Nadine T. Gaisa

Identification and validation of potential new biomarkers for prostate cancer diagnosis and prognosis using 2D-DIGE and MS.

This study was designed to identify and validate potential new biomarkers for prostate cancer and to distinguish patients with and without biochemical relapse. Prostate tissue samples analyzed by 2D-DIGE (two-dimensional difference in gel electrophoresis) and mass spectrometry (MS) revealed downregulation of secernin-1 (P < 0.044) in prostate cancer, while vinculin showed significant upregulation (P < 0.001). Secernin-1 overexpression in prostate tissue was validated using Western blot and immunohistochemistry while vinculin expression was validated using immunohistochemistry. These findings indicate that secernin-1 and vinculin are potential new tissue biomarkers for prostate cancer diagnosis and prognosis, respectively. For validation, protein levels in urine were also examined by Western blot analysis. Urinary vinculin levels in prostate cancer patients were significantly higher than in urine from nontumor patients (P = 0.006). Using multiple reaction monitoring-MS (MRM-MS) analysis, prostatic acid phosphatase (PAP) showed significant higher levels in the urine of prostate cancer patients compared to controls (P = 0.012), while galectin-3 showed significant lower levels in the urine of prostate cancer patients with biochemical relapse, compared to those without relapse (P = 0.017). Three proteins were successfully differentiated between patients with and without prostate cancer and patients with and without relapse by using MRM. Thus, this technique shows promise for implementation as a noninvasive clinical diagnostic technique.

The human urothelium consists of multiple clonal units, each maintained by a stem cell

Little is known about the clonal architecture of human urothelium. It is likely that urothelial stem cells reside within the basal epithelial layer, yet lineage tracing from a single stem cell as a means to show the presence of a urothelial stem cell has never been performed. Here, we identify clonally related cell areas within human bladder mucosa in order to visualize epithelial fields maintained by a single founder/stem cell. Sixteen frozen cystectomy specimens were serially sectioned. Patches of cells deficient for the mitochondrially encoded enzyme cytochrome c oxidase (CCO) were identified using dual‐colour enzyme histochemistry. To show that these patches represent clonal proliferations, small CCO‐proficient and ‐deficient areas were individually laser‐capture microdissected and the entire mitochondrial genome (mtDNA) in each area was PCR amplified and sequenced to identify mtDNA mutations. Immunohistochemistry was performed for the different cell layers of the urothelium and adjacent mesenchyme. CCO‐deficient patches could be observed in normal urothelium of all cystectomy specimens. The two‐dimensional length of these negative patches varied from 2–3 cells (about 30 µm) to 4.7 mm. Each cell area within a CCO‐deficient patch contained an identical somatic mtDNA mutation, indicating that the patch was a clonal unit. Patches contained all the mature cell differentiation stages present in the urothelium, suggesting the presence of a stem cell. Our results demonstrate that the normal mucosa of human bladder contains stem cell‐derived clonal units that actively replenish the urothelium during ageing. The size of the clonal unit attributable to each stem cell was broadly distributed, suggesting replacement of one stem cell clone by another. Copyright

Clonal architecture of human prostatic epithelium in benign and malignant conditions

