Endothelial HIF signaling regulates pulmonary fibrosis-associated pulmonary hypertension.

Pulmonary hypertension (PH) complicating chronic parenchymal lung disease, such as idiopathic pulmonary fibrosis, results in significant morbidity and mortality. Since the hypoxia-inducible factor (HIF) signaling pathway is important for development of pulmonary hypertension in chronic hypoxia, we investigated whether HIF signaling in vascular endothelium regulates development of PH related to pulmonary fibrosis. We generated a transgenic model in which HIF is deleted within vascular endothelial cells and then exposed these mice to chronic intraperitoneal bleomycin to induce PH associated with lung fibrosis. Although no differences in the degree of fibrotic remodeling were observed, we found that endothelial HIF-deficient mice were protected against development of PH, including right ventricle and pulmonary vessel remodeling. Similarly, endothelial HIF-deficient mice were protected from PH after a 4-wk exposure to normobaric hypoxia. In vitro studies of pulmonary vascular endothelial cells isolated from the HIF-targeted mice and controls revealed that endothelial HIF signaling increases endothelial cell expression of connective tissue growth factor, enhances vascular permeability, and promotes pulmonary artery smooth muscle cell proliferation and wound healing ability, all of which have the potential to impact the development of PH in vivo. Taken together, these studies demonstrate that vascular endothelial cell HIF signaling is necessary for development of hypoxia and pulmonary fibrosis associated PH. As such, HIF and HIF-regulated targets represent a therapeutic target in these conditions.

Clinical pharmacogenetics implementation: approaches, successes, and challenges.

Current challenges exist to widespread clinical implementation of genomic medicine and pharmacogenetics. The University of Florida (UF) Health Personalized Medicine Program (PMP) is a pharmacist-led, multidisciplinary initiative created in 2011 within the UF Clinical Translational Science Institute. Initial efforts focused on pharmacogenetics, with long-term goals to include expansion to disease-risk prediction and disease stratification. Herein we describe the processes for development of the program, the challenges that were encountered and the clinical acceptance by clinicians of the genomic medicine implementation. The initial clinical implementation of the UF PMP began in June 2012 and targeted clopidogrel use and the CYP2C19 genotype in patients undergoing left heart catheterization and percutaneous-coronary intervention (PCI). After 1 year, 1,097 patients undergoing left heart catheterization were genotyped preemptively, and 291 of those underwent subsequent PCI. Genotype results were reported to the medical record for 100% of genotyped patients. Eighty patients who underwent PCI had an actionable genotype, with drug therapy changes implemented in 56 individuals. Average turnaround time from blood draw to genotype result entry in the medical record was 3.5 business days. Seven different third party payors, including Medicare, reimbursed for the test during the first month of billing, with an 85% reimbursement rate for outpatient claims that were submitted in the first month. These data highlight multiple levels of success in clinical implementation of genomic medicine.

Deep sequencing analysis of HCV NS3 resistance-associated variants and mutation linkage in liver transplant recipients.

Viral variants with decreased susceptibility to HCV protease inhibitors (PIs) occur naturally and preexist at low levels within HCV populations. In patients failing PI monotherapy, single and double mutants conferring intermediate to high-level resistance to PIs have been selected in vivo. The abundance, temporal dynamics and linkage of naturally occurring resistance-associated variants (RAVs), however, have not been characterized in detail. Here, using high-density pyrosequencing, we analyzed HCV NS3 gene segments from 20 subjects with chronic HCV infection, including 12 subjects before and after liver transplantation. Bioinformatics analysis revealed that Q80 substitution was a dominant variant in 40% of the subjects, whereas other RAVs circulate at low levels within quasispecies populations. Low frequency mutation linkage was detectable by Illumina paired-end sequencing in as low as 0.5% of the mock populations constructed from in vitro RNA transcripts but were uncommon in vivo. We show that naturally occurring RAVs are common and can persist long term following liver transplant at low levels not readily detectable by conventional sequencing. Our results indicate that mutation linkage at low levels could be identified using the Illumina paired-end approach. The methods described here should facilitate the analysis of low frequency HCV drug resistance, mutation linkage and evolution, which may inform future therapeutic strategies in patients undergoing direct acting antiviral therapies.

