Proteomic Analysis of Urine to Identify Breast Cancer Biomarker Candidates Using a Label-Free LC-MS/MS Approach.

INTRODUCTION: Breast cancer is a complex heterogeneous disease and is a leading cause of death in women. Early diagnosis and monitoring progression of breast cancer are important for improving prognosis. The aim of this study was to identify protein biomarkers in urine for early screening detection and monitoring invasive breast cancer progression. METHOD: We performed a comparative proteomic analysis using ion count relative quantification label free LC-MS/MS analysis of urine from breast cancer patients (n = 20) and healthy control women (n = 20). RESULTS: Unbiased label free LC-MS/MS-based proteomics was used to provide a profile of abundant proteins in the biological system of breast cancer patients. Data analysis revealed 59 urinary proteins that were significantly different in breast cancer patients compared to the normal control subjects (p<0.05, fold change >3). Thirty-six urinary proteins were exclusively found in specific breast cancer stages, with 24 increasing and 12 decreasing in their abundance. Amongst the 59 significant urinary proteins identified, a list of 13 novel up-regulated proteins were revealed that may be used to detect breast cancer. These include stage specific markers associated with pre-invasive breast cancer in the ductal carcinoma in-situ (DCIS) samples (Leucine LRC36, MAST4 and Uncharacterized protein CI131), early invasive breast cancer (DYH8, HBA, PEPA, uncharacterized protein C4orf14 (CD014), filaggrin and MMRN2) and metastatic breast cancer (AGRIN, NEGR1, FIBA and Keratin KIC10). Preliminary validation of 3 potential markers (ECM1, MAST4 and filaggrin) identified was performed in breast cancer cell lines by Western blotting. One potential marker MAST4 was further validated in human breast cancer tissues as well as individual human breast cancer urine samples with immunohistochemistry and Western blotting, respectively. CONCLUSIONS: Our results indicate that urine is a useful non-invasive source of biomarkers and the profile patterns (biomarkers) identified, have potential for clinical use in the detection of BC. Validation with a larger independent cohort of patients is required in the following study.

Diagnostic evaluation of apoptosis inhibitory gene and tissue inhibitor matrix metalloproteinase-2 in patients with bladder cancer.

Bladder carcinoma is an important worldwide health problem. Both cystoscopy and urine cytology used in detecting bladder cancer suffer from drawbacks where cystoscopy is an invasive method and urine cytology shows low sensitivity in low-grade tumors. This study validates easier and less time-consuming techniques for the estimation of survivin and TIMP-2 in urine of bladder cancer patients to evaluate them in comparison with cytology. This study includes malignant (bladder cancer patients, n = 42), benign (patients with bilharzial cystitis, n = 22) and healthy (n = 21) groups. The studied groups were subjected to cystoscopic examination, detection of bilharzial antibodies, urine cytology, and estimation of urinary survivin by qualitative RT-nested PCR and TIMP-2 by ELISA. Significantly higher positivity rates of urinary survivin and TIMP-2 were observed in the malignant group compared with benign and healthy groups. On associating the two urinary markers with different clinicopathological factors, only TIMP-2 exerted significantly higher positivity rate in invasive stage (100%) than superficial stage (82.3%). Survivin showed 78.6% sensitivity, 95.3% specificity, 94.3% PPV, 82% NPV, and 87% accuracy. When combined with urine cytology, the sensitivity increased to 83.3%. While on applying the cutoff value of urinary TIMP-2 (< or =639.5 pg/mg protein), it showed 93% sensitivity, 83.7% specificity, 85% PPV, 92.3% NPV, and 88.2% accuracy. When combined with urine cytology, the TIMP-2 sensitivity remained 93%. On combining cytology with both urinary survivin and TIMP-2, the highest sensitivity was reached (98%). Survivin and TIMP-2 can be considered as potentially useful urine markers in early detection of bladder cancer.

Clinical experience with survivin as a biomarker for urothelial bladder cancer.

