A global view of the oncogenic landscape in nasopharyngeal carcinoma : an integrated analysis at the genetic and expression levels

Previous studies have reported that the tumour cells of nasopharyngeal carcinoma (NPC) exhibit recurrent chromosome abnormalities. These genetic changes are broadly assumed to lead to changes in gene expression which are important for the pathogenesis of this tumour. However, this assumption has yet to be formally tested at a global level. Therefore a genome wide analysis of chromosome copy number and gene expression was performed in tumour cells micro-dissected from the same NPC biopsies. Cellular tumour suppressor and tumour-promoting genes (TSG, TPG) and Epstein-Barr Virus (EBV)-encoded oncogenes were examined. The EBV-encoded genome maintenance protein EBNA1, along with the putative oncogenes LMP1, LMP2 and BARF1 were expressed in the majority of NPCs that were analysed. Significant downregulation of expression in an average of 76 cellular TSGs per tumour was found, whilst a per-tumour average of 88 significantly upregulated, TPGs occurred. The expression of around 60% of putative TPGs and TSGs was both up-and down-regulated in different types of cancer, suggesting that the simplistic classification of genes as TSGs or TPGs may not be entirely appropriate and that the concept of context-dependent onco-suppressors may be more extensive than previously recognised. No significant enrichment of TPGs within regions of frequent genomic gain was seen but TSGs were significantly enriched within regions of frequent genomic loss. It is suggested that loss of the FHIT gene may be a driver of NPC tumourigenesis. Notwithstanding the association of TSGs with regions of genomic loss, on a gene by gene basis and excepting homozygous deletions and high-level amplification, there is very little correlation between chromosomal copy number aberrations and expression levels of TSGs and TPGs in NPC

Samples and their properties.

<p>The names and properties of the samples are indicated, together with their EBV genome status (EBV DNA) and status of expression of the EBV genes EBNA1, BARF1, LMP1 and LMP2. Both expression array data and SNP array data were obtained from the first 13 biopsies and cell line C666-1. “SNP array only” indicates that only SNP array data were obtained from these biopsies whilst “Expression array only” signifies that only expression array data were obtained. U  =  unknown; ND  =  not determined.</p

<i>A</i><i> priori</i> defined, putative tumour promoting genes upregulated more than twofold in at least 12 (75%) samples.

<p>↓  =  downregulated, ↑  =  upregulated, NC  =  no change. Further information can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041055#pone.0041055.s002" target="_blank">Table S1</a>.</p

Chromosomal copy number changes.

<p>Panel A shows the major regions of copy number gain (red) or loss (blue) across the genome. The Y axis shows the number of cases (out of 16) at which a region was changed. Chromosomes are ordered from left to right as indicated. Panels B–D show traces of the log2 ratio of the copy number of DNA from the tumour samples compared to the normal controls. B: the homozygous deletion at the FHIT locus in C666-1; C: a hemizygous deletion in C666-1 and a homozygous deletion in tumour XY5, both at 6q22.33; D: homozygous deletions encompassing the CDKN2B locus in tumours MMAH, XY5, XY8 and HKC1. Tumour MMAH also shows a 600 Kb homozygous deletion at 9p24.1 containing the NFIB gene. The sizes of the discrete aberrations are indicated.</p

The 8p11.21 amplified region of tumour HKD1.

<p>A. Trace of the log2 ratio of the copy number of DNA from the tumour sample compared to the normal controls. The 2.5 Mb amplification is indicated. B. Heat map of the relative expression levels of the genes found within the amplified region. The samples appear in columns and the individual genes within the amplified region form the rows. High level expression is represented by the intensity of red and low level by blue. The brackets at the bottom indicate tumour samples with genome copy numbers of 2 or 3 within this region.</p

<i>A priori</i> defined, putative tumour suppressor genes downregulated more than twofold in at least 12 (75%) samples.

<p>↓  =  downregulated, ↑  =  upregulated, NC  =  no change. Further information can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041055#pone.0041055.s003" target="_blank">Table S2</a>.</p

Immunohistochemical validation of differential regulation.

<p>Panels A–F show normal epithelium on the left and tumour tissue on the right. Panel A uses frozen sections from the same samples that were used in the array analysis (MHAU; normal epithelium: XY23; NPC), Panels B–F are paired specimens from the NPC tissue array. A–C: the upregulated genes EZH2, SKIL and CD44. D–F: the downregulated genes ANXA1, LCN2 and MSH3. Panel G summarises all the tissue array staining. The Y axis shows the log₂ value of the ratio of the paired tumour:normal IHC scores. Some IHC scores were zero resulting in log₂ ratio values of plus or minus infinity. For convenience, these are represented as 4 or−4 on the figure. Except for JAK and CD44, p values were less then 0.05. Individual p values are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041055#pone.0041055.s002" target="_blank">Tables S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041055#pone.0041055.s003" target="_blank">S2</a>.</p

Evaluation of a quality improvement intervention to reduce anastomotic leak following right colectomy (EAGLE): pragmatic, batched stepped-wedge, cluster-randomized trial in 64 countries

Background Anastomotic leak affects 8 per cent of patients after right colectomy with a 10-fold increased risk of postoperative death. The EAGLE study aimed to develop and test whether an international, standardized quality improvement intervention could reduce anastomotic leaks. Methods The internationally intended protocol, iteratively co-developed by a multistage Delphi process, comprised an online educational module introducing risk stratification, an intraoperative checklist, and harmonized surgical techniques. Clusters (hospital teams) were randomized to one of three arms with varied sequences of intervention/data collection by a derived stepped-wedge batch design (at least 18 hospital teams per batch). Patients were blinded to the study allocation. Low- and middle-income country enrolment was encouraged. The primary outcome (assessed by intention to treat) was anastomotic leak rate, and subgroup analyses by module completion (at least 80 per cent of surgeons, high engagement; less than 50 per cent, low engagement) were preplanned. Results A total 355 hospital teams registered, with 332 from 64 countries (39.2 per cent low and middle income) included in the final analysis. The online modules were completed by half of the surgeons (2143 of 4411). The primary analysis included 3039 of the 3268 patients recruited (206 patients had no anastomosis and 23 were lost to follow-up), with anastomotic leaks arising before and after the intervention in 10.1 and 9.6 per cent respectively (adjusted OR 0.87, 95 per cent c.i. 0.59 to 1.30; P = 0.498). The proportion of surgeons completing the educational modules was an influence: the leak rate decreased from 12.2 per cent (61 of 500) before intervention to 5.1 per cent (24 of 473) after intervention in high-engagement centres (adjusted OR 0.36, 0.20 to 0.64; P &lt; 0.001), but this was not observed in low-engagement hospitals (8.3 per cent (59 of 714) and 13.8 per cent (61 of 443) respectively; adjusted OR 2.09, 1.31 to 3.31). Conclusion Completion of globally available digital training by engaged teams can alter anastomotic leak rates. Registration number: NCT04270721 (http://www.clinicaltrials.gov)