Detection of extracellular vesicles in plasma and urine of prostate cancer patients by flow cytometry and surface plasmon resonance imaging.

Large (> 1 µm) tumor-derived extracellular vesicles (tdEVs) enriched from the cell fraction of centrifuged whole blood are prognostic in metastatic castration-resistant prostate cancer (mCRPC) patients. However, the highest concentration of tdEVs is expected in the cell-free plasma fraction. In this pilot study, we determine whether mCRPC patients can be discriminated from healthy controls based on detection of tdEVs (< 1µm, EpCAM+) and/or other EVs, in cell-free plasma and/or urine. The presence of marker+ EVs in plasma and urine samples from mCRPC patients (n = 5) and healthy controls (n = 5) was determined by flow cytometry (FCM) and surface plasmon resonance imaging (SPRi) using an antibody panel and lactadherin. For FCM, the concentrations of marker positive (+) particles and EVs (refractive index <1.42) were determined. Only the lactadherin+ particle and EV concentration in plasma measured by FCM differed significantly between patients and controls (p = 0.017). All other markers did not result in signals exceeding the background on both FCM and SPRi, or did not differ significantly between patients and controls. In conclusion, no difference was found between patients and controls based on the detection of tdEVs. For FCM, the measured sample volumes are too small to detect tdEVs. For SPRi, the concentration of tdEVs is probably too low to be detected. Thus, to detect tdEVs in cell-free plasma and/or urine, EV enrichment and/or concentration is required. Furthermore, we recommend testing other markers and/or a combination of markers to discriminate mCRPC patients from healthy controls.

Urinary hyaluronic acid: a versatile marker of bladder cancer.

PURPOSE: To evaluate the role of urinary hyaluronic acid (HA) as a diagnostic marker in urothelial carcinoma (UCC), squamous cell carcinoma (SCC), and adenocarcinoma (ADC) of urinary bladder and compare it with urine cytology. METHODS: HA was estimated in 170 subjects divided into three groups. Group I: UCC 88 patients, 28 with SCC and 12 with ADC; group II: 34 patients with benign bladder tumors; and group III: 10 healthy bladders. HA was estimated in urine and then readjusted to creatinine (HA/Cr) and protein (HA/Pr) in urine. Urine cytology was evaluated. RESULTS: The mean ± SD level HA was higher in UCC (589 ± 72), SCC (637 ± 45), and ADC (526 ± 30) as compared with benign (476 ± 92) and normal (277 ± 44) groups regardless the grade of tumor (p < 0.0001). A cutoff value of 490 ng/ml was calculated to detect malignancy with sensitivity of 98% and specificity of 66%. PPV, NPV, and ACC were 88.6%, 94.1%, and 90%, respectively. Urine cytology showed sensitivity of, specificity, PPV, NPV, and ACC of 52.6%, 90%, 90.45, 50%, and 65.5%, respectively. HA/Pr and HA/Cr, cutoff values for detection of malignancy were 84.9 and 9.6 but with less predictive values. Histopathological type was the only independent factor affecting level of HA on multivariate analysis, (p = 0.012, Exp (B) 14.98, 95% CI 1.8-121). CONCLUSION: Combination of urinary HA and urine cytology provides reliable marker of bladder cancer.

Attenuated total reflection Fourier-transform infrared spectral discrimination in human bodily fluids of oesophageal transformation to adenocarcinoma.

Diagnostic tools for the detection of early-stage oesophageal adenocarcinoma (OAC) are urgently needed. Our aim was to develop an accurate and inexpensive method using biofluids (plasma, serum, saliva or urine) for detecting oesophageal stages through to OAC (squamous; inflammatory; Barrett's; low-grade dysplasia; high-grade dysplasia; OAC) using attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy. ATR-FTIR spectroscopy coupled with variable selection methods, with successive projections or genetic algorithms (GA) combined with quadratic discriminant analysis (QDA) were employed to identify spectral biomarkers in biofluids for accurate diagnosis in a hospital setting of different stages through to OAC. Quality metrics (Accuracy, Sensitivity, Specificity and F-score) and biomarkers of disease were computed for each model. For plasma, GA-QDA models using 15 wavenumbers achieved 100% classification for four classes. For saliva, PCA-QDA models achieved 100% for the inflammatory stage and high-quality metrics for other classes. For serum, GA-QDA models achieved 100% performance for the OAC stage using 13 wavenumbers. For urine, PCA-QDA models achieved 100% performance for all classes. Selected wavenumbers using a Student's t-test (95% confidence interval) identified a differentiation of the stages on each biofluid: plasma (929 cm-1 to 1431 cm-1, associated with DNA/RNA and proteins); saliva (1000 cm-1 to 1150 cm-1, associated with DNA/RNA region); serum (1435 cm-1 to 1573 cm-1, associated with methyl groups of proteins and Amide II absorption); and, urine (1681 cm-1 to 1777 cm-1, associated with a high frequency vibration of an antiparallel ß-sheet of Amide I and stretching vibration of lipids). Our methods have demonstrated excellent efficacy for a rapid, cost-effective method of diagnosis for specific stages to OAC. These findings suggest a potential diagnostic tool for oesophageal cancer and could be translated into clinical practice.