OBJECTIVES: This study was carried out as a prospective pilot study to evaluate the potential of survivin mRNA measurement in patients suspicious for urothelial bladder cancer (BC). Data were also analyzed for possible influences of secondary urological findings on survivin measurements. METHODS: Survivin was measured by an mRNA assay in voided urine samples of 50 patients with suspicion of new or recurrent BC prior to transurethral resection. Sample evaluation was possible in 49 cases. Histopathology revealed no malignancy in 17 (35%) and BC in 32 (65%) patients. Survivin mRNA was quantitated by real-time PCR from frozen cell pellets of centrifuged urine samples. A ROC analysis of the survivin data was performed. RESULTS: ROC analysis identified the best cut-off level at 10,000 mRNA copies, resulting in a sensitivity of 53% and a specificity of 88%. Seven of the 20 pTa tumors (35%), all four pT1 (100%) and all four muscle-invasive tumors (100%) were detected. Of four patients with carcinoma in situ (Cis), 50% could be identified. Only two patients (4%) were assessed as false positive. Histologically confirmed cystitis and concomitant urological findings (inflammatory cells in urine, microhematuria and others) had no detectable influence on survivin measurements. CONCLUSION: In present group of patients, survivin was a reliable biomarker for high-grade urothelial BC (sensitivity 83%), but not for low grade (sensitivity 35%) urothelial BC with a high specificity (88%). No confounders influencing the results of survivin measurements could be identified.

Urine-based biomarkers for the early detection and surveillance of non-muscle invasive bladder cancer.

Bladder cancer has a very high frequency of recurrence and therefore requires lifelong surveillance, traditionally consisting of serial cystoscopy and cytology. These tests are both invasive and expensive, with considerable inter-user and inter-institutional variability. In addition, the sensitivity of cytology in detecting low-grade tumors is low. Therefore, there has been active investigation into urinary biomarkers that can either supplement or supplant these tests. At this point there are only six urine-based tests that are FDA-approved in bladder cancer surveillance, but a wide variety of other biomarkers are being studied. In this review, we examine the natural history of bladder cancer as well as the rationale and performance of an ideal urinary biomarker. The authors describe the FDA-approved biomarkers such as Bladder Tumor Antigen, ImmunoCyt, Nuclear Matrix Protein-22, and Fluorescent In Situ Hybridization, as well as the most promising investigational tests (i.e., Urinary bladder cancer test, BLCA-1, BLCA-4, hyaluronic acid, hyaluronidase, Lewis X antigen, microsatellite analysis, Quanticyt, soluble Fas, Survivin, and telomerase). The biological foundation, methodologies, and diagnostic performance of the biomarkers are discussed. The characteristics of the biomarkers are compared to urine cytology. At this time, urine biomarkers are utilized in a variety of clinical situations but their role is not well defined. The goal of identifying an optimal marker that will replace cystoscopy and/or cytology is still ongoing.

Focus on urinary bladder cancer markers: a review.

Finding and development of new bladder cancer markers is still a very dynamic field. Because of the mass of all these markers it is impossible to report all of them. This paper reviews the role of bladder cancer markers in diagnosis and highlights the most important biomarkers studied and reported recently. A medline based literature search was performed to examine the field of bladder cancer markers. Major topics focus on selected bladder cancer markers from nearly all categories of the wide field of bladder cancer markers: Hematuria, FISH, FGFR3, SURVIVIN, u-PAR, TP53 mutation, HER-2/neu, TPA, NMP22, CK-19, CK-20, CYFRA 21-1. The use and clinical importance as diagnostic help are discussed. In this review a highlight to some of the most important markers was made. Further determination of recurrence and progression marker will contribute to establish better treatments for the individual patient. Molecular staging of urological tumors will allow selecting cases that will require systemic treatment. It is necessary and important to integrate under the same objectives basic and clinical research.

Serum and urine survivin levels in breast cancer.

