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Evaluation of two new urinary tumor markers: Bladder tumor fibronectin and cytokeratin 18 for the diagnosis of bladder cancer
Abstract
Our objectives were to evaluate the diagnostic value of two new urinary tumor markers, cytokeratin 18 (CK18) and bladder tumor fibronectin (BTF), for the detection and monitoring of bladder cancer. The study comprised 931 urine samples belonging to 402 subjects: 112 individuals under suspicion for a primary bladder tumor (group 1); 104 bladder cancer patients under scheduled follow-up (group 2); 109 bladder cancer patients receiving intravesical instillations (group 3); 45 patients with other urological diseases (group 4); and 32 healthy subjects (group 5). Voided urine samples were collected before cystoscopies, between them and before intravesical instillations. CK18 and BTF tests were measured by chemiluminescent immunoassays. Optimal receiver operating characteristic cutoffs of 7.4 microg/L for CK18 and 52.8 microg/liter for BTF rendered overall sensitivities of 66.2% for CK18 and 80.0% for BTF at specificities of 88.4 and 74.7%, respectively. Urinary cytology provided a sensitivity of 29.2% at a specificity of 99.1%. Sensitivities were 80.8, 74.2, and 82.3% for BTF and 71.1, 77.4, and 64.7% for CK18 for groups 1 to 3, respectively. False positive rates were higher for BTF in all groups of patients. Elevated urinary tumor markers during the monitoring of patients with bladder cancer could detect recurrence sooner than scheduled cystoscopies. Persistence of negative markers was greatly indicative of free of disease status in follow-up. CK18 and BTF in urine may eventually prove to be of benefit for specific patients with bladder carcinoma given its higher sensitivity compared with cytology. In selected patients, namely those with persistent negative urinary CK18 and BTF, the number of cystoscopies could be reduced.
... Fibronektin, antijenik özellik gösteren, proteolize dirençli, birçok fonksiyonel bölgeden oluşan, 440 kDa ağırlığında, büyük dimerik bir glikoproteindir. 77,79,80 Fibronektinin, plazma, idrar ve idrar yolları dokularında bulunduğu vurgulanmaktadır. 77,79 Fibronektinin en önemli fonksiyonu, ekstraselüler matriks ve hücreler arasındaki ilişkiyi sağlamasıdır. ...
... 77,79,80 Fibronektinin, plazma, idrar ve idrar yolları dokularında bulunduğu vurgulanmaktadır. 77,79 Fibronektinin en önemli fonksiyonu, ekstraselüler matriks ve hücreler arasındaki ilişkiyi sağlamasıdır. Bu ilişkinin sonucunda hücre-substrat adezyonu, hücre migrasyonu ve hücre morfolojisinin düzenlenmesi gibi etkiler ortaya çıkmaktadır. ...
... Bu ilişkinin sonucunda hücre-substrat adezyonu, hücre migrasyonu ve hücre morfolojisinin düzenlenmesi gibi etkiler ortaya çıkmaktadır. 77,[79][80][81][82] İdrarda fibronektin, genellikle hücre bağlayan bölgelerden oluşan fragmanlar şeklinde bulunmaktadır. Bu fragmanların ürogenital sistemin, özellikle de idrar kesesi ve prostat dokusunda bulunan ekstraselüler matriksin proteolitik yıkımı sonucunda ortaya çıktıkları düşünülmektedir. ...
Routine analysis of biomarkers, existing in blood, body fluids and tissue and being a biological molecule and indicator about normal or abnormal circumstances or disease can be performed in specific tissue and body fluid. Due to contact with urinary system ephitelium, urine contains biomarkers, releated with pathological disorders or functions of organs such as kidney, bladder and prostate. Proteins and enzymes in urine compose of most commonly used biomarkers. Proteins in urine are formed by soluble proteins in urine and protein components of solid phase compounds. Soluble proteins in urine are mostly provided from glomerular filtration. Solid-phase compounds are found in sediments consisting of castes can be obtained at the lowest centrifuge speed and spilled epithelial cells. Changes in the rate of excretion of urinary proteins can demonstrate systemic, glomerular or tubular damage. In systemic and metabolic diseases, disorders of specific organs affecting the enzymatic activity of urine or urinary tract diseases, changes such as growth or reduction also seen in urine normally absent can be in urinary enzyme. N-Acetyl-β-D-glucosaminidase (NAG), γ-glutamyl transferase (GGT), β-glucosidase can demonstrate be examples for urinary enzymes. In this review, general information is given about urinary biomarkers and it is also mentioned about biomarkers used in specific diseases of related organs such as kidney, urinary bladder and prostate gland.
... Fibronektin, antijenik özellik gösteren, proteolize dirençli, birçok fonksiyonel bölgeden oluşan, 440 kDa ağırlığında, büyük dimerik bir glikoproteindir. 77,79,80 Fibronektinin, plazma, idrar ve idrar yolları dokularında bulunduğu vurgulanmaktadır. 77,79 Fibronektinin en önemli fonksiyonu, ekstraselüler matriks ve hücreler arasındaki ilişkiyi sağlamasıdır. ...
... 77,79,80 Fibronektinin, plazma, idrar ve idrar yolları dokularında bulunduğu vurgulanmaktadır. 77,79 Fibronektinin en önemli fonksiyonu, ekstraselüler matriks ve hücreler arasındaki ilişkiyi sağlamasıdır. Bu ilişkinin sonucunda hücre-substrat adezyonu, hücre migrasyonu ve hücre morfolojisinin düzenlenmesi gibi etkiler ortaya çıkmaktadır. ...
... Bu ilişkinin sonucunda hücre-substrat adezyonu, hücre migrasyonu ve hücre morfolojisinin düzenlenmesi gibi etkiler ortaya çıkmaktadır. 77,[79][80][81][82] İdrarda fibronektin, genellikle hücre bağlayan bölgelerden oluşan fragmanlar şeklinde bulunmaktadır. Bu fragmanların ürogenital sistemin, özellikle de idrar kesesi ve prostat dokusunda bulunan ekstraselüler matriksin proteolitik yıkımı sonucunda ortaya çıktıkları düşünülmektedir. ...
ÖZET
Kanda, vücut sıvılarında veya dokularda bulunan, normal/anormal bir durumun veya hastalığın göstergesi ve biyolojik bir molekül olan biyobelirteçlerin; rutin analizleri, belirli dokularda/vücut sıvılarında yapılabilmektedir. İdrar üriner sistem epitelyumu ile temasta olması bakımından böbrek, idrar kesesi, prostat gibi organlara ait patolojik bozuklukların veya fonksiyonlarının biyobelirteçlerini bünyesinde barındırmaktadır. İdrardaki proteinler ve enzimler, en sık kullanılan biyobelirteçleri oluşturmaktadır. İdrardaki proteinleri, idrarda çözünen proteinler ve solid faz bileşiklerin protein elemanları oluşturmaktadır. İdrardaki çözünmüş proteinler, büyük oranda glomerüler filtrasyondan sağlanır. Solid faz bileşikler ise düşük santrifüj hızında elde edilebilen kastlar ve dökülmüş epitelyal hücrelerin oluşturduğu sedimentlerde bulunurlar. Üriner proteinlerin atılım oranındaki değişiklikler, sistemik, glomerüler ve/veya tübüler hasarı gösterebilir. Sistemik ve metabolik hastalıklar, idrarın enzimatik aktivitesini etkileyen belirli organların hastalıkları veya üriner sistem hastalıklarında idrar enzimlerinde, azalma ya da çoğalma veya normalde bulunmayan enzimin görülmesi gibi değişiklikler olabilir. N-Asetil-β-D-glukozaminidaz (NAG), γ-Glutamil transferaz (GGT), β-glukozidaz üriner enzimlere örnek olarak gösterilebilir. Bu derlemede, üriner biyobelirteçlerle ilgili genel bilgi verilmektedir ve böbrek, idrar kesesi ve prostat bezi gibi ilgili organlara ait hastalıklarda kullanılan çeşitli biyobelirteçlerden bahsedilmektedir.
ABSTRACT
Routine analysis of biomarkers, existing in blood, body fluids and tissue and being a biological molecule and indicator about normal or abnormal circumstances or disease can be performed in specific tissue and body fluid. Due to contact with urinary system ephitelium, urine contains biomarkers, releated with pathological disorders or functions of organs such as kidney, bladder and prostate. Proteins and enzymes in urine compose of most commonly used biomarkers. Proteins in urine are formed by soluble proteins in urine and protein components of solid phase compounds. Soluble proteins in urine are mostly provided from glomerular filtration. Solid-phase compounds are found in sediments consisting of castes can be obtained at the lowest centrifuge speed and spilled epithelial cells. Changes in the rate of excretion of urinary proteins can demonstrate systemic, glomerular or tubular damage. In systemic and metabolic diseases, disorders of specific organs affecting the enzymatic activity of urine or urinary tract diseases, changes such as growth or reduction also seen in urine normally absent can be in urinary enzyme. N-Acetyl-β-D-glucosaminidase (NAG), γ-glutamyl transferase (GGT), β-glucosidase can demonstrate be examples for urinary enzymes. In this review, general information is given about urinary biomarkers and it is also mentioned about biomarkers used in specific diseases of related organs such as kidney, urinary bladder and prostate gland.
... Interestingly, several of the proteins described above have been documented to be elevated in bladder cancer tissue (at the RNA or protein level) and/or implicated in tumor biology at some level, as summarized in Additional file 1: Table S5. Considering their biomarker potential and functional properties based on the literature (Additional file 1: Table S5), these urine proteins warrant further investigation, including, d-dimer [34], Apolipoprotein A1 [35,36], Apolipoprotein L1, Calgranulin B [37,38], complement C2 [39], Fibronectin [40][41][42][43], Ficolin-3, IL-8 [44][45][46][47][48][49], IgA [50], MMP-1 [51,52], Properdin, and Proteinase 3 [53]. A summary of previous research on these proteins can be found in Additional file 1: Table S6. ...
... Tumor cells can attach to fibronectin via integrins or other cell surface receptors [55]. Its effectiveness as a urine biomarker for BC has been explored in a variety of studies [40][41][42][43]. Here, fibronectin showed the third best discriminatory ability in identifying MIBC compared to NMIBC. ...
Background
Bladder cancer (BC) is among the most common cancers diagnosed in men in the USA. The current gold standards for the diagnosis of BC are invasive or lack the sensitivity to correctly identify the disease.
Methods
An aptamer-based screen analyzed the expression of 1317 proteins in BC compared to urology clinic controls. The top hits were subjected to systems biology analyses. Next, 30 urine proteins were ELISA-validated in an independent cohort of 68 subjects. Three of these proteins were next validated in an independent BC cohort of differing ethnicity.
Results
Systems biology analysis implicated molecular functions related to the extracellular matrix, collagen, integrin, heparin, and transmembrane tyrosine kinase signaling in BC susceptibility, with HNF4A and NFKB1 emerging as key molecular regulators. STEM analysis of the dysregulated pathways implicated a functional role for the immune system, complement, and interleukins in BC disease progression. Of 21 urine proteins that discriminated BC from urology clinic controls (UC), urine d-dimer displayed the highest accuracy (0.96) and sensitivity of 97%. Furthermore, 8 urine proteins significantly discriminated MIBC from NMIBC (AUC = 0.75–0.99), with IL-8 and IgA being the best performers. Urine IgA and fibronectin exhibited the highest specificity of 80% at fixed sensitivity for identifying advanced BC.
Conclusions
Given the high sensitivity (97%) of urine d-dimer for BC, it may have a role in the initial diagnosis or detection of cancer recurrence. On the other hand, urine IL-8 and IgA may have the potential in identifying disease progression during patient follow-up. The use of these biomarkers for initial triage could have a significant impact as the current cystoscopy-based diagnostic and surveillance approach is costly and invasive when compared to a simple urine test.
... 11,20 FN is associated with tumorigenesis and progression in bladder cancer, 21 although the underlying mechanisms remain largely unknown. At the level of urinary tract, in the case of bladder tumor, levels of FN can rise due to the degradation of the extracellular matrix, 22,23 with higher expression correlated with tumor stage and grade. 24,25 Higher FN gene expression at the level urinary tract has already been associated with poorer outcomes after radical cystectomy. ...
... Conversely, tumor grade and stage were not independently associated with FN increase. This is in slight contrast with previous studies correlating FN expression with pathological features.22,24 One possible explanation could be that our study tried to analyze FN expression on the residual urothelium after TURBT, while the above-mentioned studies evaluated urinary FN with the bladder tumor still in place. ...
Background
A marker of urothelial damage could be helpful for early detection and monitoring of local toxicity due to intravesical therapy for non-muscle invasive bladder cancer (NMIBC). The aim of the study was to investigate the correlation between fibronectin (FN) gene expression in bladder washings and local toxicity secondary to adjuvant intravesical therapy.
