Copy number analysis through shallow whole genome sequencing of cell free DNA for neuroendocrine neoplasia monitoring

Masato Ahsan; Shailesh Gohil; Rebecca Allsopp; Karen Page; Reddy Narendra L; Jacqui Shaw; Miles Levy

doi:10.1530/endoabs.114.P24

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Endocrine Abstracts (2025) 114 P24 | DOI: 10.1530/endoabs.114.P24

UKINETS2025 23rd National Conference of the UK and Ireland Neuroendocrine Tumour Society 2025 Poster Presentations (33 abstracts)

Copy number analysis through shallow whole genome sequencing of cell free DNA for neuroendocrine neoplasia monitoring

Masato Ahsan ^1,2 , Shailesh Gohil ^1,2 , Rebecca Allsopp ¹ , Karen Page ¹ , Narendra L Reddy ^1,2 , Jacqui Shaw ¹ & Miles Levy ^1,2

Author affiliations

¹University Hospitals of Leicester, Leicester, United Kingdom; ²Leicester University, Leicester, United Kingdom

Introduction: The clinical application of circulating tumour DNA (ctDNA) monitoring in neuroendocrine neoplasms (NENs) remain underexplored compared to malignancies such as breast, colorectal and lung. Here, we attempt to profile copy number alterations (CNAs) in cell free DNA (cfDNA) as a potential biomarker in NENs.

Methods: cfDNA was extracted from 4 mL of plasma using a semi-automated MagMAX™ Cell-Free DNA Isolation Kit with KingFisher™ Flex Magnetic Particle Processor. cfDNA quantity and integrity were assessed with the Agilent 4200 TapeStation System, enabling fragment size profiling and quantification. Shallow whole genome sequencing (sWGS) was performed with the ReproSeq PGS kit, and data were analysed with the ichorCNA pipeline to estimate tumour fraction and detect copy number alterations (CNAs).

Results: cfDNA was isolated from serial plasma samples from 11 patients: 7 with sporadic NENs (2–5 samples each), 4 with germline mutations (1–2 samples each), and totalling 33 plasma samples. cfDNA yields across patients were generally low (mean ~297 pg/µL), but fragment profiles indicated preserved cfDNA integrity. Fragment analysis consistently revealed a dominant peak representing mononucleosomal fragments, alongside a secondary peak indicative of dinucleosomal DNA. This confirmed high-quality cfDNA suitable for downstream analysis. CNA analysis through sWGS demonstrated flat genomic profiles across all samples, with no CNAs detectable, suggesting either the absence of ctDNA containing CNAs or lack of sensitivity to detect ctDNA in these samples by sWGS technique.

Discussion: sWGS did not identify CNAs indicating either the lack of CNA in cfDNA or ctDNA levels below the threshold of detection. Future studies should explore complementary approaches, including single nucleotide variant (SNV) analysis and DNA methylation profiling, to enhance detection sensitivity. A multi-omic liquid biopsy strategy may be necessary to establish the clinical value of cfDNA analysis in NENs.

Learning points: 1. Multi-omic liquid biopsy approaches, including SNV and methylation profiling, warrant further evaluation.