Intratumoral Heterogeneity Determines the Expression of mTOR-pathway Proteins in Prostate Cancer.

The aim of this study was to evaluate the expression of mammalian target of rapamycin (mTOR), phosphorylated-mTOR (p-mTOR), and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) in prostate cancer (PCa) in order to assess intratumoral heterogeneity and correlation with clinicopathological parameters. Tissue samples from 115 patients undergoing radical prostatectomy were included in a tissue microarray comprising (A) tissue from the tumor center, (B) malignant border of the tumor, (C) tumor-adjacent benign tissue, and (D) tumor-distant benign prostatic tissue. Immune reactive scores 0-12 were correlated with clinical data in reference to localization. A meta-analysis of studies investigating the association between biochemical recurrence (BCR) and parameters of the mTOR pathway was conducted. Regardless of the location within the tumor, cancer tissue showed higher expression of mTOR, p-mTOR, and 4EB-P1 compared to benign tissue (p < 0.01). Significant differences in expression between tissue samples from groups C and D were observed for mTOR and p-mTOR. When considering expression according to the pathological stage, we observed lower p-mTOR expression in pT3 vs. pT2 (7.9 and 6.3; p = 0.01). After a median follow-up of 74.5 months (IQR 65.0-84.0), 27 patients (23.47%) developed BCR. Weak staining of mTOR was associated with shorter time to BCR (HR: 2.0; p = 0.049) after correcting for PSA and T stage. However, a significant association of mTOR expression with BCR was found for specimens from the malignant border of the tumor (B) but not the tumor center (A) (p = 0.0034 log rank). In a meta-analysis, we found that the expressions of mTOR ((RR) = 0.70; 95% CI 0.43-1.12; p = 0.13) and 4E-BP1 ((RR) = 0.86; p = 0.53) were not statistically associated with BCR, while strong staining of p-mTOR was associated with a lower risk of BCR ((RR) = 0.57; p = 0.002). All 3 markers showed stronger expression in PCa and exhibited local gradients in relation to the border of tumor and healthy tissue. Our results suggest an important role of intratumor heterogeneity for the use of mTOR parameters as biomarkers in PCa.

Impact of variant microscopic interpretation of the uCyt+ immunocytological urine test for the detection of bladder cancer.

BACKGROUND: Urinary marker tests for bladder cancer (BC) detection and surveillance represent a desirable approach to diagnosis and follow-up. The SCIMEDX uCyt+ assay detects antigens expressed by BC cells (mucin glycoprotein and carcinoembryonic antigen) using green and red fluorescence and is interpreted according to specific manufacturer's recommendations. In the present study, we evaluated divergent approaches of numeric and morphological analysis of uCyt+ to generate a rationale for alternative test interpretation strategies. METHODS: A total of 444 patients with hematuria and without history of BC underwent uCyt+ analysis, cystoscopy and histological examination of tissue biopsies. Beside positive cells according to the manufacturer's definition (definitely positive cells, DPC), (i) cells showing borderline character (borderline cells, BLC), and (ii) cells with staining present below defined border (subliminal cells, SLC) were included into the analytical algorithm. Different cut-off levels for cell counts (>0, ≥3, ≥5) were evaluated separately with regard to their diagnostic accuracy. Moreover, the influence of clinical factors on test results were evaluated. RESULTS: Adding BLC at a cut-off of ≥3 cells resulted in Area Under the Curve (AUC) of 0.70 for green and 0.77 for red fluorescence, respectively. Adding SLC led to reduced AUC (0.62 and 0.73, respectively). Male gender was significantly associated with false positive results in the "best AUC" groups (P = .0101). No further correlations to clinical influencing factors were observed. CONCLUSIONS: Adding microscopic BLC as test-positive and adjusting cut-off level for the interpretation of uCyt+ may improve assay performance independent of clinical factors.

Evaluation of a new quantitative point-of-care test platform for urine-based detection of bladder cancer.

