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The low abundance of Hodgkin/Reed-Sternberg (HRS) cells in lymph node biopsies in classical Hodgkin lymphoma (cHL) complicates the analysis of somatic genetic alterations in HRS cells. As circulating cell-free DNA (cfDNA) contains circulating tumor DNA (ctDNA) from HRS cells, we prospectively collected cfDNA from 177 patients with newly diagnosed, mostly early-stage cHL in a monocentric study at Leuven, Belgium (n = 59) and the multicentric BREACH study by Lymphoma Study Association (n = 118). To catalog the patterns and frequencies of genomic copy number aberrations (CNAs), cfDNA was sequenced at low coverage (0.26×), and data were analyzed with ichorCNA to yield read depth-based copy number profiles and estimated clonal fractions in cfDNA. At diagnosis, the cfDNA concentration, estimated clonal fraction, and ctDNA concentration were significantly higher in cHL cases than controls. More than 90% of patients exhibited CNAs in cfDNA. The most frequent gains encompassed 2p16 (69%), 5p14 (50%), 12q13 (50%), 9p24 (50%), 5q (44%), 17q (43%), 2q (41%). Losses mostly affected 13q (57%), 6q25-q27 (55%), 4q35 (50%), 11q23 (44%), 8p21 (43%). In addition, we identified loss of 3p13-p26 and of 12q21-q24 and gain of 15q21-q26 as novel recurrent CNAs in cHL. At diagnosis, ctDNA concentration was associated with advanced disease, male sex, extensive nodal disease, elevated erythrocyte sedimentation rate, metabolic tumor volume, and HRS cell burden. CNAs and ctDNA rapidly diminished upon treatment initiation, and persistence of CNAs was associated with increased probability of relapse. This study endorses the development of ctDNA as gateway to the HRS genome and substrate for early disease response evaluation.

The low abundance of Hodgkin/Reed-Sternberg (HRS) cells in lymph node biopsies in classical Hodgkin lymphoma (cHL) complicates the analysis of somatic genetic alterations in HRS cells. As circulating cell-free DNA (cfDNA) contains circulating tumor DNA (ctDNA) from HRS cells, we prospectively collected cfDNA from 177 patients with newly diagnosed, mostly early-stage cHL in a monocentric study at Leuven, Belgium (n = 59) and the multicentric BREACH study by Lymphoma Study Association (n = 118). To catalog the patterns and frequencies of genomic copy number aberrations (CNAs), cfDNA was sequenced at low coverage (0.26×), and data were analyzed with ichorCNA to yield read depth-based copy number profiles and estimated clonal fractions in cfDNA. At diagnosis, the cfDNA concentration, estimated clonal fraction, and ctDNA concentration were significantly higher in cHL cases than controls. More than 90% of patients exhibited CNAs in cfDNA. The most frequent gains encompassed 2p16 (69%), 5p14 (50%), 12q13 (50%), 9p24 (50%), 5q (44%), 17q (43%), 2q (41%). Losses mostly affected 13q (57%), 6q25-q27 (55%), 4q35 (50%), 11q23 (44%), 8p21 (43%). In addition, we identified loss of 3p13-p26 and of 12q21-q24 and gain of 15q21-q26 as novel recurrent CNAs in cHL. At diagnosis, ctDNA concentration was associated with advanced disease, male sex, extensive nodal disease, elevated erythrocyte sedimentation rate, metabolic tumor volume, and HRS cell burden. CNAs and ctDNA rapidly diminished upon treatment initiation, and persistence of CNAs was associated with increased probability of relapse. This study endorses the development of ctDNA as gateway to the HRS genome and substrate for early disease response evaluation.

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+versus41 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.

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.

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 caused by surgical induction of diaphragmatic hernia (DH) and additional TO in the fetal rabbit model. Induction of DH was associated with 378 upregulated 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 downregulated by consecutive TO. But DH+TO was associated with an upregulation of 157 genes compared to DH and controls. When being compared to control lungs, 106 genes were downregulated 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 than 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.

Introduction.For decades, the study of genomic alterations in Hodgkin lymphoma (HL) has been hampered by the low abundance of the malignant Hodgkin Reed-Sternberg (HRS) cells in HL lymph node biopsies. Laser microdissection or flow cytometric cell sorting allow the study of purified HRS cells, but the use of these applications is restricted to specialized research centers. We have recently demonstrated proof-of-principle that copy number aberrations (CNA) in HRS cells can be retrieved in circulating cell-free DNA (cfDNA) of HL patients (Vandenberghe et al., Lancet Haematol. 2015). Taking advantage of the presence of HRS cell-derived DNA (ctDNA) in plasma, the aim of this study was to catalogue CNA in HL in a large series of prospectively recruited HL patients.

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No relevant conflicts of interest to declare.

Molecular pathogenesis of ALK-positive Anaplastic Large Cell Lymphoma (ALK+ ALCL) is not completely understood. Approximately 80% of ALK+ ALCL cases harbor the t(2;5)(p23;q35)-associated NPM1-ALKrearrangement, while several variant 2p23/ALKtranslocations involving at least nine partner genes have been identified in the remaining cases. The 5'ALKpartners play a key role in the constitutive activation of the chimeric protein by mediating its oligomerization and its subcellular localization. Their role in ALK lymphomagenesis is debated, but experimental studies showed that the partners impact a range of biological activities of ALK chimeras, including proliferation, transformation and metastatic capacities. Comparative analysis of biological properties of ALK oncoproteins, however, is hampered by the relative low frequency of different variant ALK fusions.

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Peripheral T-cell lymphoma - not otherwise specified (PTCL-NOS) is the largest, most common and very heterogeneous category of PTCL. To date three PTCL variants have been described, including follicular variant (PTCL-F), a rare and poorly understood entity. Cytogenetically, PTCL-F is associated with a recurrent t(5;9)(q33;q22) resulting in the fusion of two protein tyrosine kinase (PTK) genes, ITKand SYK, showing constitutive activation of SYK.Transforming capacities of ITK-SYK were documented both in vitroand in vivo.Involvement of other PTK genes in the pathogenesis of PTCL-F has been postulated, but is thus far, not evidenced.