Whole genome in vivo RNAi screening identifies the leukemia inhibitory factor receptor as a novel breast tumor suppressor.

Cancer is caused by mutations in oncogenes and tumor suppressor genes, resulting in the deregulation of processes fundamental to the normal behavior of cells. The identification and characterization of oncogenes and tumor suppressors has led to new treatment strategies that have significantly improved cancer outcome. The advent of next generation sequencing has allowed the elucidation of the fine structure of cancer genomes, however, the identification of pathogenic changes is complicated by the inherent genomic instability of cancer cells. Therefore, functional approaches for the identification of novel genes involved in the initiation and development of tumors are critical. Here we report the first whole human genome in vivo RNA interference screen to identify functionally important tumor suppressor genes. Using our novel approach, we identify previously validated tumor suppressor genes including TP53 and MNT, as well as several novel candidate tumor suppressor genes including leukemia inhibitory factor receptor (LIFR). We show that LIFR is a key novel tumor suppressor, whose deregulation may drive the transformation of a significant proportion of human breast cancers. These results demonstrate the power of genome wide in vivo RNAi screens as a method for identifying novel genes regulating tumorigenesis.

Quantitative and qualitative characterization of Two PD-L1 clones: SP263 and E1L3N.

BACKGROUND: Programmed Death Ligand 1 (PD-L1) is an immune modulating protein expressed on the surface of various inflammatory cells, including T Cells, B Cells, dendritic cells, and macrophages. PD-L1 represents an important diagnostic target; expression of PD-L1 on the surface of tumor cells, or within tumor-associated immune cells, is an important predictor of likely response to targeted therapies. In this study, we describe the optimization of immunohistochemistry (IHC) assays using two PD-L1 antibodies (SP263 and E1L3N) and compare the performance of the optimized assays. METHODS: Fully automated immunohistochemical assays were optimized for the VENTANA PD-L1 (SP263) Rabbit Monoclonal Antibody and the PD-L1 (E1L3N®) XP® Rabbit mAb using instruments and detection chemistries from Ventana Medical Systems, Inc. ("SP263 assay" and "E1L3N assay," respectively). Tissue microarrays (TMAs) containing formalin fixed paraffin embedded (FFPE) non-small cell lung cancer (NSCLC) cases were used for the optimization and comparison staining. H scores were used for membrane scoring whereas percent positivity was used for tumor-associated immune cell scoring. RESULTS: One-hundred NSCLC TMA case cores each stained with the SP263 and E1L3N assays were evaluated by two pathologists in a blinded study. Analysis of these specimens showed that the SP263 assay was more sensitive and had a wider dynamic range than the E1L3N assay. For sensitivity, many cases were found to be negative for membrane staining with the E1L3N assay, but had measurable staining with the SP263 assay. Dynamic range was demonstrated by the SP263 assay having well-distributed H scores while the E1L3N assay had a significantly higher proportion of cases clustered in the lowest H score bins. For tumor-associated immune cell staining, the two assays identified similar amounts of cells staining in each case, but the SP263 assay gave overall darker staining. CONCLUSION: Since PD-L1 status is important for targeted therapies, having a specific and accurate diagnostic test is crucial for identifying patients who could benefit from these treatments. Due to its staining intensity, scoring range, and pathologist preference, the SP263 IHC assay has been deemed superior to the E1L3N IHC assay. Future clinical utility remains to be determined.

Chronic hepatitis C and genotyping: the clinical significance of determining HCV genotypes.

Chronic hepatitis C, attributed to infection with hepatitis C virus (HCV), is a global health problem. The overall prevalence of viral hepatitis worldwide is estimated to be 3-5% with over 175 million people infected with HCV. Clinically, HCV can establish a persistent, chronic infection contributing to progressive liver disease, including cirrhosis and hepatocellular carcinoma (HCC), requiring intensive treatment regimens, possible liver transplantation and long-term care. Due to the chronic nature of HCV infection and the tremendous burden on healthcare resources, clinicians and laboratorians have looked for key epidemiological, pathological and viral characteristics that may provide insight into disease progression, severity and response to therapy to permit the administration of effective therapeutic regimens as well as long-term management of infected individuals. Determination of viral genotype has been identified as one parameter that could provide direction in the clinical management of patients with chronic HCV infections. The following review provides background on determination of HCV genotypes and the relevance of viral genome characterization in the current clinical setting.

