Differential expression of steroid 5 alpha-reductase isozymes and association with disease severity and angiogenic genes predict their biological role in prostate cancer

The biological role of steroid 5 alpha-reductase isozymes (encoded by the SRD5A1 and SRD5A2 genes) and angiogenic factors that play important roles in the pathogenesis and vascularization of prostate cancer (PC) is poorly understood. The sub-cellular expression of these isozymes and vascular endothelial growth factor (VEGF) in PC tissue microarrays (n=62) was examined using immunohistochemistry. The effect of SRD5A inhibition on the angiogenesis pathway genes in PC was also examined in prostate cell lines, LNCaP, PC3, and RWPE-1, by treating them with the SRD5A inhibitors finasteride and dutasteride, followed by western blot, quantitative PCR, and ELISA chip array techniques. In PC tissues, nuclear SRD5A1 expression was strongly associated with higher cancer Gleason scores (P=0.02), higher cancer stage (P=0.01), and higher serum prostate specific antigen (PSA) levels (P=0.01), whereas nuclear SRD5A2 expression was correlated with VEGF expression (P=0.01). Prostate tumor cell viability was significantly reduced in dutasteride-treated PC3 and RWPE-1 cells compared with finasteride-treated groups. Expression of the angiogenesis pathway genes transforming growth factor beta 1 (TGFB1), endothelin (EDN1), TGF alpha (TGFA), and VEGFR1 was upregulated in LNCaP cells, and at least 7 out of 21 genes were upregulated in PC3 cells treated with finasteride (25 mu M). Our findings suggest that SRD5A1 expression predominates in advanced PC, and that inhibition of SRD5A1 and SRD5A2 together was more effective in reducing cell numbers than inhibition of SRD5A2 alone. However, these inhibitors did not show any significant difference in prostate cell angiogenic response. Interestingly, some angiogenic genes remained activated after treatment, possibly due to the duration of treatment and tumor resistance to inhibitors. Endocrine-Related Cancer (2010) 17 757-770

CD133 expression predicts for non-response to chemotherapy in colorectal cancer

The cancer stem cell hypothesis may explain why conventional chemotherapies are unable to fully eradicate cancers. In this study, we examined both the prognostic and predictive significance of putative cancer stem cell markers in colorectal cancer. In this study, immunohistochemistry for three candidate cancer stem cell markers (CD133, Oct-4 and Sox-2) and for six other postulated prognostic markers (CK7, CK20, Cox-2, Ki-67, p27 and p53) were performed using tissue microarrays containing 501 primary colorectal cancer cases. Receiver-operating characteristic analysis was used to determine cut-off scores for positive protein expression. Multivariate analysis revealed that positive expression for CD133 and Oct-4 was associated with significantly worse survival in patients treated by surgery alone (P=0.023 and P

Novel breast cancer biomarkers identified by integrative proteomic and gene expression mapping

Proteomic and transcriptomic platforms both play important roles in cancer research, with differing strengths and limitations. Here, we describe a proteo-transcriptomic integrative strategy for discovering novel cancer biomarkers, combining the direct visualization of differentially expressed proteins with the high-throughput scale of gene expression profiling. Using breast cancer as a case example, we generated comprehensive two-dimensional electrophoresis (2DE)/mass spectrometry (MS) proteomic maps of cancer (MCF-7 and HCC-38) and control (CCD-1059Sk) cell lines, identifying 1724 expressed protein spots representing 484 different protein species. The differentially expressed cell-line proteins were then mapped to mRNA transcript databases of cancer cell lines and primary breast tumors to identify candidate biomarkers that were concordantly expressed at the gene expression level. Of the top nine selected biomarker candidates, we reidentified ANX1, a protein previously reported to be differentially expressed in breast cancers and normal tissues, and validated three other novel candidates, CRAB, 6PGL, and CAZ2, as differentially expressed proteins by immunohistochemistry on breast tissue microarrays. In total, close to half (4/9) of our protein biomarker candidates were successfully validated. Our study thus illustrates how the systematic integration of proteomic and transcriptomic data from both cell line and primary tissue samples can prove advantageous for accelerating cancer biomarker discovery.

