Evaluation of Alere i Influenza A&B for rapid detection of influenza viruses A and B.

Rapid and accurate diagnosis of influenza is important for infection control, as well as for patient management. Alere i Influenza A&B is an isothermal nucleic acid amplification-based integrated system for detection and differentiation of influenza virus A and influenza virus B. The performance of the Alere i Influenza A&B was screened using frozen nasopharyngeal-swab specimens collected in viral transport medium (VTM) that were originally tested fresh with the FilmArray Respiratory Panel (RP) assay during the 2012-2013 influenza outbreak. In total, 360 VTM specimens were selected for Alere i Influenza A&B testing: 40 influenza virus A H1N1-2009 (influenza virus A-1), 40 influenza virus A H3N2 (influenza virus A-3), 37 influenza virus A "equivocal" or "no subtype detected" (influenza virus A-u), 41 influenza virus B, and 202 influenza virus-negative specimens, as initially determined by the FilmArray RP assay. The Alere assay showed sensitivities of 87.2%, 92.5%, 25.0%, and 97.4% for influenza virus A-1, influenza virus A-3, influenza virus A-u, and influenza virus B, respectively, after discordant resolution by Prodesse ProFLU+ PCR. The specificities were 100% for both influenza virus A and influenza virus B. In general, the Alere i Influenza A&B provided good sensitivity, although the assay did show poorer sensitivity with samples determined to have low influenza virus A titers by Prodesse ProFlu+ PCR (a mean real-time PCR threshold cycle [CT] value of 31.9 ± 2.0), which included the majority of the samples called influenza virus A "equivocal" or "no subtype detected" by a single BioFire FilmArray RP test. The integrated, rapid, and simple characteristics of the Alere i Influenza A&B assay make it a potential candidate for point-of-care testing, with a test turnaround time of less than 15 min.

Human endometriosis is associated with plasma cells and overexpression of B lymphocyte stimulator.

Endometriosis affects 10-20% of women of reproductive age and is associated with pelvic pain and infertility, and its pathogenesis is not well understood. We used genomewide transcriptional profiling to characterize endometriosis and found that it exhibits a gene expression signature consistent with an underlying autoimmune mechanism. Endometriosis lesions are characterized by the presence of abundant plasma cells, many of which produce IgM, and macrophages that produce BLyS/BAFF/TNFSF13B, a member of the TNF superfamily implicated in other autoimmune diseases. B lymphocyte stimulator (BLyS) protein was found elevated in the serum of endometriosis patients. These observations suggest a model for the pathology of endometriosis where BLyS-responsive plasma cells interact with retrograde menstrual tissues to give rise to endometriosis lesions.

Gene expression analyses reveal molecular relationships among 20 regions of the human CNS.

Transcriptional profiling was performed to survey the global expression patterns of 20 anatomically distinct sites of the human central nervous system (CNS). Forty-five non-CNS tissues were also profiled to allow for comparative analyses. Using principal component analysis and hierarchical clustering, we were able to show that the expression patterns of the 20 CNS sites profiled were significantly different from all non-CNS tissues and were also similar to one another, indicating an underlying common expression signature. By focusing our analyses on the 20 sites of the CNS, we were able to show that these 20 sites could be segregated into discrete groups with underlying similarities in anatomical structure and, in many cases, functional activity. These findings suggest that gene expression data can help define CNS function at the molecular level. We have identified subsets of genes with the following patterns of expression: (1) across the CNS, suggesting homeostatic/housekeeping function; (2) in subsets of functionally related sites of the CNS identified by our unsupervised learning analyses; and (3) in single sites within the CNS, indicating their participation in distinct site-specific functions. By performing network analyses on these gene sets, we identified many pathways that are upregulated in particular sites of the CNS, some of which were previously described in the literature, validating both our dataset and approach. In summary, we have generated a database of gene expression that can be used to gain valuable insight into the molecular characterization of functions carried out by different sites of the human CNS.

