Exotoxin-encoding gene content in community-acquired and hospital-acquired methicillin-resistant Staphylococcus aureus

AbstractReports of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) causing hospital infections are increasing, and it is questionable whether the existing molecular definition of CA-MRSA is suitable for the characterization of all strains involved. The 821 methicillin-resistant S. aureus (MRSA) isolates recovered from patients in Health Region East, Norway during the period 1991—2006 were characterized by multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) typing, staphylococcal protein A (spa) gene typing, and their content of exotoxin-encoding genes. Cluster analysis based on exotoxin-encoding gene content was performed to separate the MRSA isolates into valid clusters with respect to microbiological characteristics. The analysis gave a four-cluster structure, and the four toxin clusters differed in the genetic lineages they included and in the diversity of the genetic lineages. A few genetic lineages were present in several toxin clusters. These results support the theory that mobile genetic elements encoding virulence genes do not move randomly among genetic lineages, but are restricted by the clonal lineages’ genetic background. Using the molecular criteria, MLST type, SCCmec type and the presence of the lucS/F-Panton–Valentine leukocidin (PVL) gene to define a CA-MRSA isolate, it was found that the CA-MRSA isolates mainly grouped together in two toxin clusters with a low prevalence of exotoxin-encoding genes. Statistical analyses supported the conclusion that toxin clusters with CA-MRSA genetic lineages were characterized by a low prevalence of exotoxin-encoding genes, whereas toxin clusters with hospital-acquired MRSA genetic lineages were characterized by a higher prevalence of exotoxin-encoding genes

DNA methylation profiling in doxorubicin treated primary locally advanced breast tumours identifies novel genes associated with survival and treatment response

<p>Abstract</p> <p>Background</p> <p>Breast cancer is the most frequent cancer in women and consists of a heterogeneous collection of diseases with distinct histopathological, genetic and epigenetic characteristics. In this study, we aimed to identify DNA methylation based biomarkers to distinguish patients with locally advanced breast cancer who may benefit from neoadjuvant doxorubicin treatment.</p> <p>Results</p> <p>We investigated quantitatively the methylation patterns in the promoter regions of 14 genes (<it>ABCB1</it>, <it>ATM</it>, <it>BRCA1</it>, <it>CDH3</it>, <it>CDKN2A</it>, <it>CXCR4</it>, <it>ESR1</it>, <it>FBXW7</it>, <it>FOXC</it>1, <it>GSTP1</it>, <it>IGF2</it>, <it>HMLH1</it>, <it>PPP2R2B</it>, and <it>PTEN</it>) in 75 well-described pre-treatment samples from locally advanced breast cancer and correlated the results to the available clinical and molecular parameters. Six normal breast tissues were used as controls and 163 unselected breast cancer cases were used to validate associations with histopathological and clinical parameters.</p> <p>Aberrant methylation was detected in 9 out of the 14 genes including the discovery of methylation at the <it>FOXC1 </it>promoter. Absence of methylation at the <it>ABCB1 </it>promoter correlated with progressive disease during doxorubicin treatment. Most importantly, the DNA methylation status at the promoters of <it>GSTP1</it>, <it>FOXC1 </it>and <it>ABCB1 </it>correlated with survival, whereby the combination of methylated genes improved the subdivision with respect to the survival of the patients. In multivariate analysis <it>GSTP1 </it>and <it>FOXC1 </it>methylation status proved to be independent prognostic markers associated with survival.</p> <p>Conclusions</p> <p>Quantitative DNA methylation profiling is a powerful tool to identify molecular changes associated with specific phenotypes. Methylation at the <it>ABCB1 </it>or <it>GSTP1 </it>promoter improved overall survival probably due to prolonged availability and activity of the drug in the cell while <it>FOXC1 </it>methylation might be a protective factor against tumour invasiveness. <it>FOXC1 </it>proved to be general prognostic factor, while <it>ABCB1 </it>and <it>GSTP1 </it>might be predictive factors for the response to and efficacy of doxorubicin treatment. Pharmacoepigenetic effects such as the reported associations in this study provide molecular explanations for differential responses to chemotherapy and it might prove valuable to take the methylation status of selected genes into account for patient management and treatment decisions.</p

Reliability of cyclin A assessment on tissue microarrays in breast cancer compared to conventional histological slides

