples from healthy patients and cancer patients from the urological ward. Nucleoside profiles were compared among the subjects. It was proved that the methods proposed were suitable for a fast and reliable determination of urinary creatinine and modified nucleoside profiles, which can be further submitted for the metabonomic analysis of cancer patients.

Evaluation of nuclear matrix protein-22 as a clinical diagnostic marker for bladder cancer.

OBJECTIVES: To evaluate prospectively the prognostic power of urinary nuclear matrix protein-22 (NMP-22) for bladder cancer in Taiwanese screening and surveillance settings. METHODS: Single voided urine samples were obtained from 68 healthy individuals, 303 patients with benign urothelial diseases, and 28 patients with urogenital tumors. The NMP-22 levels in the urine samples were measured using enzyme-linked immunosorbent assay methods. RESULTS: The median NMP-22 level in healthy individuals and patients with benign and malignant disease was 5.9, 4.8, and 7.4 U/mL, respectively. The positive NMP-22 rate in healthy individuals and patients with benign and malignant disease was 4.4%, 17.2%, and 50%, respectively. The sensitivity, specificity, positive predictive value, and negative predictive value was 50%, 82.8%, 21.2%, and 94.7%, respectively, using 7.5 U/mL as the cutoff value. CONCLUSIONS: Our data demonstrated that NMP-22 is not a good diagnostic tool for screening or follow-up surveillance of bladder cancer owing to its low sensitivity and positive predictive value. Nevertheless, it could be adopted as a tool to rule out the possibility or risk of developing bladder cancer because of its high negative predictive value in our study.

Urine detection of survivin and diagnosis of bladder cancer.

CONTEXT: Dysregulation of apoptosis may favor onset and progression of cancer and influence response to therapy. Survivin is an inhibitor of apoptosis that is selectively overexpressed in common human cancers, but not in normal tissues, and that correlates with aggressive disease and unfavorable outcomes. OBJECTIVE: To investigate the potential suitability of survivin detection in urine as a novel predictive/prognostic molecular marker of bladder cancer. DESIGN, SETTING, AND PATIENTS: Survey of urine specimens from 5 groups: healthy volunteers (n = 17) and patients with nonneoplastic urinary tract disease (n = 30), genitourinary cancer (n = 30), new-onset or recurrent bladder cancer (n = 46), or treated bladder cancer (n = 35), recruited from 2 New England urology clinics. MAIN OUTCOME MEASURES: Detectable survivin levels, analyzed by a novel detection system and confirmed by Western blot and reverse transcriptase polymerase chain reaction (RT-PCR), in urine samples of the 5 participant groups. RESULTS: Survivin was detected in the urine samples of all 46 patients with new or recurrent bladder cancer using a novel detection system (31 of 31) and RT-PCR (15 of 15) methods. Survivin was not detected in the urine samples of 32 of 35 patients treated for bladder cancer and having negative cystoscopy results. None of the healthy volunteers or patients with prostate, kidney, vaginal, or cervical cancer had detectable survivin in urine samples. Of the 30 patients with nonneoplastic urinary tract disease, survivin was detected in 3 patients who had bladder abnormalities noted using cystoscopy and in 1 patient with an increased prostate-specific antigen level. Patients with low-grade bladder cancer had significantly lower urine survivin levels than patients with carcinoma in situ (P =.002). CONCLUSIONS: Highly sensitive and specific determination of urine survivin appears to provide a simple, noninvasive diagnostic test to identify patients with new or recurrent bladder cancer.

The development of a multitarget, multicolor fluorescence in situ hybridization assay for the detection of urothelial carcinoma in urine.

The purpose of this study was to develop a multitarget, multicolor fluorescence in situ hybridization (FISH) assay for the detection of urothelial carcinoma (UC) in urine specimens. Urinary cells obtained from voided urine specimens of 21 patients with UC and 9 normal donors were analyzed with nine different centromere enumeration probes and a single locus-specific indicator probe to determine an optimal set of FISH probes for UC detection. The four probes with the greatest sensitivity for UC detection were then labeled with a unique fluorophore and combined into a single probe set. The probes with the greatest combined sensitivity for UC detection were CEP3, CEP7, CEP17, and the 9p21 (P16) LSI. This probe set was used to evaluate urine specimens acquired from 179 patients for prospective testing (46 with biopsy-proven UC). FISH slides were evaluated by scanning the slide for cells with nuclear features suggestive of malignancy and assessing the FISH signal pattern of these cells for polysomy (ie, gains of two or more different chromosomes). A receiver operator characteristic curve revealed that a cutoff of 5 cells with polysomy as the positive criterion for cancer resulted in an overall sensitivity of 84.2% for patients with biopsy-proven UC and a specificity of 91.8% among patients with genitourinary disorders but no evidence of UC. This study demonstrates that a multitarget, multicolor FISH assay containing centromeric probes to chromosomes 3, 7, and 17 and a locus-specific probe to band 9p21 has high sensitivity and specificity for the detection of UC in voided urine specimens.

DNA image analysis of urinary cytology: prediction of recurrent transitional cell carcinoma.

