ABSTRACT Background The value and optimal timing of circulating tumour DNA (ctDNA) analysis in locally advanced oesophageal adenocarcinoma (OAC) is uncertain. We hypothesised that perioperative detection would predict event-free (EFS) and overall (OS) survival, and that 3-6 months post-operative detection would predict recurrence. Methods In this prospective multi-centre cohort study, tumour-informed ctDNA assays were designed for 49 patients using whole-exome sequencing. Bloods were collected for ctDNA detection up to 8 days prior to surgery, and at 3-6 weeks and 3-6 months post-surgery, then correlated with clinicopathological characteristics, EFS and OS. Results Pre- and early post-surgery ctDNA positivity were associated with worse EFS (HRs 7.97 (95% confidence interval, CI 2.64-24.04), p<0.0001; 8.18 (95%CI 3.23-20.69), p<0.0001) and OS (HRs 7.82 (95%CI 2.22-27.54), p=0.00018; 13.69 (95%CI 4.52-41.49), p<0.0001). In pre-surgery positive patients, post-surgery ctDNA clearance associated with improved EFS and OS, and predicted better OS in those with a poor histopathological response to neoadjuvant treatment. 3-6 month ctDNA-positivity preceded standard-of-care recurrence detection by median 53.5 (interquartile range 39.3-200.0) days. Conclusions Perioperative ctDNA positivity associates with worse EFS and OS in OAC, identifying a subgroup with improved outcomes despite adverse pathological features. ctDNA testing at 3-6 months predicts recurrence earlier than standard-of-care surveillance.

Cancer generally takes years to evolve, and early diagnosis can prevent life-threatening cancer. Establishing a link between precancerous states and cancer is essential for effective screening and prevention. Esophageal adenocarcinoma (EAC) is an increasingly prevalent, poor-outcome cancer, and its presumed precursor, Barrett's esophagus (BE), characterized by intestinal metaplasia, is evident in only about half of cases. Here to test whether BE is a prerequisite to EAC, we integrated epidemiological and clinical characteristics in a prospective cohort of 3,100 patients with EAC for any evidence of BE (BE-positive and BE-negative) and compared genomic features using a subset of 710 patients with whole-genome sequencing and 87 patients (380 samples) with multiregional whole-exome sequencing. Demographic and genomic features typically associated with BE were observed across BE-positive and BE-negative EAC cases. Notably, molecular features consistent with early BE evolution were detected in both phenotypes. Advanced tumor stage was the only variable that corresponded with increased likelihood of BE-negative EAC, including in some patients with a previous BE diagnosis. Phylogenetic analyses revealed shared evolutionary trajectories, and spatial transcriptomic and proteomic analyses demonstrated intestinal metaplasia-associated lineage markers in both groups. These findings suggest a single pathway to EAC, with implications for early diagnosis and prevention strategies.

Cancer generally takes years to evolve, and early diagnosis can prevent life-threatening cancer 1,2 . Symptomatic, advanced cancers often lack evidence of a precancer at diagnosis and yet, if the link between a precancerous state and cancer can be proven, the opportunity arises for screening and prevention. Oesophageal adenocarcinoma (EAC) is an increasingly prevalent, 3 poor outcome cancer and its presumed precursor, Barrett’s oesophagus (BE) is only evident in around half of cases 4,5 . To test the hypothesis that BE is not a pre-requisite to EAC we classified a prospective cohort of 3,100 EAC patients for any evidence of BE and compared the epidemiological, clinical and genomic characteristics. Our findings demonstrate that there are specific demographic and genomic features observed in BE that also correlate strongly with EAC, regardless of the presence of a BE phenotype. Further we found evidence for the earliest features of BE evolution in both BE-negative and BE-positive EAC phenotypes. Advanced tumour stage was the only variable that corresponded with increased likelihood of BE-negative EAC, including in some patients with a prior diagnosis of Barrett’s. The role of a single BE-mediated pathway to EAC has implications for early diagnosis strategies. We anticipate that our methodology combining large epidemiological and genomic datasets with respect to phenotype could help establish the significance of other precancer states.

Multi-center collaboration is essential to achieve the sample sizes required for robust and informative research studies for less common medical conditions. Substantial logistical and governance support is needed to ensure that the clinical and molecular data generated are of high quality and benefit the international research community and patients.