Utility of liquid biopsy using urine in patients with pancreatic ductal adenocarcinoma.

In recent years, liquid biopsy for blood and body fluid in cancer patients has attracted attention. However, there have been few reports of liquid biopsy focusing on urine of pancreatic ductal adenocarcinoma (PDAC). In 56 patients with PDAC, DNA was extracted from urine and plasma prior to treatment, and KRAS mutations were analyzed with droplet digital PCR to examine the mutation detection rate. Our study showed that KRAS mutations were found in 27 cases (48%) in urine and 27 cases (48%) in plasma. The detection rate of urine KRAS mutations varied by renal functions. The rates were 70% (14/20) and 36% (13/36) in the creatinine clearance rate (CCr) < 70 mL/min group and in the CCr ≥ 70 mL/min group, respectively (P = .024). Whereas, no influence of the CCr was observed in the detection rates of plasma KRAS mutations. The rates were 50% (10/20) and 47% (17/36) in cases with the CCr < 70 mL/min group and the CCr ≥ 70 mL/min group, respectively. Although the sample size was small, this study clearly indicated a new possibility of less invasive urine liquid biopsy in PDAC patients.

Cervical cancer detection by DNA methylation analysis in urine.

Urine samples provide a potential alternative to physician-taken or self-collected cervical samples for cervical screening. Screening by primary hrHPV testing requires additional risk assessment (so-called triage) of hrHPV-positive women. Molecular markers, such as DNA methylation, have proven most valuable for triage when applied to cervical specimens. This study was set out to compare hrHPV and DNA methylation results in paired urine and cervical scrapes, and to evaluate the feasibility of DNA methylation analysis in urine to detect cervical cancer. Urine samples (n = 41; native and sediment) and paired cervical scrapes (n = 38) from cervical cancer patients, and urine from 44 female controls, were tested for hrHPV and 6 methylation markers. Results on native urine and sediment were highly comparable. A strong agreement was found between hrHPV testing on urine and scrapes (kappa = 0.79). Also, methylation levels in urine were moderately to strongly correlated to those detected in scrapes (r = 0.508-0.717). All markers were significantly increased in urine from cervical cancer patients compared to controls and showed a good discriminatory power for cervical cancer (AUC = 0.744-0.887). Our results show a good agreement of urine-based molecular analysis with reference cervical samples, and suggest that urine-based DNA methylation testing may provide a promising strategy for cervical cancer detection.

Post-treatment urinary sarcosine as a predictor of recurrent relapses in patients with prostate cancer.

To date, there has been no evidence regarding the association between urinary sarcosine content and prostate cancer survival. Our main objective was to investigate whether levels of post-treatment urinary sarcosine are associated with relapse. The inclusion criteria were (in accordance with EAU 2017) as follows: histopathologically verified adenocarcinoma in prostate biopsy cores or specimens from transurethral resection of the prostate (TURP) or prostatectomy for benign prostatic enlargement (BPE) with retained ability to urinate. The median follow-up was 53 months. In the study, we retrospectively evaluated a cohort of 511 patients with prostate cancer with various risk factors and treatment strategies. Post-treatment sarcosine levels were elevated in 266 (52%) patients and highly elevated (≥200 nmol/L) in 71 (13%) patients. Urinary sarcosine content was significantly associated with number of relapses that patients experienced, P = 0.002 for sarcosine ≥200 vs ≤30 nmol/L. Multivariate analysis revealed that sarcosine was an independent predictor of recurrent relapses (≥2 relapses with an intermediate period of remission), HR = 3.89 (95% CI 1.29-11.7) for sarcosine >200 vs <30 nmol/L. This trend was even more pronounced in a subgroup of patients who underwent radical prostatectomy, HR = 3.29 (95% CI 1.06-10.18), where (single) relapse-free survival could also be predicted by sarcosine levels, HR = 1.96 (1.05-3.66). Urinary sarcosine may become a possible predictor for patients' outcomes, because patients with elevated post-treatment sarcosine could be predicted to have recurrent relapses of the disease.