Epigenetic upregulation of HGF and c-Met drives metastasis in hepatocellular carcinoma.

Hepatocyte growth factor (HGF) and its receptor, c-Met, are important regulators of growth and differentiation of healthy hepatocytes. However, upregulation of HGF and c-Met have been associated with tumor progression and metastasis in hepatocellular carcinoma (HCC). Hematogenous dissemination is the most common route for cancer metastasis, but the role of HGF and c-Met in circulating tumor cells (CTCs) is unknown. We have isolated and established a circulating tumor cell line from the peripheral blood of a mouse HCC model. Our studies show that these CTCs have increased expression of HGF and c-Met in comparison to the primary tumor cells. The CTCs display phenotypic evidence of epithelial-mesenchymal transition (EMT) and the EMT appears to be inducible by HGF. Epigenetic analysis of the c-Met promoter identified significant loss of DNA methylation in CTCs which correlated with overexpression of c-Met and increased expression of HGF. Six specific CpG sites of c-Met promoter demethylation were identified. CTCs show significantly increased tumorigenicity and metastatic potential in a novel orthotopic syngeneic model of metastatic HCC. We conclude that during hematogenous dissemination in HCC, CTCs undergo EMT under the influence of increased HGF. This process also involves up regulation of c-Met via promoter demethylation at 6 CpG sites. Consequently, targeting HGF and c-Met expression by CTCs may be a novel non-invasive approach with potential clinical applications in HCC management.

The Role of KRAS Mutational Analysis to Determine the Site of Origin of Metastatic Carcinoma to the Lung: A Case Report.

Metastatic carcinomas involving the lung are a common specimen encountered in surgical pathology. These metastases may have different morphologic, and architectural patterns and may mimic primary pulmonary adenocarcinoma, especially the intra-alveolar (lepidic) pattern of spread which may simulate a primary pulmonary bronchioloalveolar carcinoma (adenocarcinoma in situ). We present the case of a metastatic pancreatic adenocarcinoma that morphologically mimicked bronchioloalveolar carcinoma of the lung in that the tumor had an exclusive intra-alveolar pattern of spread and had an immunophenotype that was noninformative as to the site of origin (cytokeratin 7+, cytokeratin 20-, TTF-1-). In this case, we used KRAS gene mutation analysis to support that the lung carcinoma represented a metastatic pancreatic carcinoma as they both possessed identical codon 12 KRAS mutations. We show that this method may be a useful way to prove site of origin of metastatic carcinoma-particularly if standard morphologic or immunohistochemical analysis is not definitive.

5-HTTLPR genotype and associations with intoxication and intention to drive: results from a field study of bar patrons.

The serotonin transporter promoter polymorphism (5-HTTLPR) has been linked to a number of human behavioral traits and disorders. The variants of 5-HTTLPR are commonly reported in three forms, L/L, S/L and S/S, with the latter most often associated with emotional distress and/or behavioral dysfunction. Missing from the research literature are investigations that assess event-level associations between 5-HTTLPR genotype and specific incidents of risk behavior in natural drinking settings. This study reports associations between 5-HTTLPR, alcohol intoxication and intention to drive among young adult patrons exiting on-premise drinking establishments (i.e. bars) at night. Self-report measures, breath alcohol concentration (BrAC) readings and saliva samples for DNA analysis were collected from 477 bar patrons. Analyses were performed on 225 patrons likely to be near their peak intoxication level for the night. Results from a linear regression revealed that the 5-HTTLPR genotype was associated with exiting patron BrAC, after adjusting for random and fixed effects of other variables. An interaction effect involving 5-HTTLPR and bar-sponsored drink specials also had an independent association with BrAC, suggesting that selection of price-discounted alcoholic beverages increased intoxication in patrons with an L allele. In addition, results from logistic regression indicated that patrons with the S/S genotype were three times more likely to intend to drive a motor vehicle (after drinking on the night of study participation) compared with those with the L/L genotype. The 5-HTTLPR genotype may play an important role in the etiology of problems associated with on-premise drinking establishments.