Materials and methods
Patients undergoing adjuvant intravesical therapy for NMIBC and age-matched healthy patients were enrolled. Real time polymerase chain reaction was performed to analyze FN expression in bladder washings. Local toxicity was classified as: 0–1 mild (no medical therapy), 2 moderate (medical therapy and/or instillation postponed), 3 severe (discontinuation of therapy).
Results
Seventy-two patients and 21 controls entered the study. A useful pellet was obtained in 58 patients and 18 controls. Intravesical Bacillus Calmette–Guerin (BCG), Epirubicin and Mitomycin C was offered to 69%, 13.8% and 17.2% of patients respectively. Compared with healthy controls (FN = 1.0 fold), overall median FN expression before adjuvant intravesical therapy was 1.73 fold [interquartile range (IQR) 0.8–2.3], while during therapy median FN expression increased to 3.41 (IQR: 1.6–6.1) fold. Considering 40 intermediate and high-risk patients undergoing intravesical BCG, median FN expression before adjuvant treatment was 1.92 [(IQR: 1.0–2.7) fold, increasing up to 4.1 (IQR: 1.9–6.6) during therapy. In more detail, FN increased during BCG therapy, showing a median expression of 4.22 (IQR: 2.2–5.5) and 6.16 (IQR: 2.6–8.7) fold in presence of grade 2 and 3 toxicity respectively, while remaining more or less stable in asymptomatic patients. After receiver operating characteristic curve analysis, FN value of 3.6 fold resulted, corresponding to 75% sensitivity and 69% specificity to predict grade 2–3 toxicity events (area under the curve 0.74, 95% confidence interval 0.63–0.85, p = 0.001).
Conclusion
Our study validated the correlation between FN expression and urothelial damage. BCG seems to induce a urothelial activation with FN overexpression during adjuvant intravesical therapy. Grade of toxicity was related to FN expression.
... This finding was quickly confirmed in the same year by Malmstrom, who further proved the follow-up value of urine Fn in bladder cancer patients [16]. Since then, the diagnostic value of this urine molecule in detecting bladder cancer has been discussed by various researches and some satisfactory results were reported [17][18][19][20][21][22][23][24]. Recent study showed that urine Fn has a sensitivity of 91.4% and a specificity of 87.8% in detecting residual bladder tumor after TURBT [21]. ...
... The primary search identified 741 articles from all databases. After excluding the duplicate publications, nonrelevant literatures, and articles that did not meet the inclusion criteria, eight manuscripts published between 2000 and 2013 were considered eligible for meta-analysis ( Fig. 1) [17][18][19][20][21][22][23][24]. Among the studies, five of them reported the diagnostic value of both urine Fn itself and urine Fn combined with urine cytology (Fn+Cyto) [18,20,[22][23][24]. ...
Background:
Previous researches pointed out that the measurement of urine fibronectin (Fn) could be a potential diagnostic test for bladder cancer (BCa). We conducted this meta-analysis to fully assess the diagnostic value of urine Fn for BCa detection.
Methods:
A systematic literature search in PubMed, ISI Web of Science, EMBASE, Cochrane library, and CBM was carried out to identify eligible studies evaluating the urine Fn in diagnosing BCa. Pooled sensitivity, specificity, and diagnostic odds ratio (DOR) with their 95% confidence intervals (CIs) were calculated, and summary receiver operating characteristic (SROC) curves were established. We applied the STATA 13.0, Meta-Disc 1.4, and RevMan 5.3 software to the meta-analysis.
Results:
Eight separate studies with 744 bladder cancer patients were enrolled in this meta-analysis. The pooled sensitivity, specificity, and DOR were 0.80 (95%CI = 0.77-0.83), 0.79 (95%CI = 0.73-0.84), and 15.18 (95%CI = 10.07-22.87), respectively, and the area under the curve (AUC) of SROC was 0.83 (95%CI = 0.79-0.86). The diagnostic power of a combined method (urine Fn combined with urine cytology) was also evaluated, and its sensitivity and AUC were significantly higher (0.86 (95%CI = 0.82-0.90) and 0.89 (95%CI = 0.86-0.92), respectively). Meta-regression along with subgroup analysis based on various covariates revealed the potential sources of the heterogeneity and the detailed diagnostic value of each subgroup. Sensitivity analysis supported that the result was robust. No threshold effect and publication bias were found in this meta-analysis.
Conclusions:
Urine Fn may become a promising non-invasive biomarker for bladder cancer with a relatively satisfactory diagnostic power. And the combination of urine Fn with cytology could be an alternative option for detecting BCa in clinical practice. The potential value of urine Fn still needs to be validated in large, multi-center, and prospective studies.
... The samples were ranged from 55 to 325 patients and controls were from 41 to 77. Three of the five included essays reported the mean age which were ranged from 48.3 to 66.9 [6,21,22]. However, the AUC values were not reported in ...
... The levels of BTF have been reported to increase with tumor stages and grades [20][21][22], what is more, it is much higher in the invasive bladder tumor patients than superficial groups [2]. To explore the early diagnosis role of BTF, we have written to all the authors to get early stage patients' data for further analysis. ...
Objectives:
Bladder tumor fibronectin (BTF) has been related as a promising biomarker for the early diagnosis of bladder tumor. The meta-analysis was used to establish the diagnostic value of bladder tumor fibronectin in diagnosing bladder tumor.
Methods:
Relevant literatures evaluating the value of BTF in the diagnosis of bladder tumor were searched in PubMed, Embase, China National Knowledge Infrastructure (CNKI), Technology of Chongqing (VIP), and Wan Fang Data. Summary estimates were used to evaluate the value of BTF in the diagnosis of bladder tumor by using the Meta-DiSc and STATA 11.0 statistical software.
Results:
The meta-analysis included 5 studies (649 patients, 291 controls). The summary estimates revealed that the pooled sensitivity was 81% (95% confidence interval [CI]: 74%-85.1%) and specificity was 80% (95%CI 74%-84%). In addition, the area under the summary ROC curve (AUC) was 0.86 (95%CI 0.82-0.89).
Conclusion:
BTF is a potential marker for the diagnosis of bladder tumor, and more prospective studies are needed in the future.
... Recent studies in TCC and RCC indicating a potential role of cFN in these malignancies (Kirkali et al. 2001; Sanchez-Carbayo et al. 2000;Brenner et al. 2000). So further evaluation of cFN plasma levels, based on our first findings (Hegele et al. 2004; Hegele et al. 2002), is a consequent step to prove the clinical relevance in different stages of these malignancies and close this diagnostic gap. ...
... In TCC an insoluble matrix form of FN was found along the urothelian tissue, but studies revealed diverse results of FN expression in bladder cancer tissue (Kirkali et al. 2001; Sanchez-Carbayo, 2000; Cheng et al. 1988; Zhang et al. 2004; Fleischmann et al. 1993). FN seems to play a major role in TCC and the response to intravesical immunotherapy with Bacillus Calmette-Guérin (BCG) after tumor resection. ...
Reliable markers for both renal cell carcinoma (RCC) and transitional cell carcinoma of the bladder (TCC) are lacking.
During tumor progression and invasion components of extracellular matrix (ECM) are degraded and parts of these different components are detectable in plasma. Cellular fibronectin (cFN) represents a well characterized ECM protein. In contrast to fibronectin in plasma produced by hepatocytes (FN) cFN has a total extra domain sequence and occurs in much smaller amounts in the circulation. The aim of our study was to evaluate cFN as a marker and to determine its possible role in clinical staging of TCC and RCC.
Blood samples were collected from 30 patients before they underwent transurethral resection of the bladder because of newly diagnosed TCC. Additionally samples were collected from 69 patients with RCC before therapy. Sixty patients with non-malignant urological disorders were recruited as control group. Determination of cFN in plasma was performed by using a highly sensitive time-resolved fluorescence immunoassay (TRFIA).
The control group had median cFN plasma levels of 437 ng/ml. Patients suffering from TCC or RCC showed significantly higher cFN levels. In patients with muscle invasive TCC significant higher cFN levels (p < 0.05) could be demonstrated compared to non-muscle invasive TCC. Similar results were found in RCC with significant elevated cFN levels in metastatic RCC (p < 0.005) compared to localized stage of disease. No differences were found concerning tumor grading in both malignancies.
In the face of significant elevated cFN levels in TCC and RCC our data underline the important role of cFN. For future investigations the elevated cFN levels in locally progressed and metastastic disease, indicating a clinically useful tool for preoperative staging and postoperative monitoring, are of high interest.
... Some of these genes have been well characterized in lung cancer, for instance, NAPSA 29 is a common prognostic marker for LUAD, and KRT18, KRT7, and GPRC5A have been reported to play important roles in tumor progression and chemoresistance in various types of cancers. [30][31][32][33][34][35][36][37] Finally, we decided to focus on the functions of IRX2, SPINK13, and CAPN8, which have been implicated and not fully explored. ...
Background:
Single-cell transcriptomics has been used to investigate various tumors to elucidate the molecular distinction of all cell type compositions of a complex mix.
Aims:
This study aimed to investigate malignant-cell-specific genes to explore diagnostic and therapeutic biomarkers using single-cell transcriptomic data of lung adenocarcinoma.
Materials & methods:
10X single-cell RNA-seq data of fourteen patients with lung adenocarcinoma were analyzed. Genes that expressed differentially and those with higher confidence to distinguish tumor cells from normal cells were picked out using the ROC curves. The LASSO regression method was used to select most markedly correlated genes to predict the malignancy of every single cell within a model. We also conducted further experiments to determine their roles in lung cancer in vitro.
Results:
Twenty two thousand four hundred and ninety one tumor and 181 666 normal single cells were analyzed where 369 genes were found to be specifically expressed in single malignant cells. Seventy of them, encoding secreted or membrane-bound proteins, showed involvement in cell-to-cell communications in tumor biology. KRT18 and the other six genes were identified as predictors to distinguish single malignant cells and were integrated to construct an accurate (96.1%) predicting model. Notably, IRX2, SPINK13, and CAPN8 outperformed the other four genes. Further experiments confirmed the upregulation of them in lung adenocarcinoma at both tissue and cell levels. Proliferative capacities of lung adenocarcinoma cells were attenuated by knocking-down of either of them. However, targeting CAPN8, IRX2, or SPINK13 hardly exerted a cytotoxic effect on these cells.
Discussion:
Apart from the current model, similar tools were still warranted using single-cell RNA-seq data of more types of tumors. The three genes identified as potential therapeutic targets in the present study still need to be validated in more in lung cancer.
Conclusion:
Our model can aid the analyses of single-cell sequencing data. CAPN8, IRX2, and SPINK13 may serve as novel targets of targeted and immune-based therapies in lung adenocarcinoma.
... The cases were diagnosed clinically by consultant urologists at Al-Yarmook Teaching Hospital, and Baghdad Hospital for Specialists Surgeries. Urine samples were centrifuged at 1500 rpm, for 5 minutes ,the supernatant was frozen at -20ºC until the (HSP-70) measurement by ELISA [11]. HSP70 concentrations was quantitatively determined in urine of patients and healthy control subjects by means of ELISA (Enzyme Linked Immunosorbent Assay) using ready kits manufactured by USBiological company(USA) . ...
The present study aimed to shed light on the urine HSP70 concentration of patients with urinary bladder carcinoma UBC and control subjects as new urinary biomarker. The second aim was to associate this protein concentration with UBC stage and grade in patients with UBC. A direct ELISA was used to quantify urine HSP concentrations in 58 patients with urinary bladder carcinoma UBC with different grades (G) and stages (T) all malignant of them was transitional cell carcinoma (TCC) type , 15 from patients with urinary Bladder disorders other than cancer UBD and 15 healthy subjects(control) . Urine concentrations of HSP70 were elevated in patients with UBC compared to those without UBC (healthy and UBD, P< 0.5). There was a high significant between mean level of HSP70 in patients with T2 tumor stage as compared with patients with stage T3 . Also, there was a high significant increased mean level of HSP70 in patients with G3 as compared with patients with grad1 G1.
... [12] www.wjpr.net was frozen at -20ºC until use to measure the urine level of IL-6 [13] , and the deposits were used for general urine examination using both x10 and x40 objectives. A drop of the urine samples were applied to a glass microscope slides, allowed to air dry, stained with gram stain, and examined microscopically. ...
Urinary tract infection is a common health problem, affecting millions of women especially pregnant women each year. The present study aimed to shed light on microbial infection and IL-6 urine levels proinflammotory cytokine in pregnant women. Urine samples from 70 pregnant women and 20 urine samples from non-pregnant women with urinary tract infection as a (control) were included in this study. Urinary tract infections were studied for all groups by culturing urine samples using specific culture media. The incidence ratio of positive urine culture in pregnant women was 52 (74.3 %) while 18(25.7 %) was the incidence of negative urine culture. The frequency of the different microorganisms causing UTI in patient groups were studied, in which Gram-negative, particularly Escherichia coli showed the highest incidence among them (28.9%). Also, the age group 20-29 years had the highest incidence of infection (82.8 %), and the urine levels of IL-6 were estimated by ELISA , the results showed significant different in IL-6 mean levels between trimester pregnancy groups and control group (p ≤0.01). Results indicated microbial infection was high in pregnant women of Thi-qar province, so urine microbial screening should be done in routine checkups for pregnant women.