OBJECTIVE: Several commercial point-of-care (POC) tests are available for urine-based detection of bladder cancer (BC). However, these tests are restricted to dichotomized results (positive or negative), which limits their diagnostic value. Quantitative protein-based tests offer improved risk stratification but require complex methods restricted to specialized centers. Recently, the first quantitative POC system based on the detection of cytokeratin fragments became available. The aim of the study was to evaluate the diagnostic accuracy of this quantitative POC test. PATIENTS AND METHODS: A total of 198 patients having symptoms suspicious for BC were included. All patients received urethrocystoscopy and upper-tract imaging. Urine samples were analyzed by the urine BC antigen (UBC) rapid POC system and evaluated both visually and quantitatively using the concile Omega 100 POC reader. For visual evaluation, different thresholds of band intensity for considering a test positive were applied. Moreover, the UBC enzyme-linked immunosorbent assay (ELISA), urine cytology, and the nuclear matrix protein 22 BladderChek were performed. Sensitivities and specifities were calculated by contingency analyses. Optimal cutoffs of quantitative tests were determined by receiver operating characteristic curves. RESULTS: A total of 61 patients (30.8%) were diagnosed with BC. Visual evaluation of the UBC revealed sensitivities of 38.1% to 71.4% with corresponding specificities of 54.1% to 89.1%, dependent on the threshold of band intensity applied. The quantitative UBC rapid showed a sensitivity of 60.7% and a specificity of 70.1% at optimal cutoff (area under the curve = 0.68). A constant increase of both the probability of BC and high-risk BC with increasing UBC rapid values was observed. UBC concentrations determined by the reader significantly correlated with the UBC ELISA (P<0.001). The UBC ELISA, the nuclear matrix protein22 BladderChek and cytology showed sensitivities of 48.3%, 16.4%, and 51.7% with specificities of 71.3%, 95.3%, and 78.1%, respectively. CONCLUSION: The UBC rapid in combination with a quantitative POC-reader system for the first time enables quantitative determination of a BC marker under POC conditions. Diagnostic accuracy is at least equivalent to elaborate ELISA-based measurement. The quantitative use of the UBC rapid test facilitates risk prediction compared with conventional nonquantitative dichotomized POC testing.

Altered expression of farnesyl pyrophosphate synthase in prostate cancer: evidence for a role of the mevalonate pathway in disease progression?

BACKGROUND: Preclinical studies demonstrated effects of drugs inhibiting the mevalonate pathway including nitrogen-containing bisphosphonates (N-BPs) and statins on tumor growth and progression. The exact role of this pathway in prostate cancer (PC) has not been identified yet. Herein, we evaluate the expression of farnesyl pyrophosphate synthase (FPPS), the key enzyme of the mevalonate pathway, in PC. PATIENTS AND METHODS: Prostate cancer (PC) and benign prostate tissue of 114 men who underwent radical prostatectomy were constructed to a tissue microarray. Immunohistochemical staining of FPPS was quantified by the Remmele/Stegner immunoreactivity-score. Patients' clinical follow-up was assessed. IRS was correlated to pathological and clinical data. The impact of FPPS expression on clinical course was assessed univariate and multivariate. RESULTS: Mean IRS in PC and benign tissue was 5.7 (95% CI 5.0-6.5) and 2.6 (2.1-3.0, p < 0.0001). Mean IRS in PC tissue of patients with organ-confined and locally advanced disease (pT ≥ 3) was 5.09 (4.22-5.96) and 6.87 (5.57-8.17, p = 0.035). IRS of PC tissue significantly correlated with Gleason score (p = 0.03). Patients with PC tissue IRS >3 showed shorter recurrence-free survival compared to the remaining (p = 0.01). Increased FPPS expression is an independent risk factor for early biochemical recurrence (p = 0.032). CONCLUSIONS: This is the first study on FPPS in PC specimens. The association of FPPS with established histopathological risk parameters and biochemical recurrence implicates a contribution of the mevalonate pathway to PC progression. Further functional analysis is required to explore the role of this pathway in PC and to investigate whether FPPS expression affects the response of PC cells to N-BPs.

Insulin receptor isoforms A and B as well as insulin receptor substrates-1 and -2 are differentially expressed in prostate cancer.