To evaluate the utility of image analysis in monitoring patients with transitional cell carcinoma, we studied, by cytologic means and by image analysis, 78 urinary tract specimens from 66 patients, of whom 49 (74%) had a previous history of transitional cell carcinoma. The specimens consisted of 51 (65%) voided urine specimens, 12 (15%) bladder washings, 8 (10%) ureteral washings, 3 (4%) ureteral brushings, 2 (3%) renal pelvic washings, and 2 (3%) catheterized urine specimens. DNA histograms were classified into five patterns on the basis of their DNA index and the percentage of their cells with DNA content greater than 5c: diploid (single peak in the 2c region with no cells greater than 5c), intermediate (diploid with less than 10% of cells greater than 5c), aneuploid (single peak or multiple peaks between the 2c and 4c region or more than 10% of cells greater than 5c), tetraploid (at least 10% of cells in the 4c region and a corresponding peak at 8c), and polyploid (multiple peaks in the 2c, 4c, 8c, and 10c regions). Of the 78 cases, 22 were diploid, 24 were intermediate, 29 were aneuploid, one was tetraploid, and two were polyploid. Histologic confirmation or clinical follow-up was found in 29 aneuploid cases, 13 intermediate cases, and one diploid case. Most cases of carcinoma in situ (five of six) and invasive tumors (12 of 17) were aneuploid. The sensitivity was 100%, and the specificity was 73% when cytologic and image analysis results were combined. We conclude that image analysis, when combined with cytologic examination, is a reliable noninvasive diagnostic test for monitoring patients with transitional cell carcinoma; aneuploidy is specific for malignancy; and the presence of cells greater than 5c, although frequently associated with tumor recurrence, can be seen in non-neoplastic conditions.

Urinary immunoglobulins in patients with continent urinary reservoirs and ileal conduits.

BACKGROUND: Although bowel segments are commonly used for reconstructing the urinary tract, a high incidence of bacteriuria is observed in patients with urinary intestinal diversion. The normal gastrointestinal tract possesses a potent mucosal immune system characterized by secretory immunoglobulin A (sIgA) as the major humoral defense factor. However, the significance of urinary IgA secretion as the mucosal defense mechanism in patients with urinary intestinal diversion has remained obscure. In this study, urinary levels of sIgA as well as serum-type IgA were measured in patients with continent urinary reservoirs (Kock and Indiana pouches) and ileal conduits. METHODS: Twenty-four-hour urine samples were collected in a total of 80 patients with urinary intestinal diversion (22 Kock pouch patients, 21 Indiana pouch patients and 37 ileal conduit patients). The amount of sIgA and serum-type IgA were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Urinary IgA levels showed great inter- and intra patient variability in all three groups. Indiana reservoir urine contained significantly greater amounts of sIgA (mean 32.0 mg/24 hrs) than Kock reservoir urine (11.9 mg) and conduit urine (4.9 mg), whereas Kock reservoir urine contained significantly more sIgA than conduit urine. However, the corresponding difference was not observed in regard to serum-type IgA. In none of the three modes of urinary diversion did 24-hour sIgA excretion show any correlation with the length of time after surgery. CONCLUSIONS: Since the amounts of sIgA in these patients were much greater than reported in urinary tract infection as well as in normal subjects, the major portion of urinary sIgA seemed to be secreted by the intestinal segments. Long-term sIgA secretion in urinary intestinal diversion, especially continent urinary reservoirs, may be an important host defense system.

Screening for urothelial malignancies by cytologic analysis and flow cytometry in a community urologic practice: a prospective study.

A prospective study was initiated to compare the ability of flow cytometry and cytologic analysis to detect malignant cells in urine obtained at the time of cystoscopy. The population studied consisted of patients from general urologic practices who were undergoing cystoscopy in a single community hospital. Over a 1-yr period, 335 specimens from 317 patients were studied. Nineteen biopsy-proven urothelial malignancies were identified. Cytologic examination of urine obtained at the time of cystoscopy was positive in seven of these cases, and an aneuploid population of cells was identified by flow cytometry in three cases. All three cases of high-grade transitional cell carcinoma and carcinoma in situ were correctly identified by the combination of cytologic examination and flow cytometry; however, only four of 16 low-grade superficial papillary transitional cell carcinomas were recognized cytologically, with only one being aneuploid. The combination of cytologic analysis and flow cytometry did not increase the diagnostic sensitivity above that achieved with cytologic testing alone (overall sensitivity, 37%). We conclude that flow cytometry and cytologic analysis, either individually or in combination, are too insensitive for use in a routine screening program for urothelial malignancy in a community hospital setting because of the inability of either method to detect low-grade transitional cell carcinomas reliably.

Methods of early diagnosis in genitourinary cancer.

The commonly employed methods for the early detection of urologic cancers remain the traditional techniques of a carefully performed history and physical examination. Newer developments include, among others, prostate-specific antigen and monoclonal antibodies in prostate cancer, flow cytometry in bladder cancer, computerized axial tomography (CAT) scanning in renal cancer, and ultrasound in testicular cancer. These and other new diagnostic techniques, with further testing and wider use, will hopefully permit the earlier diagnosis of genitourinary cancer.