Enteropathy-associated T cell lymphoma (EATL) is a lethal, and the most common, neoplastic complication of celiac disease. Here, we defined the genetic landscape of EATL through whole-exome ...sequencing of 69 EATL tumors.
was the most frequently silenced gene in EATL (32% of cases). The JAK-STAT pathway was the most frequently mutated pathway, with frequent mutations in
as well as
,
,
, and
We also identified mutations in
,
, and
Type I EATL and type II EATL (monomorphic epitheliotropic intestinal T cell lymphoma) had highly overlapping genetic alterations indicating shared mechanisms underlying their pathogenesis. We modeled the effects of SETD2 loss in vivo by developing a T cell-specific knockout mouse. These mice manifested an expansion of γδ T cells, indicating novel roles for SETD2 in T cell development and lymphomagenesis. Our data render the most comprehensive genetic portrait yet of this uncommon but lethal disease and may inform future classification schemes.
Inhibitor of DNA binding (Id) proteins, including Id1-4, are transcriptional regulators involved in promoting cell proliferation and survival in various cell types. Although upregulation of Id ...proteins is associated with a broad spectrum of tumors, recent studies have identified that Id3 plays a tumor-suppressor role in the development of Burkitt's lymphoma in humans and hepatosplenic T cell lymphomas in mice. In this article, we report rapid lymphoma development in
/
double-knockout mice that is caused by unchecked expansion of invariant NKT (iNKT) cells or a unique subset of innate-like CD1d-independent T cells. These populations began to expand in neonatal mice and, upon malignant transformation, resulted in mortality between 3 and 11 mo of age. The malignant cells also gave rise to lymphomas upon transfer to
-deficient and wild-type hosts, reaffirming their inherent tumorigenic potential. Microarray analysis revealed a significantly modified program in these neonatal iNKT cells that ultimately led to their malignant transformation. The lymphoma cells demonstrated chromosome instability along with upregulation of several signaling pathways, including the cytokine-cytokine receptor interaction pathway, which can promote their expansion and migration. Dysregulation of genes with reported driver mutations and the NF-κB pathway were found to be shared between
/
double-knockout lymphomas and human NKT tumors. Our work identifies a distinct premalignant state and multiple tumorigenic pathways caused by loss of function of Id2 and Id3. Thus, conditional deletion of
and
in developing T cells establishes a unique animal model for iNKT and relevant innate-like lymphomas.
A family of transcription factors known as E proteins, and their antagonists, Id proteins, regulate T cell differentiation at critical developmental checkpoints. Id proteins promote the ...differentiation of conventional αβ T cells and suppress the expansion of innate-like αβ T cells known as invariant natural killer T (iNKT) cells. However, it remains to be determined whether Id proteins differentially regulate these distinct lineage choices in early stages of T cell development. In this manuscript, we report that in Id-deficient mice, uninhibited activity of the E protein family member E2A mediates activation of genes that support iNKT cell development and function. There is also biased rearrangement in Id-deficient DP cells that promotes selection into the iNKT lineage in these mice. The observed expansion of iNKT cells is not abrogated by blocking pre-TCR signaling, which is required for conventional αβ T cell development. Finally, E2A is found to be a key transcriptional regulator of both iNKT and γδNKT lineages, which appear to have shared lineage history. Therefore, our study reveals a previously unappreciated role of E2A in coordinating the development of the iNKT lineage at an early stage, prior to their TCR-mediated selection alongside conventional αβ T cells.
Hepatosplenic T-cell lymphoma (HSTL) is a rare and lethal lymphoma; the genetic drivers of this disease are unknown. Through whole-exome sequencing of 68 HSTLs, we define recurrently mutated driver ...genes and copy-number alterations in the disease. Chromatin-modifying genes, including
, and
, were commonly mutated in HSTL, affecting 62% of cases. HSTLs manifest frequent mutations in
(31%),
(9%), and
(9%), for which there currently exist potential targeted therapies. In addition, we noted less frequent events in
, and
was the most frequently silenced gene in HSTL. We experimentally demonstrated that
acts as a tumor suppressor gene. In addition, we found that mutations in
and
activate critical signaling pathways important to cell survival in HSTL. Our work thus defines the genetic landscape of HSTL and implicates gene mutations linked to HSTL pathogenesis and potential treatment targets.
