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Differences in gastric carcinoma microenvironment stratify according to EBV infection intensity: implications for possible immune adjuvant therapy.

Strong MJ, Xu G, Coco J, Baribault C, Vinay DS, Lacey MR, Strong AL, Lehman TA, Seddon MB, Lin Z, Concha M, Baddoo M, Ferris M, Swan KF, Sullivan DE, Burow ME, Taylor CM, Flemington EK - PLoS Pathog. (2013)

Bottom Line: Epstein-Barr virus (EBV) is associated with roughly 10% of gastric carcinomas worldwide (EBVaGC).In four samples with the highest EBV coverage (hiEBVaGC - high EBV associated gastric carcinoma), transcripts from the BamHI A region comprised the majority of EBV reads.These results were confirmed in a separate cohort of 21 Vietnamese gastric carcinoma samples using qRT-PCR and on tissue samples using in situ hybridization and immunohistochemistry.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Tulane University, New Orleans, Louisiana, United States of America.

ABSTRACT
Epstein-Barr virus (EBV) is associated with roughly 10% of gastric carcinomas worldwide (EBVaGC). Although previous investigations provide a strong link between EBV and gastric carcinomas, these studies were performed using selected EBV gene probes. Using a cohort of gastric carcinoma RNA-seq data sets from The Cancer Genome Atlas (TCGA), we performed a quantitative and global assessment of EBV gene expression in gastric carcinomas and assessed EBV associated cellular pathway alterations. EBV transcripts were detected in 17% of samples but these samples varied significantly in EBV coverage depth. In four samples with the highest EBV coverage (hiEBVaGC - high EBV associated gastric carcinoma), transcripts from the BamHI A region comprised the majority of EBV reads. Expression of LMP2, and to a lesser extent, LMP1 were also observed as was evidence of abortive lytic replication. Analysis of cellular gene expression indicated significant immune cell infiltration and a predominant IFNG response in samples expressing high levels of EBV transcripts relative to samples expressing low or no EBV transcripts. Despite the apparent immune cell infiltration, high levels of the cytotoxic T-cell (CTL) and natural killer (NK) cell inhibitor, IDO1, was observed in the hiEBVaGCs samples suggesting an active tolerance inducing pathway in this subgroup. These results were confirmed in a separate cohort of 21 Vietnamese gastric carcinoma samples using qRT-PCR and on tissue samples using in situ hybridization and immunohistochemistry. Lastly, a panel of tumor suppressors and candidate oncogenes were expressed at lower levels in hiEBVaGC versus EBV-low and EBV-negative gastric cancers suggesting the direct regulation of tumor pathways by EBV.

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Detection of EBV in gastric carcinoma samples.Four gigabytes of deduplicated RNA-seq read data from each of the seventy-one gastric carcinoma samples were analyzed using RNA CoMPASS. The virome branch of the taxonomy trees for the four samples with the highest number of EBV reads (A) and two EBV-negative samples (B) were generated using the metagenome analysis tool, MEGAN 4. (C) For a more in depth analysis of EBV reads, the combined sequence read files for each sample were aligned to the EBV genome and the hg19 human genome assembly using the genome aligner, Novoalign. Of the EBV-positive samples, four samples were identified as having high numbers of EBV reads while eight were found to have low but detectable numbers of EBV reads (see Figure S2 for plot of EBV reads per 1,000,000 human mapped reads).
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ppat-1003341-g001: Detection of EBV in gastric carcinoma samples.Four gigabytes of deduplicated RNA-seq read data from each of the seventy-one gastric carcinoma samples were analyzed using RNA CoMPASS. The virome branch of the taxonomy trees for the four samples with the highest number of EBV reads (A) and two EBV-negative samples (B) were generated using the metagenome analysis tool, MEGAN 4. (C) For a more in depth analysis of EBV reads, the combined sequence read files for each sample were aligned to the EBV genome and the hg19 human genome assembly using the genome aligner, Novoalign. Of the EBV-positive samples, four samples were identified as having high numbers of EBV reads while eight were found to have low but detectable numbers of EBV reads (see Figure S2 for plot of EBV reads per 1,000,000 human mapped reads).

Mentions: RNA-seq data from The Cancer Genome Atlas (TCGA) gastric adenocarcinoma cohort (SRA035410) was first analyzed using RNA CoMPASS (Figure S1 and Xu et al., unpublished) to assess the virome for each of the 71 data sets. This initial screening was performed using a single lane of sequencing data from each patient. Most samples contained relatively low numbers of reads matching non-human viral sources (e.g. enterobacteria phage T4T) that possibly represent environmental contamination (Figure 1A–B). Of the known human viruses detected, one sample (BR-4298, Figure 1A) contained 6 reads attributed to Hepatitis C virus. Further inspection of these reads showed high homology to the human immunoglobulin light chain variable region (Table S1). These reads likely represent human sequences rather than reads derived from Hepatitis C virus. Twelve samples showed evidence of human cytomegalovirus (HCMV) with read numbers ranging from 5 to 132. Individual BLASTing of selected HCMV reads showed high homology to HCMV genomes but not to human sequences indicating that these are bona fide HCMV derived reads. The relatively low numbers of HCMV reads in these samples (relative to the numbers of EBV in some samples, see below) suggests that these reads are derived from a low number of HCMV infected cells or that the virus is not expressing substantial numbers of polyadenylated RNAs in these tumor samples.


