Genomic And Transcriptomic Analyses of Rare Lymphomas

Post-transplant lymphoproliferative disorders (PTLDs) are heterogenous and potentially fatal disorders that arise in up to 20% of organ transplant recipients. According to World Health Organization (WHO), three major categories are distinguished: benign early lesions, polymorphic PTLD, and malignant monomorphic PTLD (M-PTLD). The latter category comprises hard-to-treat lymphoma entities of which diffuse large B-cell lymphoma (DLBCL) is the most common subtype. The molecular pathogenesis of posttransplant DLBCL (PT-DLBCL) is largely unknown. DLBCL is the most common non-Hodgkin's lymphoma and typically has an aggressive behavior that if not treated would be immediately fatal. For most patients it is curable with immunotherapy but form some this therapy fails and they die. Less common M-PTLD include, among other, Burkitt lymphomas and the very rare Hepatosplenic T-cell lymphoma (HSTL). Burkitt Lymphoma (BL) is an aggressive, GCB-derived malignancy and one of its variants is the immunodeficiency-associated BL arising in patients with HIV infection or in those who underwent an organ transplant and immunosuppressive therapy. This tumor is biologically and molecularly hallmarked by IG-mediated translocation t(8q24) resulting in upregulation of the oncogene MYC. HSTL is also an aggressive lymphoma occurring predominantly in young male. Cytogenetically it is characterized by isochromosome 7q [i(7)(q10)], but the molecular consequences of this alteration remain unknown. Sporadically a ring chromosome 7 (r7) have been observed. The disease is also characterized by a rapid downhill progression and very poor responsiveness to treatment. Its clinical presentation consist of splenomegaly and hepatomegaly with no visible lymphadenopathy. The diagnostic is challenging because the symptoms can be shared by many other pathological conditions. In this PhD work we performed an integrative genomic and transcriptomic analyses of these rare lymphomas using cytogenetics (classical and molecular), SNP-arrays, expression arrays, RNA-seq and a set of bioinformatics methodologies. The results are summarized below. HSTL: We analyzed six i(7)(q10)-positive HSTL cases and three cases with ring 7 [r(7)]. Using aCGH, we profiled all cases and mapped the common deleted region (CDR) at 7p22.1p14.1 and the common gained region (CGR) at 7q22.11q31.1. The CGR spans a smaller 13 Mb region constantly amplified in cases with r(7). We found that TCRG (7p14.1) and TCRB (7q32) are involved in formation of r(7), probably a byproduct of somatic rearrangement of these loci. Transcriptomic analysis didn't identified any CDR-related tumor suppressor gene, instead, loss of 7p22.1p14.1 was associated with an enhanced expression of CHN2 (7p14.1) and the encoded β2-chimerin. Gains of 7q22.11q31.1 associated with an increased expression of several cancer-related genes including ABCB1, a multidrug resistance gene. RNA-seq did not identify any disease-defining mutation neither gene fusions, leaving chromosome 7 imbalances as the only event driving the pathogenesis in this tumor. We hypothesize that the ∆7p22.1p14.1-associated enhanced expression of CHN2/β2-chimerin leads to downmodulation of the NFAT pathway and to a proliferative response, while upregulation of the CGR-related genes provides growth advantage and chemoresistance. Our study also identifies a gene signature comprising 24 genes distinguishing HSTL from other malignancies. PT-DLBCL: we have shown previously that Epstein-Barr Virus-positive (EBV+) and -negative (EBV-) PT-DLBCL have distinct gene expression profiles, and the transcriptomic profile of EBV-PT-DLBCL is similar to that of DLBCL in immunocompetent individuals (IC-DLBCL). To study if these differences/similarities between the 3 groups also hold at the genomic level, we performed aCGH on 21 EBV+ PT-DLBCL, 6 EBV- PT-DLBCL, and 11 control IC-DLBCL. The analysis showed EBV+ and EBV- PT-DLBCL have distinct genomic profiles and shared only one recurrent imbalance. EBV- PT-DLBCL, however, displayed at least 10 aberrations which were also present in IC-DLBCL including gain of 3/3q and 18q and losses of 6q23/TNFAIP3 as well as 9p21/CDKN2A. Gain/amplification of 9p24.1 targeting PDCD1LG2/PDL2 was the most prevalent aberration in EBV+ PT-DLBCL. Our study indicate that the oncogene FOXP1 and the tumor suppressor CDKNA2 may be implicated in EBV- DLBCL but do not play critical role in the pathogenesis of EBV+ PT-DLBCL. Altogether, genomic profiling of PT-/IC-DLBCL confirms that EBV- and EBV+ PT-DLBCL are distinct entities, while EBV- PT-DLBCL has features in common with IC-DLBCL. These findings support the hypothesis that EBV- PT-DLBCL are de novo lymphomas in transplant recipients PT-BL: Recent studies of 59 molecular BL (mBL) identified a novel aberration manifested by a specific 11q-gain/loss pattern in two cases lacking MYC translocation. The aberration was subsequently detected in 15 MYC-negative high-grade B-cell lymphomas resembling BL and two high-grade B-cell lymphomas cell lines. Our study provide evidence that this 11q-gain/loss aberration is particularly frequent in BL in immunodeficient hosts, as it was identified in three out of seven patients with mBL which underwent solid organ transplantation and immunosuppressive therapy. The seven posttransplant BL cases reported here were analysed using conventional cytogenetics, aCGH, FISH, immunohistochemistry, gene expression profiling and bioinformatics. As controls, we included four cases of typical MYC-translocation-positive BL from immunocompetent hosts. Altogether, we confirmed a recurrent occurrence of the 11q-gain/loss pattern in high grade B-cell lymphoma and showed that this aberration is significantly more frequent in BL occurring in the setting of transplantation and immunosuppression than in immunocompetent patients, suggesting that immunosuppression may favor its formation. As identification of patients with the 11q-gain/loss aberration is clinically important but cytogenetically challenging, we recommend the designed 11q-MGR/MLR FISH assay as a useful diagnostic tool to evaluate both, posttransplant- and immunocompetent BL patients.status: publishe