The location of stem cells in the epithelium of the prostatic acinus remains uncertain, as does the cellular origin of prostatic neoplasia. Here, we apply lineage tracing to visualize the clonal progeny of stem cells in benign and malignant human prostates and understand the clonal architecture of this epithelium. Cells deficient for the mitochondrially‐encoded enzyme cytochrome c oxidase (CCO) were identified in 27 frozen prostatectomy specimens using dual colour enzyme histochemistry and individual CCO‐normal and ‐deficient cell areas were laser‐capture microdissected. PCR‐sequencing of the entire mitochondrial genome (mtDNA) of cells from CCO‐deficient areas found to share mtDNA mutations not present in adjacent CCO‐normal cells, thus proving a clonal origin. Immunohistochemistry was performed to visualize the three cell lineages normally present in the prostatic epithelium. Entire CCO‐deficient acini, and part‐deficient acini were found. Deficient patches spanned either basal or luminal cells, but sometimes also both epithelial cell types in normal, hyperplastic or atrophic epithelium, and prostatic intraepithelial neoplasia (PIN). Patches comprising both PIN and invasive cancer were observed. Each cell area within a CCO‐deficient patch contained an identical mtDNA mutation, defining the patch as a clonal unit. CCO‐deficient patches in benign epithelium contained basal, luminal and endocrine cells, demonstrating multilineage differentiation and therefore the presence of a stem cell. Our results demonstrate that the normal, atrophic, hypertrophic and atypical (PIN) epithelium of human prostate contains stem cell‐derived clonal units that actively replenish the epithelium during ageing. These deficient areas usually included the basal compartment indicating the basal layer as the location of the stem cell. Importantly, single clonal units comprised both PIN and invasive cancer, supporting PIN as the pre‐invasive lesion for prostate cancer. Copyright

Different immunohistochemical and ultrastructural phenotypes of squamous differentiation in bladder cancer

Besides worse prognosis of bladder cancer with squamous differentiation (pure squamous cell carcinoma (SCC) or mixed urothelial carcinoma (UC/SCC)), high-grade non-keratinising squamous differentiation is difficult to identify in haematoxylin–eosin stainings. This study aims to validate routine immunohistochemical markers for squamous differentiation in a larger cohort of patients. Tissue microarrays of 89 pure SCCs and mixed UC/SCCs, 66 urothelial carcinomas (UC), precursor lesions and normal urothelium were stained for cytokeratin (CK) 5/6, CK 5/14, CK 7, CK 20 and uroplakin III. Electron microscopy was performed to confirm the differentiation. Pure SCCs displayed staining throughout the epithelium for CK 5/6 (76.6% (36/47)) and CK 5/14 (95.8% (46/48)), focal staining for CK 7 (28.9% (13/45)) and no staining for CK 20 and uroplakin III (both 0% (0/48)). UCs exhibited a basal or diffuse staining for CK 5/6 (30.2% (16/53)) and CK 5/14 (57.1% (32/56)), focal positivity for CK 7 (83.6% (46/55)), CK 20 (50.9% (29/57)) and uroplakin III (21.8% (12/55)). Each marker discriminated SCC and UC significantly (p < 0.01). A third subgroup rarely showed full epithelial staining for CK 5/6 (14.3% (1/7)) and CK 5/14 (28.6% (2/7)), focal staining for CK 7 (85.7% (6/7)) and no staining for CK 20 and uroplakin III (both 0% (0/7)). Electron microscopy could prove both, SCC and UC characteristics, revealing a transient type. A staining pattern with CK 5/6- and CK 5/14-positivity plus CK 20- and uroplakin III-negativity identified squamous differentiation in bladder tumours and revealed a third type of squamous transdifferentiation.

Hydrogen sulfide does not increase resuscitability in a porcine model of prolonged cardiac arrest.

Treatment options to improve resuscitability and neurological prognosis after cardiac arrest (CA) are limited. Hydrogen sulfide has demonstrated remarkable improvements in outcomes in small animal models of severe hypoxia or hemorrhage. We investigated the influence of sodium sulfide (Na2S), a liquid hydrogen sulfide donor, on resuscitability, postresuscitation hemodynamics, and neurological performance in a porcine model of prolonged CA and cardiopulmonary resuscitation. Twenty-four male pigs were instrumented with arterial and pulmonary artery catheters before 10 min of CA was induced. During resuscitation, animals were randomized to receive either high-dose (1 mg/kg; n = 8) or low-dose (0.3 mg/kg; n = 8) Na2S (IK-1001; Ikaria, Clinton, NJ) or control (saline placebo; n = 8) i.v. injection and consecutive infusion. Cardiopulmonary resuscitation was performed for 6 min before defibrillation was attempted. Hemodynamic variables were taken at baseline and 10, 30, 60, 120, and 240 min after successful resuscitation. Neurological outcome was evaluated on 4 postoperative days before brains and hearts were harvested for histopathologic analysis. No differences in hemodynamic parameters were observed at baseline. Initial resuscitability was not improved by Na2S. Animals exposed to high- and low-dose Na2S showed significantly reduced cardiac output, heart rate, and pulmonary arterial pressure compared with control animals during the early postresuscitation period. Strikingly, two of the high-dose Na2S animals died during the postresuscitation period, whereas all other animals survived. High-dose Na2S significantly decreased microglial activation in striatal areas, although this did not translate into improved neurological outcome. Although animals receiving Na2S developed higher troponin T serum levels, these differences remained insignificant. In this investigation, Na2S did not improve resuscitability but significantly compromised postresuscitation hemodynamics.