Differences in gastric carcinoma microenvironment stratify according to EBV infection intensity: implications for possible immune adjuvant therapy.

Strong MJ, Xu G, Coco J, Baribault C, Vinay DS, Lacey MR, Strong AL, Lehman TA, Seddon MB, Lin Z, Concha M, Baddoo M, Ferris M, Swan KF, Sullivan DE, Burow ME, Taylor CM, Flemington EK - PLoS Pathog. (2013)

Detection of EBV in gastric carcinoma samples.Four gigabytes of deduplicated RNA-seq read data from each of the seventy-one gastric carcinoma samples were analyzed using RNA CoMPASS. The virome branch of the taxonomy trees for the four samples with the highest number of EBV reads (A) and two EBV-negative samples (B) were generated using the metagenome analysis tool, MEGAN 4. (C) For a more in depth analysis of EBV reads, the combined sequence read files for each sample were aligned to the EBV genome and the hg19 human genome assembly using the genome aligner, Novoalign. Of the EBV-positive samples, four samples were identified as having high numbers of EBV reads while eight were found to have low but detectable numbers of EBV reads (see Figure S2 for plot of EBV reads per 1,000,000 human mapped reads).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3649992&req=5

ppat-1003341-g001: Detection of EBV in gastric carcinoma samples.Four gigabytes of deduplicated RNA-seq read data from each of the seventy-one gastric carcinoma samples were analyzed using RNA CoMPASS. The virome branch of the taxonomy trees for the four samples with the highest number of EBV reads (A) and two EBV-negative samples (B) were generated using the metagenome analysis tool, MEGAN 4. (C) For a more in depth analysis of EBV reads, the combined sequence read files for each sample were aligned to the EBV genome and the hg19 human genome assembly using the genome aligner, Novoalign. Of the EBV-positive samples, four samples were identified as having high numbers of EBV reads while eight were found to have low but detectable numbers of EBV reads (see Figure S2 for plot of EBV reads per 1,000,000 human mapped reads).
Mentions: RNA-seq data from The Cancer Genome Atlas (TCGA) gastric adenocarcinoma cohort (SRA035410) was first analyzed using RNA CoMPASS (Figure S1 and Xu et al., unpublished) to assess the virome for each of the 71 data sets. This initial screening was performed using a single lane of sequencing data from each patient. Most samples contained relatively low numbers of reads matching non-human viral sources (e.g. enterobacteria phage T4T) that possibly represent environmental contamination (Figure 1A–B). Of the known human viruses detected, one sample (BR-4298, Figure 1A) contained 6 reads attributed to Hepatitis C virus. Further inspection of these reads showed high homology to the human immunoglobulin light chain variable region (Table S1). These reads likely represent human sequences rather than reads derived from Hepatitis C virus. Twelve samples showed evidence of human cytomegalovirus (HCMV) with read numbers ranging from 5 to 132. Individual BLASTing of selected HCMV reads showed high homology to HCMV genomes but not to human sequences indicating that these are bona fide HCMV derived reads. The relatively low numbers of HCMV reads in these samples (relative to the numbers of EBV in some samples, see below) suggests that these reads are derived from a low number of HCMV infected cells or that the virus is not expressing substantial numbers of polyadenylated RNAs in these tumor samples.

Bottom Line: Epstein-Barr virus (EBV) is associated with roughly 10% of gastric carcinomas worldwide (EBVaGC).In four samples with the highest EBV coverage (hiEBVaGC - high EBV associated gastric carcinoma), transcripts from the BamHI A region comprised the majority of EBV reads.These results were confirmed in a separate cohort of 21 Vietnamese gastric carcinoma samples using qRT-PCR and on tissue samples using in situ hybridization and immunohistochemistry.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Tulane University, New Orleans, Louisiana, United States of America.

ABSTRACT
Epstein-Barr virus (EBV) is associated with roughly 10% of gastric carcinomas worldwide (EBVaGC). Although previous investigations provide a strong link between EBV and gastric carcinomas, these studies were performed using selected EBV gene probes. Using a cohort of gastric carcinoma RNA-seq data sets from The Cancer Genome Atlas (TCGA), we performed a quantitative and global assessment of EBV gene expression in gastric carcinomas and assessed EBV associated cellular pathway alterations. EBV transcripts were detected in 17% of samples but these samples varied significantly in EBV coverage depth. In four samples with the highest EBV coverage (hiEBVaGC - high EBV associated gastric carcinoma), transcripts from the BamHI A region comprised the majority of EBV reads. Expression of LMP2, and to a lesser extent, LMP1 were also observed as was evidence of abortive lytic replication. Analysis of cellular gene expression indicated significant immune cell infiltration and a predominant IFNG response in samples expressing high levels of EBV transcripts relative to samples expressing low or no EBV transcripts. Despite the apparent immune cell infiltration, high levels of the cytotoxic T-cell (CTL) and natural killer (NK) cell inhibitor, IDO1, was observed in the hiEBVaGCs samples suggesting an active tolerance inducing pathway in this subgroup. These results were confirmed in a separate cohort of 21 Vietnamese gastric carcinoma samples using qRT-PCR and on tissue samples using in situ hybridization and immunohistochemistry. Lastly, a panel of tumor suppressors and candidate oncogenes were expressed at lower levels in hiEBVaGC versus EBV-low and EBV-negative gastric cancers suggesting the direct regulation of tumor pathways by EBV.

Show MeSH
Related in: MedlinePlus