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The incidence of esophageal adenocarcinoma (EAC) is rapidly rising and has a 5-year survival rate of20%. 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).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 7082× (range 2196-28 524) and ctDNA results correlated with survival.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.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.

Barrett's oesophagus with indefinite for dysplasia (BE-IND) is a subjective diagnosis with a low interobserver agreement (IOA) among pathologists and uncertain clinical implications. This study aimed to assess the utility of p53 immunohistochemistry (p53-IHC) in assessing BE-IND specimens.Archive endoscopic biopsies with a BE-IND diagnosis from two academic centres were analysed. First, haematoxylin and eosin-stained slides (HE) were reviewed by four expert gastrointestinal (GI) pathologists allocated into two groups (A and B). After a washout period of at least 8 weeks, HE slides were reassessed side-to-side with p53-IHC available. We compared the rate of changed diagnosis and the IOA for all BE grades before and after p53-IHC. We included 216 BE-IND specimens from 185 patients, 44.0 and 32.9% of which were confirmed after HE slide revision by groups A and B, respectively. More than half the cases were reclassified to a non-dysplastic BE (NDBE), while 5.6% of cases in group A and 7.4% in group B were reclassified to definite dysplasia. The IOA for NDBE, BE-IND, low-grade dysplasia (LGD) and high-grade dysplasia (HGD)/intramucosal cancer (IMC) was 0.31, 0.21, -0.03 and -0.02, respectively. Use of p53-IHC led to a40% reduction in BE-IND diagnoses (P 0.001) and increased IOA for all BE grades [κ = 0.46 (NDBE), 0.26 (BE-IND), 0.49 (LGD), 0.35 (HGD/IMC)]. An aberrant p53-IHC pattern significantly increased the likelihood of reclassifying BE-IND to definite dysplasia (odds ratio = 44.3, 95% confidence interval = 18.8-113.0).P53-IHC reduces the rate of BE-IND diagnoses and improves the IOA among pathologists when reporting BE with equivocal epithelial changes.

Background Barrett’s oesophagus (BE) is the main risk factor for the development of oesophageal adenocarcinoma (OAC), yet few patients ever go on to progress to cancer. The acquisition of events during the metaplasia-dysplasia-cancer sequence is poorly characterised. We present a large, unbiased, multi-omics analysis of a cross-sectional cohort of pre-cancer samples, with the aim of providing a comprehensive insight into the diversity and molecular changes driving the disease to cancer. Methods We generated and integrated the genomic (50x), transcriptomic and epigenomic (850K EPIC array) landscapes of snap-frozen endoscopic biopsies from 146 patients with a range of outcomes (27 long-standing non-dysplastic; 12 prior to progression to dysplasia; 14 low-grade; 25 high-grade; 21 intramucosal carcinoma; 47 cases of BE taken adjacent to OAC) and 642 person years of follow-up. All biopsies were reviewed independently by 3 pathologists and had associated annotation with detailed clinical information. Results The total number of structural variants (SV) captured the most variance between samples. Complex SVs and LINE-1 retrotransposon activity were observed even before dysplasia had developed and increased with progression. Increasing SV burden was associated with chromothripsis (12%, 18/146) and breakage-fusion bridges (BFBs; 8%, 13/146). In more than 50% of these, the BFBs were in chromosome 17, harbouring the oncogenes ERBB2 and CDK12, for which expression was significantly higher. With progression there was increased expression of genes related to cell-cycle checkpoint, DNA repair and chromosomal instability, and the epigenetic silencing of genes in WNT-signalling and cell-cycle pathways. Conclusions Genomic complexity occurs very early in the natural history of BE and increasing genomic instability appears to tip the balance towards cancer. This may inform the potential for progression to cancer beyond the clinically discernible phenotype. Efforts to better understand the triggers for chromosomal breakages and rearrangements that underly progression will aid clinical prediction and prevention strategies.

Dysplasia in Barrett's esophagus often is invisible on high-resolution white-light endoscopy (HRWLE). We compared the diagnostic accuracy for inconspicuous dysplasia of the combination of autofluorescence imaging (AFI)-guided probe-based confocal laser endomicroscopy (pCLE) and molecular biomarkers vs HRWLE with Seattle protocol biopsies.Barrett's esophagus patients with no dysplastic lesions were block-randomized to standard endoscopy (HRWLE with the Seattle protocol) or AFI-guided pCLE with targeted biopsies for molecular biomarkers (p53 and cyclin A by immunohistochemistry; aneuploidy by image cytometry), with crossover to the other arm after 6 to 12 weeks. The primary end point was the histologic diagnosis from all study biopsies (trial histology). A sensitivity analysis was performed for overall histology, which included diagnoses within 12 months from the first study endoscopy. Endoscopists were blinded to the referral endoscopy and histology results. The primary outcome was diagnostic accuracy for dysplasia by real-time pCLE vs HRWLE biopsies.Of 154 patients recruited, 134 completed both arms. In the primary outcome analysis (trial histology analysis), AFI-guided pCLE had similar sensitivity for dysplasia compared with standard endoscopy (74.3%; 95% CI, 56.7-87.5 vs 80.0%; 95% CI, 63.1-91.6; P = .48). Multivariate logistic regression showed pCLE optical dysplasia, aberrant p53, and aneuploidy had the strongest correlation with dysplasia (secondary outcome). This 3-biomarker panel had higher sensitivity for any grade of dysplasia than the Seattle protocol (81.5% vs 51.9%; Plt; .001) in the overall histology analysis, but not in the trial histology analysis (91.4% vs 80.0%; P = .16), with an area under the receiver operating curve of 0.83.Seattle protocol biopsies miss dysplasia in approximately half of patients with inconspicuous neoplasia. AFI-guided pCLE has similar accuracy to the current gold standard. The addition of molecular biomarkers could improve diagnostic accuracy.