Proteomic tissue profiling for the improvement of grading of noninvasive papillary urothelial neoplasia.

OBJECTIVES In 2004, a novel grading system for papillary non-invasive bladder cancer was introduced; low grade (LG) and high grade (HG) in lieu of the former G1, G2, G3. This change allowed for increased reproducibility as well as diminished interobserver variability in histopathological grading among individual pathologists. Matrix Assisted Laser Desorption/Ionization Time of Flight Imaging Mass Spectrometry (MALDI TOF IMS) was evaluated as an automatic and objective tool to assist grading of urothelial neoplasms and to facilitate accuracy. DESIGN AND METHODS To separate G1 (LG, n=27) and G3 (HG, n=21) papillary tumors MALDI TOF IMS was performed using an appropriate algorithm. Thereafter, the automatic assignment of a separate G2 (n=31) group was completed. RESULTS G1 (LG) and G3 (HG) tumors were separated with an overall cross validation of 97.18%. G2 tumors indicated a true positive rate of 78.3% for LG and 87.5% for HG, respectively. CONCLUSIONS MALDI TOF IMS is a powerful support tool to ascertain pathological diagnosis/grading.

Epigenetic inactivation of ITIH5 promotes bladder cancer progression and predicts early relapse of pT1 high-grade urothelial tumours

Inter-α-trypsin inhibitor heavy chain 5 (ITIH5) has been associated with tumour suppression in various cancers. However, its putative role in bladder cancer is completely unknown. Therefore, we initiated a study analysing ITIH5 expression as well as its prognostic and functional impact on human urothelial cancers (UCs). Expression analysis showed a clear down-regulation of ITIH5 mRNA in 61% (n = 45) of UCs, especially in muscle-invasive tumours (P < 0.001). ITIH5 loss in UCs was further evident on protein level (65.5%, n = 55) as detected by immunohistochemistry. DNA methylation analysis demonstrated tumour-specific ITIH5 promoter methylation in 50% of papillary none-invasive pTa (n = 30) and 68% of invasive (n = 28) UCs. Aberrant ITIH5 promoter methylation in bladder tumours was tightly linked (P < 0.001) with loss of ITIH5 mRNA expression, which was furthermore functionally confirmed by demethylation analysis in cell lines. Pyrosequencing analysis revealed that ITIH5 promoter hypermethylation was closely associated with progressive bladder cancers. Subsequently, a large cohort (n = 120) of clinically challenging pT1 high-grade UC was analysed for ITIH5 expression. Of clinical significance, we found an association between loss of ITIH5 expression and unfavourable prognosis of UC patients without distant metastasis at first diagnosis (recurrence-free survival; hazard ratio: 4.35, P = 0.048). Functionally, ITIH5 re-expression in human RT112 bladder cancer cells led to both suppression of cell migration and inhibition of colony spreading. Hence, we provide evidence that down-regulation of ITIH5 by aberrant DNA hypermethylation may provoke invasive phenotypes in human bladder cancer. Moreover, ITIH5 protein might become a prognostic biomarker for relapse risk stratification in high-grade UC patients.