Association of the PDCD5 locus with lung cancer risk and prognosis in smokers.

PURPOSE: Whole-genome scan association analysis was carried out to identify genetic variants predictive of lung cancer risk in smokers and to confirm the identified variants in an independent sample. PATIENTS AND METHODS: A case-control study was performed using two pools consisting of DNA from 322 German smoking lung cancer patients and 273 healthy smoking controls, respectively. A replication study was carried out using 254 Italian lung adenocarcinoma (ADCA) patients and 235 healthy controls. RESULTS: Patients with genotypes GG or CG for the rs1862214 single nucleotide polymorphism, 5' upstream of the programmed cell death 5 (PDCD5) gene, compared with those with the common genotype CC showed an increased risk of lung cancer (odds ratio, 1.6; 95% CI, 1.2 to 2.1) and a higher incidence of poor clinical stage disease (hazard ratio [HR], 1.9; 95% CI, 1.1 to 3.4; P = .023), nodal involvement (HR, 1.9; 95% CI, 1.1 to 3.6; P = .033), and short-term survivorship (HR, 1.8; 95% CI, 1.2 to 2.6, P = .003). PDCD5 mRNA expression levels were approximately 2.4-fold lower in lung ADCA as compared to normal lung tissue. Human NCI-H520 cancer cells transfected with PDCD5 cDNA showed decreased colony-forming ability. CONCLUSION: These results suggest that the rs1862214 polymorphism in PDCD5 is predictive for lung cancer risk and prognosis, and that PDCD5 may represent a novel tumor suppressor gene influencing lung cancer.

BRAF polymorphisms and risk of melanocytic neoplasia.

Somatic mutations of the BRAF gene are common in melanomas and nevi but the contribution of polymorphisms in this gene to melanoma or nevus susceptibility remains unclear. An Australian melanoma case-control sample was typed for 16 single nucleotide polymorphisms (SNP) within the BRAF gene, and five SNP in three neighboring genes. The sample comprised 755 melanoma cases from 740 families stratified by family history of melanoma and controls from 635 unselected twin families (2,239 individuals). Ancestry of the cases and controls was recorded, and the twins had undergone skin examination to assess total body nevus count, degree of freckling, and pigmentation phenotype. Genotyping was carried out via primer extension followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. SNP in the BRAF gene were found to be weakly associated with melanoma status but not with development of nevi or freckles. The estimated proportion of attributable risk of melanoma due to variants in BRAF is 1.6%. This study shows that BRAF polymorphisms predispose to melanoma but the causal variant has yet to be determined. The burden of disease associated with this variant is greater than that associated with the major melanoma susceptibility locus CDKN2A, which has an estimated attributable risk of 0.2%.

Genetic polymorphisms in DPF3 associated with risk of breast cancer and lymph node metastases.

BACKGROUND: Several studies have identified rare genetic variations responsible for many cases of familial breast cancer but their contribution to total breast cancer incidence is relatively small. More common genetic variations with low penetrance have been postulated to account for a higher proportion of the population risk of breast cancer. METHODS AND RESULTS: In an effort to identify genes that influence non-familial breast cancer risk, we tested over 25,000 single nucleotide polymorphisms (SNPs) located within approximately 14,000 genes in a large-scale case-control study in 254 German women with breast cancer and 268 age-matched women without malignant disease. We identified a marker on chromosome 14q24.3-q31.1 that was marginally associated with breast cancer status (OR = 1.5, P = 0.07). Genotypes for this SNP were also significantly associated with indicators of breast cancer severity, including presence of lymph node metastases (P = 0.006) and earlier age of onset (P = 0.01). The association with breast cancer status was replicated in two independent samples (OR = 1.35, P = 0.05). High-density association fine mapping showed that the association spanned about 80 kb of the zinc-finger gene DPF3 (also known as CERD4). One SNP in intron 1 was found to be more strongly associated with breast cancer status in all three sample collections (OR = 1.6, P = 0.003) as well as with increased lymph node metastases (P = 0.01) and tumor size (P = 0.01). CONCLUSION: Polymorphisms in the 5' region of DPF3 were associated with increased risk of breast cancer development, lymph node metastases, age of onset, and tumor size in women of European ancestry. This large-scale association study suggests that genetic variation in DPF3 contributes to breast cancer susceptibility and severity.

