Correction to: High resolution hemodynamic profiling of murine arteriovenous fistula using magnetic resonance imaging and computational fluid dynamics.

After publication of the original article.

Endostatin: A novel inhibitor of androgen receptor function in prostate cancer.

Acquired resistance to androgen receptor (AR)-targeted therapies compels the development of novel treatment strategies for castration-resistant prostate cancer (CRPC). Here, we report a profound effect of endostatin on prostate cancer cells by efficient intracellular trafficking, direct interaction with AR, reduction of nuclear AR level, and down-regulation of AR-target gene transcription. Structural modeling followed by functional analyses further revealed that phenylalanine-rich α1-helix in endostatin-which shares structural similarity with noncanonical nuclear receptor box in AR-antagonizes AR transcriptional activity by occupying the activation function (AF)-2 binding interface for coactivators and N-terminal AR AF-1. Together, our data suggest that endostatin can be recognized as an endogenous AR inhibitor that impairs receptor function through protein-protein interaction. These findings provide new insights into endostatin whose antitumor effect is not limited to inhibiting angiogenesis, but can be translated to suppressing AR-mediated disease progression in CRPC.

Differential Gene Expression in Ductal Carcinoma In Situ of the Breast Based on ERBB2 Status.

BACKGROUND: The molecular signature of ductal carcinoma in situ (DCIS) in the breast is not well understood. Erb-b2 receptor tyrosine kinase 2 (ERBB2 [formerly known as HER2/neu]) positivity in DCIS is predictive of coexistent early invasive breast carcinoma. The aim of this study is to identify the gene-expression signature profiles of estrogen receptor (ER)/progesterone receptor (PR)-positive, ERBB2, and triple-negative subtypes of DCIS. METHODS: Based on ER, PR, and ERBB2 status, a total of 18 high nuclear grade DCIS cases with no evidence of invasive breast carcinoma were selected along with 6 non-neoplastic controls. The 3 study groups were defined as ER/PR-positive, ERBB2, and triple-negative subtypes. RESULTS: A total of 49 genes were differentially expressed in the ERBB2 subtype compared with the ER/PR-positive and triple-negative groups. PROM1 was overexpressed in the ERBB2 subtype compared with ER/PR-positive and triple-negative subtypes. Other genes differentially expressed included TAOK1, AREG, AGR3, PEG10, and MMP9. CONCLUSIONS: Our study identified unique gene signatures in ERBB2-positive DCIS, which may be associated with the development of invasive breast carcinoma. The results may enhance our understanding of the progression of breast cancer and become the basis for developing new predictive biomarkers and therapeutic targets for DCIS.

High resolution hemodynamic profiling of murine arteriovenous fistula using magnetic resonance imaging and computational fluid dynamics.

BACKGROUND: Arteriovenous fistula (AVF) maturation failure remains a major cause of morbidity and mortality in hemodialysis patients. The two major etiologies of AVF maturation failure are early neointimal hyperplasia development and persistent inadequate outward remodeling. Although hemodynamic changes following AVF creation may impact AVF remodeling and contribute to neointimal hyperplasia development and impaired outward remodeling, detailed AVF hemodynamics are not yet fully known. Since murine AVF models are valuable tools for investigating the pathophysiology of AVF maturation failure, there is a need for a new approach that allows the hemodynamic characterization of murine AVF at high resolutions. METHODS: This methods paper presents a magnetic resonance imaging (MRI)-based computational fluid dynamic (CFD) method that we developed to rigorously quantify the evolving hemodynamic environment in murine AVF. The lumen geometry of the entire murine AVF was reconstructed from high resolution, non-contrast 2D T2-weighted fast spin echo MRI sequence, and the flow rates of the AVF inflow and outflow were extracted from a gradient echo velocity mapping sequence. Using these MRI-obtained lumen geometry and inflow information, CFD modeling was performed and used to calculate blood flow velocity and hemodynamic factors at high resolutions (on the order of 0.5 µm spatially and 0.1 ms temporally) throughout the entire AVF lumen. We investigated both the wall properties (including wall shear stress (WSS), wall shear stress spatial gradient, and oscillatory shear index (OSI)) and the volumetric properties (including vorticity, helicity, and Q-criterion). RESULTS: Our results demonstrate increases in AVF flow velocity, WSS, spatial WSS gradient, and OSI within 3 weeks post-AVF creation when compared to pre-surgery. We also observed post-operative increases in flow disturbances and vortices, as indicated by increased vorticity, helicity, and Q-criterion. CONCLUSIONS: This novel protocol will enable us to undertake future mechanistic studies to delineate the relationship between hemodynamics and AVF development and characterize biological mechanisms that regulate local hemodynamic factors in transgenic murine AVF models.

