Assessment of RainDrop BS-seq as a method for large-scale, targeted bisulfite sequencing.

We present a systematic assessment of RainDrop BS-seq, a novel method for large-scale, targeted bisulfite sequencing using microdroplet-based PCR amplification coupled with next-generation sequencing. We compared DNA methylation levels at 498 target loci (1001 PCR amplicons) in human whole blood, osteosarcoma cells and an archived tumor tissue sample. We assessed the ability of RainDrop BS-seq to accurately measure DNA methylation over a range of DNA quantities (from 10 to 1500 ng), both with and without whole-genome amplification (WGA) following bisulfite conversion. DNA methylation profiles generated using at least 100 ng correlated well (median R = 0.92) with those generated on Illumina Infinium HumanMethylation450 BeadChips, currently the platform of choice for epigenome-wide association studies (EWAS). WGA allowed for testing of samples with a starting DNA amount of 10 and 50 ng, although a reduced correlation was observed (median R = 0.79). We conclude that RainDrop BS-seq is suitable for measuring DNA methylation levels using nanogram quantities of DNA, and can be used to study candidate epigenetic biomarker loci in an accurate and high-throughput manner, paving the way for its application to routine clinical diagnostics

Proteomics identifies neddylation as a potential therapy target in small intestinal neuroendocrine tumors.

Patients with small intestinal neuroendocrine tumors (SI-NETs) frequently develop spread disease; however, the underlying molecular mechanisms of disease progression are not known and effective preventive treatment strategies are lacking. Here, protein expression profiling was performed by HiRIEF-LC-MS in 14 primary SI-NETs from patients with and without liver metastases detected at the time of surgery and initial treatment. Among differentially expressed proteins, overexpression of the ubiquitin-like protein NEDD8 was identified in samples from patients with liver metastasis. Further, NEDD8 correlation analysis indicated co-expression with RBX1, a key component in cullin-RING ubiquitin ligases (CRLs). In vitro inhibition of neddylation with the therapeutic agent pevonedistat (MLN4924) resulted in a dramatic decrease of proliferation in SI-NET cell lines. Subsequent mass spectrometry-based proteomics analysis of pevonedistat effects and effects of the proteasome inhibitor bortezomib revealed stabilization of multiple targets of CRLs including p27, an established tumor suppressor in SI-NET. Silencing of NEDD8 and RBX1 using siRNA resulted in a stabilization of p27, suggesting that the cellular levels of NEDD8 and RBX1 affect CRL activity. Inhibition of CRL activity, by either NEDD8/RBX1 silencing or pevonedistat treatment of cells resulted in induction of apoptosis that could be partially rescued by siRNA-based silencing of p27. Differential expression of both p27 and NEDD8 was confirmed in a second cohort of SI-NET using immunohistochemistry. Collectively, these findings suggest a role for CRLs and the ubiquitin proteasome system in suppression of p27 in SI-NET, and inhibition of neddylation as a putative therapeutic strategy in SI-NET

Toxicity associated with capecitabine plus oxaliplatin in colorectal cancer before and after an institutional policy of capecitabine dose reduction

BACKGROUND: Capecitabine plus oxaliplatin (CAPOX) is an established treatment option in colorectal cancer, but can be associated with severe toxicities. METHODS: Following reporting of severe diarrhoea and dehydration with capecitabine 2000 mg m(-2) per day plus oxaliplatin every 3 weeks (CAPOX 2000) in 2006, we instituted a policy change to reduce capecitabine dose to 1700 mg m(-2) per day (CAPOX 1700). We undertook a retrospective analysis comparing toxicities encountered before and after this dose change. RESULTS: Of the 400 patients treated, no significant differences were seen between the CAPOX 2000 and CAPOX 1700 in grades 3 and 4 diarrhoea (21% vs 19%; P=0.80), stomatitis (0% vs 1%; P=0.50) or grades 2-4 hand foot syndrome (16% vs 11%; P=0.18). Grades 3 and 4 neutropenia (9.5% vs 3.5%; P=0.03) and all grades hyperbilirubinaemia (60% vs 40%; P<0.0001) were significantly reduced with CAPOX 1700. Rates of hospitalisation due to toxicities were not different between two groups (13% vs 11%; P=0.53). CONCLUSIONS: No clinically or statistically significant differences in gastrointestinal toxicities or hospitalisation rate were seen after reducing our routine capecitabine dose from CAPOX 2000 to CAPOX 1700.We acknowledge National Health Service funding to the National Institute for Health Research Biomedical Research Centre

Molecular profiling of gastrointestinal neuroendocrine tumours

Small intestinal neuroendocrine tumours (SI NETs) are the most common malignancy of the small intestine, however they remain poorly characterised and the underlying pathogenic mechanisms driving disease development have yet to be elucidated. Whole genome and exome sequencing has suggested SI NETs to be mutationally quiet, with the most frequent mutation in Cyclin Dependent Kinase 1B occurring in only 8% of tumours, suggesting mechanisms other than genetic mutations may be responsible for driving SI NET tumourigenesis. Using integrated genomic and epigenomic analysis three distinct somatic copy number alteration (SCNA) profiles of SI NET were identified. The largest subgroup characterised by loss of heterozygosity at chromosome 18, negative CpG island methylator phenotype (CIMP) status, and the presence of CDKN1B mutations, is associated with improved clinical outcomes. A novel Multiple-SCNA signature has been described which defines a smaller subgroup of SI NETs and is characterized by significantly (p=0.04) reduced progression-free survival. A panel of 21 recurrently epigenetically dysregulated genes has been identified, and these represent putative novel pathogenic drivers for SI NET tumourigenesis and candidate novel biomarkers. Epigenetically dysregulated genes identified at a recurrence rate of 80-100% include gastric inhibitory polypeptide receptor (GIPR)(73.5%) – a target for novel imaging techniques in NETs, CDX1 (85.7%), CELSR3 (83.7%), FBP1 (83.7%), PCSK1 (67.3%) and TRIM15 (63.3%). The utility of methylated circulating tumour DNA analysis, and molecular profiling of circulating tumour cells as novel non-invasive biomarkers in SI NETs have been demonstrated. This is the first comprehensive integrated molecular analysis of SI NETs, providing evidence for epigenetic rather than mutational events in addition to SCNAs as drivers of SI NET development. These findings will facilitate improved patient management, treatment selection and prognostication