NMR-based metabolic profiling of urine, serum, fecal, and pancreatic tissue samples from the Ptf1a-Cre; LSL-KrasG12D transgenic mouse model of pancreatic cancer.

Pancreatic cancer is the third leading cause of cancer deaths in the United States with more than 53,000 expected to be diagnosed with the disease in 2018. The median survival time after diagnosis is four to six months. The poor survival statistics are due in part to the fact that pancreatic cancer is typically asymptomatic until it reaches advanced stages of the disease. Although surgical resection provides the best chance of survival, pancreatic cancer is rarely detected when surgery is still possible due, in part, to lack of effective biomarkers for early detection. The goal of the research reported here was to determine if it was possible to identify metabolic biomarkers for detection of pre-cancerous pancreatic intraepithelial neoplasia (PanIN) that precede pancreatic adenocarcinoma. The transgenic Ptf1a-Cre; LSL-KrasG12D mouse strain was used as a model of pancreatic cancer progression. Nuclear magnetic resonance (NMR) spectroscopy was employed to compare metabolic profiles of urine, sera, fecal extracts, and pancreatic tissue extracts collected from control and study mice aged 5, 11, and 15 months, including 47 mice with tumors. We were able to identify the following potential biomarkers: decreased 3-indoxylsulfate, benzoate and citrate in urine, decreased glucose, choline, and lactate in blood, and decreased phenylalanine and benzoate and increased acetoin in fecal extracts. Potential biomarkers were validated by p-values, PLS-DA VIP scores, and accuracies based on area under ROC curve analyses. Essentially, all of the metabolic profiling changes could be explained as being associated with the consequences of bicarbonate wasting caused by a complete substitution of the normal pancreatic acinar tissue by tissue entirely composed of PanIN. Given the nature of the mouse model used here, our results indicate that it may be possible to use NMR-based metabolic profiling to identify biomarkers for detection of precancerous PanIN that immediately precede pancreatic cancer.

Biomarkers in Urachal Cancer and Adenocarcinomas in the Bladder: A Comprehensive Review Supplemented by Own Data.

Urachal cancer (UrC) is a rare but aggressive cancer. Due to overlapping histomorphology, discrimination of urachal from primary bladder adenocarcinomas (PBAC) and adenocarcinomas secondarily involving the bladder (particularly colorectal adenocarcinomas, CRC) can be challenging. Therefore, we aimed to give an overview of helpful (immunohistochemical) biomarkers and clinicopathological factors in addition to survival analyses and included institutional data from 12 urachal adenocarcinomas. A PubMed search yielded 319 suitable studies since 1930 in the English literature with 1984 cases of UrC including 1834 adenocarcinomas (92%) and 150 nonadenocarcinomas (8%). UrC was more common in men (63%), showed a median age at diagnosis of 50.8 years and a median tumor size of 6.0 cm. No associations were noted for overall survival and progression-free survival (PFS) and clinicopathological factors beside a favorable PFS in male patients (p = 0.047). The immunohistochemical markers found to be potentially helpful in the differential diagnostic situation are AMACR and CK34ßE12 (UrC versus CRC and PBAC), CK7, ß-Catenin and CD15 (UrC and PBAC versus CRC), and CEA and GATA3 (UrC and CRC versus PBAC). Serum markers like CEA, CA19-9 and CA125 might additionally be useful in the follow-up and monitoring of UrC.

Identification of urine biomarkers associated with lung adenocarcinoma.

Lung adenocarcinoma (LAC) progression is accompanied by changes in protein levels that may be reflected in body fluids, such as urine. Urine collected from LAC patients (n=34) and healthy controls (n=36) was analyzed via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) combined with weak cationic exchange magnetic beads. The results revealed 76 urinary polypeptides significantly different between LAC patients and normal controls (P<0.05). Twenty-two of these peptides were up-regulated and 54 were down-regulated. Thirteen peptides had average peak intensities >600. Twelve of these 13 peptides were successfully identified using nano-liquid chromatography-tandem MS. Receiver operating characteristic analyses identified seven peptides with superior LAC diagnostic performances. Immunohistochemical staining in 20 paired LAC and adjacent normal tissues showed that IGKC, AAT, SH3BGRL3, osteopontin and gelsolin levels were higher in LAC tissues than in adjacent tissuesand were closely associated with LAC. Urinary peptides assessments may thus provide a novel, noninvasive, repeatable method for detecting and monitoring LAC. New, low-cost detection methods and bioinformatics tools are therefore urgently needed for the analysis of low abundance proteins and peptides in body fluids.