The protective effect of p16(INK4a) in oral cavity carcinomas: p16(Ink4A) dampens tumor invasion-integrated analysis of expression and kinomics pathways.

A large body of evidence shows that p16(INK4a) overexpression predicts improved survival and increased radiosensitivity in HPV-mediated oropharyngeal squamous cell carcinomas.(OPSCC). Here we demonstrate that the presence of transcriptionally active HPV16 in oral cavity squamous cell carcinomas does not correlate with p16(INK4a) overexpression, enhanced local tumor immunity, or improved outcome. It is interesting that HPV-mediated oropharyngeal squamous cell carcinomas can be categorized as having a 'nonaggressive' invasion phenotype, whereas aggressive invasion phenotypes are more common in HPV-negative squamous cell carcinomas. We have developed primary cancer cell lines from resections with known pattern of invasion as determined by our validated risk model. Given that cell lines derived from HPV-mediated oropharyngeal squamous cell carcinomas are less invasive than their HPV-negative counterparts, we tested the hypothesis that viral oncoproteins E6, E7, and p16(INK4a) can affect tumor invasion. Here we demonstrate that p16(INK4a) overexpression in two cancer cell lines (UAB-3 and UAB-4), derived from oral cavity squamous cell carcinomas with the most aggressive invasive phenotype (worst pattern of invasion type 5 (WPOI-5)), dramatically decreases tumor invasiveness by altering expression of extracellular matrix remodeling genes. Pathway analysis integrating changes in RNA expression and kinase activities reveals different potential p16(INK4a)-sensitive pathways. Overexpressing p16(INK4a) in UAB-3 increases EGFR activity and increases MMP1 and MMP3 expression, possibly through STAT3 activation. Overexpressing p16(INK4a) in UAB-4 decreases PDGFR gene expression and reduces MMP1 and MMP3, possibly through STAT3 inactivation. Alternatively, ZAP70/Syk might increase MUC1 phosphorylation, leading to the observed decreased MMP1 expression.

Noggin is novel inducer of mesenchymal stem cell adipogenesis: implications for bone health and obesity.

Noggin is a glycosylated-secreted protein known so far for its inhibitory effects on bone morphogenetic protein (BMP) signaling by sequestering the BMP ligand. We report here for the first time a novel mechanism by which noggin directly induces adipogenesis of mesenchymal stem cells independently of major human adipogenic signals through C/EBPδ, C/EBPα and peroxisome proliferator-activated receptor-γ. Evaluation of a possible mechanism for noggin-induced adipogenesis of mesenchymal stem cells identified the role of Pax-1 in mediating such differentiation. The relevance of elevated noggin levels in obesity was confirmed in a preclinical, immunocompetent mouse model of spontaneous obesity and in human patients with higher body mass index. These data clearly provide a novel role for noggin in inducing adipogenesis and possibly obesity and further indicates the potential of noggin as a therapeutic target to control obesity.

Ultrastructural and molecular confirmation of the trichodysplasia spinulosa-associated polyomavirus in biopsies of patients with trichodysplasia spinulosa.