AIMS/HYPOTHESIS: In different cancers types, insulin receptor isoform composition or insulin receptor substrate (IRS) isoforms are different to healthy tissue. This may be a molecular link to increased cancer risk in diabetes and obesity. Since this is yet unclear for prostate cancer, we investigated IR isoform composition and IRS balance in prostate cancer compared to benign and tumor adjacent benign prostate tissue and brought this into relation to cell proliferation. METHODS: We studied 23 benign prostate samples from radical cystectomy or benign prostatic hyperplasia surgery, 30 samples from benign tissue directly adjacent to prostate cancer foci and 35 cancer samples from different patients. RNA expression levels for insulin receptor isoforms A and B, IRS-1, IRS-2, and IGF-1 receptor were assessed by quantitative real-time RT-PCR. In addition, RNA- and protein expression of the cell cycle regulator p27(Kip1) was quantified by real-time RT-PCR and immunohistochemistry. RESULTS: Insulin receptor isoform A to B ratio was significantly higher in cancer as well as in tumor adjacent benign prostate tissue compared to purely benign prostates (p<0.05). IRS-1 to IRS-2 ratios were lower in malignant than in benign prostatic tissue (p<0.05). These altered ratios both in cancer and adjacent tissue were significantly associated with reduced p27(Kip1) content (p<0.02). Interestingly, IGF-1 receptor levels were significantly lower in patients with type 2 diabetes (p = 0.0019). CONCLUSIONS/INTERPRETATION: We found significant differences in the insulin signaling cascade between benign prostate tissue and prostate cancer. Histological benign tissue adjacent to cancer showed expression patterns similar to the malignancies. Our findings suggest a role of the insulin signaling pathway in prostate cancer and surrounding tissue and can hence be relevant for both novel diagnostic and therapeutic approaches in this malignancy.

Preliminary experience on the use of the Adnatest® system for detection of circulating tumor cells in prostate cancer patients.

BACKGROUND: The Adnatest® system combines immunomagnetic enrichment of epithelial cells with polymerase chain reaction for prostate cancer (PC)-specific transcripts for the detection circulating tumor cells (CTCs). We evaluated the Adnatest® in patients with castration-resistant PC receiving docetaxel chemotherapy. PATIENTS AND METHODS: CTCs were assessed in 16 patients with castration-resistant PC before cycles one and three of chemotherapy. Furthermore, markers of stem cells and epithelial-mesenchymal transition were assessed. Treatment response was assessed by imaging and prostate-specific antigen measurements. RESULTS: Before chemotherapy, 11 patients were Adnatest®-positive whereas five patients were Adnatest®-positive before cycle three. A positive Adnatest® correlated with radiological progression (p=0.02). Rates of disease progression in epidermal growth factor receptor (EGFR)-positive and -negative patients were 100% and 7.7% (p=0.03). CONCLUSION: In this preliminary study, the Adnatest® detected CTCs in a considerable proportion of patients with castration-resistant PC. First data on certain markers (EGFR and aldehyd dehydrogenase 1) encourage future studies investigating transcripts predicting treatment response.

XPA-210: a new proliferation marker determines locally advanced prostate cancer and is a predictor of biochemical recurrence.

PURPOSE: XPA-210 is a proliferation marker derived from Thymidine kinase-1. It is of clinical significance in kidney, breast, and bladder cancer. There are no data available for XPA-210 in prostate cancer (PC). Herein, we aim to determine the clinical usefulness of XPA-210 in PC. MATERIALS AND METHODS: In a retrospective study, cancer and benign tissue samples of 103 patients (median age 65 years, median PSA 9.04 ng/ml, median Gleason score 6) who underwent prostatectomy were constructed to a tissue micro array and stained for XPA-210. Semi-quantitative results were correlated with pathological and clinical data by Wilcoxon-Kruskall-Wallis and linear regression analysis. Expression levels in PC were correlated between the time of biochemical recurrence and the time to development of metastasis by the Kaplan-Meier method. Multivariate analysis was done to correlate those with the resection status. RESULTS: Mean staining score was 0.51-0.14 for tumor and benign tissue (P < 0.0001). Tumor staining score was significantly associated with Gleason score <6/≥6 (P < 0.0001) and T2/T >2 (P = 0.0007). When dividing the tumor score by the mean value, higher expression of XPA-210 was associated with a shorter time to biochemical recurrence (P = 0.003) and time to development of metastasis (P = 0.0061). Tumor staining (P = 0.0371) was an independent prognostic factor for biochemical relapse regardless of resection status. CONCLUSIONS: XPA-210 is a new tissue-based prognostic marker for prostate cancer histopathology. It reliably differentiates tumor and normal prostatic tissue predicting biochemical relapse and onset of metastatic disease. XPA-210 might be clinically useful for individual decision-making in PC-treatment.