Whole genome sequencing of single circulating tumor cells from neuroendocrine neoplasms

Single-cell profiling of circulating tumor cells (CTCs) as part of a minimally invasive liquid biopsy presents an opportunity to characterize and monitor tumor heterogeneity and evolution in individual patients. In this study, we aimed to compare single-cell copy number variation (CNV) data with tissue, and define the degree of intra- and inter-patient genomic heterogeneity. We performed next generation sequencing (NGS) whole genome CNV analysis of 125 single CTCs derived from seven patients with neuroendocrine neoplasms (NEN) alongside matched white blood cells (WBC), formalin fixed paraffin embedded (FFPE) and fresh frozen (FF) samples. CTC CNV profiling demonstrated recurrent chromosomal alterations in previously reported NEN copy number hotspots, including the prognostically relevant loss of chromosome 18. Unsupervised hierarchical clustering revealed CTCs with distinct clonal lineages as well as significant intra- and inter-patient genomic heterogeneity, including subclonal alterations not detectable by bulk analysis and previously unreported in NEN. Notably, we also demonstrate the presence of genomically distinct CTCs according to the enrichment strategy utilized (EpCAM-dependent versus size-based). This work has significant implications for the identification of therapeutic targets, tracking of evolutionary change and the implementation of CTC-biomarkers in cancer

Human umbilical cord perivascular cells improve human pancreatic islet transplant function by increasing vascularization

Islet transplantation is an efficacious therapy for type 1 diabetes; however, islets from multiple donor pancreata are required, and a gradual attrition in transplant function is seen. Here, we manufactured human umbilical cord perivascular mesenchymal stromal cells (HUCPVCs) to Good Manufacturing Practice (GMP) standards. HUCPVCs showed a stable phenotype while undergoing rapid ex vivo expansion at passage 2 (p2) to passage 4 (p4) and produced proregenerative factors, strongly suppressing T cell responses in the resting state and in response to inflammation. Transplanting an islet equivalent (IEQ):HUCPVC ratio of 1:30 under the kidney capsule in diabetic NSG mice demonstrated the fastest return to normoglycemia by 3 days after transplant: Superior glycemic control was seen at both early (2.7 weeks) and later stages (7, 12, and 16 weeks) versus ratios of 1:0, 1:10, and 1:50, respectively. Syngeneic islet transplantation in immunocompetent mice using the clinically relevant hepatic portal route with a marginal islet mass showed that mice transplanted with an IEQ:HUCPVC ratio of 1:150 had superior glycemic control versus ratios of 1:0, 1:90, and 1:210 up to 6 weeks after transplant. Immunodeficient mice transplanted with human islets (IEQ:HUCPVC ratio of 1:150) exhibited better glycemic control for 7 weeks after transplant versus islet transplant alone, and islets transplanted via the hepatic portal vein in an allogeneic mouse model using a curative islet mass demonstrated delayed rejection of islets when cotransplanted with HUCPVCs (IEQ:HUCPVC ratio of 1:150). The immunosuppressive and proregenerative properties of HUCPVCs demonstrated long-term positive effects on graft function in vivo, indicating that they may improve long-term human islet allotransplantation outcomes

Using high-density DNA methylation arrays to profile copy number alterations.

The integration of genomic and epigenomic data is an increasingly popular approach for studying the complex mechanisms driving cancer development. We have developed a method for evaluating both methylation and copy number from high-density DNA methylation arrays. Comparing copy number data from Infinium HumanMethylation450 BeadChips and SNP arrays, we demonstrate that Infinium arrays detect copy number alterations with the sensitivity of SNP platforms. These results show that high-density methylation arrays provide a robust and economic platform for detecting copy number and methylation changes in a single experiment. Our method is available in the ChAMP Bioconductor package: http://www.bioconductor.org/packages/2.13/bioc/html/ChAMP.html

Investigation of pathogenic mechanisms in multiple colorectal adenoma patients without germline APC or MYH/MUTYH mutations

Patients with multiple (5–100) colorectal adenomas (MCRAs) often have no germline mutation in known predisposition genes, but probably have a genetic origin. We collected a set of 25 MCRA patients with no detectable germline mutation in APC, MYH/MUTYH or the mismatch repair genes. Extracolonic tumours were absent in these cases. No vertical transmission of the MCRA phenotype was found. Based on the precedent of MYH-associated polyposis (MAP), we searched for a mutational signature in 241 adenomatous polyps from our MCRA cases. Somatic mutation frequencies and spectra at APC, K-ras and BRAF were, however, similar to those in sporadic colorectal adenomas. Our data suggest that the genetic pathway of tumorigenesis in the MCRA patients' tumours is very similar to the classical pathway in sporadic adenomas. In sharp contrast to MAP tumours, we did not find evidence of a specific mutational signature in any individual patient or in the overall set of MCRA cases. These results suggest that hypermutation of APC does not cause our patients' disease and strongly suggests that MAP is not a paradigm for the remaining MCRA patients. Our MCRA patients' colons showed no evidence of microadenomas, unlike in MAP and familial adenomatous polyposis (FAP). However, nuclear β-catenin expression was significantly greater in MCRA patients' tumours than in sporadic adenomas. We suggest that, at least in some cases, the MCRA phenotype results from germline variation that acts subsequent to tumour initiation, perhaps by causing more rapid or more likely progression from microadenoma to macroadenoma