Urinary circulating DNA detection for dynamic tracking of EGFR mutations for NSCLC patients treated with EGFR-TKIs.

PURPOSE: Changes in EGFR profiles of non small cell lung cancer (NSCLC) patients correlates to clinical outcome. Extracting quality tumor tissue remains a challenge for molecular profiling. Our study aims to ascertain the clinical relevance of urinary cell free DNA as an alternative tumor material source. METHODS: 150 patients with activating EGFR mutation and received EGFR-TKIs were recruited to participate in the serial monitoring study. Matched primary tumor samples were taken together with blood and urine specimens before the initiation of TKIs. The EGFR mutation testing was performed and quantified using ddPCR. For serial time point measurements, urine and blood samples were extracted at 1-month intervals for duration of 9 months. RESULTS: Urinary ctDNA yielded a close agreement of 88 % on EGFR mutation status when compared to primary tissue at baseline. Almost all samples detected via urine specimens were uncovered in plasma samples. Analysis of urinary cell free DNA at different time points showed a strong correlation to treatment efficacy. Interestingly, a secondary EGFR mutation T790M was detected for 53 % of the patients during monitoring. The results were corroborated with the plasma ctDNA analysis. The T790M+ group had a reduced median survival when compared to the wildtype group. CONCLUSION: Urinary cell free DNA may be a potential alternative to conventional primary tissue based EGFR mutation testing. Our findings showed that the assay sensitivity was comparable to results from blood plasma. Urinary samples being noninvasive and readily available have clinical utility for monitoring of EGFR TKI treatment.

Cranberry intervention in patients with prostate cancer prior to radical prostatectomy. Clinical, pathological and laboratory findings.

BACKGROUND AND OBJECTIVES: Recently, we described an inverse association between cranberry supplementation and serum prostate specific antigen (PSA) in patients with negative biopsy for prostate cancer (PCa) and chronic nonbacterial prostatitis. This double blind placebo controlled study evaluates the effects of cranberry consumption on PSA values and other markers in men with PCa before radical prostatectomy. METHODS: Prior to surgery, 64 patients with prostate cancer were randomized to a cranberry or placebo group. The cranberry group (n=32) received a mean 30 days of 1500 mg cranberry fruit powder. The control group (n=32) took a similar amount of placebo. Selected blood/urine markers as well as free and total phenolics in urine were measured at baseline and on the day of surgery in both groups. Prostate tissue markers were evaluated after surgery. RESULTS: The serum PSA significantly decreased by 22.5% in the cranberry arm (n=31, P<0.05). A trend to down-regulation of urinary beta-microseminoprotein (MSMB) and serum gamma-glutamyltranspeptidase, as well as upregulation of IGF-1 was found after cranberry supplementation. There were no changes in prostate tissue markers or, composition and concentration of phenolics in urine. CONCLUSIONS: Daily consumption of a powdered cranberry fruit lowered serum PSA in patients with prostate cancer. The whole fruit contains constituents that may regulate the expression of androgen-responsive genes.

The utility of urine-circulating miRNAs for detection of prostate cancer.

BACKGROUND: In this paper, the utility of urine-circulating microRNAs (miRNAs) as the potential biomarker of prostate cancer (PCa), the second most prevalent male cancer worldwide, was evaluated. METHODS: Cancerous (N=56) and non-cancerous (N=16) prostate tissues were analysed on TaqMan Low Density Array, with the initial screening of 754 miRNAs in a subset of the samples. The abundance of selected miRNAs was analysed in urine specimens from two independent cohorts of patients with PCa (N=215 overall), benign prostatic hyperplasia (BPH; N=23), and asymptomatic controls (ASC; N=62) by means of quantitative reverse transcription PCR. RESULTS: Over 100 miRNAs were found deregulated in PCa as compared with non-cancerous prostate tissue. After thorough validation, four miRNAs were selected for the analysis in urine specimens. The abundance of miR-148a and miR-375 in urine was identified as specific biomarkers of PCa in both cohorts. Combined analysis of urine-circulating miR-148a and miR-375 was highly sensitive and specific for PCa in both cohorts (AUC=0.79 and 0.84) and strongly improved the diagnostic power of the PSA test (AUC=0.85, cohort PCa1), including the grey diagnostic zone (AUC=0.90). CONCLUSIONS: Quantitative measurement of urine-circulating miR-148a and miR-375 can serve as the non-invasive tool for sensitive and specific detection of PCa.

