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Endocrine Abstracts (2023) 96 P15 | DOI: 10.1530/endoabs.96.P15

1University of Exeter, Exeter, United Kingdom; 2University of Cordoba, Cordoba, Spain; 3University College London, London, United Kingdom; 4Vall d’Hebron Hospital, Barcelona, Spain; 5University of Bristol, Bristol, United Kingdom

Neuroendocrine tumours, although considered a rare neoplasia, have been increasing in incidence in developed countries over the last few decades. Previous research has identified several genetic components1. Despite this there is a gap in our knowledge of the causal mechanisms underlying the development of these tumours, with a low background mutation rate and lack of putative variants suggesting that other mechanisms are responsible. Recent work in Pancreatic Neuroendocrine Tumours (P-NET) has highlighted the dysregulation of alternative splicing as having a plausible role2. We investigated whether similar patterns of dysregulation are present in Small Intestine Neuroendocrine tumours (SI-NET), after a pilot study using P-NET tumours. Using Oxford Nanopore long-read cDNA sequencing from 3 P-NET tumour samples and 3 pancreatic tissue healthy controls we set up a pilot study, with a follow-on investigation of 15 participants with SI-NET tumour samples with matched control samples.

Methods: Qiagen AllPrep DNA/RNA (Simultaneous Purification of Genomic DNA and Total RNA from Animal Tissues) was used to extract RNA from fresh frozen tissue. cDNA library preparation performed using the Oxford Nanopore Ligation Sequencing Kit V14 (SQK-LSK114). Sequencing performed on PromethION R10.4.1 flow cells. Long reads were aligned against GRCh38 with Minimap2, followed by transcript assembly using Stringtie in long read mode. Generated GFF annotation was then compared to reference annotation by gffcompare. Gene fusion detection was using the JAFFAL extension. Epi2Me wf-transcriptomics workflow was used for these steps.

Results: Gene fusions differences were detected in known oncogenes such as GNAS, between tumours and controls. Differences in alternate RNA isoforms between the controls and cases were also investigated. These pilot results support the value and originality of long-read sequencing analysis in the discovery of novel molecular players in NETs. 1. Karpathakis, A. et al. Prognostic impact of novel molecular subtypes of small intestinal neuroendocrine tumor. Clinical Cancer Research 22, 250–258 (2016). 2. Alors-Perez, E. et al. Dysregulated splicing factor SF3B1 unveils a dual therapeutic vulnerability to target pancreatic cancer cells and cancer stem cells with an anti-splicing drug. Journal of Experimental and Clinical Cancer Research 40, 1–21 (2021).

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