Fibroblast growth factor receptor (FGFR) gene amplifications are rare events in bladder cancer

Activating point mutations and protein overexpression of fibroblast growth factor receptors (FGFRs), especially FGFR3, are frequent events in bladder cancer. Little is known about gene amplifications, therefore we characterized amplification of FGFR1‐3 by fluorescence in‐situ hybridization (FISH).

Epigenetic inactivation of ST6GAL1 in human bladder cancer

BackgroundPosttranslational protein modifications are known to modulate key biological processes like proliferation and apoptosis. Accumulating evidence shows that ST6GAL1, an enzyme that catalyzes the transfer of sialic acid onto galactose-containing substrates, is aberrantly expressed in various cancers and may affect cell motility and invasion. This is the first study to describe ST6GAL1 expression and regulation in human bladder cancer.MethodsST6GAL1 mRNA expression levels in human cell lines (UROtsa, RT4, RT112 and J82) and tissue samples (n = 15 normal urothelium (NU), n = 13 papillary non-invasive tumors (pTa), n = 12 carcinoma in situ (CIS), n = 26 muscle invasive tumors (pT2-4)) were assessed using real-time PCR. In addition, ST6GAL1 protein expression was evaluated using immunohistochemistry. Promoter methylation analysis was performed using methylation-specific PCR (MSP) in cell lines (n = 4) and patient samples (n = 23 NU, n = 12 CIS, n = 29 pTa, n = 41 pT2-4). Epigenetic ST6GAL1 gene silencing was confirmed by in vitro demethylation of bladder cell lines. Data were validated by analysis of an independent bladder tumor data set (n = 184) based on The Cancer Genome Atlas (TCGA) portal.ResultsSemi-quantitative ST6GAL1 real-time PCR expression analysis showed two distinct trends: In muscle-invasive tumors ST6GAL1 expression was downregulation by 2.7-fold, while papillary non-invasive tumors showed an increased ST6GAL1 mRNA expression compared to normal urothelium. ST6GAL1 loss in muscle-invasive tumors was associated with increasing invasiveness. On the protein level, 69.2% (n = 45/65) of all tumors showed a weak ST6GAL1 protein staining (IRS ≤ 4) while 25.6% (16/65) exhibited a complete loss (IRS = 0) of ST6GAL1 protein. Tumor-specific DNA methylation of the ST6GAL1 promoter region was frequently found in pT2-4 tumors (53.6% (22/41)), whereas only 13.8% (4/29) of pTa tumors showed ST6GAL1 promoter methylation. Normal urothelium remained unmethylated. Importantly, we significantly revealed an inverse correlation between ST6GAL1 mRNA expression and ST6GAL1 promoter merthylation in primary bladder cancer. These findings were clearly verified by the TCGA public data set and in vitro demethylation assays functionally confirmed ST6GAL1 promoter methylation as a potential regulatory factor for ST6GAL1 gene silencing.ConclusionsOur study characterizes for the first time ST6GAL1 expression loss caused by aberrant ST6GAL1 promoter methylation potentially indicating a tumor suppressive role in bladder carcinogenesis.

Tissue and serum proteomic profiling for diagnostic and prognostic bladder cancer biomarkers

A panel of biomarkers for the early detection of bladder cancer has not yet been identified. Many different molecules, including DNA, RNA or proteins have been reported but none have provided adequate sensitivity for a single-tier screening test or a test to replace cystoscopy. Therefore, multimarker panels are discussed at present to give a more-precise answer to the biomarker quest. Mass spectrometry or 2D gel-electrophoresis have evolved greatly within recent years and are capable of analyzing multiple proteins or peptides in parallel with high sensitivity and specificity. However, transmission of screening results from one laboratory to another is still the main pitfall of those methods; a fact that emphasizes the need for consistent and standardized procedures as suggested by the Human Proteome Organization (HUPO). In this article, recent results in screening approaches and other proteomic techniques used for biomarker evaluation in bladder cancer are discussed with a focus on serum and tissue biomarkers.