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High-definition mapping of retroviral integration sites defines the fate of allogeneic T cells after donor lymphocyte infusion.

Cattoglio C, Maruggi G, Bartholomae C, Malani N, Pellin D, Cocchiarella F, Magnani Z, Ciceri F, Ambrosi A, von Kalle C, Bushman FD, Bonini C, Schmidt M, Mavilio F, Recchia A - PLoS ONE (2010)

Bottom Line: Comparison with matched random controls and with integrations obtained from CD34(+) hematopoietic stem/progenitor cells showed that integration clusters occur within chromatin regions bearing epigenetic marks associated with active promoters and regulatory elements in a cell-specific fashion.Analysis of integration sites in T cells obtained ex vivo two months after infusion showed no evidence of integration-related clonal expansion or dominance, but rather loss of cells harboring integration events interfering with RNA post-transcriptional processing.The study shows that high-definition maps of retroviral integration sites are a powerful tool to analyze the fate of genetically modified T cells in patients and the biological consequences of retroviral transduction.

View Article: PubMed Central - PubMed

Affiliation: IIT Unit of Molecular Neuroscience, Istituto Scientifico H. San Raffaele, Milan, Italy.

ABSTRACT
The infusion of donor lymphocytes transduced with a retroviral vector expressing the HSV-TK suicide gene in patients undergoing hematopoietic stem cell transplantation for leukemia/lymphoma promotes immune reconstitution and prevents infections and graft-versus-host disease. Analysis of the clonal dynamics of genetically modified lymphocytes in vivo is of crucial importance to understand the potential genotoxic risk of this therapeutic approach. We used linear amplification-mediated PCR and pyrosequencing to build a genome-wide, high-definition map of retroviral integration sites in the genome of peripheral blood T cells from two different donors and used gene expression profiling and bioinformatics to associate integration clusters to transcriptional activity and to genetic and epigenetic features of the T cell genome. Comparison with matched random controls and with integrations obtained from CD34(+) hematopoietic stem/progenitor cells showed that integration clusters occur within chromatin regions bearing epigenetic marks associated with active promoters and regulatory elements in a cell-specific fashion. Analysis of integration sites in T cells obtained ex vivo two months after infusion showed no evidence of integration-related clonal expansion or dominance, but rather loss of cells harboring integration events interfering with RNA post-transcriptional processing. The study shows that high-definition maps of retroviral integration sites are a powerful tool to analyze the fate of genetically modified T cells in patients and the biological consequences of retroviral transduction.

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Related in: MedlinePlus

Functional classification of genes targeted by integration clusters.The figure shows those function (a) and disease (b) categories significantly over-represented among the target genes of MLV integration clusters in pre-infusion (red bars) or post-infusion (blue bars) T cells, as analyzed by the Ingenuity® software, using the Ingenuity Pathways Knowledge Base gene population as background. P values were corrected for multiple testing by the Bonferroni method. Statistical significance was set at a p-value of 0.01 (2 in log scale, vertical line). (n) represents the total number of genes eligible for the analysis.
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pone-0015688-g006: Functional classification of genes targeted by integration clusters.The figure shows those function (a) and disease (b) categories significantly over-represented among the target genes of MLV integration clusters in pre-infusion (red bars) or post-infusion (blue bars) T cells, as analyzed by the Ingenuity® software, using the Ingenuity Pathways Knowledge Base gene population as background. P values were corrected for multiple testing by the Bonferroni method. Statistical significance was set at a p-value of 0.01 (2 in log scale, vertical line). (n) represents the total number of genes eligible for the analysis.

Mentions: A functional classification of the genes annotated within the integration clusters was carried out by the Ingenuity® software. Functional categories significantly over-represented in the pre-infusion dataset included cell signaling, growth and proliferation, development and death (red bars in Figure 6A), all above the threshold of statistical significance (black vertical line in Figure 6A) compared to the Ingenuity Knowledge Base background. Over-represented disease-associated categories included inflammatory response, immunological, hematological and genetic disorders (Figure 6B), well related to T cell-specific functions. All these categories were barely or not at all over-represented in the post-infusion dataset, indicating no preferential survival in vivo of cells harboring integrations in specific gene categories, particularly cell growth and proliferation and cancer (blue bars in Figure 6). To investigate the association with oncogenic transformation more directly, we annotated all cancer-related genes targeted by MLV integration clusters, using a comprehensive compilation that includes proto-oncogenes and genes associated with common insertion sites (CIS) in murine tumors. Genes cumulatively belonging to both categories accounted for 11.7 and 16.0% of the genes targeted by the clusters in pre- and post-infusion T cells respectively, a significant over-representation with respect to the frequency observed in the random integration dataset (7.8% of 15,200 genes, p<10−4). However, the difference between pre- and post-integration datasets was not significant (p = 0.11) and 71% of the cancer-associated genes targeted by post-infusion clusters (in bold in Table S1) were targeted also by pre-infusion ones, suggesting no selection in vivo for cells harboring integrations around such genes.


