Anna Karpathakis

Somatic mutation of CDKN1B in small intestine neuroendocrine tumors

The diagnosed incidence of small intestine neuroendocrine tumors (SI-NETs) is increasing, and the underlying genomic mechanisms have not yet been defined. Using exome- and genome-sequence analysis of SI-NETs, we identified recurrent somatic mutations and deletions in CDKN1B, the cyclin-dependent kinase inhibitor gene, which encodes p27. We observed frameshift mutations of CDKN1B in 14 of 180 SI-NETs, and we detected hemizygous deletions encompassing CDKN1B in 7 out of 50 SI-NETs, nominating p27 as a tumor suppressor and implicating cell cycle dysregulation in the etiology of SI-NETs.

Prognostic Impact of Novel Molecular Subtypes of Small Intestinal Neuroendocrine Tumor

Purpose: Small intestinal neuroendocrine tumors (SINET) are the commonest malignancy of the small intestine; however, underlying pathogenic mechanisms remain poorly characterized. Whole-genome and -exome sequencing has demonstrated that SINETs are mutationally quiet, with the most frequent known mutation in the cyclin-dependent kinase inhibitor 1B gene (CDKN1B) occurring in only ∼8% of tumors, suggesting that alternative mechanisms may drive tumorigenesis. The aim of this study is to perform genome-wide molecular profiling of SINETs in order to identify pathogenic drivers based on molecular profiling. This study represents the largest unbiased integrated genomic, epigenomic, and transcriptomic analysis undertaken in this tumor type. Experimental Design: Here, we present data from integrated molecular analysis of SINETs (n = 97), including whole-exome or targeted CDKN1B sequencing (n = 29), HumanMethylation450 BeadChip (Illumina) array profiling (n = 69), methylated DNA immunoprecipitation sequencing (n = 16), copy-number variance analysis (n = 47), and Whole-Genome DASL (Illumina) expression array profiling (n = 43). Results: Based on molecular profiling, SINETs can be classified into three groups, which demonstrate significantly different progression-free survival after resection of primary tumor (not reached at 10 years vs. 56 months vs. 21 months, P = 0.04). Epimutations were found at a recurrence rate of up to 85%, and 21 epigenetically dysregulated genes were identified, including CDX1 (86%), CELSR3 (84%), FBP1 (84%), and GIPR (74%). Conclusions: This is the first comprehensive integrated molecular analysis of SINETs. We have demonstrated that these tumors are highly epigenetically dysregulated. Furthermore, we have identified novel molecular subtypes with significant impact on progression-free survival. Clin Cancer Res; 22(1); 250–8. ©2015 AACR.

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.

Epigenetic dysregulation and poorer prognosis in DAXX-deficient pancreatic neuroendocrine tumours

Exome sequencing of sporadic pancreatic neuroendocrine tumours (PNETs) has identified mutually exclusive mutations in the chromatin regulators α- thalassaemia/mental retardation X-linked (ATRX) and death associated protein 6 (DAXX) genes in 43% of cases (18% and 23% of cases respectively in 68 cases studied) (Elsasser et al. 2011; Jiao et al. 2011). ATRX and DAXX are chromatin remodelers; their loss leads to alternative lengthening of telomeres (ALT) and chromosomal instability (CIN) (Heaphy et al. 2011). ALT is a telomerase independent mechanism for maintenance of telomere stabilisation. Although it was initially reported that ATRX/DAXX mutant tumours had superior 10-year survival and outcome (Jiao et al. 2011), a recent larger study on 243 tumours has demonstrated that ATRX and DAXX loss and associated ALT in PNETs correlates with CIN, advanced tumour stage, development of metastases and poorer progression free (PFS) and overall survival (OS) (Marinoni et al. 2014) ...