... [12] www.wjpr.net was frozen at -20ºC until use to measure the urine level of IL-6 [13] , and the deposits were used for general urine examination using both x10 and x40 objectives. A drop of the urine samples were applied to a glass microscope slides, allowed to air dry, stained with gram stain, and examined microscopically. ...
Urinary tract infection is a common health problem, affecting millions of women especially pregnant women each year. The present study aimed to shed light on microbial infection and IL-6 urine levels proinflammotory cytokine in pregnant women. Urine samples from 70 pregnant women and 20 urine samples from non-pregnant women with urinary tract infection as a (control) were included in this study. Urinary tract infections were studied for all groups by culturing urine samples using specific culture media. The incidence ratio of positive urine culture in pregnant women was 52 (74.3 %) while 18(25.7 %) was the incidence of negative urine culture. The frequency of the different microorganisms causing UTI in patient groups were studied, in which Gram-negative, particularly Escherichia coli showed the highest incidence among them (28.9%). Also, the age group 20-29 years had the highest incidence of infection (82.8 %), and the urine levels of IL-6 were estimated by ELISA , the results showed significant different in IL-6 mean levels between trimester pregnancy groups and control group (p ≤0.01). Results indicated microbial infection was high in pregnant women of Thi-qar province, so urine microbial screening should be done in routine checkups for pregnant women.
... In addition, a number of other solid tumors express midkine at high levels, and its expression increases with advancing tumor stage (15,33). The low benefit of midkine as a diagnostic marker for low-grade pTa and pTIS tumors is unfortunate but comparable to other urine-based protein markers, including cytokeratin 18 (34) and survivin (35). However, as 70% of patients with NMIBC have pTa tumors and recurrence is the main problem in these patients (3), urinary midkine alone is not reliable as a diagnostic marker for the surveillance of patients with a history of BCa. ...
As it has been demonstrated previously that midkine (also known as neurite growth-promoting factor 2) protein levels in urine of bladder cancer (BCa) patients are increased compared to healthy controls, the present study validated the diagnostic utility of midkine in an independent patient cohort and compared the observed values with voided urine cytology (VUC), which is the current reference standard for non-invasive diagnosis of BCa. Voided urine samples were prospectively collected from 92 BCa patients and 70 control subjects. Protein levels of midkine were assessed using a commercially available enzyme-linked immunosorbent assay and normalized to urinary creatinine. The diagnostic performance of urinary midkine was evaluated by receiver operating characteristic curves. The best combinations of sensitivities and specificities were determined by Youden’s Index. Midkine concentrations were significantly elevated in urine samples from BCa patients compared to controls (P<0.001; Mann-Whitney U Test). The level of midkine was associated with disease progression, with the highest concentrations in urine specimens of patients with pT1 and ≥pT2a, as well as high-grade tumors (P<0.001; Mann-Whitney U test). Sensitivities of urinary midkine and VUC were 69.7 and 87.6%, respectively. The corresponding specificities for midkine and VUC were 77.9 and 87.7%, respectively. The combined use of VUC and midkine improved the sensitivity to 93.3%, but reduced the specificity to 66.2%. Despite its reduced discriminatory power for low-grade and low-stage BCa, urinary midkine can be utilized for the identification of high-grade pT1 and ≥pT2a tumors. This means that midkine may potentially be suitable for the identification of patients with high risk BCa.
... The utility of urinary fibronectin for detecting bladder cancer reported in 2 unequivocal studies [14,15] is supported by 9 "equivocal" studies: 5 of these studies present moderately high sensitivities and specificities with weighted means across the studies of 82.5% and 80.2%, respectively (390 cases and 520 controls) [16][17][18][19][20]. Although there is substantial evidence that increased urinary fibronectin is indicative of bladder cancer, Alias-Melgar [21] found that urinary fibronectin is increased in urolithiasis, and Eissa [17] reported that in side-by-side comparison NMP22 slightly outperforms fibronectin. ...
For over 80 years, cystoscopy has remained the gold-standard for detecting tumours of the urinary bladder. Since bladder tumours have a tendency to recur and progress, many patients are subjected to repeated cystoscopies during long-term surveillance, with the procedure being both unpleasant for the patient and expensive for healthcare providers. The identification and validation of bladder tumour specific molecular markers in urine could enable tumour detection and reduce reliance on cystoscopy, and numerous classes of biomarkers have been studied. Proteins represent the most intensively studied class of biomolecule in this setting.
As an aid to researchers searching for better urinary biomarkers, we report a comprehensive systematic review of the literature and a searchable database of proteins that have been investigated to date. Our objective was to classify these proteins as: 1) those with robustly characterised sensitivity and specificity for bladder cancer detection; 2) those that show potential but further investigation is required; 3) those unlikely to warrant further investigation; and 4) those investigated as prognostic markers. This work should help to prioritise certain biomarkers for rigorous validation, whilst preventing wasted effort on proteins that have shown no association whatsoever with the disease, or only modest biomarker performance despite large-scale efforts at validation.
... While cytology is sensitive (70-80%) and highly specific (90-95%) for diagnosis of high-grade of bladder cancer , its sensitivity is as low as 6-38% for low grade tumors . Cancer often develops with an associated local inflammatory response [10] . Because the urine of patients with BC is in close contact with tumor cells and adjacent inflamed urothelium, we hypothesized that immune mediators in urine may serve as biomarkers for BC. ...
The present study aimed to shed light on the urine HSP70 concentration of patients with urinary bladder carcinoma UBC and control subjects as new urinary biomarker. The second aim was to associate this protein concentration with UBC stage and grade in patients with UBC. A direct ELISA was used to quantify urine HSP concentrations in 58 patients with urinary bladder carcinoma UBC with different grades (G) and stages (T) all malignant of them was transitional cell carcinoma (TCC) type , 15 from patients with urinary Bladder disorders other than cancer UBD and 15 healthy subjects(control). Urine concentrations of HSP70 were elevated in patients with UBC compared to those without UBC (healthy and UBD, P< 0.5). There was a high significant between mean level of HSP70 in patients with T2 tumor stage as compared with patients with stage T3. Also, there was a high significant increased mean level of HSP70 in patients with G3 as compared with patients with grad1 G1.
... So it was concluded that the urine fibronectin measurement is useful to differentiate normal subjects from subjects with bladder cancer. This result is consistent with the results of other studies (8,(23)(24)(25) . Although each author found its own cutoff value, most of them conclude that urine Fibronectin measurement is important to discriminate bladder cancer subjects from normal subjects. ...
Background: Accurate and sensitive detection of bladder cancer is important to diagnose this deadly disease at an early stage, estimate prognosis, prediction the response to treatment and for monitoring the recurrence. In past few years, laboratory diagnosis and surveillance of urinary bladder cancer have improved significantly. Although, urine cytology remains the gold standard test, many new urinary biochemical markers have been identified.
Objectives : To evaluate the value of fibronectin in serum and urine to detect bladder cancer in different grades and stages.
Methods : A case-control study was conducted at chemistry and biochemistry department, college of medicine, Al-Nahrain University-Baghdad-Iraq from September 2012 to August 2013. Thirty five patients diagnosed as bladder cancer with mean age 61.94±11.66 years and thirty five aged-matched healthy volunteers as control group were included in this study. Serum and urinary Fibronectin were measured by ELISA technique.
Results : The mean ±SEM serum and urine levels of fibronectin in patients with bladder cancer (33.11±1.90µg/ml; 33.08±1.12 ng/ml respectively) were significantly higher than the levels in control group (8.57±1.10µg/ml; 7.58±1.00 respectively). When using a serum fibronectin concentration of 25.65µg/ml as a cut off value for the diagnosis of bladder cancer, sensitivity was 71.4%, specificity 100%, the positive predictive value was 100% and the negative predictive value 77.78%, and the sensitivity and specificity of urine fibronectin were (94.3%, 97.1% respectively) when using a urine fibronectin concentration of 20.00ng/ml as a cut off value for the diagnosis of bladder cancer. The positive predictive value was 97.05%, and the negative predictive value was 94.44%. There was no correlation between serum and urine fibronectin with grade and stage of tumor of bladder cancer patients.
Conclusions: The measurement of fibronectin level in serum and urine is useful in discriminating bladder cancer patients from normal subjects.
... 58 Several biomarkers (such as NMP22, Calreticulin, Clusterin, CystatinB, Proepithelin, UHRF1, a-1B-Glycoprotein, PCA3, and Cathepsin D) for urinary tract carcinoma, prostate cancer, and bladder cancer have been successfully identified in urine. [59][60][61][62] An interesting phenomenon observed in frozen/thawed urine is the formation of a precipitant. 63 The precipitates, which are usually discarded before analysis, are determined to be calcium oxalate dihydrate and amorphous calcium crystals. ...
Fluid biospecimens (blood, serum, urine, saliva, cerebrospinal fluid and bronchial lavage fluid) contain not only cells and subcellular components, but also proteins, enzymes, lipids, metabolites, and peptides, which are utilized as biomarkers. Availability of high-quality biospecimens is a requirement for biomarker discovery. The quality of the biospecimens depends upon preanalytical variables (ie, collection and processing techniques, freeze/thaw stability, and storage stability), which account for >60%-90% of the diagnostic errors. Currently, millions of fluid biospecimens are stored in hundreds of biorepositories across the nation, and tens of thousands of new biospecimens are added to the pool daily. Specimen stabilization is imperative, because fluid biospecimens degrade quickly when kept untreated at room temperature. Achieving a high-quality fluid biospecimen requires understanding the effects of storage processing parameters (eg, freezing and thawing as well as cryo-/lyoprotectant additives) and storage conditions on biomarkers contained within the biospecimens. In this article, we will discuss the main issues related to the stabilization of specific biofluids by reviewing (a) the current preservation and storage practices applied in biobanks/biorepositories and (b) the sensitivity of certain biomarkers to current storage techniques.
... Studies on urinary tract tumours comprise the majority of reports concerning UFN (10)(11)(12)(13)(14)(15)(16)(17). In a rare study on other types of cancer, Katayama et al found that UFN levels were significantly elevated in patients with various types of cancer, and were extremely elevated in certain patients with distant metastasis. ...
We measured the serum and urinary levels of fibronectin (FN) in 113 patients with colorectal cancer (CRC) - 15 with synchronous hepatic metastases, 21 with metachronous hepatic metastases and 77 with no hepatic metastases - as well as in 40 controls, with the aim of determining if FN can be used as a marker of CRC invasion or metastasis and its clinical significance. Urinary FN levels were significantly higher in patients with CRC than in the controls, and both urinary and serum FN levels rose with cancer progression. Patients positive for FN tended to have a more advanced disease. High levels of FN expression in both urine and serum showed a sensitivity of 80%, specificity of 33.3%, accuracy of 66.6% and positive predictive value of 75% for the diagnosis of metachronous hepatic metastases. These results indicate that FN levels increase with the progression of CRC, that FN expression in urine and serum is a useful marker of the degree of disease advancement, and that FN may play a part in cancer growth and development.
... FGFR3 mutation detection may in the future provide a useful tool in the standard management of patients with low-grade papillary bladder tumors (228, 316, 318 ). The NACB panel recommends that this should be studied further in prospective clinical trials.321, 322 ) and human chorionic gonadotropin (HCG) -subunit and -core (protein and mRNA transcript) may also be markers for transitional cell carcinoma of the bladder (323 ). ...
Updated National Academy of Clinical Biochemistry Laboratory Medicine Practice Guidelines for the use of tumor markers in the clinic have been developed.
Published reports relevant to use of tumor markers for 4 cancer sites--liver, bladder, cervical, and gastric--were critically reviewed.
Alpha-fetoprotein (AFP) may be used in conjunction with abdominal ultrasound for early detection of hepatocellular carcinoma (HCC) in patients with chronic hepatitis or cirrhosis associated with hepatitis B or C virus infection. AFP concentrations >200 microg/L in cirrhotic patients with typical hypervascular lesions >2 cm in size are consistent with HCC. After a diagnosis of HCC, posttreatment monitoring with AFP is recommended as an adjunct to imaging, especially in the absence of measurable disease. Although several urine markers have been proposed for bladder cancer, none at present can replace routine cystoscopy and cytology in the management of patients with this malignancy. Some may, however, be used as complementary adjuncts to direct more effective use of clinical procedures. Although carcinoembryonic antigen and CA 19-9 have been proposed for use gastric cancer and squamous cell carcinoma antigen for use in cervical cancer, none of these markers can currently be recommended for routine clinical use.
Implementation of these recommendations should encourage optimal use of tumor markers for patients with liver, bladder, cervical, or gastric cancers.