Extracellular vesicles in inflammatory bowel disease: small particles, big players.

Extracellular vesicles are nanovesicles released by many cell types into the extracellular space. They are important mediators of intercellular communication, enabling the functional transfer of molecules from one cell to another. Moreover, their molecular composition reflects the physiological status of the producing cell and tissue. Consequently, these vesicles have been involved in many (patho)physiological processes such as immunomodulation and intestinal epithelial repair, both key processes involved in inflammatory bowel disease. Given that these vesicles are present in many body fluids, they also provide opportunities for diagnostic, prognostic and therapeutic applications. In this review, we summarize functional roles of extracellular vesicles in health and disease, with a focus on immune regulation and intestinal barrier integrity, and review recent studies on extracellular vesicles and inflammatory bowel disease. We also elaborate on their clinical potential in inflammatory bowel disease.status: Published onlin

Posttransplant molecular Burkitt lymphomas are frequently MYC-negative and characterized by the 11q-gain/loss pattern

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Rearrangement of NOTCH1 or BCL3 can independently trigger progression of CLL

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Pulmonary transcriptome analysis in the rabbit model of surgically-induced diaphragmatic hernia treated with fetal tracheal occlusion

Congenital diaphragmatic hernia (CDH) is a malformation leading to pulmonary hypoplasia which can be treated in utero by fetal tracheal occlusion (TO). However the changes of gene expression induced by TO remain largely unknown but could be used to further improve the clinically used prenatal treatment of this devastating malformation. Therefore we aimed to investigate the pulmonary transcriptome changes due to surgical induction of diaphragmatic hernia (DH) and additional tracheal occlusion in the fetal rabbit model.Induction of DH was associated with 378 up-regulated genes compared to controls when allowing a false discovery rate (FDR) of 0.1 and a Fold Change (FC) of 2. Those genes were again down-regulated by consecutive TO. But DH+TO was associated with an up-regulation of 157 genes compared to DH and controls. When being compared to control lungs, 106 genes were down-regulated in the DH group and were not changed by TO. Therefore, the overall pattern of gene expression in DH+TO is more similar to the control group then to the DH group. In this study we further provide a database of gene expression changes induced by surgical creation of DH and consecutive TO in the rabbit model. Future treatment strategies could be developed using this dataset. We also discuss the most relevant genes that are involved in CDH.status: publishe

