Erratum: Publisher Correction: Somatic mutations in DROSHA and DICER1 impair microRNA biogenesis through distinct mechanisms in Wilms tumours.

[This corrects the article DOI: 10.1038/ncomms5802.].

Somatic mutations in DROSHA and DICER1 impair microRNA biogenesis through distinct mechanisms in Wilms tumours.

Wilms tumour is the most common childhood kidney cancer. Here we report the whole-exome sequencing of 44 Wilms tumours, identifying missense mutations in the microRNA (miRNA)-processing enzymes DROSHA and DICER1, and novel mutations in MYCN, SMARCA4 and ARID1A. Examination of tumour miRNA expression, in vitro processing assays and genomic editing in human cells demonstrates that DICER1 and DROSHA mutations influence miRNA processing through distinct mechanisms. DICER1 RNase IIIB mutations preferentially impair processing of miRNAs deriving from the 5'-arm of pre-miRNA hairpins, while DROSHA RNase IIIB mutations globally inhibit miRNA biogenesis through a dominant-negative mechanism. Both DROSHA and DICER1 mutations impair expression of tumour-suppressing miRNAs, including the let-7 family, important regulators of MYCN, LIN28 and other Wilms tumour oncogenes. These results provide new insights into the mechanisms through which mutations in miRNA biogenesis components reprogramme miRNA expression in human cancer and suggest that these defects define a distinct subclass of Wilms tumours.

Immunohistochemical expression of GLUT1 and its correlation with unfavorable histology and TP53 codon 72 polymorphism in Wilms tumors.

Reprogramming of energy metabolism, such as increased glycolysis, is a hallmark of cancer cells. One mechanism by which cancer cells fuel glycolysis is through increased uptake of glucose across cell membranes via the glucose transporter GLUT1. One of the transcriptional repressors of GLUT1 is wild-type TP53, and cancer-associated loss of function mutations within the DNA-binding domain of TP53 impairs the repressive effect of TP53 on transcriptional activity of the GLUT1 gene promoter. Because TP53 mutations are associated with unfavorable histology (diffuse anaplasia) in Wilms tumors, we hypothesized increased expression of GLUT1 in these tumors. To evaluate this hypothesis, we performed tissue microarray-based immunohistochemistry for GLUT1 in a set of 50 Wilms tumors, including 5 with unfavorable histology. In a subset of 16 favorable histology Wilms tumors, we compared the GLUT1 immunoexpression with TP53 codon 72 polymorphism status. We found consistently stronger immunoexpression of GLUT1 in unfavorable histology Wilms tumors compared to favorable histology Wilms tumors (P  =  0.04). We noted that the favorable histology Wilms tumors with a proline residue at position 72 of TP53 tended to have higher immunoexpression of GLUT1, although this immunoexpression did not reach statistical significance in this small set of cases. In summary, our finding of strong GLUT1 immunoexpression in unfavorable histology Wilms tumors indicates that these tumors are likely to be 2-deoxy-2-((18)F)fluoro-d-glucose avid and that GLUT1 should be evaluated as a therapeutic target for these tumors that otherwise show resistance to conventional therapy.

TP53 codon 72 polymorphisms in favorable histology Wilms tumors.

In Wilms tumor (WT), mutations in the gene encoding p53, TP53, are correlated with anaplasia; however TP53 variants have not been studied in favorable histology (FH) WTs. A single nucleotide polymorphism of TP53 encoding either arginine or proline at codon 72 is suggested to alter in vitro p53 behavior. Therefore, we analyzed tissue from 23 consecutive patients with FHWT to determine allelic and genotypic frequencies of Pro72 and Arg72 variants and correlate this with clinical outcomes. Interestingly, our cohort showed a statistically significant over-representation of the Arg allele and Arg/Arg genotype. However, the genotypic and allelic frequencies showed no significant correlation with age, stage, or disease recurrence.

Evaluation of Maxwell® 16 for automated DNA extraction from whole blood and formalin-fixed paraffin embedded (FFPE) tissue.

BACKGROUND: The aim of the study was to assess the performance of Promega, Maxwell® 16 for the extraction of genomic DNA from whole blood and FFPE tissue. METHODS: DNA was extracted from 10 whole blood and 10 FFPE specimens using six different commercial kits. RESULTS: For whole blood, the mean DNA concentration obtained by Maxwell® 16 was significantly greater than either easyMAG® (p<0.0001) or QIAamp® Blood DNA kit (p<0.001). For FFPE, the mean DNA concentration obtained by the AllPrep® FFPE specific DNA/RNA kit was significantly greater than either the Maxwell® 16 (p<0.0001) or the general AllPrep® DNA/RNA kit (p<0.0001). CONCLUSIONS: Comparative evaluation of the six DNA extraction kits indicated that the semi-automated Maxwell® 16 was superior for whole blood extraction while the manual AllPrep® FFPE DNA/RNA kit (Qiagen) performed better for FFPE DNA extraction in terms of quantity of DNA obtained. All six extraction methods (blood and FFPE) performed well in terms of purity. Although there were variances in the quantity of DNA obtained, there were no significant differences in the efficiency of these methods in yielding amplifiable DNA extracts, as demonstrated by ß-actin for whole blood specimens. In evaluation of FFPE DNA extraction methods, the Qiagen AllPrep® FFPE DNA/RNA Mini Kit was the best for applications requiring larger amplicons, but for smaller amplicons the Maxwell was most consistent.

Papillary thyroid carcinoma shows elevated levels of 2-hydroxyglutarate.