Trichodysplasia spinulosa (TS) is a rare and only recently characterized cutaneous disease occurring in immunocompromised patients. The disease is characterized by spiny follicular papules on clinical examination and by the presence of viral inclusions at ultrastructural examination. In the last year, this virus has been identified as a new member of the polyomavirus family and designated as TS-associated polyomavirus (TSPyV). We report two organ transplant patients with this disease in which we were able to identify the TSPyV at ultrastructural and molecular level from formalin-fixed paraffin-embedded biopsies of lesional skin. Similar to prior described cases, the patients presented with follicular papules which were concentrated on the central face and associated with alopecia. Histopathology of both cases showed dilated follicular infundibula plugged with cornified eosinophilic cells containing large trichohyaline granules. Transmission electron microscopy on paraffin-embedded tissue in case 1 showed 28-nm intracellular viral particles morphologically consistent with polyoma virus. For both cases the presence of TSPyV was confirmed by polymerase chain reaction with virus-specific primers followed by identification by direct sequencing. These two cases show the presence of the newly described TSPyV in TS further establishing its association with this distinctive disease.

Tumoristatic effects of endostatin in prostate cancer is dependent on androgen receptor status.

BACKGROUND: Although anti-angiogenic therapy is a promising new line of therapy for prostate cancer, we recently reported that stable expression of endostatin arrested the progression of prostate cancer to poorly differentiated state and distant metastasis in TRAMP mice. However, the same therapy failed to provide any benefit when given either during or after the onset of metastatic switch. The present study determined the possible mechanisms behind the selective advantage of endostatin therapy in early-stage disease. METHODS: Angiogenesis-related gene expression analysis was performed to identify target genes and molecular pathways involved in the therapy effects. Based on the results from in vivo studies, and recapitulation of the in vivo data in vitro using tumorigenic and non-tumorigenic human prostate cancer cells that are either androgen-sensitive or androgen-independent, analyses of possible mechanisms of the selective advantage of early treatment were performed using assays for cell proliferation, apoptosis, migration, and cell signaling. The identified mechanisms were further confirmed in vivo. RESULTS: Results indicated that cells with high androgen receptor (AR) expression were more sensitive to endostatin treatment than androgen-independent cells with low or no AR expression. Endostatin was found to significantly downregulate the expression of growth factors, receptor tyrosine kinases, proteases, and AR both in vitro and in vivo only when the cells express high-levels of AR. Cell proliferation was not influenced by endostatin treatment but migration was significantly affected only in androgen-sensitive cells. Targeted downregulation of AR prior to endostatin treatment in androgen-sensitive cells and overexpression of AR in androgen-independent cells indicated that the effect of endostatin via AR downregulation is mediated by a non-genotropic mechanism on Ras and RhoA pathways, and independently of AR on MAPK/ERK pathway. CONCLUSIONS: These data indicate that systemically stable endostatin expression delays the onset of metastatic switch by acting on multiple pathways involving AR.

High Cks1 expression in transgenic and carcinogen-initiated mammary tumors is not always accompanied by reduction in p27Kip1.

Cks1 plays an essential role in SCFSkp2-mediated ubiquitination, and consequently turnover, of the cdk2 inhibitor and tumor supressor p27Kip1. High Cks1 expression is associated with aggressive breast tumors and correlates with low p27Kip1 levels in some cases, although it is also an independent prognostic marker for survival, and provides predictive information in addition to that provided by p27Kip1 alone. In this report we demonstrate that Cks1 protein and mRNA are elevated to very high levels in mammary tumors initiated by erbB2, c-myc and polyoma middle-T (PyMT) in transgenic mice, whereas Cks1 protein is hardly detectable in the normal mammary epithelium. Cks1 is also highly upregulated in rat mammary tumors initiated by methylnitrosourea (MNU). Despite high levels of Cks1 expression, p27Kip1 levels were not reduced, and were in fact slightly higher in mammary tumors initiated by erbB2, PyMT and MNU. In contrast mammary tumors from MMTV-c-myc mice did exhibit low p27Kip1 and higher levels of Skp2. Together, these data suggest that deregulated Cks1 expression might play roles in oncogene and carcinogen-initiated mammary tumorigenesis independent of p27Kip1 turnover in certain tumors. Stable overexpression of Cks1 in human breast carcinoma MCF-7 cells did not significantly reduce p27Kip1 expression, although it conferred resistance to Faslodex (ICI 182780)-mediated inhibition of colony outgrowth in these cells. In contrast, Cks1-depleted MCF-7 cells formed fewer colonies in estrogen-containing medium. Therefore, our studies also suggest that Cks1 levels regulate the responsiveness of ER+ breast cancers to estrogens and anti-estrogens.