Identification of distinct subgroups of EBV-positive post-transplant diffuse large B-cell lymphoma

Post-transplantation lymphoproliferative disorder is an aggressive complication of transplantation, most frequently of diffuse large B-cell lymphoma morphology and associated with Epstein-Barr virus (EBV) infection/reactivation. In this study the microenvironment of EBV(+) (n=23) and EBV(-) (n=9) post-transplant non-germinal center B-cell diffuse large B-cell lymphoma was characterized. Of EBV(+) cases somatic hypermutation analysis, gene expression profiling, and extensive phenotyping were performed. Our results demonstrated variable cytotoxic T-cell infiltration and significantly increased CD163(+) M2 macrophage infiltration in EBV(+) compared with EBV(-) post-transplant diffuse large B-cell lymphoma. On the basis of IgM staining and hypermutation analysis, two EBV(+) post-transplant diffuse large B-cell lymphoma subgroups were identified: IgM(+) tumors lacking somatic hypermutations and IgM(-) tumors harboring somatic hypermutations. IgM(-) tumors arose late following transplantation (median interval: 16 months), mainly in kidney recipients. IgM(+) tumors on the other hand arose early (median interval: 3 months, P-value=0.0032), almost exclusively following stem cell transplantation and were associated with worse outcome (median survival 1 month for IgM(+) versus 41 months for IgM(-) tumors, log-rank/Wilcoxon P-value 0.07/0.04). Notably, IgM(+) tumors were characterized by plasma cell features (monotypic kappa/lambda expression, high MUM1 expression, and partial CD138 expression) and a high proliferation index. Consistent with the plasma cell phenotype, unfolded protein response signaling was upregulated. In contrast, IgM(-) EBV(+) post-transplant diffuse large B-cell lymphoma did not express kappa, lambda, IgD, or CD138 and expressed limited MUM1. In these tumors T-cell signaling was enhanced associated with increased T-cell infiltration compared with IgM(+) cases. Overall, our results allow further molecular classification of EBV(+) post-transplant diffuse large B-cell lymphoma and provide a rationale for the use of subtype-specific-targeted therapies (eg, bortezomib in IgM(+) tumors). Our findings also provide a biological basis for the clinical differences between post-transplant lymphoproliferative disorder following solid organ and stem cell transplantation, which are regarded as different disorders.Modern Pathology advance online publication, 6 January 2017; doi:10.1038/modpathol.2016.199.status: publishe

EBV-Positive and EBV-Negative Posttransplant Diffuse Large B Cell Lymphomas Have Distinct Genomic and Transcriptomic Features

The molecular pathogenesis of posttransplant diffuse large B cell lymphoma (PT-DLBCL) is largely unknown. We have recently shown that Epstein-Barr virus-positive (EBV(+)) and -negative (EBV(-)) PT-DLBCL have distinct gene expression profiles, and the transcriptomic profile of EBV(-) PT-DLBCL is similar to that of DLBCL in immunocompetent individuals (IC-DLBCL). To validate these observations at the genomic level, we performed array-comparative genome hybridization (aCGH) analysis of 21 EBV(+) PT-DLBCL, 6 EBV(-) PT-DLBCL, and 11 control IC-DLBCL, and subsequently combined genomic and transcriptomic data. The analysis showed that EBV(+) and EBV(-) PT-DLBCL have distinct aCGH profiles and shared only one recurrent imbalance. EBV(-) PT-DLBCL, however, displayed at least 10 aberrations recurrent in IC-DLBCL, among which characteristic gain of 3/3q and 18q, and loss of 6q23/TNFAIP3 as well as 9p21/CDKN2A. The most prevalent aberration in EBV(+) PT-DLBCL was gain/amplification of 9p24.1 targeting PDCD1LG2/PDL2. Our data indicate that the FOXP1 oncogene and the tumor suppressor CDKNA2 implicated in EBV(-) DLBCL, do not play a critical role in the pathogenesis of EBV(+) PT-DLBCL. Altogether, genomic profiling of PT-/IC-DLBCL confirms that EBV(-) and EBV(+) PT-DLBCL are distinct entities, while EBV(-) PT-DLBCL has features in common with IC-DLBCL. These findings support the hypothesis that EBV(-) PT-DLBCL are de novo lymphomas in transplant recipients.status: publishe