Abstract 218: Somatic mutations in single-cell derived alveolar organoids
Authors: Przybilla Moritz Jakob , Ammar Amany , Khaw Ryan Chuen , Lawson Andrew R. , Nicola Pantelis , Davies Kate , Frazer Zoe , Gowers Kate H. , Butler Timothy M. , Clarke Sarah E. , Martincorena Inigo , Janes Sam M. , Campbell Peter J. 
Published: April 2023

Abstract
The diversity in somatic variation across human tissues outshining human cancers has only recently been started to be appreciated. In particular, healthy individuals carry large numbers of cells and clonal expansions with mutations in driver genes commonly observed in cancer. We previously demonstrated that the majority of epithelial cells in the proximal airway accumulate DNA damage proportionally to an individual's smoking habits. However, the analysis of whole genomes indicated the abundance of a population of cells with a near-normal mutation burden, mirroring cellular populations found in never-smoking individuals. In contrast to the proximal airway, the genomics of cells in the distal airway of healthy individuals with diverse smoking histories, and thus the foundation of lung adenocarcinoma (LUAD), has not been investigated yet. To assess the influence of smoking on the distal airway and to compare the genomics of proximal and distal airway in the lung, we established an experimental procedure to create single-cell derived alveolar type II (AT2) organoids from primary human tissue of 9 patients. In particular, we focus on AT2 cells, given their hypothesised role as a potential cell of origin for LUAD.
Whole genome sequencing (WGS) of &gt;400 organoids was utilised to assess the landscape of somatic variation, including single nucleotide variants (SNVs), insertions and deletions (indels), copy number and structural variation (CNVs, SVs). Focusing on SNVs, we found that the mutation burden of never-smokers is significantly lower compared to ex- and current-smokers. Emphasising the effect of tobacco smoking on the distal airway, this difference was driven by the presence of mutations related to SBS4 and SBS92, mutational signatures associated with tobacco smoking. Interestingly, and in contrast to the proximal airway, we did not find a difference in the mutation burden of ex- and current-smokers, suggesting that smoking damage to alveolar cells is more severe and long-term. Lastly, to characterise the driver mutation landscape in depth, we used targeted single-molecule sequencing of 250 known cancer genes to profile 15 AT2 single-cell suspensions as well as 22 frozen parenchyma biopsies. To date, our preliminary results indicate positive selection on lineage defining mutations including SFTPB and SFTPC, coding for surfactant as well as TP53. As we begin to explore these data further, we expect this work to provide unprecedented insights into the contribution of somatic and driver mutations to the earliest stages of lung cancer development.
Citation Format: Moritz Jakob Przybilla, Amany Ammar, Ryan Chuen Khaw, Andrew R. Lawson, Pantelis Nicola, Kate Davies, Zoe Frazer, Kate H. Gowers, Timothy M. Butler, Sarah E. Clarke, Inigo Martincorena, Sam M. Janes, Peter J. Campbell. Somatic mutations in single-cell derived alveolar organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 218.