Chronic hepatitis B and viral genotype: the clinical significance of determining HBV genotypes.

The global health challenge posed by the hepatitis B virus (HBV) centres around the widespread distribution and the serious complications as a result of persistent infection with the virus. As with other chronic diseases mediated by pathogens such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV), clinicians are searching for epidemiological, pathological and viral characteristics of HBV infection that may lead to more effective management of patients with chronic infection. Unlike HCV, the role of HBV genotype in disease progression, severity, response to therapy and drug resistance is still under investigation and just beginning to be clarified. This review examines the potential role of HBV genotype determination in the clinic with emphasis on how this genetic information may used to provide effective management for the treatment of patients with chronic hepatitis B.

The role of SATB1 in breast cancer pathogenesis.

BACKGROUND: SATB1 has been previously proposed as a key protein that controls the development and progression of breast cancer. We explored the potential of the SATB1 protein as a therapeutic target and prognostic marker for human breast cancer. METHODS: We used aggressive (MDA-MB-231 and BT549) and nonaggressive (SKBR3 and MCF7) breast cancer cell lines to investigate the potential of SATB1 as a therapeutic target. SATB1 mRNA expression was silenced in aggressive cells by use of short hairpin RNAs against SATB1. SATB1 was overexpressed in nonaggressive cells by use of SATB1 expression vectors. We assessed the effect of modifying SATB1 expression on the transformed phenotype by examining anchorage-independent cell proliferation, acinar morphology on matrigel, and migration by wound healing in cultured cells. We examined tumor formation and metastasis, respectively, by use of orthotopic mammary fat pad and tail vein xenograft mouse models (mice were used in groups of six, and in total, 96 mice were used). SATB1 mRNA expression was compared with outcome for patients with primary breast cancer from six previous microarray studies that included a total of 1170 patients. All statistical tests were two-sided. RESULTS: The transformed phenotype was not suppressed by SATB1 silencing in aggressive cells and was not enhanced by ectopic expression of SATB1 in nonaggressive cells. Modifying SATB1 expression did not alter anchorage-independent cell proliferation, invasive acinar morphology, or cell migration in cultured cells and did not affect tumor formation or metastasis in xenograft mouse models. In addition, SATB1 expression was not associated with decreased overall survival of patients with primary breast cancer in six previous independent microarray studies (overall odds ratio = 0.80, 95% confidence interval = 0.62 to 1.03, P = .10). CONCLUSION: In contrast to previous studies, we found that SATB1 expression did not promote breast cancer progression and was not associated with breast cancer outcome.

Rapid and specific detection of Mycobacterium tuberculosis from acid-fast bacillus smear-positive respiratory specimens and BacT/ALERT MP culture bottles by using fluorogenic probes and real-time PCR.

A real-time PCR assay using the LightCycler (LC) instrument for the specific identification of Mycobacterium tuberculosis complex (MTB) was employed to detect organisms in 135 acid-fast bacillus (AFB) smear-positive respiratory specimens and in 232 BacT/ALERT MP (MP) culture bottles of respiratory specimens. The LC PCR assay was directed at the amplification of the internal transcribed spacer region of the Mycobacterium genome with real-time detection using fluorescence resonance energy transfer probes specific for MTB. The results from the respiratory specimens were compared to those from the Amplicor M. tuberculosis PCR test. Specimens from MP culture bottles were analyzed by Accuprobe and conventional identification methods. MTB was cultured from 105 (77.7%) respiratory AFB smear-positive specimens; 103 of these samples were positive by LC PCR and Amplicor PCR. Two samples negative in the LC assay contained rare numbers of organisms; both were positive in the Amplicor assay. Two separate samples negative by Amplicor PCR contained low and moderate numbers of AFB, respectively, and both of these were positive in the LC assay. There were 30 AFB smear-positive respiratory specimens that grew mycobacteria other than tuberculosis (MOTT), and all tested negative in both assays. Of the 231 MP culture bottles, 114 cultures were positive for MTB and all were positive by the LC assay. The remaining 117 culture bottles were negative in the LC assay and grew various MOTT. This real-time MTB assay is sensitive and specific; a result was available within 1 h of having a DNA sample available for testing.