Targets of genome copy number reduction in primary breast cancers identified by integrative genomics

The identification of specific oncogenes and tumor suppressor genes in regions of recurrent aneuploidy is a major challenge of molecular cancer research. Using both oligonucleotide single-nucleotide polymorphism and mRNA expression arrays, we integrated genomic and transcriptional information to identify and prioritize candidate cancer genes in regions of increased and decreased chromosomal copy number in a cohort of primary breast cancers. Confirming the validity of this approach, several regions of previously-known copy number (CN) alterations in breast cancer could be successfully reidentified. Focusing on regions of decreased CN, we defined a prioritized list of eighteen candidate genes, which included ARPIN, FBNI, and LZTSI, previously shown to be associated with cancers in breast or other tissue types, and novel genes such as P29, MORF4LI, and TBCID5. One such gene, the RUNX3 transcription factor, was selected for further study. We show that RUNX3 is present at reduced CNs in proportion to the rest of the tumor genome and that RUNX3 CN reductions can also be observed in a breast cancer series from a different center. Using tissue microarrays, we demonstrate in an independent cohort of over 120 breast tissues that RUNX3 protein is expressed in normal breast epithelium but not fat and stromal tissue, and widely down-regulated in the majority of breast cancers (> 85%). In vitro, RUNX3 overexpression suppressed the invasive potential of MDA-MB-231 breast cancer cells in a matrigel assay. Our results demonstrate the utility of integrative genomic approaches to identify novel potential cancer-related genes in primary tumors. This article contains Supplementary Material available at http:// www.interscience.wiley.com/jpages/1045-2257/suppmat. (c) 2006 Wiley-Liss, Inc.

Ultra-fast processing of gigapixel Tissue MicroArray images using high performance computing.

BACKGROUND:
tissue MicroArrays (TMAs) are a valuable platform for tissue based translational research and the discovery of tissue biomarkers. The digitised TMA slides or TMA Virtual Slides, are ultra-large digital images, and can contain several hundred samples. The processing of such slides is time-consuming, bottlenecking a potentially high throughput platform.
METHODS:
a High Performance Computing (HPC) platform for the rapid analysis of TMA virtual slides is presented in this study. Using an HP high performance cluster and a centralised dynamic load balancing approach, the simultaneous analysis of multiple tissue-cores were established. This was evaluated on Non-Small Cell Lung Cancer TMAs for complex analysis of tissue pattern and immunohistochemical positivity.
RESULTS:
the automated processing of a single TMA virtual slide containing 230 patient samples can be significantly speeded up by a factor of circa 22, bringing the analysis time to one minute. Over 90 TMAs could also be analysed simultaneously, speeding up multiplex biomarker experiments enormously.
CONCLUSIONS:
the methodologies developed in this paper provide for the first time a genuine high throughput analysis platform for TMA biomarker discovery that will significantly enhance the reliability and speed for biomarker research. This will have widespread implications in translational tissue based research.

Prognostic Significance of TRAIL Signaling Molecules in Stage II and III Colorectal Cancer

Purpose: We previously found that cellular FLICE-inhibitory protein (c-FLIP), caspase 8, and tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) receptor 2 (DR5) are major regulators of cell viability and chemotherapy-induced apoptosis in colorectal cancer. In this study, we determined the prognostic significance of c-FLIP, caspase 8, TRAIL and DR5 expression in tissues from patients with stage II and III colorectal cancer.

Experimental Design: Tissue microarrays were constructed from matched normal and tumor tissue derived from patients (n = 253) enrolled in a phase III trial of adjuvant 5-fluorouracil–based chemotherapy versus postoperative observation alone. TRAIL, DR5, caspase 8, and c-FLIP expression levels were determined by immunohistochemistry.

Results: Colorectal tumors displayed significantly higher expression levels of c-FLIP (P < 0.001), caspase 8 (P = 0.01), and DR5 (P < 0.001), but lower levels of TRAIL (P < 0.001) compared with matched normal tissue. In univariate analysis, higher TRAIL expression in the tumor was associated with worse overall survival (P = 0.026), with a trend to decreased relapse-free survival (RFS; P = 0.06), and higher tumor c-FLIP expression was associated with a significantly decreased RFS (P = 0.015). Using multivariate predictive modeling for RFS in all patients and including all biomarkers, age, treatment, and stage, we found that the model was significant when the mean tumor c-FLIP expression score and disease stage were included (P < 0.001). As regards overall survival, the overall model was predictive when both TRAIL expression and disease stage were included (P < 0.001).

Conclusions: High c-FLIP and TRAIL expression may be independent adverse prognostic markers in stage II and III colorectal cancer and might identify patients most at risk of relapse.