Progressive epigenetic dysregulation in neuroendocrine tumour liver metastases

The incidence of small intestinal neuroendocrine tumours (SINETs) is increasing and distant metastases are present at diagnosis in 70% of cases, the liver being the commonest site of metastasis (Yao et al. 2008). Despite this, our understanding of the mechanisms underlying metastatic progression of SINETs is currently limited, and prior studies of the molecular biology of SINET liver metastases (LM) have been performed predominantly in small cohorts utilising candidate-based techniques. SINETs have a low rate of mutations compared to most cancers. The most frequently mutated gene is CDKN1B (encoding p27, a cell cycle regulator); however, mutations in this gene occur in only 8% of tumours, and there is no characteristic mutational hotspot (Francis et al. 2013). Furthermore, mutation of CDKN1B does not correlate with the expression of p27 (Crona et al. 2015). We previously identified that SINETs are epigenetically dysregulated, and a panel of candidate driver epimutation genes has been identified (Karpathakis et al. 2016). Therefore, we postulated that metastatic progression in SINETs may also be epigenetically regulated. Here, we present the findings from the largest molecular profiling study of SINET LM performed to date, integrating copy number variance (CNV), DNA methylation, and RNA expression profiling to characterise the mechanisms underlying metastatic progression. Experimental details of DNA methylation, CNV and RNA expression profiling are as previously published (Karpathakis et al. 2016). Patients provided informed consent for their tissue to be analysed in this study, which was Research Ethics Committee approved (Ref: 09/H0722/27). All cases were reviewed by two expert NET histopathologists (TVL/MN). Nucleic acids were extracted using standard methods (Qiagen, QIAamp DNA Mini kit; Roche, High Pure RNA Paraffin kit). H&E-stained sections were evaluated to ensure >80% purity of tumour specimens. Methylation profiling was performed on the HumanMethylation450 BeadChip (HM450) (Illumina). Methylation data were analysed using ChAMP pipeline (https://www.bioconductor.org/packages/release/bioc/ html/ChAMP.html). Whole-genome methylation profiling using methylated DNA immunoprecipitation sequencing (MeDIP) was performed as previously described. MeDIP data were analysed using the custom pipeline MeDUSA, v2.0 (https://www.ucl.ac.uk/cancer/research/departmentcancer-biology/medical-genomics-group/past-projects/ medusa-project). Gene expression levels were quantified using the whole genome cDNA-mediated annealing, selection and ligation (DASL) (Illumina) assay. Expression data were analysed using the ‘LIMMA’ package in R (https://bioconductor.org/packages/release/bioc/html/ limma.html). Raw data from this study has previously been deposited in GEO (Accession number: GSE73832) (Karpathakis et al. 2016). In summary, n = 90 samples underwent array-based DNA methylation analysis, n = 26 samples underwent methylation-specific immunoprecipitation followed by DNA sequencing and n = 49 underwent array-based RNA expression analysis. Of cases with relevant clinical data, 93% had received no systemic treatment prior to specimen collection (27/29 cases). The CNV profile of SINET LM (n = 20) mirrors that of primary tumours with the most frequent alteration of chr18 LOH seen in 79% of cases. A greater proportion of LM demonstrates amplification of chr20 (42%) and deletion of chr19 (35%), whereas gain of 17q is found only in LM (21%). A trend of increased incidence of CNVs was seen in LM compared to that of SINET primary tumours (SINET primary: median 78 mbp; LM: median 114 mbp, P = 0.08). Comparison of methylation profiles of SINET LM to those of primary SINETs identified 29,263 methylation

Hitting the target: where do molecularly targeted therapies fit in the treatment scheduling of neuroendocrine tumours?

Neuroendocrine tumours (NETs) are a rare and heterogeneous group of tumours whose incidence is increasing and their prevalence is now greater than that of any other upper gastrointestinal tumour. Diagnosis can be challenging, and up to 25% of patients present with metastatic disease. Following the recent FDA approval of two new molecularly targeted therapies for the treatment of advanced pancreatic NETs (pNETs), the first in 25 years, we review all systemic therapies and suggest where these newer targeted therapies fit in the treatment schedule for these challenging tumours. Clinical trial data relating to the routine use of sunitinib and everolimus in low-intermediate-grade pNETs are summarised alongside newer molecularly targeted agents undergoing clinical assessment in NETs. We particularly focus on the challenge of optimal scheduling of molecularly targeted treatments around existing systemic and localised treatment such as chemotherapy or radiotargeted therapy. We also discuss application of current evidence to subgroups of patients who have not so far been directly addressed such as those with poorer performance status or patients receiving radical surgery who may benefit from adjuvant treatment.