Cross-clade inhibition of HIV-1 replication and cytopathology by using RNase P-associated external guide sequences.

RNase P complexes have been proposed as a novel RNA-based gene interference strategy to inhibit gene expression in human malignancies and infectious diseases. This approach is based on the sequence-specific design of an external guide sequence (EGS) RNA molecule that can specifically hybridize to almost any complementary target mRNA and facilitate its cleavage by the RNase P enzyme component. We designed a truncated RNase P-associated EGS molecule to specifically recognize the U5 region of HIV-1 mRNA and mediate cleavage of hybridized mRNA by the RNase P enzyme. Genes encoding for this U5-EGS (560) molecule, as well as a U5 EGS (560D) antisense control, were cloned into retroviral plasmids and transferred into a CD4(+) T cell line. Transfected cells were exposed to increasing concentrations of HIV-1 clinical isolates from clades A, B, C, and F. Heterogeneous cultures of CD4(+) T cells expressing the U5 EGS (560) molecule were observed to maintain CD4 levels, were devoid of cytopathology, and did not produce HIV p24 gag antigen through 30 days after exposure to all HIV-1 clades at a multiplicity of infection of 0.01. Identical cells expressing the U5 EGS (560D) antisense control molecule underwent a loss of CD4 expression, produced elevated levels of HIV-1, and formed large syncytia similar to untreated cells. When the viral inoculum was increased at the time of exposure (multiplicity of infection = 0.05), the inhibitory effect of the U5 EGS (560) molecule was overwhelmed, but viral-mediated cytopathology and particle production were delayed compared with control cell populations. Viral replication and cytopathology associated with infection of multiple HIV-1 clades can be effectively inhibited in CD4(+) cells expressing the RNase P-associated U5 EGS (560) molecule.

Multicenter evaluation of the VERSANT hepatitis B virus DNA 3.0 assay.

The VERSANT hepatitis B virus (HBV) 3.0 Assay (branched DNA [bDNA]) (referred to herein as VERSANT 3.0) was evaluated at four external sites for analytical sensitivity, specificity, reproducibility, linearity of quantification, and limits of detection. In addition, each of the test evaluation sites provided HBV DNA-positive clinical samples that were previously analyzed by one of three commercially available HBV DNA quantitative tests: Digene Hybrid Capture II HBV DNA Test (Digene); VERSANT HBV DNA 1.0 Assay (bDNA) (VERSANT 1.0); and COBAS AMPLICOR HBV Monitor Test (COBAS AMPLICOR). These samples were reexamined using VERSANT 3.0. The results from these studies showed that VERSANT 3.0 has high specificity (99.3%), excellent reproducibility (between-run coefficient of variation [CV] = 1.6 to 9.4%; within-run CV = 6.5 to 20.7%), and a broad linear range of quantification (2.0 x 10(3) to 1.0 x 10(8) HBV DNA copies/ml) that facilitate the monitoring of HBV DNA levels at diagnosis and throughout the course of treatment. In general, correlation was very good between results obtained from clinical samples analyzed by VERSANT 3.0 and the comparative HBV DNA quantitative assays (VERSANT 1.0, R(2) = 0.900; Digene, R(2) = 0.985; COBAS AMPLICOR, R(2) = 0.771). The greatest differences in comparative quantitation occurred at HBV DNA levels approaching the limits of the dynamic ranges for the comparative assays. The performance characteristics of the new VERSANT 3.0 assay demonstrated that it provides a reliable and robust method for routinely monitoring serum HBV DNA levels in assessing disease activity and determining response to antiviral treatment.