Viral expression and molecular profiling in liver tissue versus microdissected hepatocytes in hepatitis B virus - associated hepatocellular carcinoma.

Background: The molecular mechanisms whereby hepatitis B virus (HBV) induces hepatocellular carcinoma (HCC) remain elusive. We used genomic and molecular techniques to investigate host-virus interactions by studying multiple areas of the same liver from patients with HCC. Methods: We compared the gene signature of whole liver tissue (WLT) versus laser capture-microdissected (LCM) hepatocytes along with the intrahepatic expression of HBV. Gene expression profiling was performed on up to 17 WLT specimens obtained at various distances from the tumor center from individual livers of 11 patients with HCC and on selected LCM samples. HBV markers in liver and serum were determined by real-time polymerase chain reaction (PCR)and confocal immunofluorescence. Results: Analysis of 5 areas of the liver showed a sharp change in gene expression between the immediate perilesional area and tumor periphery that correlated with a significant decrease in the intrahepatic expression of HB surface antigen (HBsAg). The tumor was characterized by a large preponderance of down-regulated genes, mostly involved in the metabolism of lipids and fatty acids, glucose, amino acids and drugs, with down-regulation of pathways involved in the activation of PXR/RXR and PPARα/RXRα nuclear receptors, comprising PGC-1α and FOXO1, two key regulators critically involved not only in the metabolic functions of the liver but also in the life cycle of HBV, acting as essential transcription factors for viral gene expression. These findings were confirmed by gene expression of microdissected hepatocytes. Moreover, LCM of malignant hepatocytes also revealed up-regulation of unique genes associated with cancer and signaling Pathways, including two novel HCC-associated cancer testis antigen genes, NUF2 and TTK. Conclusions: Integrated gene expression profiling of whole liver tissue with that of microdissected hepatocytes demonstrated that HBV-associated HCC is characterized by a metabolism switch-off and by a significant reduction in HBsAg. LCM proved to be a critical tool to validate gene signatures associated with HCC and to identify genes that may play a role in hepatocarcinogenesis, opening new perspectives for the discovery of novel diagnostic markers and therapeutic targets

An exfoliation and enrichment strategy results in improved transcriptional profiles when compared to matched formalin fixed samples

<p>Abstract</p> <p>Background</p> <p>Identifying the influence formalin fixation has on RNA integrity and recovery from clinical tissue specimens is integral to determining the utility of using archival tissue blocks in future molecular studies. For clinical material, the current gold standard is unfixed tissue that has been snap frozen. Fixed and frozen tissue however, both require laser capture microdissection to select for a specific cell population to study. The recent development of a sampling method capable of obtaining a viable, enriched cell population represents an alternative option in procuring cells from clinical material for molecular research purposes. The expression profiles of cells obtained by using this procurement approach, in conjunction with the profiles from cells laser capture microdissected from frozen tissue sections, were compared to the expression profiles from formalin fixed cells to determine the influence fixation has on expression profiles in clinical material.</p> <p>Methods</p> <p>Triplicate samples of non-neoplastic colonic epithelial cells were recovered from a hemicolectomy specimen using three different procurement methods from the same originating site: 1) an exfoliation and enrichment strategy 2) laser capture microdissection from formalin fixed tissue and 3) laser capture microdissection from frozen tissue. Parameters currently in use to assess RNA integrity were utilized to assess the quality of recovered RNA. Additionally, an expression microarray was performed on each sample to assess the influence each procurement technique had on RNA recovery and degradation.</p> <p>Results</p> <p>The exfoliation/enrichment strategy was quantitatively and qualitatively superior to tissue that was formalin fixed. Fixation negatively influenced the expression profile of the formalin fixed group compared to both the frozen and exfoliated/enrichment groups.</p> <p>Conclusion</p> <p>The exfoliation/enrichment technique represents a superior alternative in tissue procurement and RNA recovery relative to formalin fixed tissue. None of the deleterious effects associated with formalin fixation are encountered in the exfoliated/enriched samples because of the absence of its use in this protocol. The exfoliation/enrichment technique also represents an economical alternative that will yield comparable results to cells enriched by laser capture microdissection from frozen tissue sections.</p

