DICER1 is an endoribonuclease central to the generation of microRNAs (miRNAs) and short interfering RNAs (siRNAs). Germline mutations in DICER1 have been associated with a pleiotropic tumour predisposition syndrome and Wilms tumour (WT) is a rare manifestation of this syndrome. Three WTs, each in a child with a deleterious germline DICER1 mutation, were screened for somatic DICER1 mutations and were found to bear specific mutations in either the RNase IIIa (n = 1) or the RNase IIIb domain (n = 2). In the two latter cases, we demonstrate that the germline and somatic DICER1 mutations were in trans, suggesting that the two-hit hypothesis of tumour formation applies for these examples of WT. Among 191 apparently sporadic WTs, we identified five different missense or deletion somatic DICER1 mutations (2.6%) in four individual WTs; one tumour had two very likely deleterious somatic mutations in trans in the RNase IIIb domain (c.5438A>G and c.5452G>A). In vitro studies of two somatic single-base substitutions (c.5429A>G and c.5438A>G) demonstrated exon 25 skipping from the transcript, a phenomenon not previously reported in DICER1. Further we show that DICER1 transcripts lacking exon 25 can be translated in vitro. This study has demonstrated that a subset of WTs exhibits two 'hits' in DICER1, suggesting that these mutations could be key events in the pathogenesis of these tumours.No conflicts of interest were declared. pleuropulmonary blastoma (PPB) syndrome (OMIM 601200) [7,8]. Several genes have been identified as being somatically mutated in WT, including WT1 , CTNNB1 , IGF2 , TP53 , WTX , DIS3L2 , FBXW7 and MYCN [5,9]. There are overlapping distributions of molecular abnormalities at 11p15, WTX , WT1 and CTNNB1 in WT [9,10], but other genes could be contributing to the aetiology of WT [3,[11][12][13]. WT histopathology is heterogeneous and tumours associated with different types of nephrogenic rests and histologies tend to show different underlying genetic changes [14,15]. A revised model for WT ontogeny takes into account genetic data (WT1 status, 11p15 imprint control region 1 methylation status, gene expression patterns of other genes) and histological data [14]. Nevertheless, each WT is thought to be of monoclonal origin [16].

Invasive lobular carcinoma (ILC) of the breast is believed to develop from in situ lesions, atypical lobular hyperplasia (ALH), and lobular carcinoma in situ (LCIS). Down-regulation of the cell-cell adhesion protein E-cadherin is a defining feature of lobular breast cancer (LBC) and already occurs in ALH and LCIS. Apart from mutational mechanisms, epigenetic silencing of the E-cadherin gene (CDH1 ) is thought to be involved in E-cadherin downregulation and has been observed at a high frequency in ILC. Whether CDH1 promoter methylation is already present in in situ lesions and thus contributes to the initiation of LBC is not established. We thus examined microdissected archived tissue from 20 LBCs by methylation-specific PCR to determine the CDH1 methylation status of lobular lesions. Nineteen of the 20 LBCs had a hypermethylated CDH1 promoter, including 13/14 ILCs and 13/13 ALHs or LCIS. Bisulphite sequencing indicated that methylation was complete within the investigated promoter fragment. Intriguingly, CDH1 methylation was likewise present in 8/8 adjacent non-neoplastic epithelia, but not in 6/6 mammary epithelia from healthy subjects. E-cadherin protein and mRNA were down-regulated in in situ lesions relative to adjacent epithelia. Together, these results indicate that CDH1 promoter methylation occurs in LBC prior to E-cadherin down-regulation and neoplastic formation. We thus propose that epigenetic silencing represents the first of the two hits required to silence both CDH1 alleles for LBC to develop. Because promoter methylation is in principle reversible, our findings suggest that chemoprevention of LBC by epigenetic drugs should be feasible. Furthermore, the presence of CDH1 methylation in pre-neoplastic epithelia suggests the existence of mammary regions with increased disease susceptibility, providing an explanation for the often multifocal presentation of LBC.