Esophageal adenocarcinoma is a prominent example of cancer characterized by frequent amplifications in oncogenes. However, the mechanisms leading to amplicons that involve breakage-fusion-bridge cycles and extrachromosomal DNA are poorly understood. Here, we use 710 esophageal adenocarcinoma cases with matched samples and patient-derived organoids to disentangle complex amplicons and their associated mechanisms. Short-read sequencing identifies ERBB2, MYC, MDM2, and HMGA2 as the most frequent oncogenes amplified in extrachromosomal DNAs. We resolve complex extrachromosomal DNA and breakage-fusion-bridge cycles amplicons by integrating of de-novo assemblies and DNA methylation in nine long-read sequenced cases. Complex amplicons shared between precancerous biopsy and late-stage tumor, an enrichment of putative enhancer elements and mobile element insertions are potential drivers of complex amplicons' origin. We find that patient-derived organoids recapitulate extrachromosomal DNA observed in the primary tumors and single-cell DNA sequencing capture extrachromosomal DNA-driven clonal dynamics across passages. Prospectively, long-read and single-cell DNA sequencing technologies can lead to better prediction of clonal evolution in esophageal adenocarcinoma. Esophageal adenocarcinoma is characterised by frequent amplifications in oncogenes. Here, the authors use short- and long-read sequencing approaches to analyze primary tumor samples and tumour-derived organoids and to investigate the mechanisms underlying complex amplifications.

BackgroundEsophageal cancer [esophagogastric adenocarcinoma (OGA)] shows heterogeneity at the molecular level, leading to lower efficacy rates and highlighting the need for personalized treatment strategies. We have developed a computational model that uses both mechanistic and statistical approaches to integrate a patient’s genomic aberrations, revealing signaling pathway dysregulation and variable drug response. The model output, Therapy Response Index (TRI), has been used to predict therapeutic outcomes in this study.DesignTRI’s ability to predict patient outcomes was retrospectively evaluated in a prospectively collected cohort of patients with operable OGA from the UK Oesophageal Cancer Clinical and Molecular Stratification (OCCAMS) consortium receiving neoadjuvant chemotherapy. Stratified random sampling was used to split the data into training and validation subsets. Multivariate Cox proportional hazard and proportional odds models were used to predict survival and pathological response as a function of TRI and clinical thresholds compared with clinical factors.ResultsA total of 270 patients with OGA were selected who had 50× whole genome sequencing carried out on tissue derived from either biopsy or resection. Patients were treated with chemotherapy drugs or regimens according to UK clinical guidelines. The association of TRI with overall survival (OS) was significant above and beyond standard clinical factors (P = 0.0012). A significant association was also observed with disease-free survival (DFS; P = 0.0288). A TRI optimized for tumor regression grade also displayed a significant association (P = 0.0011).ConclusionsTRI was predictive of OS and DFS beyond clinical factors. These positive results suggest the potential utility of personalized biosimulation-informed therapy selection and that further assessment in prospective clinical studies is warranted.

Most cancer types exhibit aberrant transcriptional activity, including derepression of retrotransposable elements (RTEs). However, the degree, specificity and potential consequences of RTE transcriptional activation may differ substantially among cancer types and subtypes. Representing one extreme of the spectrum, we characterize the transcriptional activity of RTEs in cohorts of esophageal adenocarcinoma (EAC) and its precursor Barrett's esophagus (BE) from the OCCAMS (Oesophageal Cancer Clinical and Molecular Stratification) consortium, and from TCGA (The Cancer Genome Atlas). We found exceptionally high RTE inclusion in the EAC transcriptome, driven primarily by transcription of genes incorporating intronic or adjacent RTEs, rather than by autonomous RTE transcription. Nevertheless, numerous chimeric transcripts straddling RTEs and genes, and transcripts from stand-alone RTEs, particularly KLF5- and SOX9-controlled HERVH proviruses, were overexpressed specifically in EAC. Notably, incomplete mRNA splicing and EAC-characteristic intronic RTE inclusion was mirrored by relative loss of the respective fully-spliced, functional mRNA isoforms, consistent with compromised cellular fitness. Defective RNA splicing was linked with strong transcriptional activation of a HERVH provirus on Chr Xp22.32 and defined EAC subtypes with distinct molecular features and prognosis. Our study defines distinguishable RTE transcriptional profiles of EAC, reflecting distinct underlying processes and prognosis, thus providing a framework for targeted studies.