... Urine is also an important source of information for bladder and prostate cancers. The levels of several proteins in urine are mea-sured as markers for bladder cancers as well as those in blood, such as bladder tumor antigen (BTA) [9], nuclear matrix protein 22 [10], urinary fibronectin (FN) fragments [11,12], and fibrinogen degradation products [13]. In view of the above considerations, the main goal of urinary proteomics is the individuation of a disease and/or of biological markers; the comparison of a normal proteome with the proteome from patients with a defined disease can detect proteins expressed differentially from one another. ...
Protein analysis in biological fluids, such as urine, by means of mass spectrometry (MS) still suffers for insufficient standardization in protocols for sample collection, storage and preparation. In this work, the influence of these variables on healthy donors human urine protein profiling performed by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was studied. A screening of various urine sample pre-treatment procedures and different sample deposition approaches on the MALDI target was performed. The influence of urine samples storage time and temperature on spectral profiles was evaluated by means of principal component analysis (PCA). The whole optimized procedure was eventually applied to the MALDI-TOF-MS analysis of human urine samples taken from prostate cancer patients. The best results in terms of detected ions number and abundance in the MS spectra were obtained by using home-made microcolumns packed with hydrophilic-lipophilic balance (HLB) resin as sample pre-treatment method; this procedure was also less expensive and suitable for high throughput analyses. Afterwards, the spin coating approach for sample deposition on the MALDI target plate was optimized, obtaining homogenous and reproducible spots. Then, PCA indicated that low storage temperatures of acidified and centrifuged samples, together with short handling time, allowed to obtain reproducible profiles without artifacts contribution due to experimental conditions. Finally, interesting differences were found by comparing the MALDI-TOF-MS protein profiles of pooled urine samples of healthy donors and prostate cancer patients. The results showed that analytical and pre-analytical variables are crucial for the success of urine analysis, to obtain meaningful and reproducible data, even if the intra-patient variability is very difficult to avoid. It has been proven how pooled urine samples can be an interesting way to make easier the comparison between healthy and pathological samples and to individuate possible differences in the protein expression between the two sets of samples.
... As above, urinary MK levels were found to increase in about 70% of patients with various carcinomas. In case of bladder carcinomas, urinary tumor markers have been frequently used: they are urinary bladder carcinoma antigen, CYFR 21-1, nuclear matrix protein, telomerase , cytokeratin 18, bladder fibronectin, matrix metalloproteases, and proteins detected by mass spec- trometry20212223242526. In addition, urinary metabolites are used as markers of neuroblastomas [27] and some markers of bone metastasis are present [28,29] . ...
Midkine (MK) is a heparin-binding growth factor, which promotes growth, migration, and survival of various cells, and MK expression is increased in many human carcinomas. We determined the urinary MK level by enzyme-linked immunoassay. Taking 311pg/mg creatinine as a cut-off level, 70% of patients with various carcinomas (n=142) gave positive values, while only 5.5% of healthy volunteers (n=330) did. In case of gastric carcinoma, 17 out of 21 patients with stage 1 tumor were positive. Urinary MK levels are expected to become a convenient marker as an aid in detection of tumors.
Simple Summary
Bladder cancer (BC) is one of the most common cancers worldwide. Early-stage diagnosis is associated with better survival rates and, as such, the timely referral of suspected cases is paramount. Urinary biomarkers have been developed to aid diagnosis, and are largely tested in patients who have been referred for further investigation. Evidence, however, on their diagnostic performance for both detecting and ruling out BC, especially in the general population, is limited. In this review, we systematically identified studies reporting on the diagnostic performance of biomarkers suitable for use in primary and community care settings. Three biomarkers, with relatively little difference in diagnostic performance between them, and some novel biomarkers were identified showing potential to be used as a triage tool in such settings. While promising, further validation studies in the general population are needed.
Abstract
Evidence on the use of biomarkers to detect bladder cancer in the general population is scarce. This study aimed to systematically review evidence on the diagnostic performance of biomarkers which might be suitable for use in community and primary care settings [PROSPERO Registration: CRD42021258754]. Database searches on MEDLINE and EMBASE from January 2000 to May 2022 resulted in 4914 unique citations, 44 of which met inclusion criteria. Included studies reported on 112 biomarkers and combinations. Heterogeneity of designs, populations and outcomes allowed for the meta-analysis of three biomarkers identified in at least five studies (NMP-22, UroVysion, uCyt+). These three biomarkers showed similar discriminative ability (adjusted AUC estimates ranging from 0.650 to 0.707), although for NMP-22 and UroVysion there was significant unexplained heterogeneity between included studies. Narrative synthesis revealed the potential of these biomarkers for use in the general population based on their reported clinical utility, including effects on clinicians, patients, and the healthcare system. Finally, we identified some promising novel biomarkers and biomarker combinations (N < 3 studies for each biomarker/combination) with negative predictive values of ≥90%. These biomarkers have potential for use as a triage tool in community and primary care settings for reducing unnecessary specialist referrals. Despite promising emerging evidence, further validation studies in the general population are required at different stages within the diagnostic pathway.
Background:
Prostate cancer is the most common cancer in American men that ranges from low risk states amenable to active surveillance to high-risk states that can be lethal especially if untreated. There is a critical need to develop relatively non-invasive and clinically useful methods for screening, detection, prognosis, disease monitoring, and prediction of treatment efficacy. In this review, we focus on important advances as well as future efforts needed to drive clinical innovation in this area of urine biomarker research for prostate cancer detection and prognostication.
Methods:
We provide a review of current literature on urinary biomarkers for prostate cancer. We evaluate the strengths and limitations of a variety of approaches that vary in sampling strategies and targets measured; discuss reported urine tests for prostate cancer with respect to their technical, analytical, and clinical parameters; and provide our perspectives on critical considerations in approaches to developing a urine-based test for prostate cancer.
Results:
There has been an extensive history of exploring urine as a source of biomarkers for prostate cancer that has resulted in a variety of urine tests that are in current clinical use. Importantly, at least three tests have demonstrated high sensitivity (~90%) and negative predictive value (~95%) for clinically significant tumors; however, there has not been widespread adoption of these tests.
Conclusions:
Conceptual and methodological advances in the field will help to drive the development of novel urinary tests that in turn may lead to a shift in the clinical paradigm for prostate cancer diagnosis and management.
The urinary tract is one of the most frequent sites for happening of bacterial infections, especially in women. Common bacteria related with urinary tract infection were isolated and identified from 400 women patients aged from 14 to 60 years in Azadi Teaching Hospital and General Kirkuk Hospital. The levels of immunoglobulins (IgM, IgG and IgA) and complement protein C3 were evaluated in women infected by Escherichia coli and non infected women with UTI. The results refer to the dominance of Escherichia coli and high significant (P ≤ 0.001) was observed for levels of immunoglobulins IgM, IgG and IgA that ranged (295.91 ± 12.51, 1758.77 ± 28.52 and 473 ± 14.67) mg/dl respectively. Also there was significant increased (P ≤ 0.001) in level of complement protein C3 ranged (195.13 ± 10.53) mg/dl.
Objectives:
To investigate the relationship between chronic kidney disease and primary non-muscle-invasive bladder cancer.
Methods:
Disease outcomes were analyzed in 418 patients treated with transurethral resection for primary non-muscle-invasive bladder cancer, and were correlated to traditional risk factors as well as chronic kidney disease stage according to estimated glomerular filtration rate: ≥60 (G1-2), 45-59 (G3a) or <45 (G3b-5).
Results:
The median follow-up time was 40.0 months. There were 287 (68.7%), 98 (23.4%), and 33 (7.9%) patients with G1-2, G3a and G3b-5 chronic kidney disease, respectively. T1 tumor was present in 29.6% of G1-2, 43.9% of G3a and 51.4% of G3b-5 chronic kidney disease (P = 0.004). The proportion of histological grade 3 non-muscle-invasive bladder cancer was higher in G3a and G3b-5 than G1-2 (P < 0.001). Higher chronic kidney disease stage was associated with worse recurrence-free (P < 0.001) and progression-free survival (P = 0.017). In multivariable analysis, G3b-5 was found to be an independent predictor for recurrence (hazard ratio 1.87; P = 0.004) and progression (hazard ratio 2.96; P = 0.019). Chronic kidney disease stage was also strongly associated with the European Association of Urology bladder cancer risk groups (P < 0.001), and with shorter time to recurrence and progression in each group.
Conclusions:
Chronic kidney disease predicts the clinical outcome of primary non-muscle-invasive bladder cancer. Adding chronic kidney disease to the conventional risk factors might increase the accuracy of risk stratification.
Objective: To investigate the clinical significance of cFN concentrations in gastric carcinoma and precancerous diseases of stomach, and the relationship between the serum cFN concentrations and HP infection. Methods: ELISA was used to detect the serum cFN concentrations in 1177 normal subjects, precancerous diseases of stomach and gastric cancer, and to detect the serum antibodies concentrations and anti-HP in all kinds of gastric diseases. Results: The median concentrations of the serum cFN of gastric cancer were significantly high than normal subjects and gastric benign diseases (P<0.05). There were significantly different between the differentiated gastric cancer and the undifferentiated gastric cancer and were not significantly different between the positive and negative groups of lymphatic metastasis. The difference of the serum cFN median concentrations was not significant between IG-HP negative group and IG-HP positive group in all kinds gastric diseases. But the serum cFN median concentrations of the IG-HP positive group were higher than the IG-HP negative group and gastric cancer group was opposite to the above results. Conclusions: The serum cFN concentrations had a clinically useful potential value for identification between gastric cancer and gastric precancerous diseases. The serum cFN concentrations were related with differentiated degree of gastric cancer and were no statistically significant with metastasis of lymphatic node. No statistical difference is found between the IG-HP negative and the HP positive group in all kinds of gastric diseases. But the serum cFN median concentration in IG-HP positive group is higher than in IG-HP negative group gastric benign diseases group, and gastric cancer group was opposite to the above result. It showed that HP played a difference role in the step of the gastric precancer diseases and gastric cancer, and this will be the focus of further studies.
Zusammenfassung
In diesem Artikel wird die aktuelle Rolle der Tumormarker beim Harnblasentumor in Diagnostik und Therapie vorgestellt. Die derzeit wichtigsten und interessantesten Tumormarker werden besonders hervorgehoben und ihr Einsatz für den klinischen Alltag diskutiert. Eine Medline-basierte Literaturrecherche wurde auf diesem Gebiet durchgeführt. Weitere Entwicklungen von Tumormarkern bei Rezidivtumoren und progressiven Krankheitsverläufen werden es in Zukunft möglich machen, Therapieoptionen für den einzelnen Patienten zu entwickeln. Molekulares Staging urologischer Tumoren kann Fälle selektieren, die von systemischen Therapien am besten profitieren. Es ist notwendig und wichtig, Grundlagenforschung und klinische Studien unter denselben Gesichtspunkten zu vereinen.
This article outlines the role of bladder cancer tumor markers in diagnosis and therapy with a particular focus on the most important biomarkers. A MEDLINE based literature search was performed to examine the field of bladder cancer markers. Further determination of recurrence and progression markers will contribute to establish better treatments for the individual patient. Molecular staging of urological tumors will allow selecting cases that will require systemic treatment. Therapeutically, knowledge of cancer progression pathways allows for drug therapies against specific tumor targets. It is necessary and important to integrate under the same objectives basic translational and clinical research.
Purpose/Objectives: To review the clinical use of tumor markers in select cancers and highlight future directions in tumor marker development. Data Sources: Guidelines from national and international societies, scientific literature, and Internet resources. Data Synthesis: Tumor markers are important tools in the management of cancer. Sequencing of the human genome has led to new tumor marker development in the fields of proteomics and DNA microarray technologies. Conclusions: Tumor marker technology is expanding rapidly; almost a dozen tumor markers currently are being used in the oncology arena, with many more in development. The use of tumor markers can be controversial, particularly because guidelines have not been established for all of the markers. Implications for Nursing: Oncology nurses need to be well versed in the use of tumor markers to educate and counsel patients with cancer.