Silencing of transforming growth factor-beta1 in situ by RNA interference for breast cancer: implications for proliferation and migration in vitro and metastasis in vivo.

PURPOSE: Overexpression of transforming growth factor (TGF)-beta has been implicated in promoting immune suppression, tumor angiogenesis, tumor cell migration, and invasion in many cancers, including carcinoma of the breast. Thus, targeted down-regulation of TGF-beta1 expression in breast cancer in situ and determination of its implications would provide new treatment approaches for disease management. EXPERIMENTAL DESIGN: Small interfering RNA constructs targeting TGF-beta1 were validated and used to develop clonal derivatives of the metastatic breast cancer cell line MDA-MB-435. The cells were used in several in vitro analyses, including migration, invasion, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, apoptosis, and signaling assays. A wound-healing assay was used to determine migration of the cells in culture and a Boyden chamber transwell assay was used for invasion. Further, the clones were used in an in vivo mouse model for the kinetics of tumor growth and gene expression in the primary site and in lungs upon metastasis. RESULTS: Inhibition of TGF-beta1 expression in MDA-MB-435 cells showed a 35% decrease in migration and a 55% decrease in invasion in vitro, with a 50% increase in proliferation and no effect on apoptosis. In vivo analysis indicated a 90% decrease in the number of mice bearing macroscopic lung metastases; however, the primary tumors did not show any difference in the growth kinetics when compared with the parental MDA-MB-435 cells. Analysis of TGF-beta signaling pathways in the clonal derivatives showed a decrease in Smad2 activation and an increase in AKT and extracellular signal-regulated kinase activation. Interestingly, analysis of TGF-beta receptor expression showed a decrease in both receptor I and II expression in TGF-beta1 silenced cells. These results suggest that inhibition of TGF-beta1 ligand may act as a negative feedback loop to disrupt the function of all TGF-beta isoforms. CONCLUSIONS: Therapies targeting the TGF-beta signaling pathway may be more effective in late-stage disease to prevent organ metastasis but not primary tumor formation and may be combined with other tumor-targeted therapies normally limited by increased circulating TGF-beta levels.

Systemic osteoprotegerin gene therapy restores tumor-induced bone loss in a therapeutic model of breast cancer bone metastasis.

Enhanced production of receptor activator of nuclear factor-kappaB ligand (RANKL) and its binding to RANK on the osteoclasts have been associated with osteolysis in breast cancer bone metastasis. Osteoprotegerin (OPG) is a decoy receptor that prevents RANKL-RANK interaction. This study determined the effects of sustained expression of OPG using a recombinant adeno-associated viral (rAAV) vector in mouse model of osteolytic breast cancer. Bone metastasis was established by intracardiac injection of the human breast cancer cell line MDA-MB-435. Following this, mice were administered a one-time intramuscular injection of rAAV encoding either OPG.Fc (OPG) or green fluorescent protein (GFP). Mice were killed 1 month later and the effects of therapy on tumor growth and bone remodeling were evaluated. Bioluminescence imaging showed significant reduction of tumor growth in bone of OPG.Fc-treated mice. Micro-computed tomography (microCT) analysis and histomorphometry of the tibia indicated significant protection of trabecular and cortical bones after OPG.Fc therapy. Despite the prevention of bone loss and tumor growth in bone, OPG.Fc therapy failed to provide long-term survival. OPG.Fc-treated mice developed more bone than age-matched normal mice, indicating a requirement for regulated transgene expression. Results of this study indicate the potential of rAAV-OPG therapy for reducing morbidity and mortality in breast cancer patients with osteolytic bone damage.