A variety of mutational processes drive cancer development, but their dynamics across the entire disease spectrum from pre-cancerous to advanced neoplasia are poorly understood. We explore the mutagenic processes shaping oesophageal adenocarcinoma tumorigenesis in 997 instances comprising distinct stages of this malignancy, from Barrett Oesophagus to primary tumours and advanced metastatic disease. The mutational landscape is dominated by the C[T > C/G]T substitution enriched signatures SBS17a/b, which are linked with TP53 mutations, increased proliferation, genomic instability and disease progression. The APOBEC mutagenesis signature is a weak but persistent signal amplified in primary tumours. We also identify prevalent alterations in DNA damage repair pathways, with homologous recombination, base and nucleotide excision repair and translesion synthesis mutated in up to 50% of the cohort, and surprisingly uncoupled from transcriptional activity. Among these, the presence of base excision repair deficiencies show remarkably poor prognosis in the cohort. In this work, we provide insights on the mutational aetiology and changes enabling the transition from pre-neoplastic to advanced oesophageal adenocarcinoma.

There is concern that the incidence of oesophageal adenocarcinoma (OAC) in patients under 50, described as early onset (EO) OAC, may be rising steadily. Epidemiological studies suggest that EO-OAC patients present with higher-stage disease compared to late-onset patients, receive more aggressive treatment but may have poorer survival outcomes. We utilised Whole Genome Sequencing (WGS) and RNA-seq to investigate if EO-OAC represents a distinct biological entity compared to late-onset disease. We obtained WGS and clinical data for stage and treatment matched OAC patients who received neoadjuvant chemotherapy prior to surgical resection (n=29 patients ≤50 years and n=144 patients ≥70 years) from the OCCAMS consortium. We performed statistical analysis between age groups using chi-squared testing with FDR correction for copy number aberrations (CNA), insertions and deletions (INDELS) and single nucleotide variants (SNV), assessing gene-level mutations and mutational burden considering coding genes only, prior to assessing 76 OAC driver genes as described by Frankell et al., 2019. Biological pathways between age groups were assessed for n=66 patients using available RNASeq data and Gene Set Enrichment Analysis (GSEA). Our results indicate that EO-OAC had a lower combined mutational burden (Wilcoxon p=0.0204) and lower indel counts (Wilcoxon p=0.0031) compared to late-onset patients. When considering gene-level mutations for SNV, CNA, INDELS and 76 OAC driver genes (Frankell et al., 2019), multiple analyses showed genes at a nominal p-value <0.05 between age groups yet none reached significance at FDR p-value <0.25. GSEA indicated 179 enriched pathways, several related to immune signalling for EO-OAC at nominal p-value <0.05 and a higher DNA Damage Immune Response (DDIR) assay score in EO-OAC (p=0.012). Our comparison of early and late-onset OAC using WGS and RNA-seq data highlights the possibility of EO-OAC as a separate biological entity, with potentially enhanced immune signalling. Future work in larger sample cohorts and the investigation of clinically relevant gene and mutational signatures is required to further assess molecular differences between early and late onset OAC.

4064 Background: Computational biological modeling reveals many dysregulated signaling pathways responsible for hallmark behaviors of cancer and variable drug response in the population. A mechanistic model created for each patient using comprehensive genomic inputs can biosimulate downstream molecular effects of cell signaling and drugs for each patient’s personalized in silico virtual disease model. Singula TRI is designed to predict the outcome of specific therapies with a continuous TRI Score, 0 to 100, for each patient’s unique genomic network. Methods: TRI’s ability to predict Overall Survival (OS), Disease Free Survival (DFS) and Mandard – tumor regression grade (TRG) was prospectively evaluated in a retrospective cohort of gastroesophageal adenocarcinoma (GEA) from UK OCCAMS consortium. Random sampling stratified by clinical factors was used to split the data into independent training (N = 140) and validation (N = 131) subsets. Multivariate Cox Proportional Hazard (PH) and Proportional Odds models were used to predict survival and pathological response as a function of the pre-defined TRI and clinical thresholds compared with standard clinical factors. Results: 271 GEA patients were selected who had pre-chemo treated biopsies with 50x whole genome sequencing from the OCCAMS International Cancer Genome Consortium study. The median age was 65.6 years, 234 male and 30 female, with deceased median OS of 21.9 months and living of 49.9 months. There were 35 T2, 215 T3, 70 N0, 126 N1, 62 N2 and 266 M0. Patients were treated with physician prescribed chemotherapy treatments (PPT) according to UK clinical guidelines (SC). Biosimulation revealed that 99% of these tumors had deficiency in DNA repair genes. Other pathways included amplification of multi-drug resistance pumps, TP53 mutations and aberrations of the PI3K/AKT pathway genes. The table shows that TRI provides additional predictive information for OS and DFS beyond PPT and standard clinical factors. TRI was also predictive of TRG in univariate analysis. TRI scores were also generated for 82 alternate therapies for each patient enabling selection of optimal therapies with estimates of improvements in median OS and DFS compared to SC. Conclusions: In this cohort of patients, Cellworks Singula TRI was predictive of survival and TRG beyond clinical factors. These positive results suggest the utility of biosimulation-informed therapy selection to improve survival of GEA and validation in prospective clinical studies is warranted.[Table: see text]

