Cancer genome sequencing studies have identified numerous driver genes but the relative timing of mutations in carcinogenesis remains unclear. The gradual progression from pre-malignant Barrett’s esophagus to esophageal adenocarcinoma (EAC) provides an ideal model to study the ordering of somatic mutations. We identified recurrently-mutated genes and assessed clonal structure using whole-genome sequencing and amplicon-resequencing of 112 EACs. We next screened a cohort of 109 biopsies from two key transition points in the development of malignancy; benign metaplastic never-dysplastic Barrett’s esophagus (NDBE, n=66), and high-grade dysplasia (HGD, n=43). Unexpectedly, the majority of recurrently mutated genes in EAC were also mutated in NDBE. Only TP53 and SMAD4 were stage-specific, confined to HGD and EAC, respectively. Finally, we applied this knowledge to identify high-risk Barrett’s esophagus in a novel non-endoscopic test. In conclusion, mutations in EAC driver genes generally occur exceptionally early in disease development with profound implications for diagnostic and therapeutic strategies.
The molecular genetic relationship between esophageal adenocarcinoma (EAC) and its precursor lesion, Barrett’s esophagus, is poorly understood. Using whole-genome sequencing on 23 paired Barrett’s esophagus and EAC samples, together with one in-depth Barrett’s esophagus case-study sampled over time and space, we have provided new insights on the following aspects: i) Barrett’s esophagus is polyclonal and highly mutated even in the absence of dysplasia; ii) when cancer develops, copy number increases and heterogeneity persists such that the spectrum of mutations often shows surprisingly little overlap between EAC and adjacent Barrett’s esophagus; and iii) despite differences in specific coding mutations the mutational context suggests a common causative insult underlying these two conditions. From a clinical perspective, the histopathological assessment of dysplasia appears to be a poor reflection of the molecular disarray within the Barrett’s epithelium and a molecular Cytosponge™ technique overcomes sampling bias and has capacity to reflect the entire clonal architecture.
Background: The incidence of esophageal adenocarcinoma (EAC) is rapidly rising and has a 5-year survival rate of <20%. Beyond TNM (tumorenodeemetastasis) 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 7082Â (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% cNþ. 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 ¼ 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.
We have previously identified a region containing 16 CpGs within the MGMT CpG islands which is critical for the transcriptional control of MGMT (Malley, Acta Neuropathol 2011). To investigate the patterns and incidence of MGMT methylation in astrocytic and oligodendroglial tumors, we quantitatively assessed methylation at these 16 CpGs using bisulfite modification followed by pyrosequencing of 362 gliomas not treated with temozolomide, and correlated the findings with previously identified patterns of genetic abnormalities, patients' age and survival. The MGMT gene was considered to be methylated when the mean methylation of the 16 CpGs was 10% or higher. This cut-off value distinguished diffuse astrocytomas with high and low MGMT expression. Within each tumor type, the patterns of methylation were highly variable and also highly heterogeneous across the 16 CpGs. A high incidence of MGMT methylation was observed in all subtypes of gliomas included in this study. Among a subset of 97 tumors where conventional methylation-specific PCR (MSP) was also applied, methylation was detected by both methods in 54 tumors, while the pyrosequencing results identified a further 17 tumors. No additional cases were found using MSP alone, indicating that pyrosequencing is a robust method for methylation analysis. All tumors with IDH1/IDH2 mutations except two had MGMT methylation, while there were many tumors with MGMT methylation, particularly primary glioblastomas, which had no mutations of IDH1/2. We suggest that MGMT methylation may be one of the earliest events in the development of astrocytic and oligodendroglial tumors.Gliomas are the most common group of primary brain tumors consisting of clinically and biologically distinct subtypes. The three main subgroups of glioma are astrocytic, oligodendroglial and mixed gliomas, which are further malignancy graded into up to four grades according to the WHO classification. 1 Glioblastomas (WHO grade IV) are the most malignant and common astrocytic brain tumor in adults, the overall median survival being only just over 14 months despite modern combined therapies. 2 Most glioblastomas are primary glioblastoma (pGB), while secondary glioblastoma (sGB) generally develops by progression from astrocytomas WHO grade II or III. Patients with astrocytic or oligodendroglial tumors WHO grade II and III survive longer, however they always recur and are ultimately fatal.Among the many genetic and epigenetic abnormalities commonly found in adult gliomas, three have been reported to predict response to treatment and/or prognosis: IDH1/ IDH2 mutations, concurrent deletion of the entire 1p and 19q chromosomal arms (total 1p/19q loss) and MGMT methylation. 3,4 With the exception of pGB, mutations of either IDH1 or IDH2 occur in more than 50% of adult astrocytic, oligodendroglial and oligoastrocytic tumors. [5][6][7][8] It has been shown that the presence of IDH1/2 mutations is associated with longer patient survival. 5-7,9,10 IDH1/2 mutations have been proposed as the earliest genetic changes in a ...
