Anna Paterson

Integrative analysis of array comparative genomic hybridisation and matched gene expression profiling data reveals novel genes with prognostic significance in oesophageal adenocarcinoma

Background and aims The incidence of oesophageal adenocarcinoma (OAC) has been increasing rapidly with a dismal survival rate of less than 20%. Understanding the genomic aberrations and biology of this cancer may enhance disease interventions. This study aimed to use genome-wide genomic and expression data to enhance the understanding of OAC pathogenesis and identify groups with differential outcomes. Methods Array-comparative genomic hybridisation (aCGH) analysis was carried out on 56 fresh frozen OAC resection samples with long-term clinical follow-up data. Samples with aberrations were further analysed with whole-genome single-nucleotide polymorphism arrays to confirm aCGH findings. Matched gene expression microarray data were used to identify genes with high copy number–expression correlations. Nested-multiplex PCR on DNA from microdissected specimens and fluorescence in situ hybridisation assays were used for target validation. Immunohistochemistry on the same cohort and independent samples (n=371) was used for subsequent validation. Kaplan–Meier survival analyses were performed based on aCGH data after unsupervised K-means clustering (K=5, 50 iterations) and immunohistochemistry data. Results aCGH identified 17 common regions (>5% samples) of gains and 11 common regions of losses, including novel regions in OAC (loci 11p13 and 21q21.2). Integration of aCGH data with matched gene expression microarray data highlighted genes with high copy number–expression correlations: two deletions (p16/CDKN2A, MBNL1) and four gains (EGFR, WT1, NEIL2, MTMR9). Immunohistochemistry demonstrated protein over-expression of targets with gains: EGFR (10%), WT1 (20%), NEIL2 (14%) and MTMR9 (25%). These targets individually (p<0.060) and in combination had prognostic significance (p=0.008). On the genomic level, K-means clustering identified a cluster (32% of cohort) with differential log2 ratios of 16 CGH probes (p<4×10-7) and a worse prognosis (median survival=1.37 years; p=0.015). Conclusions Integration of aCGH and gene expression data identified copy number aberrations and novel genes with prognostic potential in OAC.

Co-amplification of 8p12 and 11q13 in breast cancers is not the result of a single genomic event.

Epithelial cancers frequently have multiple amplifications, and particular amplicons tend to occur together. These co‐amplifications have been suggested to result from amplification of pre‐existing junctions between two chromosomes, that is, translocation junctions. We investigated this hypothesis for two amplifications frequent in breast cancer, at 8p12 and 11q13, which had been reported to be associated in Southern blot studies. We confirmed that both genomic amplification and expression of genes was correlated between the frequently‐amplified regions of 8p and 11q, in array CGH and microarray expression data, supporting the importance of co‐amplification. We examined by FISH the physical structure of co‐amplifications that we had identified by array CGH, in five breast cancer cell lines (HCC1500, MDA‐MB‐134, MDA‐MB‐175, SUM44, and ZR‐75‐1), four breast tumors, and a pancreatic cancer cell line (SUIT2). We found a variety of arrangements: amplification of translocation junctions; entirely independent amplification of the two regions on separate chromosomes; and separate amplification of 8p and 11q sequences in distinct sites on the same rearranged chromosome. In this last arrangement, interphase nuclei often showed intermingling of FISH signals from 8p12 and 11q13, giving a false impression that the sequences were interdigitated. We conclude that co‐amplification of the main 8p and 11q amplicons in breast tumors is not usually the result of a preceding translocation event but most likely reflects selection of clones that have amplified both loci. This article contains supplementary material available at http://www.interscience.wiley.com/jpages/1045‐2257/suppmat.

A systematic approach to therapeutic target selection in oesophago-gastric cancer

Objective The success of personalised therapy depends on identification and inhibition of the oncogene(s) on which that tumour is dependent. We aimed to determine whether a receptor tyrosine kinase (RTK) array could be used to select the most effective therapeutic strategies in molecularly heterogeneous oesophago-gastric adenocarcinomas. Design Gene expression profiling from oesophago-gastric tumours (n=75) and preinvasive stages (n=57) identified the active signalling pathways, which was confirmed using immunohistochemistry (n=434). RTK arrays on a cell line panel (n=14) determined therapeutic targets for in vitro cytotoxic testing. Feasibility of this personalised approach was tested in tumour samples (n=46). Results MAPK was the most frequently activated pathway (32/75 samples (42.7%)) with progressive enrichment in preinvasive disease stages (p<0.05) and ERK phosphorylation in 148/434 (34.3%) independent samples. Cell lines displayed a range of RTK activation profiles. When no RTKs were activated, tyrosine kinase inhibitors (TKIs) and a Mek inhibitor were not useful (MKN1). In lines with a dominant phosphorylated RTK (OE19, MKN45 and KATOIII), selection of this TKI or Mek in nM concentrations induced cytotoxicity and inhibited Erk and Akt phosphorylation. In cells lines with complex activation profiles (HSC39 and OE33), a combination of TKIs or Mek inhibition (in nM concentrations) was necessary for cytotoxicity and inhibition of Erk and Akt phosphorylation. Human tumours demonstrated diverse activation profiles and 65% of cases had two or more active RTKs. Conclusions The MAPK pathway is commonly activated in oesophago-gastric cancer following activation of a variety of RTKs. Molecular phenotyping can inform a rational choice of targeted therapy.

Characterization of the timing and prevalence of receptor tyrosine kinase expression changes in oesophageal carcinogenesis.

Despite being common in epithelial malignancies, the timing of receptor tyrosine kinase (RTK) up‐regulation is poorly understood and therefore hampers the identification of the receptor to target for effective treatment. We aimed to determine if RTK expression changes were early events in carcinogenesis. Oesophageal adenocarcinoma and its pre‐invasive lesion, Barretts oesophagus, were used for immunohistochemical analysis of the RTK panel, EGFR, ErbB2, ErbB3, Met, and FGFR2, by utilizing a cohort of patients with invasive disease (

Mobile element insertions are frequent in oesophageal adenocarcinomas and can mislead paired-end sequencing analysis.

n = 367

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

) and two cohorts with pre‐invasive disease, one cross‐sectional (

Range of pathologies diagnosed using a minimally invasive capsule sponge to evaluate patients with reflux symptoms

n = 110

A UK feasibility and validation study of the VE1 monoclonal antibody immunohistochemistry stain for BRAF-V600E mutations in metastatic melanoma

) and one longitudinal in time (

Biomarkers in Barrett's oesophagus and oesophageal adenocarcinoma

n = 91

PWE-171 Mobile element insertions are frequent and potentially mutagenic events in oesophageal carcinogenesis

). The results demonstrated that 51% of oesophageal adenocarcinomas overexpressed at least one of the RTK panel, with 21% of these overexpressing multiple receptors. Up‐regulation of RTK expression was an early event corresponding with low‐grade dysplasia development (25% in areas without dysplasia versus 63% in low‐grade dysplasia,