Oesophageal adenocarcinoma (OAC) provides an ideal case study to characterize large-scale rearrangements. Using whole genome short-read sequencing of 383 cases, for which 214 had matched whole transcriptomes, we observed structural variations (SV) with a predominance of deletions, tandem duplications and inter-chromosome junctions that could be identified as LINE-1 mobile element (ME) insertions. Complex clusters of rearrangements resembling breakage-fusion-bridge cycles or extrachromosomal circular DNA accounted for 22% of complex SVs affecting known oncogenes. Counting SV events affecting known driver genes substantially increased the recurrence rates of these drivers. After excluding fragile sites, we identified 51 candidate new drivers in genomic regions disrupted by SVs, including ETV5, KAT6B and CLTC. RUNX1 was the most recurrently altered gene (24%), with many deletions inactivating the RUNT domain but preserved the reading frame, suggesting an altered protein product. These findings underscore the importance of identification of SV events in OAC with implications for targeted therapies.

Background: The incidence of esophageal adenocarcinoma (EAC) is rapidly rising and has a 5-year survival rate of <20%. Beyond TNM (tumor-node-metastasis) staging, no reliable risk stratification tools exist and no large-scale studies have profiled circulating tumor DNA (ctDNA) at relapse in EAC. Here we analyze the prognostic potential of ctDNA dynamics in EAC, taking into account clonal hematopoiesis with indeterminate potential (CHIP). Patients and methods: A total of 245 samples from 97 patients treated with neoadjuvant chemotherapy and surgery were identified from the prospective national UK Oesophageal Cancer Clinical and Molecular Stratification (OCCAMS) consortium data set. A pan-cancer ctDNA panel comprising 77 genes was used. Plasma and peripheral blood cell samples were sequenced to a mean depth of 7082x (range 2196-28 524) and ctDNA results correlated with survival. Results: Characteristics of the 97 patients identified were as follows: 83/97 (86%) male, median age 68 years (SD 9.5 years), 100% cT3/T4, 75% cN+. EAC-specific drivers had higher variant allele fractions than passenger mutations. Using stringent quality criteria 16/79 (20%) were ctDNA positive following resection; recurrence was observed in 12/16 (75%) of these. As much as 78/97 (80%) had CHIP analyses that enabled filtering for CHIP variants, which were found in 18/78 (23%) of cases. When CHIP was excluded, 10/63 (16%) patients were ctDNA positive and 9/10 of these (90%) recurred. With correction for CHIP, median cancer-specific survival for ctDNA-positive patients was 10.0 months versus 29.9 months for ctDNA-negative patients (hazard ratio 5.55, 95% confidence interval 2.42-12.71; P = 0.0003). Similar outcomes were observed for disease-free survival. Conclusions: We demonstrate in a large, national, prospectively collected data set that ctDNA in plasma following surgery for EAC is prognostic for relapse. Inclusion of peripheral blood cell samples can reduce or eliminate false positives from CHIP. In future, post-operative ctDNA could be used to risk stratify patients into high- and low-risk groups for intensification or de-escalation of adjuvant chemotherapy.