Cyclin A has in some studies been associated with poor breast cancer survival, although all studies have not confirmed this. Its prognostic significance in breast cancer needs evaluation in larger studies. Tissue microarray (TMA) technique allows a simultaneous analysis of large amount of tumours on a single microscopic slide. This makes a rapid screening of molecular markers in large amount of tumours possible. Because only a small tissue sample of each tumour is punched on an array, the question has arisen about the representativeness of TMA when studying markers that are expressed in only a small proportion of cells. For this reason, we wanted to compare cyclin A expression on TMA and on traditional large sections. Two breast cancer TMAs were constructed of 200 breast tumours diagnosed between 1997–1998. TMA slides and traditional large section slides of these 200 tumours were stained with cyclin A antibody and analysed by two independent readers. The reproducibility of the two readers' results was good or even very good, with kappa values 0.71–0.87. The agreement of TMA and large section results was good with kappa value 0.62–0.75. Cyclin A overexpression was significantly (P<0.001) associated with oestrogen receptor and progesterone receptor negativity and high grade both on TMA and large sections. Cyclin A overexpression was significantly associated with poor metastasis-free survival both on TMA and large sections. The relative risks for metastasis were similar on TMA and large sections. This study suggests that TMA technique could be useful to study histological correlations and prognostic significance of cyclin A on breast cancer on a large scale

Genomic instability and proliferative activity as risk factors for distant metastases in breast cancer

The role of genomic instability and proliferative activity for development of distant metastases in breast cancer was analysed, and the relative contribution of these two risk factors was quantified. A detailed quantitative comparison was performed between Ki67 and cyclin A as proliferative markers. The frequency of Ki67 and cyclin A-positive cells was scored in the same microscopic areas in 428 breast tumours. The frequency of Ki67-positive cells was found to be highly correlated with the frequency of cyclin A-positive cells, and both proliferation markers were equally good to predict risk of distant metastases. The relative contribution of degree of aneuploidy and proliferative activity as risk markers for developing distant metastases was studied independently. Although increased proliferative activity in general was associated with an increased risk of developing distant metastases, ploidy level was found to be an independent and even stronger marker when considering the group of small (T1) node negative tumours. By combining proliferative activity and ploidy level, a large group of low risk breast tumours (39%) could be identified in which only a few percentage of the tumours (5%) developed distant metastases during the 9-year follow-up time period

Two novel connexin32 mutations cause early onset X-linked Charcot-Marie-Tooth disease

<p>Abstract</p> <p>Background</p> <p>X-linked Charcot-Marie Tooth (CMT) is caused by mutations in the connexin32 gene that encodes a polypeptide which is arranged in hexameric array and form gap junctions.</p> <p>Methods</p> <p>We describe two novel mutations in the connexin32 gene in two Norwegian families.</p> <p>Results</p> <p>Family 1 had a c.225delG (R75fsX83) which causes a frameshift and premature stop codon at position 247. This probably results in a shorter non-functional protein structure. Affected individuals had an early age at onset usually in the first decade. The symptoms were more severe in men than women. All had severe muscle weakness in the legs. Several abortions were observed in this family. Family 2 had a c.536 G>A (C179Y) transition which causes a change of the highly conserved cysteine residue, i.e. disruption of at least one of three disulfide bridges. The mean age at onset was in the first decade. Muscle wasting was severe and correlated with muscle weakness in legs. The men and one woman also had symptom from their hands.</p> <p>The neuropathy is demyelinating and the nerve conduction velocities were in the intermediate range (25–49 m/s). Affected individuals had symmetrical clinical findings, while the neurophysiology revealed minor asymmetrical findings in nerve conduction velocity in 6 of 10 affected individuals.</p> <p>Conclusion</p> <p>The two novel mutations in the connexin32 gene are more severe than the majority of previously described mutations possibly due to the severe structural change of the gap junction they encode.</p

Expression levels of uridine 5'-diphospho-glucuronosyltransferase genes in breast tissue from healthy women are associated with mammographic density