We report the first systematic application of whole-exome sequencing to define the genetic basis of HSTL, a rare but lethal disease. Our work defines
as a tumor suppressor gene in HSTL and implicates genes including
and
in the disease.
.
Follicular lymphoma (FL) is the most common low grade lymphoma in adults, but uncommon in children. While the two diseases are similar in morphology and immunophenotype, pediatric-type FLs manifest ...higher grade histology with most cases being localized and curable with standard therapy. Pediatric-type FLs are thought arise from germinal center B cells and typically demonstrate higher expression of the germinal center B cell markers BCL6 and CD10.
While the genetic basis of adult FL has been studied previously, the genetic basis of pediatric-type FL remains largely unknown. In this study, we sought to define the gene coding mutations occurring in pediatric FL and compared them to those of 48 adult FLs, as well as other adult lymphomas derived from germinal center B cells including 88 GCB DLBCLs and 67 cases of BL that we, and others, have published previously.
We obtained a total of 32 pediatric-type FLs along with paired normal tissue. The median age wa 8 years with 62% male patients. We performed whole-exome sequencing for these cases using the Agilent solution-based system of exon capture, which uses RNA baits to target all protein coding genes (CCDS database), as well as ~700 human miRNAs from miRBase (v13). In all, we generated over 6 GB of sequencing data using high throughput sequencing on the Illumina platform.
Using bioinformatics approaches that we have described previously, we identified 26 candidate cancer genes that were recurrently somatically mutated in pediatric FL. We found that the genes mutated in pediatric FLs were from three predominant ontologies/pathways. These included DNA damage repair (40%, ATM, RB1), chromatin modification (34%, SETD2, SMARCA4 and TET2) and alternative splicing (12%, SF3B1). We also noted IRF4 translocations and TNFRSF14 mutations in 10% of the cases as has been described previously.
We further compared pediatric FLs to other germinal center B cell derived lymphomas and, in particular, FLs occurring in adults. We found that the mutations that characterize adult FLs including MLL2, CREBBP, and GNA13 were absent in pediatric FLs. There was only one pediatric FL case with an EZH2 mutation, which otherwise occur commonly in germinal center B cell derived lymphomas.
Thus, pediatric FLs are genetically unlike both adult FLs and other germinal center B cell derived lymphomas. Our data provide new clues to the distinctive genetic basis of these tumors and their clinical behavior.
No relevant conflicts of interest to declare.
Peripheral T cell lymphomas (PTCL) of γδ T cell origin including subsets of hepatosplenic T cell lymphoma (HSTL) and enteropathy associated T cell lymphoma (EATL) are characterized by short survival ...with multiagent chemotherapy programs compared to other PTCLs. Additionally, the molecular and genetic events underlying the development of HSTL and EATL are poorly understood, thus hampering development of more effect novel therapies in these lymphoma subtypes. Given this problem, we sought to define and characterize genetic events associated with HSTL/EATL.