Effects of sustained antiangiogenic therapy in multistage prostate cancer in TRAMP model.

Antiangiogenic therapy is a promising alternative for prostate cancer growth and metastasis and holds great promise as an adjuvant therapy. The present study evaluated the potential of stable expression of angiostatin and endostatin before the onset of neoplasia and during the early and late stages of prostate cancer progression in transgenic adenocarcinoma of mouse prostate (TRAMP) mice. Groups of 5-, 10-, and 18-week-old male TRAMP mice received recombinant adeno-associated virus-6 encoding mouse endostatin plus angiostatin (E+A) by i.m. injection. The effects of therapy were determined by sacrificing groups of treated mice at defined stages of tumor progression and following cohorts of similarly treated mice for long-term survival. Results indicated remarkable survival after recombinant adeno-associated virus-(E+A) therapy only when the treatment was given at an earlier time, before the onset of high-grade neoplasia, compared with treatment given for invasive cancer. Interestingly, early-stage antiangiogenic therapy arrested the progression of moderately differentiated carcinoma to poorly differentiated state and distant metastasis. Immunohistochemical analysis of the prostate from treated mice indicated significantly lower endothelial cell proliferation and increased tumor cell apoptosis. Vascular endothelial growth factor receptor (VEGFR)-2 expression was significantly down-regulated in tumor endothelium after treatment but not VEGFR-1. Analysis of the neuroendocrine marker synaptophysin expression indicated that antiangiogenic therapy given at an early-stage disease reduced neuroendocrine transition of the epithelial tumors. These studies indicate that stable endostatin and angiostatin gene therapy may be more effective for minimally invasive tumors rather than advanced-stage disease.

The effect of endothelial nitric oxide synthase on the hemodynamics and wall mechanics in murine arteriovenous fistulas.

Creation of a hemodialysis arteriovenous fistula (AVF) causes aberrant vascular mechanics at and near the AVF anastomosis. When inadequately regulated, these aberrant mechanical factors may impede AVF lumen expansion to cause AVF maturation failure, a significant clinical problem with no effective treatments. The endothelial nitric oxide synthase (NOS3) system is crucial for vascular health and function, but its effect on AVF maturation has not been fully characterized. We hypothesize that NOS3 promotes AVF maturation by regulating local vascular mechanics following AVF creation. Here we report the first MRI-based fluid-structure interaction (FSI) study in a murine AVF model using three mouse strains: NOS3 overexpression (NOS3 OE) and knockout (NOS3-/-) on C57BL/6 background, with C57BL/6 as the wild-type control (NOS3+/+). When compared to NOS3+/+ and NOS3-/-, AVFs in the OE mice had larger lumen area. AVFs in the OE mice also had smoother blood flow streamlines, as well as lower blood shear stress at the wall, blood vorticity, inner wall circumferential stretch, and radial wall thinning at the anastomosis. Our results demonstrate that overexpression of NOS3 resulted in distinct hemodynamic and wall mechanical profiles associated with favorable AVF remodeling. Enhancing NOS3 expression may be a potential therapeutic approach for promoting AVF maturation.

Author Correction: The effect of endothelial nitric oxide synthase on the hemodynamics and wall mechanics in murine arteriovenous fistulas.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Micro-RNA Profiling as a Predictor of Clinical Outcomes for Head and Neck Cancer Patients.

Head and neck cancer is one of the leading malignancies worldwide. Due to the lack of symptoms in the early stage of the disease, about two thirds of patients present with locally advanced disease at the time of diagnosis. Even with significantly improved survival rates over the past two decades due to advanced imaging and treatment modalities, locoregional recurrence rates in patients with advanced disease ranges from 16% to 35%. Alternative therapeutic targets are being developed to improve survival outcomes. MicroRNAs (miRNA or miRs) are a family of small non-coding RNA species that have been demonstrated to regulate all cellular, physiological and developmental processes. Recently, there has been an exponential increase in the number of studies suggesting that miRNA is involved in regulating tumor metastasis, chemoresistance, radioresistance and survival outcomes. MiRNA candidates have been identified as potential prognostic biomarkers to diagnose cancer stages and progression, as well as to monitor follow-up treatment. In this review, we will discuss the miRNA profile in each stage of head and neck patients' therapy, with an emphasis on its application to clinical outcome prognosis.