OBJECTIVE To identify whether integrin1 subunit is responsible for the resistance of bladder cancer cell to the therapeutic drug mitomycin-C (MMC), when grown on fibronectin (FN). MATERIALS AND METHODS The expression of integrin1 on bladder cancer T24 and 5637 cells was examined by the flow cytometer. The adhesion of cells to plates with the absence or presence of FN was determined. Analysis of apoptosis induced by MMC was assessed using the flow cytometer in combination with an integrin1-blocking antibody or siRNA targeting the coding region of integrin1. Western blot was used to study the expression change of integrin1 and its downstream molecules. RESULTS Bladder cancer T24 and 5637 cells express high level of integrin1 (87.3% 2.3 and 90.1% 1.9, respectively). Cellular adhesion to FN was significantly reduced by the blocking of integrin1. Blocking or silencing of integrin1 significantly abolished the drug resistance of cells grown on FN to MMC (P .05) and inhibited the activation of survival signals phosphoinosi-tide-3 kinase (PI3-K)/Akt. CONCLUSION Integrin1-mediated cellular adhesion to FN confers drug resistance to MMC on bladder cancer cells. Knockdown of integrin1 may abolish the drug resistance phenotype and sensitize bladder cancer cells to MMC. UROLOGY 79: 638 – 643, 2012. © 2012 Elsevier Inc. B ladder cancer is a common malignancy, accounting for 7% of all cancers and presenting the eighth highest cancer-related mortality rate in American men. In 2009, approximately 70,980 cases were newly di-agnosed with bladder tumor, and there were about 14,330 deaths caused by this disease in the United States. 1 Al-though the incidence of bladder cancer has been gradually rising over the past 3 decades, nearly 80% of diagnosed patients present with the superficial form, 2 now described as non–muscle-invasive bladder cancer (NMIBC). These pa-tients are generally treated by transurethral resection for diagnosis and local control followed by intravesical chemo-therapy or immunotherapy for elimination of residual tumor and prevention of recurrence. Several chemotherapeutic agents, such as thiotepa, mitomycin-C (MMC), doxorubi-cin, and epirubicin, have been proven to reduce intravesical recurrence when administrated intravesically. 3 Of those, MMC, an antitumor antibiotic whose mode of action is mainly attributable to the formation of cross-linking with DNA, is a well-known chemotherapeutic agent for the intravesical therapy of NMIBC. The efficacy of MMC in-stillation therapy varies significantly among several large studies. 4-6 In 2 large-scale studies, the recurrence rate and the progression rate was as high as 46.4% 4 and 9.44% 7 in the patients treated with MMC, respectively. Drug resis-tance remains the major obstacle in adjuvant chemotherapy of bladder cancer. In a previous study, we showed that cell adhesion to fibronectin (FN), an extracellular matrix pro-tein, protects bladder cancer T24 cells from apoptosis in-duced by chemotherapeutic agent MMC. 8 Although the intracellular signal transduction has been fully elucidated, it remains unclear which subunit of the integrin located in the cell membrane initiated the signal pathway. In the present study we provided evidence that integrin1-mediated cell adhesion to FN protects bladder cancer cells from MMC-induced apoptosis. Further, silencing of integrin1 gene– sensitized bladder cancer cells to adjuvant chemotherapy may be a novel strategy.
Up to now markers for transitional cell carcinoma of the bladder (TCC) are missing. Fibronectin (FN) seems to play a key role in progression and invasion of malignant tumors. The aim of this study was to assess the value of cellular FN (cFN), a more specific subform of produced FN, in different stages of TCC.
cFN was determined using a highly sensitive immunoassay which we developed. Blood samples were taken of 45 patients with the first diagnosis of TCC before undergoing TUR-B and 6 patients with metastatic TCC before chemotherapy; 70 patients with nonmalignant urological disorders served as a control group.
Patients with TCC showed significantly elevated cFN plasma levels compared to controls (p<0.05). Patients with muscle-invasive disease (n=15) showed significantly higher cFN plasma levels compared to the group with superficial TCC. Patients with metastatic TCC showed the highest, but not significantly elevated cFN plasma levels compared to patients with muscle-invasive TCC.
The elevated cFN plasma levels in TCC underline the important role of cFN for tumor progression and its potential role as a marker for TCC. Upcoming investigations are necessary to prove the value of the potential marker cFN during follow-up and its impact as a prognostic factor for recurrence and progression of TCC.
To identify whether integrinβ1 subunit is responsible for the resistance of bladder cancer cell to the therapeutic drug mitomycin-C (MMC), when grown on fibronectin (FN).
The expression of integrinβ1 on bladder cancer T24 and 5637 cells was examined by the flow cytometer. The adhesion of cells to plates with the absence or presence of FN was determined. Analysis of apoptosis induced by MMC was assessed using the flow cytometer in combination with an integrinβ1-blocking antibody or siRNA targeting the coding region of integrinβ1. Western blot was used to study the expression change of integrinβ1 and its downstream molecules.
Bladder cancer T24 and 5637 cells express high level of integrinβ1 (87.3% ± 2.3 and 90.1% ± 1.9, respectively). Cellular adhesion to FN was significantly reduced by the blocking of integrinβ1. Blocking or silencing of integrinβ1 significantly abolished the drug resistance of cells grown on FN to MMC (P <.05) and inhibited the activation of survival signals phosphoinositide-3 kinase (PI3-K)/Akt.
Integrinβ1-mediated cellular adhesion to FN confers drug resistance to MMC on bladder cancer cells. Knockdown of integrinβ1 may abolish the drug resistance phenotype and sensitize bladder cancer cells to MMC.
Up to now markers for transitional cell carcinoma of the bladder (TCC) are missing. Fibronectin (FN) seems to play a key role in progression and invasion of malignant tumors. The aim of this study was to assess the value of cellular FN (cFN), a more specific subform of produced FN, in different stages of TCC.cFN was determined using a highly sensitive immunoassay which we developed. Blood samples were taken of 45 patients with the first diagnosis of TCC before undergoing TUR-B and 6 patients with metastatic TCC before chemotherapy; 70 patients with nonmalignant urological disorders served as a control group.Patients with TCC showed significantly elevated cFN plasma levels compared to controls (p<0.05). Patients with muscle-invasive disease (n=15) showed significantly higher cFN plasma levels compared to the group with superficial TCC. Patients with metastatic TCC showed the highest, but not significantly elevated cFN plasma levels compared to patients with muscle-invasive TCC.The elevated cFN plasma levels in TCC underline the important role of cFN for tumor progression and its potential role as a marker for TCC. Upcoming investigations are necessary to prove the value of the potential marker cFN during follow-up and its impact as a prognostic factor for recurrence and progression of TCC.
The major advantages of urine-based assays are their noninvasive character and ability to monitor prostate cancer with heterogeneous foci. Almost all urine-detectable prostate-specific markers have been recently reviewed. For this reason, we focus here on only a few promising markers which have been independently evaluated (in particular PCA3, fusion genes, TERT, AMACR, GSTP1, MMP9 and VEGF) and very recent ones (ANXA3 and sarcosine). The emphasis is also on multiplex biomarker analysis and on microarray-based analysis of fusion genes. A combination of multiple urine biomarkers may be valuable in the case of men with persistently elevated serum prostate-specific antigen and a history of negative biopsies. The emerging urine tests should help in both early diagnosis of prostate cancer and identifying aggressive tumors for radical treatment.
To assess the expression rate of a cancer-related gene, hepatoma-up-regulated protein (HURP), in the tumor tissue of transitional cell carcinoma (TCC), and to assess the potential suitability of using this gene as a novel molecular marker for detecting TCC in voided urine.
Total RNA was extracted from 80 TCC tissue samples of the urinary tract. Forty-five of the 80 tumor-adjacent tissue samples were from the same patients and 15 were from control subjects (patients with benign prostatic hyperplasia). The expression levels of HURP mRNA in these specimens were examined using reverse transcriptase-polymerase chain reaction. The HURP mRNA transcripts in voided urine pellets from 14 additional patients were determined using the same method. The messages were normalized to beta-actin mRNA.
The detection of HURP expression in the TCC tissue samples had a sensitivity of 88.8% (71 of 80) and a specificity of 100% (15 of 15). Ten of the 45 grossly tumor-adjacent tissue samples expressed HURP mRNA, which may indicate subtle genetic changes in tissue adjacent to tumor. All seven urine specimens from the patients with TCC revealed HURP expression; however, no specimens from patients with nonmalignant diseases did so.
A potential molecular marker for detecting TCC with tissue specimens and voided urine samples has been found. Although the real clinical application of this marker requires additional evaluation, the high sensitivity and specificity of the HURP gene amplification method warrants more investigation in the future.
We evaluate the diagnostic efficacy of nuclear matrix protein-22 (NMP22, Matritech, Newton, Massachusetts), fibronectin and urinary bladder cancer antigen (UBC, IDL Biotech, Borlange, Sweden) compared with voided urine cytology in the detection of bladder cancer.
A total of 168 patients provided a single voided urine sample for NMP22, fibronectin an ideal monoclonal for urinary bladder cancer and cytology before cystoscopy. Cystoscopy was done for all patients as the reference standard for identification of bladder cancer. Biopsy of any suspicious lesion was performed for histopathological examination. Of the 168 cases 100 were histologically diagnosed as bladder cancer, whereas the remaining 68 had benign urological disorders. A group of 47 healthy volunteers were also enrolled in this study. Voided urine was evaluated by NMP22, fibronectin and UBC, and their values were expressed relative to mg. creatinine.
The optimal threshold values for NMP22, fibronectin and UBC were calculated by receiver operator characteristics curves as 27 units per mg. creatinine, 198 mg./mg. creatinine and 13 ng./mg. creatinine, respectively. The levels and positive rates of the 3 parameters were significantly higher in the malignant group compared to either the benign group or normal controls. Of the entire group NMP22, fibronectin and UBC were positive in 93.2%, 91% and 68.2%, respectively in bladder cancer cases with positive cytology. Moreover, these positive rates were significantly higher in bilharzial bladder cancer cases (58.8%, 67.5%, 58.8%, respectively) compared to nonbilharzial cases (35.6%, 36.3%, 31.1%). Overall sensitivity and specificity were 85% and 91.3% for NMP22, 83% and 82.6% for fibronectin, 67% and 80.8% for UBC and 44% and 100% for voided urine cytology. Combined sensitivity of voided urine cytology with the 3 biomarkers together was higher than either combined sensitivity of voided urine cytology with 1 of the biomarkers or than that of the biomarker alone.
Our data indicate that NMP22 and fibronectin had superior sensitivities compared to UBC and voided urine cytology, while NMP22 and voided urine cytology had the highest specificities. The combined use of markers increased the sensitivity of cytology from 44% to 95.3%. The higher sensitivities of markers in bilharzial than nonbilharzial bladder cancer highlight their clinical use in screening patients with urinary bilharziasis.
Numerous markers have been described to correlate to some extent with tumor stage and prognosis of patients with bladder cancer. The power of many of these biomarkers in detecting superficial disease or predicting the clinical outcome of individual tumors is limited, and alternative markers are still in demand. High-throughput microarrays represent novel means for cancer research and tumor marker discovery.
The aim of this report was to discuss the application of DNA technologies to provide novel biomarkers for bladder cancer.
Specific bladder tumor subtypes have distinct gene expression profiles. The use of high-throughput DNA microarrays allows identification of the most prevalent and relevant alterations within bladder tumors. Clusters of differentially expressed genes will become biomarkers to discriminate subgroups of patients with different histopathology or clinical outcome. Additionally, the identified individual molecular targets might be further validated and developed into novel serum or urinary biomarkers for the diagnosis and/or as prognostic factors to be applied in clinical practice. The diagnosis and prognosis of bladder cancer would be enhanced by the use of such markers, and the marker itself may constitute a therapeutic target when studied in appropriate patients and control groups.
Expression profiling with high-throughput DNA microarrays has the potential of providing critical clues for the management of bladder cancer patients. As the quality, standardization, and ease of use of the technology increase and the costs decrease, DNA microarrays will move from being a technology restricted to research to clinical laboratories in the near future.
Various tumor markers for transitional cell carcinoma (TCC) of the bladder have been described, but none of them are used in clinical routine. Fibronectin, a glycoprotein, seems to play a very important role in both the progression and invasion of cancer. The aim of this study was to evaluate cellular fibronectin (cFN) in the urine and blood of patients with TCC of the bladder and to determine its possible role as a tumor marker and prognostic factor. Morning urine samples and blood were collected from 20 patients (8 women, 12 men, mean age 69.9 years) before they underwent transurethral resection of bladder tumors (TURB). Twenty patients (10 women, 10 men, mean age 63.4 years) with nonmalignant urological disorders were recruited as the control group. Determination of cFN in plasma and urine was performed by using a newly developed time-resolved fluorescence immunoassay (TRFIA). Patients with nonmalignant diseases had mean cFN plasma levels of 404 ng/ml (range 181-746 ng/ml). Patients with TCC of the bladder showed significantly higher cFN plasma levels of 686 ng/ml (range 274-1999 ng/ml, p<0.05). Subdivided according to the TNM system, muscle-invasive bladder tumors (n=5) demonstrated higher cFN plasma levels (mean 944 ng/ml) than superficial bladder tumors (n=15, mean 463 ng/ml). There were no differences of plasma cFN concentrations concerning tumor grade and also no differences in urine levels between the different groups. We found a significant difference (p<0.04) of cFN plasma levels between patients with TCC of the bladder and the control group. The difference in cFN plasma levels between pTa/pT1 and >or=pT2 tumors indicates a clinically useful potential of this tumor marker for preoperative staging and postoperative follow-up. Our data underline the important but still unclear role of cFN as a tumor marker in TCC, and this will be the focus of future studies.