Genomic approaches to research in lung cancer

The medical research community is experiencing a marked increase in the amount of information available on genomic sequences and genes expressed by humans and other organisms. This information offers great opportunities for improving our understanding of complex diseases such as lung cancer. In particular, we should expect to witness a rapid increase in the rate of discovery of genes involved in lung cancer pathogenesis and we should be able to develop reliable molecular criteria for classifying lung cancers and predicting biological properties of individual tumors. Achieving these goals will require collaboration by scientists with specialized expertise in medicine, molecular biology, and decision-based statistical analysis

An NIH intramural percubator as a model of academic-industry partnerships: from the beginning of life through the valley of death

In 2009 the NIH publicly announced five strategic goals for the institutes that included the critical need to translate research discoveries into public benefit at an accelerated pace, with a commitment to find novel ways to engage academic investigators in the process. The emphasis on moving scientific advancements from the laboratory to the clinic is an opportune time to discuss how the NIH intramural program in Bethesda, the largest biomedical research center in the world, can participate in this endeavor. Proposed here for consideration is a percolator-incubator program, a 'percubator' designed to enable NIH intramural investigators to develop new medical interventions as quickly and efficiently as possible

Increased matrix metalloproteinase activation in esophageal squamous cell carcinoma

<p>Abstract</p> <p>Background</p> <p>Esophageal squamous cell carcinomas (ESCC) are usually asymptomatic and go undetected until they are incurable. Cytological screening is one strategy to detect ESCC at an early stage and has shown promise in previous studies, although improvement in sensitivity and specificity are needed. Proteases modulate cancer progression by facilitating tumor invasion and metastasis. In the current study, matrix metalloproteinases (MMPs) were studied in a search for new early detection markers for ESCC.</p> <p>Methods</p> <p>Protein expression levels of MMPs were measured using zymography in 24 cases of paired normal esophagus and ESCC, and in the tumor-associated stroma and tumor epithelium in one sample after laser capture microdissection (LCM). MMP-3 and MMP-10 transcripts in both the epithelium and stroma in five cases were further analyzed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR).</p> <p>Results</p> <p>Gelatin zymography showed bands corresponding in size to MMP-2, MMP-3, MMP-9, and MMP-10 enzymes in each of the 24 cancer cases. MMP levels tended to be higher in tumors than paired normal tissue; however, only the 45 kDa band that corresponds to the activated form of MMP-3 and MMP-10 was strongly expressed in all 24 tumors with little or no expression in the paired normal foci. LCM-based analysis showed the 45 kDA band to be present in both the stromal and epithelial components of the tumor microenvironment, and that MMP-3 and MMP-10 mRNA levels were higher in tumors than paired normal tissues for each compartment.</p> <p>Conclusions</p> <p>Increased levels of MMPs occur in ESCC suggesting their up-regulation is important in esophageal tumorigenesis. The up-regulated gene products have the potential to serve as early detection markers in the clinic.</p

Identification of unique expression signatures and therapeutic targets in esophageal squamous cell carcinoma

<p>Abstract</p> <p>Background</p> <p>Esophageal squamous cell carcinoma (ESCC), the predominant histological subtype of esophageal cancer, is characterized by high mortality. Previous work identified important mRNA expression differences between normal and tumor cells; however, to date there are limited <it>ex vivo </it>studies examining expression changes occurring during normal esophageal squamous cell differentiation versus those associated with tumorigenesis. In this study, we used a unique tissue microdissection strategy and microarrays to measure gene expression profiles associated with cell differentiation versus tumorigenesis in twelve cases of patient-matched normal basal squamous epithelial cells (NB), normal differentiated squamous epithelium (ND), and squamous cell cancer. Class comparison and pathway analysis were used to compare NB versus tumor in a search for unique therapeutic targets.</p> <p>Results</p> <p>As a first step towards this goal, gene expression profiles and pathways were evaluated. Overall, ND expression patterns were markedly different from NB and tumor; whereas, tumor and NB were more closely related. Tumor showed a general decrease in differentially expressed genes relative to NB as opposed to ND that exhibited the opposite trend. FSH and IgG networks were most highly dysregulated in normal differentiation and tumorigenesis, respectively. DNA repair pathways were generally elevated in NB and tumor relative to ND indicating involvement in both normal and pathological growth. PDGF signaling pathway and 12 individual genes unique to the tumor/NB comparison were identified as therapeutic targets, and 10 associated ESCC gene-drug pairs were identified. We further examined the protein expression level and the distribution patterns of four genes: ODC1, POSTN, ASPA and IGF2BP3. Ultimately, three genes (ODC1, POSTN, ASPA) were verified to be dysregulated in the same pattern at both the mRNA and protein levels.</p> <p>Conclusions</p> <p>These data reveal insight into genes and molecular pathways mediating ESCC development and provide information potentially useful in designing novel therapeutic interventions for this tumor type.</p