This study was conducted to investigate the serum and urine levels of survivin in patients with breast cancer and the relationships with known prognostic parameters and therapy. Forty-three patients with breast cancer and 21 healthy control subjects were investigated. Serum samples were obtained on the first admission before adjuvant and metastatic treatment were given and after two cycles of chemotherapy. Serum and urine survivin levels were determined using enzyme immunometric assay (EIA) and enzyme-linked immunosorbent assay (ELISA), respectively. There was no significant difference in the baseline serum and urine levels between patients with breast carcinoma and healthy controls (p = 0.19 and p = 0.84, respectively). None of the prognostic parameters analyzed were significantly correlated with the urine survivin concentrations. This was also true for serum survivin values, except for nodal involvement. Serum survivin levels were significantly higher in the patients with nodal involvement compared with node negatives (p = 0.043). However, serum survivin levels were not influenced by the number of involved nodes (p = 0.77). No significant correlation was found between the serum and urine levels of survivin (r = 0.15, p = 0.27). Serum and urine levels did not change significantly after chemotherapy (p = 0.59 and p = 0.50, respectively). In conclusion, the result of this study suggested that serum survivin level could be a sensitive marker for detecting metastases in lymph nodes from breast cancer patients. However, much research continues in this field, and exciting new knowledge will ultimately emerge.

Contribution of the prostate limits the usefulness of survivin for the detection of bladder cancer.

PURPOSE: We determined if urinary survivin is influenced by the prostate and analyzed survivin levels in a healthy control population. MATERIALS AND METHODS: This was a blinded, retrospective analysis of frozen urine samples. Three groups were designated. Group 1 patients were diagnosed with prostate cancer and samples were collected immediately prior to radical retropubic prostatectomy. Group 2 patients had already undergone radical retropubic prostatectomy at least 3 months prior to providing a urine sample. Group 3 comprised healthy controls. Groups 1 and 2 patients were recruited at an institutional practice. Group 3 patients were recruited from the local community. RESULTS: The frequency of high survivin scores was much lower in patients who did not have a prostate (p < 0.0002). The survivin score was 2 in 35% and 44% of the patients in groups 1 and 3, respectively, while only 9% of those in group 2 had a survivin score of 2 (p < 0.0002). Of healthy controls 50% had a false-positive survivin score. Urinary survivin predicted prostate cancer poorly with 52% sensitivity and 50% specificity, and it did not predict any biological features of prostate cancer. CONCLUSIONS: Urinary survivin markedly decreases after prostatectomy. There is a 50% false-positive rate when using urinary survivin in controls. These 2 features make urinary survivin a poor bladder cancer biomarker.

Efficacy of BTA stat, cytology, and survivin in bladder cancer surveillance over 5 years in patients with spinal cord injury.

OBJECTIVES: To evaluate three urine markers, BTA stat, cytology, and urinary survivin levels, in the spinal cord injury (SCI) population. The incidence of bladder cancer in patients with SCI is up to 20 times greater than in the general population. However, bladder cancer biomarkers have not been assessed in this population. METHODS: Between April 1999 and April 2004, 457 patients with SCI enrolled at the HealthSouth Harmarville Rehabilitation Spinal Cord Clinic donated their urine to our SCI urine repository. BTA stat tests and the survivin assay were performed according to published standards. Cytology specimens were sent to our cytopathology laboratory for analysis. RESULTS: A total of 1075 urine specimens from 457 patients were analyzed. Of the 1073 BTA stat tests, 119 showed positive reactions and 954 were negative. In the survivin assays, 47 samples had a score of 1, 38 a score of 2, and 9 a score of 3. No cytology specimens were noted to have malignant cells. During the past 5 years, 3 patients were diagnosed with bladder cancer by cystoscopy and treated for superficial disease. For these patients, none of these three tests (BTA stat, survivin assay, and cytology) was positive before the diagnosis of bladder cancer. CONCLUSIONS: The BTA stat, survivin assay, and urine cytology were unable to predict bladder cancer cases in our cohort of patients with SCI. Cystoscopy, therefore, remains the gold standard for bladder cancer surveillance in patients with SCI.

Urine detection of survivin is a sensitive marker for the noninvasive diagnosis of bladder cancer.