Bladder cancer is the fourth most common malignant neoplasm in men and the tenth most common in women. Cystoscopy presents the gold standard for detection and monitoring of bladder cancer. However, it is an invasive and expensive procedure. Therefore, development of biomarkers for the purposes of screening, diagnosis and prediction of the prognosis in bladder cancer is required. Bladder tumor fibronectin is one of the new urinary tumor markers. The aim of this study is to evaluate the diagnostic performance of urinary bladder tumor fibronectin in detecting and monitoring bladder cancer. A total of 75 patients with the diagnosis of bladder cancer, 20 patients with the diagnosis of benign prostatic hyperplasia, 7 patients with the diagnosis of prostate cancer between the years 1996-2000, and 28 age-matched healthy individuals, were enrolled in the study. The patients were diagnosed by cystoscopy, with histopathological evaluation of the tumor, as having superficial or invasive bladder cancer. Patients were followed-up clinically with data pertinent to disease recurrence and progression. Bladder tumor fibronectin (BTF; ng/ml) was determined by solid phase, two-site chemiluminescent immunometric commercial diagnostic assay developed for the Immulite automated immunoassay system (Diagnostic Products Corporation, Los Angeles, CA, USA). All measured values were normalized by urinary creatinine, and the obtained data were evaluated by receiver-operating characteristics (ROC) curve analysis. Optimal cut-off was established at 43.4 ng/mg. This cut-off rendered overall sensitivities of 72% and specificity of 82.1%. The analytical evaluation of the BTF test displayed promising results in terms of a non-invasive in vitro test in the diagnosis of bladder cancer. Although it was not satisfactory in prediction of recurrence or progression of the disease, it correlated well with the stage, one of the most reliable prognostic factors. In conclusion, the urinary bladder tumor fibronectin test warrants further clinical evaluation.
Transitional cell carcinoma of bladder is one of the most common tumors of genitourinary tract. Cystoscopy along with cytology is the mainstay for diagnosing bladder cancer. Cytology is specific for diagnosis of bladder carcinomas but less sensitive particularly in low-grade disease. Cystoscopy on the other hand is invasive and relatively costly technique and may also be inconclusive at times particularly in case of cystitis. A simple noninvasive marker for detecting bladder cancer would be helpful. A clinically useful urinary marker should be easy to perform, should have minimum requirements for sample preparations and should have high sensitivity and specificity in diagnosis. In this review we discussed the various urinary markers and their role in detection of bladder cancer.
We reviewed the literature on urinary markers and tests that may have clinical usefulness.
Several urinary markers and tests such as BTA Stat, BTA TRAK, NMP22, telomerase, HA and HAse tests, Immunocyt, Quanticyt, FDP, BLCA-4, FISH, CYFRA-21-1 have enough potential for future clinical use. BTA stat, NMP22 (bladder check)and AccuDX (FDP) tests are presently point of care tests. The rest of the tests are laboratory-based and may need trained technicians. Majorities of the urinary markers have higher sensitivity and specificity than cytology. However, voided urinary cytology has the highest specificity.
Till now there is no urinary marker or test that can replace the need of cystoscopy. However, some markers have the potential for future clinical use.
To evaluate the diagnostic efficacy of the analysis of fibronectin in the urine samples of patients with bladder cancer.
The study included 123 subjects: one group of 68 patients with bladder cancer confirmed by transurethral resection; a second group of 10 patients with benign urologic disease, and a third group of 45 healthy subjects. We carried out the analysis of bladder tumor fibronectin (BTF), cytology, and creatinine in urine, and calculated the BTF/creatinine (BTF/CREA) ratio. For the determination of BTF, we used a solid-phase chemoluminescent immunometric test.
The receiver operating characteristic curves showed an optimal cutoff point of 25.6 microg/L and 36.9 microg/g for BTF and the BTF/CREA ratio, respectively, with a sensitivity of 78% and 75%, respectively, and specificity of 80% for both. The sensitivity of urinary cytology was only 55%, but the specificity was 100%. The patients with bladder cancer had significantly greater levels of BTF and the BTF/CREA ratio than did the healthy subjects (P <0.001) and, in the case of BTF without correcting for creatinine, than did the patients with benign urologic disease (P <0.05). We also found significant differences in the levels of BTF and the BTF/CREA ratio among tumor stages, degree of differentiation, tumor size, multifocal nature, and macroscopic appearance.
Determination of fibronectin could be a useful test in the diagnosis of bladder tumors. The association between BTF and all known prognostic parameters implies its potential value in making therapeutic decisions. Nevertheless, the utility of BTF needs to be studied in a wider way in the presence of other pathologic features concurrent with bladder cancer.
Proteome analysis with protein separation by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and mass spectrometric measurement is utilized for comprehensive identification of proteins in various biological samples. However, this technique has not been widely applied for the analysis of body fluid for clinical assessments, because large amounts of proteins in fluid such as albumin and globulin hinder the detection of small amounts of proteins with high clinical values. The aim of the present study is to develop a method of proteomic analysis for urine of patients with bladder cancers.
Urine samples collected from 40 patients with bladder cancers, 32 healthy volunteers, and 7 old volunteers with benign prostate hypertrophy were treated with gelatin-beads as a group-specific affinity carrier. The gelatin-bound proteins were subjected to the proteome analysis.
Various amounts of matrix metalloproteinase-2 (MMP-2), -9 (MMP-9), fibronectin (FN), and their fragments were identified on 2-D PAGE gels from cancer-bearing patients, but not from normal individuals. Importantly, not only total amounts of these proteins, but also patterns of their distributions on the gels were well matched with the extent of invasion diagnosed with histopathological examinations. Thus, the score including such qualitative patterning of these proteins predicts pathological stages of the cancer.
The proteomic analysis with gelatin-affinity purification of urine samples is useful not only for the diagnosis of bladder cancers, but also for estimating the extent of tumor invasion.
Combining cell surface phenotyping with functional analysis, human CD8+ T cells have been divided into several subsets which are being studied extensively in diverse physiological situations, such as viral infection, cancer and ageing. In particular, so-called terminally differentiated effector cells possess a CD45RA+ CCR7- CD27- CD28- phenotype, contain perforin and, in different models, have been shown to exert direct ex vivo killing and to release interleukins upon both antigen-nonspecific and -specific stimulation. Using HLA class I multimers, we have identified a high frequency of peripheral CD8+ T cells that recognize a peptide derived from the self protein cytokeratin 18 presented by the HLA-A*0201 molecule. These cells can be detected in approximately 15% of the HLA-A2-positive healthy donors tested. A detailed analysis revealed that they must have divided extensively in vivo, have an effector cell phenotype and express various natural killer cell-associated receptors. Interestingly, however, they remained unresponsive to antigen-specific stimulation in vitro in terms of cytotoxicity and cytokine secretion. Thus, cytokeratin 18-specific cells constitute a frequently encountered, new CD8+ T lymphocyte subpopulation without classical effector status and with so far unknown function.
In recent years, an increasing number of urine-based tests have been proposed as potential screening tests for prostate cancer. The goal of this review was to summarize the current status of evidence regarding performance characteristics of the proposed tests and their practicality under screening conditions.
Relevant articles published up to and including May 2005 were identified in the PubMed database. At least 10 cases and 10 controls had to be analyzed for a study to be included in the review. Data concerning the study population, performance characteristics, and the collection and processing of urine samples were extracted from the reviewed articles.
In all, 34 retrospective studies evaluating 21 different markers complied with the inclusion criteria. Most of the studies were rather small and included heterogeneous clinical study populations. Promising results were reported for a few markers in single studies, but they have often not been replicated in subsequent larger studies. Some of the more promising results were obtained with 24-h urines or with specimen-handling procedures that might be difficult to perform under screening conditions.
Larger studies with a prospective design are required to confirm promising findings regarding performance characteristics of some novel markers recently reported in mostly small studies. Future studies should also pay particular attention to the practicality of the markers under screening conditions.
One of the most effective ways to combat different types of cancer is through early diagnosis and administration of effective treatment, followed by efficient monitoring that will allow physicians to detect relapsing disease and treat it at the earliest possible time. Apoptosis, a normal physiological form of cell death, is critically involved in the regulation of cellular homeostasis. Dysregulation of programmed cell death mechanisms plays an important role in the pathogenesis and progression of cancer as well as in the responses of tumours to therapeutic interventions. Many members of the BCL2 (B-cell CLL/lymphoma 2; Bcl-2) family of apoptosis-related genes have been found to be differentially expressed in various malignancies, and some are useful prognostic cancer biomarkers. We have recently cloned a new member of this family, BCL2L12, which was found to be differentially expressed in many tumours. Most of the BCL2 family genes have been found to play a central regulatory role in apoptosis induction. Results have made it clear that a number of coordinating alterations in the BCL2 family of genes must occur to inhibit apoptosis and provoke carcinogenesis in a wide variety of cancers. However, more research is required to increase our understanding of the extent to which and the mechanisms by which they are involved in cancer development, providing the basis for earlier and more accurate cancer diagnosis, prognosis and therapeutic intervention that targets the apoptosis pathways. In the present review, we describe current knowledge of the function and molecular characteristics of a series of classic but also newly discovered genes of the BCL2 family as well as their implications in cancer development, prognosis and treatment.
The urothelial carcinoma is the most frequent malignancy of the urinary bladder (UBC). The transition into invasive growth is accompanied by several histological changes including an oncofoetal reorganization of the extracellular matrix. Recently, the occurrence of oncofoetal fibronectin with an O-linked glycosylation in the IIICS region (oncf Fn) was shown to be present in urine from UBC patients and was recommended as a tumour marker. Until now there are no data available regarding the source and distribution of oncf Fn in UBC and its value for the assessment of invasiveness.
oncf Fn was analysed in noninvasive and invasive UBC using immunohistochemistry and western blot. Additionally, the mRNA expression of the IIICS splicing region was evaluated by quantitative real time RT-PCR.
Immunohistochemical results reveal a highly significant correlation of oncf Fn to invasiveness. Papillary tumours regularly show no positivity. In western blot, invasive UBC show a strongly increased amount of the 250 kDa oncf Fn. Additionally, several smaller bands could be shown suggesting a proteolytic processing of Fn. The mRNA of the IIICS region shows a 21.5-fold increase in invasive UBC compared with noninvasive carcinomas.
In summary, immunohistochemistry of oncf Fn is a valuable histological marker for invasiveness of urothelial carcinoma of the urinary bladder. The restricted and invasion-associated tissue distribution of immunoreactivity enables to monitor the recurrence of invasive UBC by a quantitative evaluation of IIICS O-linked glycosylated Fn in urine.
To determine whether the level of urinary fibronectin predicts the residual tumour load after transurethral resection (TUR) of bladder transitional cell carcinoma (TCC).
Urine samples were collected from 167 consecutive patients with suspected bladder cancer admitted for TUR. Samples were taken both before and after surgery. Bladder tumour fibronectin (BTF) was analysed using a solid-phase chemiluminescent immunometric test. Creatinine in urine was also determined and the BTF/creatinine ratio calculated.
Patients were divided into a control group of 41 whose previous diagnosis was negative for BT and another of 126 with a positive diagnosis for BT, which was further subdivided into those with and without residual tumour, according to findings from specimens obtained during the second procedure (repeat TUR or cystectomy). After the second procedure, 68 patients (56%) had no residual tumour, whereas 54 (44%) did. Four patients with BT who did not have the second procedure were excluded from the study. The median BTF and BTF/creatinine value in the control group was 33.2 microg/L and 51.4 microg/g, respectively, before the first TUR, and 29.6 microg/L and 46.7 microg/g, respectively, after the first TUR. There were no statistically significant changes in BTF and BTF/creatinine ratio (P = 0.61 and 0.79, respectively). In the group with TCC, the BTF decreased from 211.9 to 97.3 microg/L (P = 0.02) and the BTF/creatinine ratio from 281.6 to 146.5 microg/g (P = 0.009) for those with residual tumour, while it decreased from 195.1 to 34.0 microg/L (P = 0.007) and the BTF/creatinine ratio decreased from 249.1 to 53.7 microg/g (P = 0.003) for those with no residual tumour. After initial TUR, the patients with residual tumour had significantly greater levels of BTF and BTF/creatinine than did those with no residual tumour (P = 0.004 and 0.006, respectively). The receiver operating characteristic curves showed an optimum threshold of 67.8 microg/L and 81.3 microg/g for BTF and the BTF/creatinine in detecting residual tumour, respectively, with a sensitivity of 91.4% and 89.0%, respectively, and a specificity of 87.8% and 85.6%, respectively.
Urinary fibronectin, in addition to being one of the best markers for diagnosing bladder carcinoma, can be used to determine the presence of residual tumour load after TUR of bladder TCC.