BACKGROUND & AIMS:Esophageal adenocarcinomas (EACs) are heterogeneous and often preceded by Barrett's esophagus (BE). Many genomic changes have been associated with development of BE and EAC, but little is known about epigenetic alterations. We performed epigenetic analyses of BE and EAC tissues and combined these data with transcriptome and genomic data to identify mechanisms that control gene expression and genome integrity. METHODS:In a retrospective cohort study, we collected tissue samples and clinical data from 150 BE and 285 EAC cases from the Oesophageal Cancer Classification and Molecular Stratification consortium in the United Kingdom. We analyzed methylation profiles of all BE and EAC tissues and assigned them to subgroups using non-negative matrix factorization with k-means clustering. Data from whole-genome sequencing and transcriptome studies were then incorporated; we performed integrative methylation and RNA-sequencing analyses to identify genes that were suppressed with increased methylation in promoter regions. Levels of different immune cell types were computed using single-sample gene set enrichment methods. We derived 8 organoids from 8 EAC tissues and tested their sensitivity to different drugs. RESULTS:BE and EAC samples shared genome-wide methylation features, compared with normal tissues (esophageal, gastric, and duodenum; controls) from the same patients and grouped into 4 subtypes. Subtype 1 was characterized by DNA hypermethylation with a high mutation burden and multiple mutations in genes in cell cycle and receptor tyrosine signaling pathways. Subtype 2 was characterized by a gene expression pattern associated with metabolic processes (ATP synthesis and fatty acid oxidation) and lack methylation at specific binding sites for transcription factors; 83% of samples of this subtype were BE and 17% were EAC. The third subtype did not have changes in methylation pattern, compared with control tissue, but had a gene expression pattern that indicated immune cell infiltration; this tumor type was associated with the shortest time of patient survival. The fourth subtype was characterized by DNA hypomethylation associated with structure rearrangements, copy number alterations, with preferential amplification of CCNE1 (cells with this gene amplification have been reported to be sensitive to CDK2 inhibitors). Organoids with reduced levels of MGMT and CHFR expression were sensitive to temozolomide and taxane drugs. CONCLUSIONS:In a comprehensive integrated analysis of methylation, transcriptome, and genome profiles of more than 400 BE and EAC tissues, along with clinical data, we identified 4 subtypes that were associated with patient outcomes and potential responses to therapy.

Response of EAC to immune-checkpoint blockade (ICB) is modest and clinicians lack biomarkers for pt selection. EAC's mutational signature profile suggest 'mutagenic' & 'DDR' subtypes might respond to ICB. Furthermore, ctDNA can detect treatment & genomic variations before other tests. Prospective, open-label, pilot trial of D (1500mg/4wk) for advanced EAC. Intensive blood sampling (PBMCs, plasma) & tumor biopsies are taken at pre-defined time-points (screening/wk12/disease progression). Tumour-derived variant allele fractions (VAF) & copy-number alterations (CN) are analyzed using Guardant360 assay. Primary objective: determine if early changes (wk4/7) in ctDNA (VAF, CN) correlates with long-term radiological responses (wk26). Enrolment of 19 evaluable pts is planned. Translational studies will analyze D on the immuno-genomic landscape of EAC. We report the interim data. 7 pts were recruited (all male, median age: 71.7 yr, 1 patient HER2+), 6 received D. Prior chemotherapy: perioperative only in 2/6, ≥ 1 line in 4/6. Three pts received ≥ 3 doses of D. Adverse events (AE): 68% Grade 1-2, 32% (8/25) Grade 3 (none drug-related). Two drug-related AE (hyperamylasemia, hypothyroidism) led to dose delay and withdrawal, respectively. Both resolved. Primary endpoint: 'changes in “total-ctDNA fraction" and RECIST1.1 response' were evaluable in 4/6 pts:Table: 1940PPtRelative change in total ctDNA fraction (scr to wk 7)Response11.85PD22PD30.31Partial response (PR)41.07Stable Disease Open table in a new tab . Also, individual gene changes reflect RECIST responses: -Pt3 had PR & ≥10% relative reduction in VAFs (PI3KCA, TP53, APC, ERBB2) & CNs (CDK4, FGFR2, CCNE1). -Pt2 had PD & ≥10% relative increase of VAFs (TP53, BRCA1, ATM) & CN (KRAS, EGRF). D was well tolerated with potential activity in EAC. Data suggest a relationship between changes in ctDNA & RECIST response. Mature data is required. Recruitment & translational studies are ongoing, mutational signature profile (WGS data) of 4pts will be presented.