Molecular Diagnostic of Prostate Cancer From Body Fluids Using Methylation-Specific PCR (MS-PCR) Method.

BACKGROUND: Worldwide prostate cancer (PCa) represents the 2nd leading cause of cancer related deaths among men. Currently, the screening for early detection of PCa is based on determination of serum prostate-specific antigen (PSA) levels. But this biomarker presents some disadvantages related to its specificity and sensitivity. In our study, we want to determine if methylation levels of the glutathione S-transferase P1 (GSTP1) gene could be used as a new biomarker for the early detection of PCa and to distinguish between malignant and benign pros-tatic lesions. METHODS: To determine the methylation levels of the GSTP1 gene, 31 men with histopathological diagnosis of prostate adenocarcinoma and 34 men with the histopathological diagnosis of benign prostatic hyperplasia (BPH) as controls were included in the study group. The genomic DNA was extracted from urine samples. We analyzed the methylation levels of the GSTP1 gene by methylation-specific polymerase chain reaction (MS-PCR) method. RESULTS: In prostate cancer patients 27 of 31 (87%) presented hypermethylated levels of the GSTP1 gene, whereas 4 of 34 (11.8%) BPH patients had hypermethylated levels of the GSTP1 gene. Further, in the case of these four patients a second biopsy was done, which confirmed the diagnosis of prostate adenocarcinoma. Using the receiver operating curve (ROC), we obtained a specificity of 87% and a sensitivity of 98% for the GSTP1 gene. CONCLUSIONS: We can conclude that GSTP1 represents a new molecular biomarker which can aid in early detection of PCa and be used to discriminate between benign and malignant prostatic lesions from body fluids by noninvasive methods.

Updated therapeutic outcome for patients with periampullary and pancreatic cancer related to recent translational research.

Chemotherapy with improved effect in patients with metastatic pancreatic cancer has recently been established, launching a new era for patients with this very aggressive disease. FOLFIRINOX and gemcitabine plus nab-paclitaxel are different regimens, both capable of stabilizing the disease, thus increasing the number of patients who can reach second line and even third line of treatment. Concurrently, new windows of opportunity open for nutritional support and other therapeutic interventions, improving quality of life. Also pancreatic surgery has changed significantly during the latest years. Extended operations, including vascular/multivisceral resections are frequently performed in specialized centers, pushing borders of resectability. Potentially curative treatment including neoadjuvant and adjuvant chemotherapy is offered new patient groups. Translational research is the basis for the essential understanding of the ongoing development. Even thou biomarkers for clinical management of patients with periampullary tumors have almost been lacking, biomarker driven trials are now in progress. New insight is constantly made available for clinicians; one recent example is selection of patients for gemcitabine treatment based on the expression level of the human equilibrium nucleoside transporter 1. An example of new diagnostic tools is identification of early pancreatic cancer patients by a three-biomarker panel in urine: The proteins lymphatic vessel endothelial hyaluronan receptor 1, regenerating gene 1 alpha and translation elongation factor 1 alpha. Requirement of treatment guideline revisions is intensifying, as combined chemotherapy regimens result in unexpected advantages. The European Study Group for Pancreatic Cancer 4 trial outcome is an illustration: Addition of capecitabine in the adjuvant setting improved overall survival more than expected from the effect in advanced disease. Rapid implementation of new treatment options is mandatory when progress finally extends to patients with this serious disease.

Histidine decarboxylase and urinary methylimidazoleacetic acid in gastric neuroendocrine cells and tumours.

