John V. Pearson

Oesophageal adenocarcinoma (OAC) incidence continues to rise in developed countries. Despite advances in deciphering cancer genomes and oncogenic pathways, precision therapies for OAC remain limited. An expansion of precision therapy repertoire in OAC is urgently required. We assembled 109 actionable and/or potentially actionable genes based on pan-cancer drug information from open-source compendia and clinical trials. To discover approved treatments and potentially targetable alterations (PTAs), we re-analysed whole genome sequencing data of 217 OAC patients and clinically evaluated somatic mutations in these genes using international cancer curation guidelines. Additionally, matching RNA-seq from 210 of the patients was interrogated for select immune checkpoint inhibitor (ICI) targets (n = 59), with expression findings validated in an independent OAC cohort (n = 114). Approved actionable targets were revealed in 23% of patients; including HER-2 and MET antagonists, and immunotherapy directed by microsatellite instability and high tumour mutation burden. PTAs were noted in a further 72.4% of patients, although the highest frequency was in TP53 which is a challenging target for treatment. Other PTAs were noted in APC, ARID1A, BRCA1, BRCA2, CCNE1, CDKN2A, EGFR, KRAS, MDM2, PIK3CA, PTEN, SMARCB1 and TSC2. Candidate co-occurring alterations included co-occurrence of PTEN loss and ERBB2 amplification and co-occurring variants in KRAS and TP53. For the remaining 4.6% of patients, no PTAs were detected. We identified and validated high expression of four genes encoding candidate ICI targets for OAC: CD24, VEGFA, PVR, and SLC3A2. These PTAs and ICI targets will help to inform future clinical trials to facilitate precision therapy for patients with OAC.

Background: Long-read sequencing (LRS) improves genome alignment and facilitates resolving variants in genomic regions of low complexity, making it a promising approach for cancer variant detection and biomarker discovery. Results: Here, we evaluate the performance of LRS using Oxford Nanopore sequencing to detect somatic variants across different tumour purities and sequencing depths by comparing to somatic variants from short-read sequencing. We generated experimental mixtures of cancer cell lines and matched normal cell lines to simulate 10 tumour purities (ranging from 0 to 100% tumour content). This resulted in 22 samples, which were sequenced using whole-genome LRS to a targeted depth of 60x, and somatic variants were identified. We down-sampled data to explore the optimal read depth for somatic variant detection. Our results show long-read variant calling tools achieve recall rates comparable to short-read gold standards. While tumour sequencing depth of 30x–60x is generally sufficient for detecting common variants, particularly structural variants, sequencing the matched normal sample at adequate depth is crucial for accuracy. Notably, we found variants unique to LRS that may represent real and previously undetected events. Conclusion: This study highlights key factors for optimising cancer genome sequencing with LRS and provides a comprehensive dataset of cell line mixtures for future research and tool development.

Kaplan–Meier curves of OS in (A) main tumor groups (n = 582)—MBOT, MOC, LGI, and UGI; (B) patients with MOC by FIGO stage (n = 184); (C) patients with MOC by pattern of invasion in all stages (n = 178); and (D) patients with stage I MOC (n = 134), by pattern of invasion.

Heat map of unsupervised clustering analysis. Contains all samples with a concordant pathology diagnosis (n = 497), with MOC grouped by FIGO stage. Labels show main clusters and diagnoses. Gene-expression values are normalized and logarithm base 2 transformed. dx, diagnosis.

Associations between gene expression and stage group and OS in MOC.

Overall survival in MOC by pattern of invasion.

Schema of study numbers for each analysis to describe different cohort numbers due to pathology review and missing data. MOC, mucinous ovarian carcinoma; MBOT, mucinous borderline ovarian tumor; LGI, lower gastrointestinal; UGI, upper gastrointestinal; SISH, silver in situ hybridization.