Abstract 369: In silico investigation of hypoxia and its association with disease outcome and ecDNA detection in TRACERx
Authors: Sharma Natasha , Bailey Chris , Richard Corentin , Kittel Jeanette , Liu Wing Kin , Frankell Alexandar M. , Satvrou Georgia , Moore David , Sivakumar Monica Sivakumar , Hanjani Mariam Jamal , Swanton Charles , Kanu Nnennya 
Published: March 2024

Abstract
Tumors exhibit dynamic gradients of oxygen diffusion and consumption leading to regions of hypoxia. The presence of hypoxia is associated with poor clinical prognosis and elevated genomic instability. Genes can be amplified extrachromosomally as ecDNA, which are acentric circular structures that can rapidly generate large genomic copy number amplifications. The amplification of oncogenes on ecDNA is unstable thus providing cells with an advantage to adjust and survive the changing environment. Here, we have analysed the effect of hypoxia on disease outcome and ecDNA formation in the multiregional longitudinal NSCLC TRACERx (TRAcking Cancer Evolution through therapy (Rx)) cohort. We have analysed next generation sequencing, clinical and histopathological data available from the tumours that were harvested at the time of surgery from patients recruited into the lung TRACERx study. Hypoxia was first quantified using the mRNA-based Buffa hypoxia gene signature. Complexheatmap from R-Bioconductor package was used for clustering based on mRNA expression of gene signature. Moreover, hypoxia scores for each region were established using the Gene Set Variation Analysis (GSVA) from R- Bioconductor package. The impact of hypoxia on overall and disease-free survival was performed using the survminer package. Histopathological data was used to study the association between hypoxia and necrosis. The genetic instability was analyzed using whole exome sequencing. The amplicon architect tool was used to study the association of hypoxia with ecDNA detection. Our preliminary results demonstrate that the extent of hypoxia varies widely both within and between primary tumours, suggestive of the presence of inter and intratumour hypoxia heterogeneity. High hypoxia status is associated with worse overall and disease-free survival in adenocarcinoma but not in squamous-cell carcinoma. The presence of necrosis in the primary tumour was associated with higher hypoxia. Moreover, higher hypoxia scores correlate with circulating tumour DNA (ctDNA) shedding in pre-surgery plasma samples (p value&lt; 0.05). Higher hypoxia was significantly associated with increased wGII scores, indicative of genomic instability. Also, hypoxia resulted in increased detection of ecDNA in both lung adenocarcinoma and squamous cell carcinoma. In conclusion, there is inter and intratumoral heterogeneity in hypoxia levels in lung TRACERx. Moreover, the presence of hypoxia is associated with the detection of necrosis and ctDNA shedding. The increased genomic instability and detection of ecDNA in this study could be associated with the poor prognosis that is observed with hypoxic tumours and could merit further investigation.
Citation Format: Natasha Sharma, Chris Bailey, Corentin Richard, Jeanette Kittel, Wing Kin Liu, Alexandar M. Frankell, Georgia Satvrou, David Moore, Monica Sivakumar Sivakumar, Mariam Jamal Hanjani, Charles Swanton, Nnennya Kanu. In silico investigation of hypoxia and its association with disease outcome and ecDNA detection in TRACERx [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 369.