Well-differentiated bronchial neuroendocrine tumors: Clinical management and outcomes in 105 patients.

Bronchial neuroendocrine tumors (NETs) are rare tumors representing approximately 20%‐30% of all neuroendocrine tumors and 2%‐3% of all adult lung cancers. Here, they present a large case series of well‐differentiated bronchial NETs with the aim of investigating the behavior of these tumors and long‐term outcomes.

1133OMOLECULAR PROFILING OF SMALL INTESTINAL NEUROENDOCRINE TUMOURS

ABSTRACT Aim: Aberrant DNA methylation is known to play an important role in the pathogenesis of many human cancers, however little is known about its role in small intestinal neuroendocrine tumour (SI NET) development. We report the first unbiased genome wide DNA methylation analysis of a large cohort of SI NET, aiming to identify key epigenetic changes specific to SI NET which may contribute to tumorigenesis. Methods: Illumina Infinium HumanMethylation450 Array (interrogating 99% of RefSeq genes) analysis was performed on DNA extracted from macrodissected SI NET primary tumours (n = 49) and normal small intestine (SI)(n = 21). Publicly available methylation data on >600 samples from The Cancer Genome Atlas was also assessed for comparison (colorectal, pancreatic and gastric adenocarcinoma and healthy tissue). Gene expression was determined using the Illumina DASL array on RNA extracted from SI NET primary tumours (n = 32) and normal SI (n = 6). Analysis was performed using ChAMP and limma R packages. A Bonferroni adjusted significance threshold value of p Results: Comparison of SI NET with normal SI identified a total of 130,083 significant methylation variable positions, including 1841 sites hypermethylated by >30% in tumour compared to normal tissue. 626 genes were found to have significant >3fold differential expression between SI NET and normal SI. Integrated analysis identified a group of 11 candidate genes where altered methylation and expression was significant and concordant (downregulated: CDX1, FBP1, C20orf54, GATA5; upregulated: PTPRN, PCSK1, PRLHR, CELSR3, GIPR, LMX1B, SCGN). Hypermethylation of GIPR (gastic inhibitory polypeptide receptor) was most significant (SI NET median methylation 0.67 vs normal SI 0.29 p Conclusions: This study is the first comprehensive analysis of the epigenetic profile of SI NET and identifies hypermethylation of GIPR as a potential novel biomarker. Novel radioligands targeting GIPR have been developed for use as imaging tools and it is a promising target for novel therapeutic agents. Disclosure: All authors have declared no conflicts of interest.

Pancreatic adenocarcinoma in a patient with multiple endocrine neoplasia 1 syndrome.

To the Editor We present the rare occurrence of a concurrent pancreatic neuroendocrine tumor (pNET) and pancreatic ductal adenocarcinoma (PDAC) in a patient with multiple endocrine neoplasia 1 (MEN1) syndrome. It is important for clinicians to consider the possibility of PDAC in patients with MEN1 because aggressive early surgical intervention provides the only chance of cure. Multiple endocrine neoplasia 1 is characterized by parathyroid adenomas, pNETs, and anterior pituitary tumors.1 Multiple endocrine neoplasia 1–associated pNETs are usually slowly progressive and associated with low malignant potential. In the context of MEN1, pancreatic NET size correlates with prognosis and the presence of metastases,2 and lesions of more than 2 cm are associated with greater genetic instability and malignant behaviour.3 The European Neuroendocrine Tumour Society recommendations for management of pancreatic lesions in a patient with MEN1 state that early diagnosis and surgical excision of MEN1-related pNET improve survival, preventing or delaying the development of distant metastases.4 It is mandatory to operate on MEN1-related nonfunctioning pancreatic tumors with metastases, size of more than 2 cm, or yearly increased size of more than 0.5 cm. Management of pNETs of less than 2 cm is controversial, current recommendation being intensive surveillance to avoid repeated intervention where lesions are typically multiple and behave in an indolent fashion.