Maria Secrier

Mutational signatures in esophageal adenocarcinoma define etiologically distinct subgroups with therapeutic relevance

Esophageal adenocarcinoma (EAC) has a poor outcome, and targeted therapy trials have thus far been disappointing owing to a lack of robust stratification methods. Whole-genome sequencing (WGS) analysis of 129 cases demonstrated that this is a heterogeneous cancer dominated by copy number alterations with frequent large-scale rearrangements. Co-amplification of receptor tyrosine kinases (RTKs) and/or downstream mitogenic activation is almost ubiquitous; thus tailored combination RTK inhibitor (RTKi) therapy might be required, as we demonstrate in vitro. However, mutational signatures showed three distinct molecular subtypes with potential therapeutic relevance, which we verified in an independent cohort (n = 87): (i) enrichment for BRCA signature with prevalent defects in the homologous recombination pathway; (ii) dominant T>G mutational pattern associated with a high mutational load and neoantigen burden; and (iii) C>A/T mutational pattern with evidence of an aging imprint. These subtypes could be ascertained using a clinically applicable sequencing strategy (low coverage) as a basis for therapy selection.

Molecular effects of Lapatinib in the treatment of HER2 overexpressing oesophago-gastric adenocarcinoma

Background:Lapatinib, a dual EGFR and HER2 inhibitor has shown disappointing results in clinical trials of metastatic oesophago-gastric adenocarcinomas (OGAs), and in vitro studies suggest that MET, IGFR, and HER3 confer resistance. This trial applied Lapatinib in the curative neoadjuvant setting and investigated the feasibility and utility of additional endoscopy and biopsy for assessment of resistance mechanisms ex vivo and in vivo.Methods:Patients with HER2 overexpressing OGA were treated for 10 days with Lapatinib monotherapy, and then in combination with three cycles of Oxaliplatin and Capecitabine before surgery. Endoscopic samples were taken for molecular analysis at: baseline including for ex vivo culture +/− Lapatinib to predict in vivo response, post-Lapatinib monotherapy and at surgery. Immunohistochemistry (IHC) and proteomic analysis was performed to assess cell kinetics and signalling activity.Results:The trial closed early (n=10) due to an anastomotic leak in two patients for which a causative effect of Lapatinib could not be excluded. The reduction in Phosphorylated-HER2 (P-HER2) and P-EGFR in the ex vivo-treated biopsy demonstrated good correlation with the in vivo response at day 10. Proteomic analysis pre and post-Lapatinib demonstrated target inhibition (P-ERBB2, P-EGFR, P-PI3K, P-AKT, and P-ERK) that persisted until surgery. There was also significant correlation between the activation of MET with the level of P-Erk (P=0.0005) and P-PI3K : T-PI3K (total PI3K) ratio (P=0.0037). There was no significant correlation between the activation status of IGFR and HER3 with downstream signalling molecules.Conclusions:Additional endoscopy and biopsy sampling for multiple biomarker endpoints was feasible and confirmed in vitro data that MET is likely to be a significant mechanism of Lapatinib resistance in vivo.

A comparative analysis of whole genome sequencing of esophageal adenocarcinoma pre- and post-chemotherapy

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.

Whole-genome sequencing of nine esophageal adenocarcinoma cell lines.

Esophageal adenocarcinoma (EAC) is highly mutated and molecularly heterogeneous. The number of cell lines available for study is limited and their genome has been only partially characterized. The availability of an accurate annotation of their mutational landscape is crucial for accurate experimental design and correct interpretation of genotype-phenotype findings. We performed high coverage, paired end whole genome sequencing on eight EAC cell lines—ESO26, ESO51, FLO-1, JH-EsoAd1, OACM5.1 C, OACP4 C, OE33, SK-GT-4—all verified against original patient material, and one esophageal high grade dysplasia cell line, CP-D. We have made available the aligned sequence data and report single nucleotide variants (SNVs), small insertions and deletions (indels), and copy number alterations, identified by comparison with the human reference genome and known single nucleotide polymorphisms (SNPs). We compare these putative mutations to mutations found in primary tissue EAC samples, to inform the use of these cell lines as a model of EAC.

Whole-genome sequencing analysis of structural variants in oesophageal adenocarcinoma

Abstract Background The incidence of oesophageal adenocarcinoma is increasing substantially. Sequencing studies have highlighted that this tumour is highly heterogeneous, with few recurrent point-mutations. Structural variants (SVs) are large mutations that can be inferred from whole-genome sequencing data and are emerging as a major component of the mutational landscape of this cancer. The aim of this study was to carry out a detailed characterisation of SVs in oesophageal cancer. Methods Snap-frozen endoscopic biopsy samples and surgical specimens were collected from 289 patients with oesophageal cancer in a nationwide collaborative effort (Oesophageal Cancer Clinical and Molecular Stratification Collaboration). A single library was created for each sample, and paired-end sequencing was performed (Illumina, San Diego, CA, USA) to a typical depth of at least 50 ×. Structural variants were detected by an in-house clustering tool that clusters discordant and split reads (v0.55). Only somatic structural variants with four supporting reads were used in the analysis. Findings We identified 48 different genes that were hit by SVs in more than 29 patients (>10%) and as many as 273 hits in more than 15 (5%). PCR verification confirmed the presence of the predicted SVs in more than 90% of cases in a subset of 98 analysed rearrangements. By contrast, in this cancer only seven genes have been reported as recurrently mutated by point-mutations in over 10% of cases, making structural variation the most common mechanism for recurrent mutations of oesophageal adenocarcinoma. Among the recurrently mutated genes, we found significant enrichment for two main pathways: cell–cell communication and cell–extracellular matrix organisation (p −7 ) and ErbB signalling/FGFR (p −3 ). In particular, we identified a subset of 20 genes including master regulator of desmosome ( JUP ), integrins ( PTK2 ), and catenins ( CTNNA3 and CTNNA3 ). We also identified SVs in CTNNA3 as a prognostic marker of poor survival in our cohort (p=0·003). Interpretation SVs are a common mechanism for recurrent mutations in oesophageal adenocarcinoma. Genes recurrently mutated by SVs point to a small number of pathways that are potentially relevant to disease progression and prognosis. Funding National Institute for Health Research, Cancer Research UK.

Erratum: Corrigendum: Mutational signatures in esophageal adenocarcinoma define etiologically distinct subgroups with therapeutic relevance

Nat. Genet.; 10.1038/ng.3659; corrected online 19 September 2016 In the version of this article initially published online, the mutation signature illustrations for S1 and S2 in Figure 3a were switched. Additionally, in the Online Methods, the text originally stated that structural variants were called using BWA-MEM, when it should have stated that these were called using BWA.

Corrigendum: A comparative analysis of whole genome sequencing of esophageal adenocarcinoma pre- and post-chemotherapy

Corrigendum: A comparative analysis of whole genome sequencing of esophageal adenocarcinoma preand post-chemotherapy Ayesha Noorani, Jan Bornschein, Andy G. Lynch, Maria Secrier, Achilleas Achilleos, Matthew Eldridge, Lawrence Bower, Jamie M.J. Weaver, Jason Crawte, Chin-Ann Ong, Nicholas Shannon, Shona MacRae, Nicola Grehan, Barbara Nutzinger, Maria O’Donovan, Richard Hardwick, Simon Tavaré, Rebecca C. Fitzgerald, on behalf of the Oesophageal Cancer Clinical and Molecular Stratification (OCCAMS) Consortium