Funder: Biomedical Research Centre; Id: http://dx.doi.org/10.13039/100014461, AIMS: Barrett's oesophagus with indefinite for dysplasia (BE-IND) is a subjective diagnosis with a low interobserver agreement (IOA) among pathologists and uncertain clinical implications. This study aimed to assess the utility of p53 immunohistochemistry (p53-IHC) in assessing BE-IND specimens. METHODS AND RESULTS: Archive endoscopic biopsies with a BE-IND diagnosis from two academic centres were analysed. First, haematoxylin and eosin-stained slides (H&E) were reviewed by four expert gastrointestinal (GI) pathologists allocated into two groups (A and B). After a washout period of at least 8 weeks, H&E slides were reassessed side-to-side with p53-IHC available. We compared the rate of changed diagnosis and the IOA for all BE grades before and after p53-IHC. We included 216 BE-IND specimens from 185 patients, 44.0 and 32.9% of which were confirmed after H&E slide revision by groups A and B, respectively. More than half the cases were reclassified to a non-dysplastic BE (NDBE), while 5.6% of cases in group A and 7.4% in group B were reclassified to definite dysplasia. The IOA for NDBE, BE-IND, low-grade dysplasia (LGD) and high-grade dysplasia (HGD)/intramucosal cancer (IMC) was 0.31, 0.21, -0.03 and -0.02, respectively. Use of p53-IHC led to a >40% reduction in BE-IND diagnoses (P < 0.001) and increased IOA for all BE grades [κ = 0.46 (NDBE), 0.26 (BE-IND), 0.49 (LGD), 0.35 (HGD/IMC)]. An aberrant p53-IHC pattern significantly increased the likelihood of reclassifying BE-IND to definite dysplasia (odds ratio = 44.3, 95% confidence interval = 18.8-113.0). CONCLUSION: P53-IHC reduces the rate of BE-IND diagnoses and improves the IOA among pathologists when reporting BE with equivocal epithelial changes.

Recent studies show that aneuploidy and driver gene mutations precede cancer diagnosis by many years1–4. We assess whether these genomic signals can be used for early detection and pre-emptive cancer treatment using the neoplastic precursor lesion Barrett’s esophagus as an exemplar5. Shallow whole-genome sequencing of 777 biopsies, sampled from 88 patients in Barrett’s esophagus surveillance over a period of up to 15 years, shows that genomic signals can distinguish progressive from stable disease even 10 years before histopathological transformation. These findings are validated on two independent cohorts of 76 and 248 patients. These methods are low-cost and applicable to standard clinical biopsy samples. Compared with current management guidelines based on histopathology and clinical presentation, genomic classification enables earlier treatment for high-risk patients as well as reduction of unnecessary treatment and monitoring for patients who are unlikely to develop cancer. Longitudinal molecular profiling of copy number alterations in patients with Barrett’s esophagus can identify patients at higher risk of developing esophageal cancer.

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.

Recent studies show that aneuploidy and driver gene mutations precede cancer diagnosis by many years

SummaryCancer arises through a process of somatic evolution and recent studies have shown that aneuploidies and driver gene mutations precede cancer diagnosis by several years to decades1–4 Here, we address the question whether such genomic signals can be used for early detection and pre-emptive cancer treatment. To this end we study Barrett’s oesophagus, a genomic copy number driven neoplastic precursor lesion to oesophageal adenocarcinoma5. We use shallow whole genome sequencing of 777 biopsies sampled from 88 patients in surveillance for Barrett’s oesophagus over a period of up to 15 years. These data show that genomic signals exist that distinguish progressive from stable disease with an AUC of 0.87 and a sensitivity of 50% even ten years prior to histopathological disease transformation. These finding are validated on two independent cohorts of 75 and 248 patients. Compared against current patient management guidelines genomic risk classification enables earlier treatment for high risk patients as well as reduction of unnecessary treatment and monitoring for patients who are unlikely to develop cancer.

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.