Role of ICAM1 in invasion of human breast cancer cells.

We identified previously a region on chromosome 19p13.2 spanning the genes encoding the intercellular adhesion molecules (ICAM), ICAM1, ICAM4 and ICAM5 as a breast cancer susceptibility locus. Genetic variants in this region were also associated with indicators of disease severity, including higher rates of metastases to other organs. Based on this association, we set out to explore the role of ICAM1 in proliferation and invasion of human breast cancer cells. We observed that ICAM1 downregulation at the mRNA and protein levels led to a strong suppression of human breast cell invasion through a matrigel matrix. Under the same conditions, no significant effect on cell proliferation in vitro was seen. Incubation of cells with an antibody against ICAM1 blocked invasion of the highly metastatic MDA-MB-435 cell line in a dose-dependent manner without affecting cell migration. We also demonstrated that the level of ICAM1 protein expression on the cell surface positively correlated with metastatic potential of five human breast cancer cell lines and that ICAM1 mRNA levels were elevated in breast tumor compared with adjacent normal tissue. These results corroborate our previous genetic finding that variations in the ICAM region are associated with the occurrence of metastases and establish a causal role of ICAM1 in invasion of metastatic human breast carcinoma cell lines.

Suppression of RAD21 gene expression decreases cell growth and enhances cytotoxicity of etoposide and bleomycin in human breast cancer cells.

A genome-wide case-control association study done in our laboratory has identified a single nucleotide polymorphism located in RAD21 as being significantly associated with breast cancer susceptibility. RAD21 is believed to function in sister chromatid alignment as part of the cohesin complex and also in double-strand break (DSB) repair. Following our initial finding, expression studies revealed a 1.25- to 2.5-fold increased expression of this gene in several human breast cancer cell lines as compared with normal breast tissue. To determine whether suppression of RAD21 expression influences cellular proliferation, RNA interference technology was used in breast cancer cell lines MCF-7 and T-47D. Proliferation of cells treated with RAD21-specific small inhibitory RNA (siRNA) was significantly reduced as compared with mock-transfected cells and cells transfected with a control siRNA (Lamin A/C). This inhibition of proliferation correlated with a significant reduction in the expression of RAD21 mRNA and with an increased level of apoptosis. Moreover, MCF-7 cell sensitivity to two DNA-damaging chemotherapeutic agents, etoposide and bleomycin, was increased after inhibition of RAD21 expression with a dose reduction factor 50 (DRF50) of 1.42 and 3.71, respectively. At the highest concentrations of etoposide and bleomycin administered, cells transfected with a single siRNA duplex targeted against RAD21 showed 57% and 60% survival as compared with control cells, respectively. Based on these findings, we conclude that RAD21 is a novel target for developing cancer therapeutics that can potentially enhance the antitumor activity of chemotherapeutic agents acting via induction of DNA damage.

Association of the NuMA region on chromosome 11q13 with breast cancer susceptibility.

