1O - The somatic mutation landscape of the normal and malignant pancreas

Background

Somatic mutations occur throughout life and are implicated in carcinogenesis. Studying somatic mutagenesis in normal tissues is challenging as mutations are often in clones too small for detection with conventional DNA sequencing. Single DNA molecule sequencing (NanoSeq) overcomes the need for large clonal expansions, enabling the study of polyclonal tissues, such as the normal pancreas.

Methods

We utilised laser capture microdissection (LCM), whole exome sequencing (WES), and NanoSeq to characterise the somatic mutations across 169 pancreatic samples from 118 donors without pancreatic ductal adenocarcinoma (PDAC), and 29 biopsies from two patients with PDAC.

Results

We performed WES on 924 microdissected ducts, acini, islets, and incidental premalignant lesions from 76 donors. Canonical PDAC drivers were rare, although an acinus with a TP53 driver was identified. KRAS hotspot variants were exclusively in premalignant lesions. Positive selection for loss of USP9X, a deubiquitinase, was observed in normal ducts. In a healthy donor with a germline APC deletion, numerous independent somatic APC second hits were associated with microscopic acinar hyperplasia. LCM-NanoSeq estimated that healthy acini accumulate 25 substitutions per year, resembling other normal tissues. Targeted NanoSeq in bulk pancreatic tissue and purified islets from 118 donors revealed 22 genes under positive selection, implicating chromatin remodelling, Wnt signalling, and cell cycle regulation. Recurrent indels were also observed in highly expressed genes. WES on 174 microdissections from two malignant pancreata highlighted a higher mutation burden in malignant histologies compared to normal histologies, driven by C>T transitions. Multi-region tumour sampling revealed independent KRAS driver acquisition (G12D, G12V, G12R), a SMAD4 frameshift, TP53loss-of-heterozygosity, and a discrete tumour compartment with BAP1 loss.

Conclusions

Here we employ novel sequencing approaches in the healthy and malignant pancreas to illuminate a previously unseen driver landscape, giving insight into pancreatic development, maintenance, and carcinogenesis. As we seek personalised therapies in the pancreas, these data provide a foundation to understand the pancreas in health and disease.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

Wellcome Sanger Institute.

Funding

Wellcome & Cancer Research UK.

Disclosure

M. Stratton, I. Martincorena: Financial Interests, Personal, Other, Co-founder, stockholder, and consultant: Quotient Therapeutics Ltd. All other authors have declared no conflicts of interest.