BackgroundAberrant DNA methylation profiles are a characteristic of all known cancer types, epitomized by the CpG island methylator phenotype (CIMP) in colorectal cancer (CRC). Hypermethylation has been observed at CpG islands throughout the genome, but it is unclear which factors determine whether an individual island becomes methylated in cancer.MethodsDNA methylation in CRC was analysed using the Illumina HumanMethylation450K array. Differentially methylated loci were identified using Significance Analysis of Microarrays (SAM) and the Wilcoxon Signed Rank (WSR) test. Unsupervised hierarchical clustering was used to identify methylation subtypes in CRC.ResultsIn this study we characterized the DNA methylation profiles of 94 CRC tissues and their matched normal counterparts. Consistent with previous studies, unsupervized hierarchical clustering of genome-wide methylation data identified three subtypes within the tumour samples, designated CIMP-H, CIMP-L and CIMP-N, that showed high, low and very low methylation levels, respectively. Differential methylation between normal and tumour samples was analysed at the individual CpG level, and at the gene level. The distribution of hypermethylation in CIMP-N tumours showed high inter-tumour variability and appeared to be highly stochastic in nature, whereas CIMP-H tumours exhibited consistent hypermethylation at a subset of genes, in addition to a highly variable background of hypermethylated genes. EYA4, TFPI2 and TLX1 were hypermethylated in more than 90% of all tumours examined. One-hundred thirty-two genes were hypermethylated in 100% of CIMP-H tumours studied and these were highly enriched for functions relating to skeletal system development (Bonferroni adjusted p value =2.88E-15), segment specification (adjusted p value =9.62E-11), embryonic development (adjusted p value =1.52E-04), mesoderm development (adjusted p value =1.14E-20), and ectoderm development (adjusted p value =7.94E-16).ConclusionsOur genome-wide characterization of DNA methylation in colorectal cancer has identified 132 genes hypermethylated in 100% of CIMP-H samples. Three genes, EYA4, TLX1 and TFPI2 are hypermethylated in >90% of all tumour samples, regardless of CIMP subtype.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-017-3226-4) contains supplementary material, which is available to authorized users.

Introduction Mounting molecular evidence suggests that invasive lobular carcinoma (ILC) is developing from in situ lesions, atypical lobular hyperplasia (ALH), and lobular carcinoma in situ (LCIS). However, little is known about the mechanisms promoting the progression of lobular breast cancer (LBC) to invasive disease. Here, we investigated whether c-Src kinase, an established inducer of invasive states, contributes to the progression from ALH/LCIS to ILC.

Objectives To compare the Roche Cell-Free DNA Collection Tubes ® against the Streck Cell-Free DNA BCT ® s for sample stability using Cell Free DNA (cfDNA) from healthy volunteers (n = 20). Design & methods Whole blood was drawn into five Roche and five Streck tubes per volunteer, stored at room temperature and processed at five different time points (Days 0, 4, 7, 10 and 14). One volunteer had blood drawn into ×10 K 3 EDTA tubes to observe the effect of no preservation buffer on White Blood Cell (WBC) lysis. DNA was extracted from the plasma and the concentration (ng/μL) measured using the Qubit Fluorometer ® at each time point. The eluted DNA was further analysed by capillary electrophoresis to determine the proportion of cfDNA and gDNA contamination in the samples over the 14 days. Results There was no difference in individual ( p = 0.097) and median paired ( p = 0.26) DNA concentration across the five time points between the two tubes. However, a difference was observed for samples in the Roche tubes for pair days 0–7 ( p = 0.01), 0 to 10 ( p = 0.046) and 0 to 14 ( p = 0.0016) in contrast to the Streck tubes after adjustment for multiple testing. Conclusion The findings of this study indicate that the Roche Cell-Free DNA Collection Tubes ® are a suitable alternative for sample collection and storage at room temperature, albeit for a duration of less than 7 days.