High-definition mapping of retroviral integration sites defines the fate of allogeneic T cells after donor lymphocyte infusion.

Cattoglio C, Maruggi G, Bartholomae C, Malani N, Pellin D, Cocchiarella F, Magnani Z, Ciceri F, Ambrosi A, von Kalle C, Bushman FD, Bonini C, Schmidt M, Mavilio F, Recchia A - PLoS ONE (2010)

Functional classification of genes targeted by integration clusters.The figure shows those function (a) and disease (b) categories significantly over-represented among the target genes of MLV integration clusters in pre-infusion (red bars) or post-infusion (blue bars) T cells, as analyzed by the Ingenuity® software, using the Ingenuity Pathways Knowledge Base gene population as background. P values were corrected for multiple testing by the Bonferroni method. Statistical significance was set at a p-value of 0.01 (2 in log scale, vertical line). (n) represents the total number of genes eligible for the analysis.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3008730&req=5

pone-0015688-g006: Functional classification of genes targeted by integration clusters.The figure shows those function (a) and disease (b) categories significantly over-represented among the target genes of MLV integration clusters in pre-infusion (red bars) or post-infusion (blue bars) T cells, as analyzed by the Ingenuity® software, using the Ingenuity Pathways Knowledge Base gene population as background. P values were corrected for multiple testing by the Bonferroni method. Statistical significance was set at a p-value of 0.01 (2 in log scale, vertical line). (n) represents the total number of genes eligible for the analysis.
Mentions: A functional classification of the genes annotated within the integration clusters was carried out by the Ingenuity® software. Functional categories significantly over-represented in the pre-infusion dataset included cell signaling, growth and proliferation, development and death (red bars in Figure 6A), all above the threshold of statistical significance (black vertical line in Figure 6A) compared to the Ingenuity Knowledge Base background. Over-represented disease-associated categories included inflammatory response, immunological, hematological and genetic disorders (Figure 6B), well related to T cell-specific functions. All these categories were barely or not at all over-represented in the post-infusion dataset, indicating no preferential survival in vivo of cells harboring integrations in specific gene categories, particularly cell growth and proliferation and cancer (blue bars in Figure 6). To investigate the association with oncogenic transformation more directly, we annotated all cancer-related genes targeted by MLV integration clusters, using a comprehensive compilation that includes proto-oncogenes and genes associated with common insertion sites (CIS) in murine tumors. Genes cumulatively belonging to both categories accounted for 11.7 and 16.0% of the genes targeted by the clusters in pre- and post-infusion T cells respectively, a significant over-representation with respect to the frequency observed in the random integration dataset (7.8% of 15,200 genes, p<10−4). However, the difference between pre- and post-integration datasets was not significant (p = 0.11) and 71% of the cancer-associated genes targeted by post-infusion clusters (in bold in Table S1) were targeted also by pre-infusion ones, suggesting no selection in vivo for cells harboring integrations around such genes.

Bottom Line: Comparison with matched random controls and with integrations obtained from CD34(+) hematopoietic stem/progenitor cells showed that integration clusters occur within chromatin regions bearing epigenetic marks associated with active promoters and regulatory elements in a cell-specific fashion.Analysis of integration sites in T cells obtained ex vivo two months after infusion showed no evidence of integration-related clonal expansion or dominance, but rather loss of cells harboring integration events interfering with RNA post-transcriptional processing.The study shows that high-definition maps of retroviral integration sites are a powerful tool to analyze the fate of genetically modified T cells in patients and the biological consequences of retroviral transduction.

View Article: PubMed Central - PubMed

Affiliation: IIT Unit of Molecular Neuroscience, Istituto Scientifico H. San Raffaele, Milan, Italy.

ABSTRACT
The infusion of donor lymphocytes transduced with a retroviral vector expressing the HSV-TK suicide gene in patients undergoing hematopoietic stem cell transplantation for leukemia/lymphoma promotes immune reconstitution and prevents infections and graft-versus-host disease. Analysis of the clonal dynamics of genetically modified lymphocytes in vivo is of crucial importance to understand the potential genotoxic risk of this therapeutic approach. We used linear amplification-mediated PCR and pyrosequencing to build a genome-wide, high-definition map of retroviral integration sites in the genome of peripheral blood T cells from two different donors and used gene expression profiling and bioinformatics to associate integration clusters to transcriptional activity and to genetic and epigenetic features of the T cell genome. Comparison with matched random controls and with integrations obtained from CD34(+) hematopoietic stem/progenitor cells showed that integration clusters occur within chromatin regions bearing epigenetic marks associated with active promoters and regulatory elements in a cell-specific fashion. Analysis of integration sites in T cells obtained ex vivo two months after infusion showed no evidence of integration-related clonal expansion or dominance, but rather loss of cells harboring integration events interfering with RNA post-transcriptional processing. The study shows that high-definition maps of retroviral integration sites are a powerful tool to analyze the fate of genetically modified T cells in patients and the biological consequences of retroviral transduction.

Show MeSH
Related in: MedlinePlus