The development of breast cancer is a complex process that involves multiple genes at many stages, from initial cell cycle dysregulation to disease progression. To identify genetic variations that influence this process, we conducted a large-scale association study using a collection of German cases and controls and >25,000 SNPs located within 16,000 genes. One of the loci identified was located on chromosome 11q13 [odds ratio (OR)=1.85, P=0.017]. The initial association was subsequently tested in two independent breast cancer collections. In both sample sets, the frequency of the susceptibility allele was increased in the cases (OR=1.6, P=0.01). The susceptibility allele was also associated with an increase in cancer family history (P=0.1). Fine mapping showed that the region of association extends approximately 300 kb and spans several genes, including the gene encoding the nuclear mitotic apparatus protein (NuMA). A nonsynonymous SNP (A794G) in NuMA was identified that showed a stronger association with breast cancer risk than the initial marker SNP (OR=2.8, P=0.005 initial sample; OR=2.1, P=0.002 combined). NuMA is a cell cycle-related protein essential for normal mitosis that is degraded in early apoptosis. NuMA-retinoic acid receptor alpha fusion proteins have been described in acute promyelocytic leukemia. Although the potential functional relevance of the A794G variation requires further biological validation, we conclude that variations in the NuMA gene are likely responsible for the observed increased breast cancer risk.

Large-scale association study identifies ICAM gene region as breast and prostate cancer susceptibility locus.

We conducted a large-scale association study to identify genes that influence nonfamilial breast cancer risk using a collection of German cases and matched controls and >25,000 single nucleotide polymorphisms located within 16,000 genes. One of the candidate loci identified was located on chromosome 19p13.2 [odds ratio (OR) = 1.5, P = 0.001]. The effect was substantially stronger in the subset of cases with reported family history of breast cancer (OR = 3.4, P = 0.001). The finding was subsequently replicated in two independent collections (combined OR = 1.4, P < 0.001) and was also associated with predisposition to prostate cancer in an independent sample set of prostate cancer cases and matched controls (OR = 1.4, P = 0.002). High-density single nucleotide polymorphism mapping showed that the extent of association spans 20 kb and includes the intercellular adhesion molecule genes ICAM1, ICAM4, and ICAM5. Although genetic variants in ICAM5 showed the strongest association with disease status, ICAM1 is expressed at highest levels in normal and tumor breast tissue. A variant in ICAM5 was also associated with disease progression and prognosis. Because ICAMs are suitable targets for antibodies and small molecules, these findings may not only provide diagnostic and prognostic markers but also new therapeutic opportunities in breast and prostate cancer.

The adaptive imbalance in base excision-repair enzymes generates microsatellite instability in chronic inflammation.

Chronic infection and associated inflammation are key contributors to human carcinogenesis. Ulcerative colitis (UC) is an oxyradical overload disease and is characterized by free radical stress and colon cancer proneness. Here we examined tissues from noncancerous colons of ulcerative colitis patients to determine (a) the activity of two base excision-repair enzymes, AAG, the major 3-methyladenine DNA glycosylase, and APE1, the major apurinic site endonuclease; and (b) the prevalence of microsatellite instability (MSI). AAG and APE1 were significantly increased in UC colon epithelium undergoing elevated inflammation and MSI was positively correlated with their imbalanced enzymatic activities. These latter results were supported by mechanistic studies using yeast and human cell models in which overexpression of AAG and/or APE1 was associated with frameshift mutations and MSI. Our results are consistent with the hypothesis that the adaptive and imbalanced increase in AAG and APE1 is a novel mechanism contributing to MSI in patients with UC and may extend to chronic inflammatory or other diseases with MSI of unknown etiology.

3-Methyladenine DNA glycosylase-deficient Aag null mice display unexpected bone marrow alkylation resistance.

Most cells deficient in 3-methyladenine (3MeA) DNA glycosylase become sensitive to the lethal and clastogenic effects of alkylating agents. Surprisingly, myeloid progenitor bone marrow (BM) cells derived from Aag -/- mice were more resistant than those from wild-type mice to the cytotoxic effects of several alkylating agents. Moreover, an alkylation-resistant phenotype was observed in vivo using the BM micronucleus assay as a measure of chromosome damage. Flow cytometry indicated that in vivo alkylation resistance in Aag null BM cells may be restricted to cells of the myeloid lineage. These results show that in particular cell types, the initiation of base excision repair is more lethal to the cell than leaving the damaged bases unrepaired by Aag.