Several companies have developed commercial kits to measure plasma fibronectin rapidly and inexpensively with readily available laboratory equipment. In two of these kits (Cooper Biomedical and Boehringer-Mannheim) an immunoturbidimetric method is used. In a third kit (Biomedical Technologies, Inc.) an enzyme immunoassay method is used. To evaluate these commercial kits for fibronectin assay, we selected nephelometry as a comparison method for ranking the kits with regard to precision and accuracy. We also compared antibody and fibronectin cross reactivity. The antibodies from various manufacturers appear similar, but the fibronectin standards from different sources showed significant variation. Rate nephelometry and the Boehringer-Mannheim kit had the best within-run precision (CVs of 0.38% and 5.5% respectively). Between-run precision for nephelometry was excellent (CV = 1.9%) and somewhat high for the Boehringer-Mannheim kit (CV = 15.4%). This study demonstrates a need for further standardization of antigen (fibronectin) and antibody in commercial kits and the development of suitable stable quality-control material.
This experimental study investigated the potential role of Tissue Polypeptide-Specific Antigen (TPS) in comparison with Prostate-Specific Antigen (PSA) in the diagnosis and the clinical and pathological staging of prostate cancer. Serum TPS and PSA levels were determined in 128 patients (pts) with benign prostatic hypertrophy (BPH; Group 1) and in 92 pts with prostate cancer (Group 2). TPS was also measured in a control group of 100 healthy subjects. Normal cutoff values of 85 U/l for TPS and 4 ng/ml for PSA were determined on the basis of ROC curve analysis. The sensitivity, specificity and accuracy in the diagnosis of prostate cancer were 49%, 95% and 76% for TPS, and 84%, 90% and 87% for PSA. The combination of the two markers provided a higher accuracy (88%), improving the sensitivity of PSA, since 47% of patients with normal PSA had pathological levels of TPS. TPS showed an increase in sensitivity from low to higher stages of disease and, in patients with skeletal involvement, from small to larger numbers of bone metastases (Kruskal Wallis p < 0.0001). Nevertheless, TPS serum levels are not useful in the clinical staging of prostate cancer as they have a poorer performance than PSA. TPS was ineffective (ROC curve area=0.68) in predicting extraprostatic disease and demonstrated a reduced ability (area = 0.78) to identify skeletal involvement. Moreover, the combination of the two markers did not significantly improve the performance of PSA alone. The serum concentration of TPS in patients with localized tumors was not related to the degree of tumor cell differentiation evaluated by the Gleason score.
Conclusion
Our preliminary experience suggests that TPS in association with PSA may be useful at the time of diagnosis of prostate cancer. However, these preliminary data have to be confirmed by larger clinical trials and the role of this association in the clinical setting needs to be analyzed with an adequate evaluation of the cost-effectiveness ratio.
A human DNA-binding protein, designated MAD-2, has recently been found to be elevated in the serum from patients with malignant diseases. MAD-2 has been purified approximately 500-fold from peritoneal and pleural fluids collected from cancer patients. Immunodiffusion studies have indicated that MAD-2 is immunochemically identical to human plasma fibronectin. The purified material has been resolved by sodium dodecyl sulfate gel electrophoresis into two major protein chains with molecular weights in the range of 200,000 to 210,000 in either the presence or absence of disulfide bond-reducing agents. These results suggest that MAD-2 is a fibronectin fragment which has been generated through proteolysis. A quantitative assay system capable of detecting ng quantities of MAD-2 has been developed and used to verify the presence of elevated MAD-2 levels in DNA-binding protein fractions isolated from the serum of individuals with malignant diseases.
We found that urinary fibronectin (UFN) in cancer patients was almost all fragmented and consisted mainly of the cell-binding domain. We developed a two-site immunoenzymometric assay for UFN, using two monoclonal antibodies that both recognize this domain of fibronectin. The amount of UFN was expressed as arbitrary units per milligram of creatinine. Some 2% of the 623 healthy subjects had UFN above the clinical cutoff point (200 arb. units/mg creatinine), as did 14% of the 271 patients with nonmalignant diseases. In contrast, concentrations of UFN exceeded the cutoff point in 59% of the 589 patients with cancer. In 37 patients with gastrointestinal cancer tested for UFN and for one or more of three established serum tumor markers, UFN was found in 25, significantly more often than the other markers. These results indicated that UFN is a marker that may be helpful in evaluating many kinds of cancer.
The morphologic discrimination between low and high grade malignant tumor cells arising in the urinary bladder is a major diagnostic problem in cytopathology. Using immunochemical peroxidase staining of cytokeratins (CKs) of human bladder exfoliative cytology specimens, we have been able to discriminate between transitional cell carcinoma cells, atypical cells and normal bladder cells. Commercially available monoclonal antibodies used in this study were: anti-CK 13 (Sigma K8.12), anti-CK 5, 7 and 8 (Sigma K8.13), anti-CK 19 (Sigma K4.62), and anti-CK 18 (Transformation Res. 1091). When normal bladder cells are stained with these antibodies, CK 18 is specific for superficial cells; CK 13 is specific for the basal type cells and CKs 5, 7, 8 and 19 are expressed by all urothelial cell types. Four cases diagnosed by cytopathological criteria as 'atypical' and 7 diagnosed as 'positive' (various grades) were used in this study. Cytologic diagnosis was confirmed by histopathology in 7 cases. Tissue was not available for histopathology in 4 cases. Malignant cells with a 'basal' or 'immature' phenotype preferentially stained with CK 13 and were associated with increased metastatic or malignant potential. Patients with higher grade tumors had more cells positive for CK 13 than did patients with atypical or lower grade malignancies. Patients with well-differentiated, low grade tumors predominantly shed cells that expressed CK 18 and CK 19. High grade, invasive bladder lesions were characterized by many cells expressing CK 13, and fewer cells expressing CK 19. The combination of diagnosis by morphologic criteria on Papanicolaou-stained specimens with immunochemical characterization of the same cells for CKs facilitate an accurate diagnosis of bladder lesions.
To study the biochemical mechanisms of bladder tumor invasion, we analyzed specimens of invasive transitional cell carcinoma cell line EJ and non-invasive transitional cell carcinoma cell line RT4 which had been implanted into the bladders of nude mice. Using immunoprobes specific to basement membrane laminin, we observed that superficial but not invasive tumors were surrounded by intact laminin. With immunoprobes specific to cathepsin B, a cysteine proteinase which has the ability to degrade laminin, we demonstrated that cathepsin B is localized in discrete cytoplasmic granules in the non-invasive tumors, and in a more diffuse cytoplasmic pattern in the invasive tumors. Subcellular fractionation followed by immunoblot analysis and enzymatic analysis confirmed that the invasive EJ cells had active cathepsin B localized to its plasma membrane, while non-invasive RT4 cells had cathepsin B confined to lysosomes. Furthermore, immunoblot analysis revealed that invasive EJ cells had the mature form of cathepsin B with a molecular weight of 25 kD, while non-invasive RT4 cells had predominantly precursor forms with molecular weights between 30 and 35 kD. In vitro degradation assays with plasma membrane fractions isolated from invasive EJ cells and non-invasive RT4 cells demonstrated that the plasma membrane of EJ cells but not that of the RT4 cells had the ability to degrade purified laminin, and that the degradative products were similar to those obtained with purified cathepsin B. We conclude that invasive tumor cells have enhanced cathepsin B in their plasma membranes which may be used to degrade basement membrane components such as laminin and thereby facilitate tumor invasion.
We surveyed the tumor-related proteins present in the urine specimens of 118 bladder cancer patients to seek a possible marker enabling future diagnosis and prognosis of this disease. We identified a protein of 180 kDa. by sodium dodecyl sulfate polyacrylamide gel electrophoresis in urine samples subjected to prior adsorption by protein-A conjugated to a sepharose bead. This protein appears to be a glycoprotein because it binds to concanavalin A-conjugated sepharose and can be eluted by alpha-methyl D-mannoside. It does not react immunochemically with antibodies prepared against either carcinoembryonic antigen or epidermal growth factor receptor, both of which have an apparent molecular weight close to 180 kDa. We found this protein in the urine of 74.3% of the patients with transitional cell carcinoma. It was not present in age-matched controls, patients with benign prostatic hyperplasia or patients with 10 other cancers. There was 1 false positive result in a patient with prostate cancer. It does not appear to be associated with urinary tract infection, blood contamination, premedication or anesthesia.
The peripheral stromal fibronectin (FN) staining patterns of invasive breast carcinomas (IBC) from 77 women were compared to the aggressivity of the tumors, which in each case had been determined through a complete clinical follow-up and autopsy investigation. Polyclonal, monospecific rabbit antibody to human FN was applied on formalin-fixed, paraffin-embedded tissue sections using the peroxidase-antiperoxidase staining technique. An FN-positive staining reaction was defined as a constant, diffuse, or pericellular demarcation of FN-positive fibers surrounding tumor cells at the invasive border. In lack of such a staining pattern, FN-negative staining was recorded. The FN-positive staining reaction was significantly associated with a low metastatic potential and appeared in a multivarians analysis to be an excellent prognostic factor, which surpassed other known parameters, such as clinical stage, histological type or grade, and lymph node status. 27 out of 31 women, who died without evidence of metastatic spread, had FN-positive IBC (87%) in contrast to women with metastatic disease, where only 15 out of 46 had FN-positive tumors (33%). An extensive histopathological examination of the contralateral breast revealed in this latter group a high rate of second primaries (22/46), which might have been responsible for the metastatic spread. If these women with bilateral IBC were excluded, only three metastasizing tumors with a FN-positive staining pattern remained, suggesting that the prognostic value of the FN-staining pattern along the invasive border of IBC might be even higher than anticipated from this study.
Two monoclonal anti-fibronectin antibodies that inhibit fibronectin-mediated cell adhesion have been established and characterized. One antibody, FN12-8, inhibited attachment of rat kidney fibroblasts on the fibronectin-coated substrate in a concentration-dependent manner, attaining a maximal inhibition of greater than 85% at 850 micrograms/ml. Another antibody, FN30-8, caused about 70% inhibition at a concentration as low as 0.85 microgram/ml, although further increase of the antibody concentration did not significantly augment the inhibitory effect. Immunoblot analysis with defined proteolytic fragments revealed that both antibodies are directed to the cell-binding domain of fibronectin. The epitopes for these antibodies were further narrowed down using recombinant cell-binding fragments expressed in Escherichia coli. FN12-8 recognized the 11.5-kDa cell-binding fragment previously characterized by Pierschbacher et al. (1981, Cell 26, 259-267), suggesting that FN12-8 blocks the Arg-Gly-Asp (RGD) cell adhesion signal. FN30-8 could not bind this fragment but did recognize a longer cell-binding fragment containing additional greater than 111 amino acid residues upstream of the 11.5-kDa fragment. Since the RGD-dependent cell adhesion seems to require another signal located at a region 50-160 residues upstream of the 11.5-kDa fragment for full activity, FN30-8 may exert its inhibitory effect by blocking the latter signal.
Fibronectin is a large, adhesive glycoprotein which is found in a number of locations, most notably on cell surfaces, in extracellular matrixes, and in blood. Fibronectin had been detected in all vertebrates tested and in many invertebrates. Its presence in sponges is significant because this suggests that fibronectin may have appeared very early in evolution, possibly with the most primitive multicellular organisms. Cellular and plasma fibronectins have many striking similarities. However, the locations of the polypeptide chain differences between these two proteins indicate that plasma fibronectin cannot be derived from cellular fibronectin by means of simple post-translational proteolysis. Instead, these different types of fibronectin may be products of different genes or of differentially spliced messenger RNA molecules. Amniotic fluid fibronectin is possibly a third form of the protein. Cellular and plasma fibronectins are composed of at least six protease-resistant domains which contain specific binding sites for actin, gelatin, heparin, Staphylococcus aureus, transglutaminase, fibrin, DNA, and a cell surface receptor. The relative locations of these domains have been mapped in the primary structure of fibronectin. The cell surface receptor for fibronectin has not been positively identified, but may be a glycoprotein, a glycolipid, or a complex of the two. Although cell-substratum adhesion is mediated by fibronectin, the locations of the areas of closest approach of the cell to the substratum (the adhesion plaques) and fibronectin are not coincident under conditions of active cell growth. Under conditions of cell growth arrest in low serum concentrations, some fibronectin may become localized at the adhesion plaques. Models describing the domain structure of fibronectin and the molecular organization of the adhesion plaque area are presented.
Plasma fibronectin was measured in patients with breast cancer, colon cancer, and acute leukemia. In the patients with solid tumors, mean levels were significantly elevated above the mean level of age- and sex-matched normals whether the disease was thought to be metastatic or not (P less than 0.001). It did not make a difference whether the determinations were done prior to or during chemotherapy. Fibronectin was measured serially in eight hospitalized patients with leukemia during intensive induction chemotherapy. Normal concentrations were found prior to therapy. However, fibronectin concentration fell on the day following chemotherapy in nine of 12 episodes (P less than 0.05), and during sepsis in 13 of 13 episodes (P less than 0.001). Thus, the concentration was influenced by at least two factors: recent chemotherapy and sepsis. Because fibronectin concentration is sensitive to clinical events other than the status of the malignancy, it seems unsuitable as a tumor marker, at least as a single isolated measurement.