AIM: To study histidine decarboxylase (HDC) expression in normal and neoplastic gastric neuroendocrine cells in relationship to the main histamine metabolite. METHODS: Control tissues from fundus (n = 3) and corpus (n = 3) mucosa of six patients undergoing operations for gastric adenocarcinoma, biopsy and/or gastric surgical specimens from 64 patients with primary gastric neuroendocrine tumours (GNETs), as well as metastases from 22 of these patients, were investigated using conventional immunohistochemistry and double immunofluorescence with commercial antibodies vs vesicular monoamine transporter 2 (VMAT-2), HDC and ghrelin. The urinary excretion of the main histamine metabolite methylimidazoleacetic acid (U-MeImAA) was determined using high-performance liquid chromatography in 27 of the 64 patients. RESULTS: In the gastric mucosa of the control tissues, co-localization studies identified neuroendocrine cells that showed immunoreactivity only to VMAT-2 and others with reactivity only to HDC. A third cell population co-expressed both antigens. There was no co-expression of HDC and ghrelin. Similar results were obtained in the foci of neuroendocrine cell hyperplasia associated with chronic atrophic gastritis type A and also in the tumours. The relative incidence of the three aforementioned markers varied in the tumours that were examined using conventional immunohistochemistry. All of these GNETs revealed both VMAT-2 and HDC immunoreactivity, and their metastases showed an immunohistochemical pattern and frequency similar to that of their primary tumours. In four patients, increased U-MeImAA excretion was detected, but only two of the patients exhibited related endocrine symptoms. CONCLUSION: Human enterochromaffin-like cells appear to partially co-express VMAT-2 and HDC. Co-expression of VMAT-2 and HDC might be required for increased histamine production in patients with GNETs.

Urinary kidney injury molecule-1 and monocyte chemotactic protein-1 are noninvasive biomarkers of cisplatin-induced nephrotoxicity in lung cancer patients.

PURPOSE: Acute kidney injury (AKI) is a common and serious adverse effect of cisplatin-based chemotherapy. However, traditional markers of kidney function, such as serum creatinine, are suboptimal, because they are not sensitive measures of proximal tubular injury. We aimed to determine whether the new urinary biomarkers such as kidney injury molecule-1 (KIM-1), monocyte chemotactic protein-1 (MCP-1), and neutrophil gelatinase-associated lipocalin (NGAL) could detect cisplatin-induced AKI in lung cancer patients in comparison with the conventional urinary proteins such as N-acetyl-ß-D-glucosaminidase (NAG) and ß2-microglobulin. METHODS: We measured KIM-1, MCP-1, NGAL, NAG, and ß2-microglobulin concentrations in urine samples from 11 lung cancer patients, which were collected the day before cisplatin administration and on days 3, 7, and 14. Subsequently, we evaluated these biomarkers by comparing their concentrations in 30 AKI positive (+) and 12 AKI negative (-) samples and performing receiver operating characteristic (ROC) curve analyses. RESULTS: The urinary levels normalized with urine creatinine of KIM-1 and MCP-1, but not NGAL, NAG, and ß2-microglobulin in AKI (+) samples were significantly higher than those in AKI (-) samples. In addition, ROC curve analyses revealed that KIM-1 and MCP-1, but not NGAL, could detect AKI with high accuracy (area under the curve [AUC] = 0.858, 0.850, and 0.608, respectively). The combination of KIM-1 and MCP-1 outperformed either biomarker alone (AUC = 0.871). CONCLUSIONS: Urinary KIM-1 and MCP-1, either alone or in combination, may represent biomarkers of cisplatin-induced AKI in lung cancer patients.

Identification of a Three-Biomarker Panel in Urine for Early Detection of Pancreatic Adenocarcinoma.

PURPOSE: Noninvasive biomarkers for early detection of pancreatic ductal adenocarcinoma (PDAC) are currently not available. Here, we aimed to identify a set of urine proteins able to distinguish patients with early-stage PDAC from healthy individuals. EXPERIMENTAL DESIGN: Proteomes of 18 urine samples from healthy controls, chronic pancreatitis, and patients with PDAC (six/group) were assayed using GeLC/MS/MS analysis. The selected biomarkers were subsequently validated with ELISA assays using multiple logistic regression applied to a training dataset in a multicenter cohort comprising 488 urine samples. RESULTS: LYVE-1, REG1A, and TFF1 were selected as candidate biomarkers. When comparing PDAC (n = 192) with healthy (n = 87) urine specimens, the resulting areas under the receiver-operating characteristic curves (AUC) of the panel were 0.89 [95% confidence interval (CI), 0.84-0.94] in the training (70% of the data) and 0.92 (95% CI, 0.86-0.98) in the validation (30% of the data) datasets. When comparing PDAC stage I-II (n = 71) with healthy urine specimens, the panel achieved AUCs of 0.90 (95% CI, 0.84-0.96) and 0.93 (95% CI, 0.84-1.00) in the training and validation datasets, respectively. In PDAC stage I-II and healthy samples with matching plasma CA19.9, the panel achieved a higher AUC of 0.97 (95% CI, 0.94-0.99) than CA19.9 (AUC = 0.88; 95% CI, 0.81-0.95, P = 0.005). Adding plasma CA19.9 to the panel increased the AUC from 0.97 (95% CI, 0.94-0.99) to 0.99 (95% CI, 0.97-1.00, P = 0.04), but did not improve the comparison of stage I-IIA PDAC (n = 17) with healthy urine. CONCLUSIONS: We have established a novel, three-protein biomarker panel that is able to detect patients with early-stage pancreatic cancer in urine specimens.