Characterization of Anti-HCV Antibodies in IL-10-Treated Patients.

There is limited information on the direct role of the neutralizing antibody responses against hepatitis C virus (HCV) infection or methodologies to study them. Previously we have demonstrated that interleukin-10 (IL-10) administered to chronic hepatitis patients led to a decrease in disease activity, but an increase in HCV viral burden. The mechanism behind this is unknown. The objective of this study was to examine the antibody response in IL-10-treated patients. To establish a neutralization antibody assay, HCV-positive and HCV-negative sera were collected and incubated with HCV strain JFH-1 particles before culture with Huh 7.5 cells. Viral replication was measured a week later by either indirect immunofluorescence assay (iIFA) or real-time reverse transcriptase polymerase chain reaction (RT-PCR). After validation of the methodology, the sera from 30 previously-described subjects of a group previously treated with IL-10 were tested for the neutralization capacity of their antibodies. The amount of total anti-HCV antibody in the sera was also measured by direct staining of HCV full-length replicon cells. With this validated neutralization assay for anti-HCV antibodies we found that HCV-neutralizing antibodies are universally present, but with significantly different titers. In patients who were treated with IL-10, the total anti-HCV antibody titers appear to be constant, but with significantly decreased antibody neutralization activity. Our study validates an assay to quantitatively determine the presence and strength of HCV-specific neutralizing antibodies. We have found that IL-10-treated patients have significantly lower HCV antibodies, but maintain the total anti-HCV antibody titer, suggesting a novel mechanism by which IL-10 treatment increases viral load in patients.

Identification of c-kit gene mutations in primary adenoid cystic carcinoma of the salivary gland.

The CD117 (KIT) protein is overexpressed in many human neoplasms including adenoid cystic carcinoma of salivary glands. To evaluate the function of c-kit-activating mutations in adenoid cystic carcinoma of the salivary gland, we studied 14 cases (13 primary, 1 cervical lymph node metastasis) from our institution. KIT protein expression was evaluated by immunohistochemistry using formalin-fixed paraffin-embedded tissue. Mutational analyses of c-kit extracellular (exon 9), juxtamembrane (exon 11) and tyrosine kinase domains (exons 13 and 17) were performed by polymerase chain reaction, clonal selection and DNA sequencing. All 14 cases demonstrated strong KIT expression by immunohistochemistry. Molecular analysis was successful in 8 of 14 cases, and c-kit missense point mutations were detected in seven of eight cases (88%) including seven in exon 11, two in exon 9, two in exon 13 and two in exon 17. Eight silent point mutations were detected in five cases. Two cases contained missense mutations in more than one exon. Different mutations were found in the primary tumor and the cervical lymph node metastasis of one patient. Point mutations in domains similar to those described in gastrointestinal stromal tumors were detected, including Pro551Leu and Lys558Glu (5' end of exon 11), Leu576Phe (3' end of exon 11), Val643Ala (exon 13) and Asn822Ser (exon 17). Additional novel point mutations in exons 9, 11, 13 and 17 were also identified. This study is the first to report c-kit gene mutations in primary adenoid cystic carcinoma of the salivary gland. Identification of such potential gain-of-function mutations in exon 11, and less frequently in exons 9, 13 and 17, suggests that KIT may be involved in the pathogenesis of adenoid cystic carcinoma of salivary glands. Our study raises a prospect of correlation of c-kit mutation and a potential treatment of adenoid cystic carcinoma with tyrosine kinase inhibitor (imatinib).