Genome-wide loss of heterozygosity and copy number alteration in esophageal squamous cell carcinoma using the Affymetrix GeneChip Mapping 10 K array

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a common malignancy worldwide. Comprehensive genomic characterization of ESCC will further our understanding of the carcinogenesis process in this disease. RESULTS: Genome-wide detection of chromosomal changes was performed using the Affymetrix GeneChip 10 K single nucleotide polymorphism (SNP) array, including loss of heterozygosity (LOH) and copy number alterations (CNA), for 26 pairs of matched germ-line and micro-dissected tumor DNA samples. LOH regions were identified by two methods – using Affymetrix's genotype call software and using Affymetrix's copy number alteration tool (CNAT) software – and both approaches yielded similar results. Non-random LOH regions were found on 10 chromosomal arms (in decreasing order of frequency: 17p, 9p, 9q, 13q, 17q, 4q, 4p, 3p, 15q, and 5q), including 20 novel LOH regions (10 kb to 4.26 Mb). Fifteen CNA-loss regions (200 kb to 4.3 Mb) and 36 CNA-gain regions (200 kb to 9.3 Mb) were also identified. CONCLUSION: These studies demonstrate that the Affymetrix 10 K SNP chip is a valid platform to integrate analyses of LOH and CNA. The comprehensive knowledge gained from this analysis will enable improved strategies to prevent, diagnose, and treat ESCC

High-throughput genomic technology in research and clinical management of breast cancer. Plasma-based proteomics in early detection and therapy

Protein-based breast cancer biomarkers are a promising resource for breast cancer detection at the earliest and most treatable stages of the disease. Plasma is well suited to proteomic-based methods of biomarker discovery because it is easily obtained, is routinely used in the diagnosis of many diseases, and has a rich proteome. However, due to the vast dynamic range in protein concentration and the often uncertain tissue and cellular origin of plasma proteins, proteomic analysis of plasma requires special consideration compared with tissue and cultured cells. This review briefly touches on the search for plasma-based protein biomarkers for the early detection and treatment of breast cancer

Delineation of prognostic biomarkers in prostate cancer

Prostate cancer is the most frequently diagnosed cancer in American men(1,2). Screening for prostate-specific antigen (PSA) has led to earlier detection of prostate cancer(3), but elevated serum PSA levels may be present in non-malignant conditions such as benign prostatic hyperlasia (BPH). Characterization of gene-expression profiles that molecularly distinguish prostatic neoplasms may identify genes involved in prostate carcinogenesis, elucidate clinical biomarkers, and lead to an improved classification of prostate cancer(4-6). Using microarrays of complementary DNA, we examined gene-expression profiles of more than 50 normal and neoplastic prostate specimens and three common prostate-cancer cell lines. Signature expression profiles of normal adjacent prostate (NAP), BPH, localized prostate cancer, and metastatic, hormone-refractory prostate cancer were determined. Here we establish many associations between genes and prostate cancer. We assessed two of these genes-hepsin, a transmembrane serine protease, and pim-1, a serine/threonine kinase-at the protein level using tissue microarrays consisting of over 700 clinically stratified prostate-cancer specimens. Expression of hepsin and pim-1 proteins was significantly correlated with measures of clinical outcome. Thus, the integration of cDNA microarray, high-density tissue microarray, and linked clinical and pathology data is a powerful approach to molecular profiling of human cancer.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62849/1/412822a0.pd