A TMA De-Arraying Method for High Throughput Biomarker Discovery in Tissue Research

Background: Tissue MicroArrays (TMAs) represent a potential high-throughput platform for the analysis and discovery of tissue biomarkers. As TMA slides are produced manually and subject to processing and sectioning artefacts, the layout of TMA cores on the final slide and subsequent digital scan (TMA digital slide) is often disturbed making it difficult to associate cores with their original position in the planned TMA map. Additionally, the individual cores can be greatly altered and contain numerous irregularities such as missing cores, grid rotation and stretching. These factors demand the development of a robust method for de-arraying TMAs which identifies each TMA core, and assigns them to their appropriate coordinates on the constructed TMA slide.

Methodology: This study presents a robust TMA de-arraying method consisting of three functional phases: TMA core segmentation, gridding and mapping. The segmentation of TMA cores uses a set of morphological operations to identify each TMA core. Gridding then utilises a Delaunay Triangulation based method to find the row and column indices of each TMA core. Finally, mapping correlates each TMA core from a high resolution TMA whole slide image with its name within a TMAMap.

Conclusion: This study describes a genuine robust TMA de-arraying algorithm for the rapid identification of TMA cores from digital slides. The result of this de-arraying algorithm allows the easy partition of each TMA core for further processing. Based on a test group of 19 TMA slides (3129 cores), 99.84% of cores were segmented successfully, 99.81% of cores were gridded correctly and 99.96% of cores were mapped with their correct names via TMAMaps. The gridding of TMA cores were also extensively tested using a set of 113 pseudo slide (13,536 cores) with a variety of irregular grid layouts including missing cores, rotation and stretching. 100% of the cores were gridded correctly.

Genome-wide profiling of methylation identifies novel targets with aberrant hyper-methylation and reduced expression in low-risk myelodysplastic syndromes

Gene expression profiling signatures may be used to classify the subtypes of Myelodysplastic syndrome (MDS) patients. However, there are few reports on the global methylation status in MDS. The integration of genome-wide epigenetic regulatory marks with gene expression levels would provide additional information regarding the biological differences between MDS and healthy controls. Gene expression and methylation status were measured using high-density microarrays. A total of 552 differentially methylated CpG loci were identified as being present in low-risk MDS; hypermethylated genes were more frequent than hypomethylated genes. In addition, mRNA expression profiling identified 1005 genes that significantly differed between low-risk MDS and the control group. Integrative analysis of the epigenetic and expression profiles revealed that 66.7% of the hypermethylated genes were underexpressed in low-risk MDS cases. Gene network analysis revealed molecular mechanisms associated with the low-risk MDS group, including altered apoptosis pathways. The two key apoptotic genes BCL2 and ETS1 were identified as silenced genes. In addition, the immune response and micro RNA biogenesis were affected by the hypermethylation and underexpression of IL27RA and DICER1. Our integrative analysis revealed that aberrant epigenetic regulation is a hallmark of low-risk MDS patients and could have a central role in these diseases.

TMAX – a Database System Integrating Tissue Biomarker & Genomic Data

Background: Popular approaches in human tissue-based biomarker discovery include tissue microarrays (TMAs) and DNA Microarrays (DMAs) for protein and gene expression profiling respectively. The data generated by these analytic platforms, together with associated image, clinical and pathological data currently reside on widely different information platforms, making searching and cross-platform analysis difficult. Consequently, there is a strong need to develop a single coherent database capable of correlating all available data types.

Method: This study presents TMAX, a database system to facilitate biomarker discovery tasks. TMAX organises a variety of biomarker discovery-related data into the database. Both TMA and DMA experimental data are integrated in TMAX and connected through common DNA/protein biomarkers. Patient clinical data (including tissue pathological data), computer assisted tissue image and associated analytic data are also included in TMAX to enable the truly high throughput processing of ultra-large digital slides for both TMAs and whole slide tissue digital slides. A comprehensive web front-end was built with embedded XML parser software and predefined SQL queries to enable rapid data exchange in the form of standard XML files.

Results & Conclusion: TMAX represents one of the first attempts to integrate TMA data with public gene expression experiment data. Experiments suggest that TMAX is robust in managing large quantities of data from different sources (clinical, TMA, DMA and image analysis). Its web front-end is user friendly, easy to use, and most importantly allows the rapid and easy data exchange of biomarker discovery related data. In conclusion, TMAX is a robust biomarker discovery data repository and research tool, which opens up the opportunities for biomarker discovery and further integromics research.