The poor outcomes in esophageal adenocarcinoma (EAC) prompted us to interrogate the pattern and timing of metastatic spread. Whole-genome sequencing and phylogenetic analysis of 388 samples across 18 individuals with EAC showed, in 90% of patients, that multiple subclones from the primary tumor spread very rapidly from the primary site to form multiple metastases, including lymph nodes and distant tissues—a mode of dissemination that we term ‘clonal diaspora’. Metastatic subclones at autopsy were present in tissue and blood samples from earlier time points. These findings have implications for our understanding and clinical evaluation of EAC. Whole-genome sequencing and phylogenetic analysis of nearly 400 esophageal adenocarcinoma samples suggest a model of rapid subclone spreading from the primary tumor to multiple metastatic sites, which the authors term ‘clonal diaspora’.

Over half of Esophageal Adenocarcinoma (EAC) patients treated with curative intent relapse. In clinical practice risk stratification is limited to TNM staging, which highlights the need for additional methods. Circulating tumor DNA (ctDNA) following surgical resection is prognostic across different tumor types. However, the sensitivity and specificity of tumor-naive ctDNA panels are limited in the minimal residual disease (MRD) setting. Additionally, EAC is known to be a low shedding tumor type, thus, a personalized, tumor-informed approach for ctDNA analysis is ideal for MRD detection after treatment and for providing prognostic value in EAC patients. Using the prospectively collected multi-centre UK OCCAMS dataset we identified patients (n=12) with pre- and post-surgical plasma samples (n=26). Mutational profiles derived from tumor tissue were used to design assays targeting patient-specific somatic variants (Signatera™ bespoke multiplex-PCR NGS assay). The personalized assays were used to determine the presence of ctDNA in the plasma samples of EAC patients. Additional patients are currently being processed and will be presented during the meeting. The cohort consisted of 12 patients with a median age of 62.8 (48.9 – 75.8) years, of which 83% were male and were T3 at diagnosis. All patients were treated with neoadjuvant chemotherapy, of which 2 also received radiotherapy. Minimal residual disease post-surgery was detected down to a mean variant allele frequency of 0.001%. Post-operative ctDNA analysis detected clinical relapse in 4 patients with a median lead time of 196 days giving a sensitivity and specificity of 100%. In this pilot study, we showed that bespoke multiplex-PCR assays for esophageal samples achieve with high sensitivity and specificity to detect recurrence in this low shedding cancer type. This result is a vast improvement over other ctDNA assays, which show <40% EAC sensitivity. Further prospective studies are warranted to investigate the clinical utility of the bespoke ctDNA assay as a modern risk stratification tool in this cancer type.

Background Oesophageal cancer is the eighth most frequently diagnosed form of cancer and has a dismal 5-year survival rate of 12%, with Oesophageal adenocarcinoma (OAC) being the most common sub-type in western countries (Ferlay et al., 2012). Previous studies in many cancer types have demonstrated potential clinical utility of circulating tumour DNA (ctDNA) for detecting minimal residual disease and tumour evolution through therapy, but there has been little investigation of this technology in large cohorts of OAC patients. Consequently, the aim of this study is to evaluate the clinical potential of longitudinal liquid biopsy sampling using 100 OAC cases. Methods Our approach uses the Roche-Avenio expanded panel and ultra-deep sequencing to detect genomic aberrations across 77 cancer genes in the ctDNA of OAC cases. All cases are late disease stage (T3 /T4) and have at least one plasma sample taken before surgery and on average two other samples taken at time points of clinical interest. One third of cases have associated whole genome sequencing of the primary tumour. Results Our results demonstrate that in spite of OAC being a low ctDNA shedding cancer type, ctDNA can be consistently detected in late disease cases. At least one mutation in OAC driver genes was identified in 69% of cases, some of which include TP53, SMAD4, and CDKN2A (Frankell et al., 2019). TP53 was the most frequently mutated gene, which is also observed in whole-genome sequencing (WGS) data. In addition, other OAC driver genes were mutated to a similar extent to the WGS data. Clinically relevant variants were identified in the plasma samples, such as SMAD4 which has been shown to be a prognostic biomarker in OAC. Therapeutically actionable targets not detected in the primary tumour were also captured, including ALK and PIK3CA. Moreover, minimal residual disease was detected after surgery in the majority of patients that went on to relapse. Conclusions These data suggest that ctDNA can be routinely detected in OAC and that clinically relevant alterations can also be identified at late stage of disease, which is when the majority of patients present. Overall our results indicate that there is potential for using targeted sequencing of ctDNA in the clinic. Legal entity responsible for the study The authors. Funding Educational grant from Roche for the library prep kits. Disclosure All authors have declared no conflicts of interest.