Elevated levels of D: -2-hydroxyglutarate (D: -2-HG) occur in gliomas and myeloid leukemias associated with mutations of IDH1 and IDH2. L: -2-Hydroxyglutaric aciduria, an inherited metabolic disorder, predisposes to brain tumors. Therefore, we asked whether sporadic cancers, without IDH1 or IDH2 hot-spot mutations, show elevated 2-hydroxyglutarate levels. We retrieved 15 pairs of frozen papillary thyroid carcinoma (PTC) and adjacent non-neoplastic thyroid, and 14 pairs of hyperplastic nodule (HN) and adjacent non-hyperplastic thyroid. In all lesions, exon 4 sequencing confirmed the absence of known mutations of IDH1 and IDH2. We measured 2-hydroxyglutarate by liquid chromatography-tandem mass spectrometry. Compared to normal thyroid, PTCs had significantly higher D: -2-HG and L: -2-hydroxyglutarate (L: -2-HG) levels, and compared to HNs, PTCs had significantly higher D: -2-HG levels. D: -2-HG/L: -2-HG levels were not significantly different between HNs and normal thyroid. Further studies should clarify if elevated 2-hydroxyglutarate in PTC may be useful as cancer biomarker and evaluate the role of 2-hydroxyglutarate in cancer biology.

Respiratory syncytial virus (RSV) RNA loads in peripheral blood correlates with disease severity in mice.

BACKGROUND: Respiratory Syncytial Virus (RSV) infection is usually restricted to the respiratory epithelium. Few studies have documented the presence of RSV in the systemic circulation, however there is no consistent information whether virus detection in the blood correlates with disease severity. METHODS: Balb/c mice were inoculated with live RSV, heat-inactivated RSV or medium. A subset of RSV-infected mice was treated with anti-RSV antibody 72 h post-inoculation. RSV RNA loads were measured by PCR in peripheral blood from day 1-21 post-inoculation and were correlated with upper and lower respiratory tract viral loads, the systemic cytokine response, lung inflammation and pulmonary function. Immunohistochemical staining was used to define the localization of RSV antigens in the respiratory tract and peripheral blood. RESULTS: RSV RNA loads were detected in peripheral blood from day 1 to 14 post-inoculation, peaked on day 5 and significantly correlated with nasal and lung RSV loads, airway obstruction, and blood CCL2 and CXCL1 expression. Treatment with anti-RSV antibody reduced blood RSV RNA loads and improved airway obstruction. Immunostaining identified RSV antigens in alveolar macrophages and peripheral blood monocytes. CONCLUSIONS: RSV RNA was detected in peripheral blood upon infection with live RSV, followed a time-course parallel to viral loads assessed in the respiratory tract and was significantly correlated with RSV-induced airway disease.

Respiratory syncytial virus persistence in the lungs correlates with airway hyperreactivity in the mouse model.

BACKGROUND: Previous studies in mice showed that respiratory syncytial virus (RSV) infection was associated with RSV RNA persistence. This study was designed to characterize the significance of RSV RNA persistence and its relation to RSV-induced chronic airway disease. METHODS: Mice were inoculated with live RSV, UV light-treated RSV, heat-inactivated RSV, or medium. Bronchoalveolar lavage fluid samples were obtained and lung specimens were harvested on days 1, 5, and 42 after inoculation to assess lung inflammation, lung mRNA expression of interleukin (IL)-4, IL-5, IL-15, and interferon (IFN)-gamma; RSV loads were assessed by culture and real-time polymerase chain reaction (PCR) and correlated with pulmonary function. RESULTS: During the acute phase of infection, RSV loads as indicated by culture and PCR were significantly higher in mice inoculated with live RSV. On day 42, RSV RNA remained detectable only in mice inoculated with live or UV light-treated RSV. Lung inflammation, IFN-gamma:IL-4 mRNA expression ratios, airway obstruction (AO), and airway hyperreactivity (AHR) were significantly increased in mice inoculated with live RSV. AO on day 5 and AHR on day 42 were significantly correlated with RSV RNA copy number in lung samples. CONCLUSIONS: Infection with live RSV induced acute and chronic airway disease that was associated with a predominantly Th-1 immune response and RSV RNA persistence that significantly correlated with pulmonary function abnormalities.

Differential effects of p63 mutants on transactivation of p53 and/or p63 responsive genes.

p63, known to play a role in development, has more recently also been implicated in cancer progression. Mutations in p63 have been shown to be responsible for several human developmental diseases. Differential splicing of the p63 gene gives rise to p63 isoforms, which can act either as tumor suppressors or as oncogene. In this report, we studied the effects of naturally occurring TAp63gamma mutants on the regulation of p53/p63 and p63 specific target genes. We observed significant differences among p63 mutants to regulate the p53/p63 and p63 specific target genes. Additionally, we observed a differential effect of p63 mutants on wildtype-p63-mediated induction of p53/p63 and p63 specific target genes. We also demonstrated that these mutants differentially regulate the binding of wildtype p63 to the promoter of target genes. Furthermore, the effects of these mutants on cell death and survival were consistent with their ability to regulate the downstream targets when compared to wildtype TAp63gamma. In summary, our data demonstrate that p63 mutants exhibit differential effects on p63 and p53/p63 specific target genes and on the induction of apoptosis, and provide further insight into the function of p63.

Patterned silk films cast from ionic liquid solubilized fibroin as scaffolds for cell growth.

Silk is an attractive biomaterial for use in tissue engineering applications because of its slow degradation, excellent mechanical properties, and biocompatibility. In this report, we demonstrate a simple method to cast patterned films directly from silk fibroin dissolved in an ionic liquid. The films cast from the silk ionic liquid solution were found to support normal cell proliferation and differentiation. The versatility of the silk ionic liquid solutions and the ability to process large amounts of silk into materials with controlled surface topography directly from the dissolved silk ionic liquid solution could enhance the desirability of biomaterials such as silk for a variety of applications.