Silencing of TGF-ß1 in tumor cells impacts MMP-9 in tumor microenvironment.

Transforming growth factor (TGF)-ß1 contributes to autocrine and paracrine functions in the tumor microenvironment (TME). The present study examined the effects of TGF-ß1 crosstalk in TME and its role in mediating tumor formation and progression by targeted abrogation of TGF-ß1 expression in metastatic cells in situ. Using species-specific primers, we found a significant increase in MMP-9 gene expression in the tumor-reactive stroma during late-stage metastasis in the lung. This effect was also confirmed in cancer-associated fibroblasts (CAFs) when co-cultured with the tumor cells. Knockdown of TGF-ß1 expression in the tumor cells negatively affected matrix metalloproteinase (MMP)-9 gene expression. Fibroblasts, cultured in the presence of tumor cells with intact TGF-ß1, showed a significant increase in proliferation rate, as well as expression of VEGF, bFGF, and SDF-1, which was not seen when TGF-ß1 expression was abrogated in tumor cells. Absence of TGF-ß1 in tumor cells also failed to result in myofibroblast differentiation. Co-implantation of CAFs and tumor cells with either intact TGF-ß1 expression or devoid of TGF-ß1 in vivo showed a significant increase in tumor growth kinetics in both cell types, suggesting a possible activation TGF-ß receptor signaling in tumor cells in response to TGF-ß from the TME.

Recombinant adeno-associated virus 2-mediated antiangiogenic prevention in a mouse model of intraperitoneal ovarian cancer.

PURPOSE: In the present study, we sought to determine the potential of sustained transgene expression by a single i.m. administration of recombinant adeno-associated virus 2 (rAAV) encoding angiostatin and endostatin in inhibiting i.p. ovarian cancer growth and dissemination in a preclinical mouse model. EXPERIMENTAL DESIGN: Cohorts of female athymic nude mice received either no virus or 1.2 x 10(11) particles of rAAV encoding green fluorescence protein or endostatin plus angiostatin, i.m. Three weeks later, the mice were i.p. injected with 10(6) human epithelial ovarian cancer cell line SKOV3.ip1. As a measure of effectiveness of the therapy, tumor weight, abdominal distension, ascites volume and vascular endothelial growth factor level, and tumor weight were determined. Immunohistochemistry was done to determine tumor cell apoptosis and endothelial cell proliferation following the therapy. Tumor-free survival was recorded as the end point. RESULTS: Results indicated a significant tumor-free survival (P < 0.003) following therapy with rAAV encoding endostatin and angiostatin compared with untreated or rAAV-green fluorescence protein-treated mice. Ascites volume in rAAV endostatin and angiostatin-treated mice was significantly lower than naive mice and contained less hemorrhage and tumor conglomerates. The level of vascular endothelial growth factor in the ascites of antiangiogenic vector treated mice was also significantly less compared with the untreated mice. Immunohistochemical analyses indicated increased tumor cell apoptosis and decreased blood vasculature following rAAV endostatin and angiostatin treatment. CONCLUSION: The results indicate that antiangiogenic genetic prevention from stable systemic levels of angiostatin and endostatin by i.m. administration of rAAV can be used for the treatment of i.p. ovarian cancer growth and dissemination.

Antiangiogenic cancer gene therapy by adeno-associated virus 2-mediated stable expression of the soluble FMS-like tyrosine kinase-1 receptor.