Molecular similarity between myelodysplastic form of chronic myelomonocytic leukemia and refractory anemia with ring sideroblasts

Chronic myelomonocytic leukemia is similar to but a separate entity from both myeloproliferative neoplasms and myelodysplastic syndromes, and shows either myeloproliferative or myelodysplastic features. We ask whether this distinction may have a molecular basis. We established the gene expression profiles of 39 samples of chronic myelomonocytic leukemia (including 12 CD34-positive) and 32 CD34-positive samples of myelodysplastic syndromes by using Affymetrix microarrays, and studied the status of 18 genes by Sanger sequencing and array-comparative genomic hybridization in 53 samples. Analysis of 12 mRNAS from chronic myelomonocytic leukemia established a gene expression signature of 122 probe sets differentially expressed between proliferative and dysplastic cases of chronic myelomonocytic leukemia. As compared to proliferative cases, dysplastic cases over-expressed genes involved in red blood cell biology. When applied to 32 myelodysplastic syndromes, this gene expression signature was able to discriminate refractory anemias with ring sideroblasts from refractory anemias with excess of blasts. By comparing mRNAS from these two forms of myelodysplastic syndromes we derived a second gene expression signature. This signature separated the myelodysplastic and myeloproliferative forms of chronic myelomonocytic leukemias. These results were validated using two independent gene expression data sets. We found that myelodysplastic chronic myelomonocytic leukemias are characterized by mutations in transcription/epigenetic regulators (ASXL1, RUNX1, TET2) and splicing genes (SRSF2) and the absence of mutations in signaling genes. Myelodysplastic chronic myelomonocytic leukemias and refractory anemias with ring sideroblasts share a common expression program suggesting they are part of a continuum, which is not totally explained by their similar but not, however, identical mutation spectrum. © 2013 Ferrata Storti Foundation.

Overexpression of neurone glial-related cell adhesion molecule is an independent predictor of poor prognosis in advanced colorectal cancer

A downstream target of the Wnt pathway, neurone glial-related cell adhesion molecule (Nr-CAM) has recently been implicated in human cancer development. However, its role in colorectal cancer (CRC) pathobiology and clinical relevance remains unknown. In this study, we examined the clinical significance of Nr-CAM protein expression in a retrospective series of 428 CRCs using immunohistochemistry and tissue microarrays. Cox proportional hazards regression was used to calculate hazard ratios (HR) of mortality according to various clinicopathological features and molecular markers. All CRC samples were immunoreactive for Nr-CAM protein expression, compared to 10 / 245 (4%) matched normal tissue (P <0.0001). Of 428 CRC samples, 97 (23%) showed Nr-CAM overexpression, which was significantly associated with nodal (P = 0.012) and distant (P = 0.039) metastasis, but not with extent of local invasion or tumor size. Additionally, Nr-CAM overexpression was associated with vascular invasion (P = 0.0029), p53 expression (P = 0.036), and peritoneal metastasis at diagnosis (P = 0.013). In a multivariate model adjusted for other clinicopathological predictors of survival, Nr-CAM overexpression correlated with a significant increase in disease-specific (HR 1.66; 95% confidence interval 1.11-2.47; P = 0.014) and overall mortality (HR 1.57; 95% confidence interval 1.07-2.30; P = 0.023) in advanced but not early stage disease. Notably, 5-fluorouracil-based chemotherapy conferred significant survival benefit to patients with tumors negative for Nr-CAM overexpression but not to those with Nr-CAM overexpressed tumors. In conclusion, Nr-CAM protein expression is upregulated in CRC tissues. Nr-CAM overexpression is an independent marker of poor prognosis among advanced CRC patients, and is a possible predictive marker for non-beneficence to 5-fluorouracil- based chemotherapy.