Plasma fibronectin was determined in 121 normal adults and in 149 patients. Fibronectin levels in normals were strongly influenced by sex and age. The mean value of the protein in cancer patients did not differ from that in normal controls; however, patients with cryofibrinogenaemia or extensive liver metastases had lower values whereas those with obstructive jaundice due to pancreatic carcinoma had higher values than normal controls. Fibronectin levels were greatly increased in patients with primary biliary cirrhosis and moderately elevated in nephrotic syndrome. In patients with severe infection or sepsis, plasma fibronectin did not show a consistent pattern. Patients with overt disseminated intravascular coagulation, irrespective of its cause, had the lowest plasma fibronectin concentrations.
The levels of fibronectin in urine from patients with prostatic cancer, from patients with benign urologic disease, and from healthy control individuals were determined by the use of a gelatin affinity chromatography procedure. The assay does not seem to give false positive results, inasmuch as evaluation of 16 patients with benign urologic disease showed urinary fibronectin levels in the same range as those found in healthy controls. For a single determination, the levels in 42 per cent of prostatic cancer patients were elevated above control levels; when prostatic cancer patients were evaluated sequentially, the determination of urinary fibronectin levels over three sampling times approached a 100 per cent correlation with presence of disease. Inasmuch as levels of urinary fibronectin episodically elevate in patients with prostatic carcinoma, the differential frequency and magnitude of urinary fibronectin elevations may be useful markers to assess tumor aggressiveness and to monitor the impact of a therapeutic modality.
The commercially available tumor marker tests TPA, TPS, TPACYK and CYFRA 21-1 react with simple epithelium keratins. From clinical studies it can be deduced that the pattern of keratin recognition must be different for each of these tests. We therefore studied the reactivity of the keratin fragment combinations K8/K18 and K8/K19 in the different tests and determined the reactivity of the corresponding soluble antibodies with purified keratin 8, 18 and 19 in immunoblots. TPS and CYFRA 21-1 were found to distinguish clearly between the keratin fragment combinations K8/K18 (TPS) and K8/K19 (CYFRA 21-1). TPA and TPACYK reacted with both combinations, however, with different intensities. On immunoblots the CYFRA 21-1 antibodies reacted exclusively with K19, whereas the antibodies of the other assays reacted with at least 2 of the keratins investigated.
Soluble forms of keratins in human sera seem to be useful analytes for the monitoring of cancer patients. CYFRA 21-1 is a new test measuring keratin 19 in human blood. The test was developed as sandwich ELISA based on two monoclonal antibodies, both reacting with keratin 19. The epitopes recognised by the antibodies are located on the rod region of the molecule. In sera from malignant patients CYFRA 21-1 detects immunoreactive compounds which appear to be larger than keratin 19 itself, indicating the presence of oligomers in these sera. In a comparison study the reactivity of other keratin tests (TPA, TPS, TPAcyk) with the keratins 8, 18 and 19 were measured both in solution and on immunoblots. The reactivity pattern was found to be different what may explain the different diagnostic properties of the individual tests.
The CA 15-3 assay could play, probably in combination with cytokeratin markers, a role in the detection of early recurrence and in monitoring therapy in metastatic breast cancer patients. The CA 15-3 assay is, however, not useful for early detection of breast cancer and has no prognostic significance. The analyte is not clearly defined and a primary reference material cannot be synthesized. A reference measurement procedure is not available and many commercial assays use the original Centocor IRMA as such. The comparability of different CA 15-3 assays is not sufficient according to the results of EQAS. The general recommendations of analytical requirements for tumour marker determinations specified by the working group "Quality Control and Standardization" (Hamburger Symposia on Tumour Markers) includes intra-assay and inter-assay precision, drift, carry-over, dilution effect and high-dose hook effect. From a medical point of view reference limits should be established. Specificity/sensitivity profiles should preferentially be represented by ROCcurves. Confounding factors should be analyzed. To improve the clinical value of CA 15-3, the following needs for improvement have been identified: > Standardization of response criteria during treatment; > Definition of significant increase at recurrence; > Investigation of the biological variance during follow-up; > Definition of the analyte; > Preparation of reference material; > Development of an accepted reference method; > Integration of different international activities and > Optimizing decision-making criteria and clinical application.
The levels of fibronectin in urine from 106 patients with urinary bladder cancer, from 13 patients with benign urological disease and from 24 healthy control individuals were determined by an enzyme-linked immunosorbent assay (ELISA). The fibronectin levels in urine from patients with bladder cancer were higher than in patients with benign urothelial disease and in healthy controls. In 9 patients with bladder cancer, sampling was done both pre- and post-operatively. In these cases the fibronectin levels after operation were significantly lower than they had been before. Among 14 patients treated with BCG intravesically for superficial bladder tumours, those with complete remission of disease had less urinary fibronectin than those who did not respond to treatment. The data suggest that urinary fibronectin may be a useful marker for detecting urinary bladder cancer and for selecting patients for BCG treatment.
Monoclonal antibodies (MoAbs) recognizing the distinct domains of human fibronectin had previously been established and they were used to construct several sandwich immunoenzymometric assays (IEMAs) for the structural analysis of fibronectin found in the urine of cancer patients. Urinary fibronectin (UFN) was immunodetectable only with FN12-8 and FN30-8 MoAbs against cell-binding domains and was less reactive with other IEMAs using MoAbs directed to terminal domains, indicating that UFN was almost completely fragmented and consisted mainly of cell-binding regions. The IEMA using MoAbs against cell-binding domains had sufficient immunoreactivities with the antigen fragmented by artificial proteolysis, but these fragments could hardly be detected by other IEMAs. UFN levels were significantly elevated in various cancer patients and extremely elevated in some patients with distant metastasis. It is presumed that UFN fragments which increase in cancer patients are generated by extracellular matrix destruction. Thus UFN levels and the ratio of the fragmented UFN level to the non-fragmented UFN level appear to be informative clinical indicators of tumor malignancy or metastatic ability in cancer patients.
Fibronectin (FN) is a major component of the glomerular extracellular matrix and it is also abundant in plasma. In physiologic conditions, FN fragments are excreted into urine. Increased urinary FN excretion has been observed in renal diseases raising the question whether urinary FN could reflect changes in the renal extracellular matrix. This study examines whether urinary FN is filtered from plasma or derived from the kidney and it attempts to specify the potential renal source of urinary FN.
(a) To investigate whether urinary FN is filtered from plasma, labeled, biotinylated FN (b-FN) was injected into rats and urine samples were specifically analyzed for b-FN by an immunoblot procedure. (b) To specify the renal source of urinary FN and to evaluate possible alterations of this protein during passage into final urine, tubular fluid was collected from distinct localizations of the nephron by micropuncture techniques. The samples were analyzed for endogenous FN by a highly sensitive immunoblot system, and the pattern was compared with that normally found in final urine.
(a) Urine samples collected over a period of 5 days after injection of b-FN contained no labeled FN. Control experiments in rats with highly elevated glomerular permeability confirmed that the plasma levels of injected b-FN were sufficiently high since b-FN was detected in urine in this condition. (b) The FN fragment pattern, with two protein bands at 75 and 45 kilodaltons, that is normally found in final urine, was already present in samples taken from the early proximal tubule and the distal tubule.
The fibronectin fragments normally found in urine originate from the kidney and are not derived from plasma. Since these fragments are already observed in early proximal tubular fluid, the glomerulus is the probable source of urinary FN. The FN fragment pattern of early proximal tubular fluid is not substantially altered during passage into final urine, thus reflecting glomerular FN release in urine. It is suggested that examination of urinary FN excretion could be helpful in the assessment of altered glomerular extracellular matrix in pathologic conditions.
We investigated the tissue concentration of sialic acid and fibronectin in patients with prostatic cancer. The mean sialic acid and fibronectin levels in patients with prostatic cancer were 19.02 +/- 6.30 micrograms/mg protein, respectively versus 13.01 +/- 4.53 micrograms/mg protein and 11.77 +/- 6.74 micrograms/mg protein for normal prostatic tissues. Sialic acid and fibronectin levels in cancerous patients were significantly higher than in the control group (P < 0.05).
The mechanism of human bladder cancer cell invasion is not clear, but it appears that extracellular matrix components, such as fibronectin, may be involved. To investigate the role of fibronectin in tumor cell invasion and progression, we used an in vitro invasion assay to define the motility stimulating fragment of fibronectin for invasive human bladder cancer T24 cells. Using a modified Boyden chamber assay and purified fragments of fibronectin, we demonstrated that both the 120 kDa chymotrypsin generated fragment of fibronectin (containing the cell attachment RGD motif and additional sequences towards the carboxyl-terminal heparin binding domain), as well as the trypsin generated 60 kDa fragment of fibronectin (containing the carboxyl-terminal heparin binding domain and additional sequences towards the cell attachment RGD motif), were able to stimulate the migration of invasive human bladder cancer T24 cells. Control fragments containing only the amino-terminal gelatin binding region of fibronectin did not stimulate the motility of the human bladder cancer T24 cells. To determine the molecular mechanism in which these fragments may stimulate the migration of the T24 cells, we assayed for intracellular signal transduction pathway protein kinase C (PKC). We demonstrated that both the 120 kDa and the 60 kDa fragments were able to stimulate the activation of protein kinase C. Non-motility stimulating fragments of fibronectin were not able to activate protein kinase C. We conclude that the PKC signal transduction pathway may be involved in matrix mediated motility, and suggest that the inhibition of such pathway(s) may alter the malignant phenotype of human bladder cancer.
We determined the levels of TPA in 133 patients with superficial bladder cancer. 79 cases were Ta stages, and 54 cases T1 stages. 53 of the tumors were well differentiated (I), 65 moderately differentiated (II) and 15 undifferentiated (III). The average follow-up time of these patients was 8.3 months; the standard deviation being 5.2 months (median value 7 months). In 43 cases a relapse of the tumor was observed. We detected high TPA levels in 25% of the patients, without observing meaningful differences in tumor invasion or in its differentiation degree. The usefulness of the TPA, the degree of tumor invasion, and the degree of cell differentiation were evaluated by means of single-variate and multivariate analysis in order to forecast tumor relapse. Only the TPA had a prognostic value, the relative risk of developing relapse being 2.03 times higher in patients with high TPA than patients with normal TPA.
The determination of tumor markers in urine samples has been proposed as an effective diagnostic tool in bladder cancer. The aim of the present investigation was to validate in urine samples the assay of the CYFRA21.1 cytokeratin-related marker, the serum concentrations of which showed promising diagnostic utility in patients with bladder cancer. First-voided urine samples were collected from patients with different malignancies. CYFRA21.1 was assayed with a commercially available enzyme immunoassay (Boehringer Mannheim). Different centrifugation patterns, the use of different buffers and nonionic detergents, and pH variations were evaluated. We demonstrated that: (a) cells and cell debris contain a large amount of CYFRA21.1 and must be eliminated by centrifugation; (b) storage at -20 degrees C causes amorphous precipitate, which may aspecifically bind CYFRA21.1; (c) the latter behavior may be prevented by diluting fresh urine samples with phosphate buffer with nonionic detergent added; (d) pH variations within the range 4.9-8.2 do not significantly affect CYFRA21.1 assay results. Provided that samples are diluted with buffer containing nonionic detergent, the CYFRA21.1 assay showed good precision and accuracy characteristic in urine samples. We therefore propose a standard protocol for the collection of urine samples for CYFRA21.1 assay. In a preliminary clinical evaluation, CYFRA21.1 concentrations in 16 patients with primary bladder cancer were higher than in healthy subjects. In the urine collected in the follow-up of patients treated for bladder cancer, CYFRA21.1 tended to be higher in relapsed patients than in those without evidence of disease. These preliminary data induced us to extend the clinical trial to establish the actual role of this assay in routine use.
The sera of 154 cancer patients were analyzed at primary diagnosis before any therapy to find out the clinical importance of CYFRA 21-1 (detecting cytokeratin 19-fragments) compared with the polyclonal TPA-IRMA and the monoclonal TPA-LIA-mat-assay (both measuring fragments of cytokeratin 8, 18 and 19). The reference group consisted of 100 healthy persons as well as 78 patients with exclusively benign urological diseases. We defined the cut-off values based on 95% specificity versus benign urological disorders. For CYFRA 21-1 the cut-off value was found to be 2.5 ng/ml, for TPA-IRMA 165 U/L, and for TPA-LIA-mat 136 U/L. Taking into account all stages CYFRA 21-1 showed a sensitivity of 31% versus 20% and 16% for TPA-IRMA and TPA-LIA-mat, respectively. Considering only the muscle invasive carcinomas 52% sensitivity for CYFRA 21-1 vs. 39% and 33% for TPA-IRMA and TPA-LIA-mat could be found. All three markers correlate with the stage of disease, CYFRA 21-1 to the highest degree (stage O: 16%, stage IV: 71%). CYFRA 21-1 shows the best sensitivity-specificity-profile and seems to be a recommendable marker for the follow-up of urinary bladder cancers except for the Ta-tumors which only rarely develop into muscle invasive cancers.