[Diagnostic significance of protein NMP22 in bladder cancer]. / Znaczenie diagnostyczne bialka NMP22 w raku pecherza moczowego.

Bladder cancer is a malignancy that affects mainly the elderly and males. Up to 90% of these cancers originate from urothelial epithelial cells and therefore they are called Transitional (Urothelial) Cell Carcinoma (TCC). Another types are: Squamous Cell Carcionoma (SCC), which involves about 5% of cases and Adenocarcinoma (less than 2%). The factors that may lead to the development of bladder cancer include: genetic disorders, molecular changes, environmental exposures, industrial carcinogens, chemical contaminants and chronic cystitis. This article depicts the current state of diagnostics of bladder cancer, with particular focus on urine-based tests. Although many markers with different structure are under research, only the following have gained FDA approval for bladder cancer screening: BTAstat, BTA TRAK, UroVysion and NMP22 BladderChek. For follow-up NMP22 ELISA and Immunocyt (uCyt+) are approved. This work is mainly focused on mainly on evaluating the diagnostic value of nuclear matrix protein NMP22 for bladder cancer in terms of the outlined researches among people susceptible to environmental toxins. A review of the current literature depicts that no research on correlation between NMP22 and genetic susceptibility has been conducted so far. There is some evidence that NMP22 protein is particularly important in high-risk groups, e.g. among tobacco smokers. The work also describes the methods of detecting NMP22 protein and factors that may influence the results. The review of current literature showed that NMP22 cannot replace invasive cystoscopy neither in screening for bladder cancer nor in follow-up. The NMP22 test could be useful for determining the frequency of cystoscopy and for early detection of high-grade tumors. Research focused on improving the specificity of this marker seems to be crucial, e.g. through the correlation between NMP22 and other parameters (e.g. other laboratory tests), which is confirmed by preliminary data about combining various markers.

Novel non-invasive biomarkers that distinguish between benign prostate hyperplasia and prostate cancer.

BACKGROUND: The objective of this study was to discover and to validate novel noninvasive biomarkers that distinguish between benign prostate hyperplasia (BPH) and localized prostate cancer (PCa), thereby helping to solve the diagnostic dilemma confronting clinicians who treat these patients. METHODS: Quantitative iTRAQ LC/LC/MS/MS analysis was used to identify proteins that are differentially expressed in the urine of men with BPH compared with those who have localized PCa. These proteins were validated in 173 urine samples from patients diagnosed with BPH (N = 83) and PCa (N = 90). Multivariate logistic regression analysis was used to identify the predictive biomarkers. RESULTS: Three proteins, ß2M, PGA3, and MUC3 were identified by iTRAQ and validated by immunoblot analyses. Univariate analysis demonstrated significant elevations in urinary ß2M (P < 0.001), PGA3 (P = 0.006), and MUC3 (P = 0.018) levels found in the urine of PCa patients. Multivariate logistic regression analysis revealed AUC values ranging from 0.618 for MUC3 (P = 0.009), 0.625 for PGA3 (P < 0.008), and 0.668 for ß2M (P < 0.001). The combination of all three demonstrated an AUC of 0.710 (95% CI: 0.631 - 0.788, P < 0.001); diagnostic accuracy improved even more when these data were combined with PSA categories (AUC = 0.812, (95% CI: 0.740 - 0.885, P < 0.001). CONCLUSIONS: Urinary ß2M, PGA3, and MUC3, when analyzed alone or when multiplexed with clinically defined categories of PSA, may be clinically useful in noninvasively resolving the dilemma of effectively discriminating between BPH and localized PCa.