Development of a Portable Tissue Micro Array Instrument

Tissue micro array (TMA) is based on the idea of applying miniaturization and a high throughput approach to hybridization-based analyses of tissues. It facilitates biomedical research on a large scale in a single experiment; thus representing one of the most commonly used technologies in translational research. A critical analysis of the existing TMA instruments indicates that there are potential constraints in terms of portability, apart from costs and complexity. This paper will present the development of an affordable, configurable, and portable TMA instrument to allow an efficient collection of tissues, especially in instrument-to-tissue scenarios. The purely mechanical instrument requires no energy sources other than the user, is light weight, portable, and simple to use. [DOI: 10.1115/1.4004922]

Multiplex biotoxin surface plasmon resonance method for marine biotoxins in algal and seawater samples

A multiplex surface plasmon resonance (SPR) biosensor method for the detection of paralytic shellfish poisoning (PSP) toxins, okadaic acid (and analogues) and domoic acid was developed. This method was compared to enzyme-linked immunosorbent assay (ELISA) methods. Seawater samples (n?=?256) from around Europe were collected by the consortia of an EU project MIcroarrays for the Detection of Toxic Algae (MIDTAL) and evaluated using each method. A simple sample preparation procedure was developed which involved lysing and releasing the toxins from the algal cells with glass beads followed by centrifugation and filtering the extract before testing for marine biotoxins by both multi-SPR and ELISA. Method detection limits based on IC20 values for PSP, okadaic acid and domoic acid toxins were 0.82, 0.36 and 1.66 ng/ml, respectively, for the prototype multiplex SPR biosensor. Evaluation by SPR for seawater samples has shown that 47, 59 and 61 % of total seawater samples tested positive (result greater than the IC20) for PSP, okadaic acid (and analogues) and domoic acid toxins, respectively. Toxic samples were received mainly from Spain and Ireland. This work has demonstrated the potential of multiplex analysis for marine biotoxins in algal and seawater samples with results available for 24 samples within a 7 h period for three groups of key marine biotoxins. Multiplex immunological methods could therefore be used as early warning monitoring tools for a variety of marine biotoxins in seawater samples.

Monitoring a toxic bloom of Alexandrium minutum using novel microarray and multiplex surface plasmon resonance biosensor technology

Blooms of Alexandrium occur annually during the summer months in the North Channel of Cork Harbour on the south coast of Ireland. This study monitored an extensive bloom of the toxin producing Alexandrium minutum during the summer of 2011 with the use of the MIDTAL (Microarrays for the Detection of Toxic Algae) microarray and a prototype multiplex surface plasmon resonance (multi SPR) biosensor. Microarray signal intensities and toxin results from three testing platforms of the prototype multi SPR biosensor, commercial (CER) enzyme-linked immunosorbent assay (ELISA) and high performance liquid chromatography (HPLC) were compared against light microscopy counts. The main aim was to demonstrate the use of these methodologies to support national monitoring agencies by providing a faster and more accurate means of identifying and quantifying the harmful phytoplankton community and their toxins in natural water samples. Both the microarray signals and multi SPR biosensor results followed a significant trend with light microscopy results and both techniques indicated detection limits of <4000 cells of A. minutum in natural seawater samples.

The prognostic significance of the aberrant extremes of p53 immunophenotypes in breast cancer

Aims: The utility of p53 as a prognostic assay has been elusive. The aims of this study were to describe a novel, reproducible scoring system and assess the relationship between differential p53 immunohistochemistry (IHC) expression patterns, TP53 mutation status and patient outcomes in breast cancer.

Methods and Results: Tissue microarrays were used to study p53 IHC expression patterns: expression was defined as extreme positive (EP), extreme negative (EN), and non-extreme (NE; intermediate patterns). Overall survival (OS) was used to define patient outcome. A representative subgroup (n = 30) showing the various p53 immunophenotypes was analysed for TP53 hotspot mutation status (exons 4-9). Extreme expression of any type occurred in 176 of 288 (61%) cases. As compared with NE expression, EP expression was significantly associated (P = 0.039) with poorer OS. In addition, as compared with NE expression, EN expression was associated (P = 0.059) with poorer OS. Combining cases showing either EP or EN expression better predicted OS than either pattern alone (P = 0.028). This combination immunophenotype was significant in univariate but not multivariate analysis. In subgroup analysis, six substitution exon mutations were detected, all corresponding to extreme IHC phenotypes. Five missense mutations corresponded to EP staining, and the nonsense mutation corresponded to EN staining. No mutations were detected in the NE group.

Conclusions: Patients with extreme p53 IHC expression have a worse OS than those with NE expression. Accounting for EN as well as EP expression improves the prognostic impact. Extreme expression positively correlates with nodal stage and histological grade, and negatively with hormone receptor status. Extreme expression may relate to specific mutational status.