Introduction Mammographic density (MD), as assessed from film screen mammograms, is determined by the relative content of adipose, connective and epithelial tissue in the female breast. In epidemiological studies, a high percentage of MD confers a four to six fold risk elevation of developing breast cancer, even after adjustment for other known breast cancer risk factors. However, the biologic correlates of density are little known. Methods Gene expression analysis using whole genome arrays was performed on breast biopsies from 143 women; 79 women with no malignancy (healthy women) and 64 newly diagnosed breast cancer patients, both included from mammographic centres. Percent MD was determined using a previously validated, computerized method on scanned mammograms. Significance analysis of microarrays (SAM) was performed to identify genes influencing MD and a linear regression model was used to assess the independent contribution from different variables to MD. Results SAM-analysis identified 24 genes differentially expressed between samples from breasts with high and low MD. These genes included three uridine 5'-diphospho-glucuronosyltransferase (UGT) genes and the oestrogen receptor gene (ESR1). These genes were down-regulated in samples with high MD compared to those with low MD. The UGT gene products, which are known to inactivate oestrogen metabolites, were also down-regulated in tumour samples compared to samples from healthy individuals. Several single nucleotide polymorphisms (SNPs) in the UGT genes associated with the expression of UGT and other genes in their vicinity were identified. Conclusions Three UGT enzymes were lower expressed both in breast tissue biopsies from healthy women with high MD and in biopsies from newly diagnosed breast cancers. The association was strongest amongst young women and women using hormonal therapy. UGT2B10 predicts MD independently of age, hormone therapy and parity. Our results indicate that down-regulation of UGT genes in women exposed to female sex hormones is associated with high MD and might increase the risk of breast cancer

Expression of p21WAF1 in Astler–Coller stage B2 colorectal cancer is associated with survival benefit from 5FU-based adjuvant chemotherapy

In several, but not all, previous studies, positive p21WAF1 expression has been suggested as an indicator of a good prognosis in patients with stage III/IV colorectal cancer. However, it is not known whether the same is true for stage B2 patients. The purpose of this study is to assess the influence of p21WAF1 expression in tumor cells on disease-free survival (DFS) and overall survival (OS) of Astler–Coller stage B2 and C patients with colorectal cancer who underwent 5-fluorouracil-based adjuvant chemotherapy. Nuclear p21WAF1 was detected by immunohistochemistry in tissue microarrays from 275 colorectal cancers. The expression of p21WAF1 was associated with DFS (p = 0.025) and OS (p = 0.008) in the subgroup of stage B2 patients that was treated with adjuvant chemotherapy. In multivariate analysis, it remained the only independent prognostic parameter in relation to DFS and OS (p = 0.035 and p = 0.02, respectively). In the subgroup of 72 stage B2 patients with positive p21WAF1 expression but not in the subgroup of 61 stage B2 patients with negative p21WAF1 expression, adjuvant chemotherapy was associated with better DFS (85% 5-year survival versus 65% without chemotherapy, p = 0.03) and OS (96% versus 82%, p = 0.014). In the combined stage B2 and C group of patients treated with adjuvant chemotherapy, positive p21WAF1 expression was also associated with better DFS and OS (p = 0.03, p = 0.002, respectively). Expression of p21WAF1 in colorectal tumor cells identifies a subgroup of Astler–Coller stage B2 patients who could benefit significantly from 5FU-based chemotherapy and may improve the selection of patients for adjuvant chemotherapy

Gene expression profiles of breast biopsies from healthy women identify a group with claudin-low features

Background Increased understanding of the variability in normal breast biology will enable us to identify mechanisms of breast cancer initiation and the origin of different subtypes, and to better predict breast cancer risk. Methods Gene expression patterns in breast biopsies from 79 healthy women referred to breast diagnostic centers in Norway were explored by unsupervised hierarchical clustering and supervised analyses, such as gene set enrichment analysis and gene ontology analysis and comparison with previously published genelists and independent datasets. Results Unsupervised hierarchical clustering identified two separate clusters of normal breast tissue based on gene-expression profiling, regardless of clustering algorithm and gene filtering used. Comparison of the expression profile of the two clusters with several published gene lists describing breast cells revealed that the samples in cluster 1 share characteristics with stromal cells and stem cells, and to a certain degree with mesenchymal cells and myoepithelial cells. The samples in cluster 1 also share many features with the newly identified claudin-low breast cancer intrinsic subtype, which also shows characteristics of stromal and stem cells. More women belonging to cluster 1 have a family history of breast cancer and there is a slight overrepresentation of nulliparous women in cluster 1. Similar findings were seen in a separate dataset consisting of histologically normal tissue from both breasts harboring breast cancer and from mammoplasty reductions. Conclusion This is the first study to explore the variability of gene expression patterns in whole biopsies from normal breasts and identified distinct subtypes of normal breast tissue. Further studies are needed to determine the specific cell contribution to the variation in the biology of normal breasts, how the clusters identified relate to breast cancer risk and their possible link to the origin of the different molecular subtypes of breast cancer