PURPOSE: In a preliminary study urine detection of survivin, an integrator of cell death and mitosis, accurately detected bladder cancer. The objectives of this study were to confirm these findings in a large cohort of subjects undergoing cystoscopy, to assess the diagnostic performance of urine survivin and to test whether evaluation of urine survivin adds independent value to urine NMP22 (Matritech, Cambridge, Massachusetts) and cytology for the detection of bladder cancer. MATERIALS AND METHODS: Urine survivin was measured using a Bio-Dot microfiltration detection system (Bio-Rad, Hercules, California) in voided urine specimens collected before cystoscopy in 117 cases and 92 controls. Bladder washout samples for cytology were collected in 174 subjects. Urine levels of NMP22 were measured using a commercially available enzyme-linked immunosorbent assay. RESULTS: Higher levels of urine survivin were associated with an increased risk of bladder cancer (p <0.001) and tumors of higher grade (p = 0.037), but not with invasive stage, after adjustment for the effects of urine cytology, NMP22 and age. The sensitivity, specificity, and positive and negative predictive values of survivin for the diagnosis of bladder cancer (64%, 93%, 92% and 67%, respectively), are superior to those of NMP22 and cytology. Survivin had the highest specificity and positive predictive value for the detection of bladder cancer across each tumor stage and grade. CONCLUSIONS: Urine survivin was a strong, independent predictor of the presence of bladder cancer and higher tumor grade. Urine detection of survivin is an accurate diagnostic test for bladder cancer that retains its efficiency regardless of cancer stage and grade.

Detection of urine survivin in 40 patients with bladder cancer.

PURPOSE: We investigated whether urine survivin, an inhibitor of the apoptosis protein, is useful for diagnosing bladder tumor. METHOD: We measured urine survivin levels in 40 patients with bladder tumors and 9 healthy volunteers. RESULTS: The average urine survivin levels in the 40 patients and 9 healthy controls were not significantly different (3.802+/-8.669 and 1.127+/-1.529, respectively (p=0.3646) ). However, significantly high urine survivin levels were observed in 3 of the 40 patients, but not in healthy volunteers. Urine Cyfra 21-1 was not elevated (1.3 ng/ml) in one patient with a significantly elevated urine survivin level (33.54 ng/ml), while in two patients with elevated Cyfra (320 ng/ml and 240 ng/ml), the urine survivin level was not detectable. CONCLUSION: With improvements in the sensitivity of our Elisa system for urine survivin and combined use of urine Cyfra 21-1, it is possible that urine survivin will be a useful tumor marker in detecting both new-onset and recurrent bladder tumors.

Effect of intravesical treatment of transitional cell carcinoma with bacillus Calmette-Guerin and mitomycin C on urinary survivin levels and outcome.

PURPOSE: Urine survivin is a predictive/prognostic molecular marker that detects transitional cell carcinoma (TCC) with high specificity and sensitivity. The presence of urine survivin in patients with TCC who receive intravesical instillation of bacillus Calmette-Guerin or mitomycin C may predict recurrence. MATERIALS AND METHODS: Urine from 25 subjects receiving 27 intravesical treatments of bacillus Calmette-Guerin or mitomycin C for TCC were collected prior to, during and after treatment. Urinary survivin levels were compared with outcome, as assessed by cytology and cystoscopy with or without biopsy 1 month and up to 12 months after the completion of treatment. RESULTS: Pretreatment survivin levels were higher in subjects in whom TCC recurred following treatment compared with those who achieved remission. Survivin levels increased several-fold during treatment with the highest survivin levels measured in subjects with recurrence. Median posttreatment values of survivin were zero in those who achieved remission and 1.0 ng/ml urine in subjects in whom TCC recurred. CONCLUSIONS: The presence of urinary survivin 1 month after the completion of treatment predicts TCC recurrence with 100% sensitivity and 78% specificity. Specificity to predict TCC recurrence increases to 92% after 1 year. No TCC recurred for 1 year in 12 of the 14 subjects with a posttreatment survivin level of 0.1 ng or less per ml urine. Three of the 4 subjects who were survivin positive but in remission 1 month after the completion of treatment had recurrent TCC within 1 year. Subjects who have urinary survivin after the completion of intravesical instillation have a high likelihood of TCC recurrence.