Data AvailabilitySequencing data that support the findings of this paper have been deposited in the European Genome-phenome Archive with the accession code EGAD00001005434. Code AvailabilityAll code required to reproduce the analysis outline in this manuscript can be found in the main and supplementary methods. There are no restrictions to the accessibility of this code. Author ContributionsAN designed and implemented the rapid autopsy study, collected the samples, performed the experiments, analyzed data and wrote the manuscript. MG and S.D.P contributed expertise in pathology and sample collection for the rapid autopsy study. ID-B and NG assisted in study implementation, and along with JC, assisted with sample collection at autopsy. M.S performed the structural variant analysis. M.D.E performed genomic data generation and QC. LB conducted data management. XL, PL-S and JW were involved with autopsy sample collection, advice on experiments and data analysis, and XL contributed to experiments, paper writing, and figure design. LA and IM assisted with data analysis. NG assisted with study Implementation. SMac coordinated the sequencing of samples from the OCCAMS project and contributed to paper writing. SM and AM provided pathology data. TT, SG, LP and DG assisted in implementation and ethical conduct of the autopsy study. R.H.H and AH were involved in surgical sample collection and providing surgical expertise. M.R.S contributed to critical evaluation of the study data and manuscript. D.C.W was responsible for data analysis, paper writing, and assuring integrity of data. The OCCAMS consortium was the vehicle through which the infrastructure and funding was obtained to support the study and the consortium contributed to discussions on the ICGC data and the clinical ramifications. R.C.F provided grant funding and was responsible for study design, supervision of the project, writing the paper and assuring integrity of the data.
The scientific community has avoided using tissue samples from patients that have been exposed to systemic chemotherapy to infer the genomic landscape of a given cancer. Esophageal adenocarcinoma is a heterogeneous, chemoresistant tumor for which the availability and size of pretreatment endoscopic samples are limiting. This study compares whole-genome sequencing data obtained from chemo-naive and chemo-treated samples. The quality of whole-genomic sequencing data is comparable across all samples regardless of chemotherapy status. Inclusion of samples collected post-chemotherapy increased the proportion of late-stage tumors. When comparing matched pre-and post-chemotherapy samples from 10 cases, the mutational signatures, copy number, and SNV mutational profiles reflect the expected heterogeneity in this disease. Analysis of SNVs in relation to allele-specific copy-number changes pinpoints the common ancestor to a point prior to chemotherapy. For cases in which pre-and post-chemotherapy samples do show substantial differences, the timing of the divergence is near-synchronous with endoreduplication. Comparison across a large prospective cohort (62 treatment-naive, 58 chemotherapy-treated samples) reveals no significant differences in the overall mutation rate, mutation signatures, specific recurrent point mutations, or copy-number events in respect to chemotherapy status. In conclusion, whole-genome sequencing of samples obtained following neoadjuvant chemotherapy is representative of the genomic landscape of esophageal adenocarcinoma. Excluding these samples reduces the material available for cataloging and introduces a bias toward the earlier stages of cancer.
Cancer cachexia is common in patients with oesophagogastric cancer (OG) and is linked to overall survival (OS). One of the key components of cachexia is anorexia; it is not known whether anorexia impacts on OS and there is no method of routine screening in current practice. Diagnosis relies on patients describing the symptoms, clinicians diagnosing anorexia and acting upon it. Patients with oesophageal/gastroesophageal junction or gastric cancer were assessed using the Functional Assessment of Anorexia Cachexia Therapy Anorexia/Cachexia Subscale (FAACT A/CS). FAACT A/CS includes 12 questions validated previously to diagnose anorexia in patients with cancer. Of the 182 patients included, 69% scored ≤37/48 and were considered to be anorexic; FAACT A/CS was a better predictor of OS in metastatic patients than body mass index or weight loss in the six months prior to cancer diagnosis. The median OS of patients with FAACT A/CS scores of >37 was longer than patients with scores of ≤37 (19.3 months vs 6.7 months, Hazard Ratio [HR] 2.9, 95% Confidence Interval [CI] 1.4–6.0, p<0.0001). Patients with performance status (PS) 0–2 and FAACT A/CS >37 had substantially longer OS than those with PS 0–2 and FAACT A/CS ≤37 (18.7 months vs 7.9 months, HR 2.5 (95% CI 1.2–5.1, P<0.0001). The FAACT A/CS questionnaire allows clinicians to identify patients with anorexia who may benefit from early nutrition interventions. Importantly, this is the first study to show the association between anorexia and survival in patients with metastatic OG cancers. This will form the basis of future interventional studies to improve patient outcomes.
BackgroundMobile elements are active in the human genome, both in the germline and cancers, where they can mutate driver genes.ResultsWhile analysing whole genome paired-end sequencing of oesophageal adenocarcinomas to find genomic rearrangements, we identified three ways in which new mobile element insertions appear in the data, resembling translocation or insertion junctions: inserts where unique sequence has been transduced by an L1 (Long interspersed element 1) mobile element; novel inserts that are confidently, but often incorrectly, mapped by alignment software to L1s or polyA tracts in the reference sequence; and a combination of these two ways, where different sequences within one insert are mapped to different loci. We identified nine unique sequences that were transduced by neighbouring L1s, both L1s in the reference genome and L1s not present in the reference. Many of the resulting inserts were small fragments that include little or no recognisable mobile element sequence. We found 6 loci in the reference genome to which sequence reads from inserts were frequently mapped, probably erroneously, by alignment software: these were either L1 sequence or particularly long polyA runs. Inserts identified from such apparent rearrangement junctions averaged 16 inserts/tumour, range 0–153 insertions in 43 tumours. However, many inserts would not be detected by mapping the sequences to the reference genome, because they do not include sufficient mappable sequence. To estimate total somatic inserts we searched for polyA sequences that were not present in the matched normal or other normals from the same tumour batch, and were not associated with known polymorphisms. Samples of these candidate inserts were verified by sequencing across them or manual inspection of surrounding reads: at least 85 % were somatic and resembled L1-mediated events, most including L1Hs sequence. Approximately 100 such inserts were detected per tumour on average (range zero to approximately 700).ConclusionsSomatic mobile elements insertions are abundant in these tumours, with over 75 % of cases having a number of novel inserts detected. The inserts create a variety of problems for the interpretation of paired-end sequencing data.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1685-z) contains supplementary material, which is available to authorized users.
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