Antiangiogenic gene transfer has the potential to be more efficacious than protein-based therapies or pharmacotherapies for the control of solid tumor growth, invasion and metastasis. For a sustained antiangiogenic effect, a vector capable of long-term expression without vector-associated immunity or toxicity is advantageous. The present study evaluated the potential of a recombinant adeno-associated virus-2 (rAAV) encoding the human soluble FMS-like tyrosine kinase receptor 1 (sFlt-1), which functions by both sequestering vascular endothelial growth factor (VEGF) and forming inactive heterodimers with other membrane-spanning VEGF receptors, in vitro and in vivo. Results indicated significant growth inhibitory activity of the transgenic factor in a human umbilical vein endothelial cell proliferation assay in vitro and protection against the growth of an angiogenesis-dependent human ovarian cancer cell line, SKOV3.ip1, xenograft in vivo with increased disease-free survival. Stable expression of the secretory factor and transgene persistence were confirmed by immunohistochemistry and in situ hybridization analyses, respectively. Increased therapeutic effects on both the growth index of the implanted tumor cells and tumor-free survival also correlated with an increasing dose of the vector used. These studies indicate that rAAV-mediated sFlt-1 gene therapy may be a feasible approach for inhibiting tumor angiogenesis, particularly as an adjuvant/therapy.

Anti-angiogenic gene therapy for cancer (review).

Angiogenesis is a prerequisite for the growth and metastasis of solid tumors. Studies have confirmed that in the absence of angiogenesis, tumors rarely have the ability to develop beyond a few millimeters in diameter. Tumor-associated endothelium is activated by the production of soluble factors by tumor and stromal cells. Tumor-associated endothelial cells are physiologically homogeneous and divide more frequently. Thus, targeting the proliferation of tumor neovasculature will form an effective anti-cancer therapy. With the identification of drug and biological molecules that inhibit the growth of tumor endothelium, several attempts have been made in preclinical and clinical research to evaluate the potential of anti-angiogenic therapy. Although several drugs and purified proteins have shown promise, their wide-spread application is limited by half-life, side effects and cost involved. Gene therapy approaches, on the other hand, have the potential to overcome these limitations. Further, genetic transfer of anti-angiogenic genes can be combined with other treatments including radiation therapy or chemotherapy for synergistic effects. In this review, we provide a comprehensive account of factors and mechanisms underlying tumor angiogenesis, the potential and limitations of available gene therapy vectors for anti-angiogenesis and current status of preclinical and clinical gene therapy studies targeting tumor angiogenesis.

Detection of Merkel cell polyomavirus in formalin-fixed, paraffin-embedded tissue of Merkel cell carcinoma and correlation with prognosis.

Merkel cell carcinoma (MCC) is a rare, but highly aggressive primary cutaneous malignancy, showing neuroendocrine differentiation. In 2008, a novel member of the polyomavirus family, named Merkel cell polyomavirus (MCPyV) was identified in the genome of MCC tumors raising the possibility of an involvement in its pathogenesis. Due to the rarity of this tumor and current pathology practices, the most readily available tissue is archival formalin-fixed, paraffin-embedded (FFPE) material. In this study, we evaluated the presence of MCPyV in FFPE tissue and correlated its presence with tumor progression. Representative FFPE specimens from 18 tumors belonging to 14 patients with a diagnosis of MCC spanning the period from 2003 to 2008 were retrieved. Following DNA extraction, we performed PCR amplification and sequencing with four different MCPyV-specific primer pairs mapping within the T antigen and VP1 region. Overall, we detected MCPyV amplicons in 8/18 (44.4%) analyzed tumors from 7/14 (50%) cases. Two-year survival rate and median survival for the MCPyV-positive MCCs were 48% and 22.5 months, respectively and for the negative ones 69% and 51.3 months, respectively; however, the difference did not reach statistical significance (p=0.8). There was no significant correlation between the presence of the virus and the stage at presentation; however, tumors in the head and neck area had a lower frequency of viral positivity compared to those arising in the extremities suggesting a MCPyV-independent oncogenetic pathway perhaps, dependent on UV exposure, in a subset of these cases.