Through whole exome sequencing of 150 HSTL/EATL cases along with paired normal control tissues we found the histone methyltransferase SETD2 to be the most frequently mutated tumor suppressor in HSTL and EATL (>40% of cases in both entities). Conversely, SETD2 mutation was found to be rare in other PTCL subtypes. HSTL/EATL SETD2 mutations frequently occurred as nonsense and frameshift mutations and more than half had evidence of biallelic mutation, suggesting these mutations constitute a loss of function phenotype. Accordingly, we found that overexpression of representative mutant SETD2 (c.T4361C/F1544S and c.7571_7572delGT/C2524*) in HEK293 cells resulted in decreased downstream H3K36me3 compared to wild type SETD2 controls. We further investigated the ramifications of SETD2 functional loss in the only known HSTL cell line, DERL2. DERL2 cells were transduced with lentiviral constructs bearing inducible SETD2 (N=2) or non-silencing control shRNAs. SETD2 shRNA induction in DERL2 cells resulted in >75% loss of SETD2 mRNA expression compared to non-silencing controls by quantitative PCR and resulted in potent depletion of both SETD2 and H3K36me3 protein by Western blotting. We then performed RNA sequencing of DERL2 cells after SETD2 knockdown compared to non-silencing controls. Gene expression analysis and gene set enrichment analysis (GSEA) revealed that SETD2 knockdown was associated dysregulation of multiple oncogenic pathways involved in cell cycle, mitotic, G2/M and NF-kB pathways (FDR <0.1 for each). DERL2 cells post SETD2-knockdown also revealed increased cellular proliferation in in vitro culture and in methylcellulose colony forming assays as compared to non-silencing controls (p<0.01). This effect was not noted in T lymphoblast Jurkat cells engineered in similar fashion for SETD2 loss, suggesting SETD2 loss favors γδ T cell expansion specifically in a context-specific manner. Overall these experiments thus confirm an association between the acquisition of oncogenic gene expression pathways with SETD2/H3K36me3 loss and enhanced cell survival and proliferation in malignant γδ T cells.
To understand effects of SETD2 loss in vivo, we quantitated progenitor and mature T cell subsets in T cell specific SETD2 (Setd2fl/fl, lck-cre) knockout mice. Sorted T cells from 6-8 week old mice but not other tissues derived from Setd2fl/fl, lck-cre mice showed lack of SETD2 protein and low H3K36me3/2 ratios consistent with lineage-specific SETD2 loss. In this model, we found 2.2 fold expansion (p=0.016) of γδ T cells in mesenteric lymph nodes and Peyer patches of Setd2fl/fl, lck-cre compared to controls. Analysis of gene expression in murine T cell progenitors in normal mice without SETD2 loss revealed upregulation of SETD2 mRNA in thymic double positive cells, prior to αβ lineage commitment. Taken together with the results of our in vitro experiments, this suggests that SETD2/H3K36me3 may control normal T cell progenitor fate and γδ T cell expansion through regulation of specific gene expression programs.
In summary, we describe SETD2 functional loss resulting in attenuation of H3K36 trimethylation as a frequent event in gamma delta T cell derived lymphomas. By gene expression analysis and functional studies in murine and human cancer cell line models, we show that SETD2 is important both in the regulation of γδ versus αβ T lymphocyte developement and has a direct role in fostering proliferation of malignant cells derived from the γδ T cell compartment. This appears in part due to loss of epigenetic control of diverse gene expression programs. This work has direct translational importance as we define a novel role of the SETD2/H3K36me3 axis in controlling global T cell differentiation and we identify a potential target for synthetic lethal approaches in malignancies such as HSTL and EATL.
No relevant conflicts of interest to declare.
Introduction
Enteropathy-associated T cell lymphoma (EATL) is an intestinal tumor of the intraepithelial T lymphocytes, with a median survival time of less than 1 year. It is a rare disease in ...general and has two main subtypes described. Type 1 EATL is a complication in patients with celiac disease, a chronic gluten-sensitive enteropathy. Type 2 EATL, characterized by smaller monomorphic lymphocytes, typically occurs sporadically in patients without celiac disease. Very little is known about the genetic mutations and gene expression signatures that define this disease, or the extent to which the two types of EATL are genetically distinct. It has been suggested that the two types of EATLs should be reclassified as separate diseases in future WHO categories.
Methods
In this study, we performed whole exome sequencing to 100-fold depth of 41 EATL tumors including 23 type 1 cases and 18 type 2 cases. Both alpha-beta (65%) and gamma-delta (35%) T cell receptor rearrangements were seen among these cases. Paired normal DNA was sequenced in most (N=30) cases. We defined somatic mutations, copy number alterations, and HLA genotypes in these cases from sequencing data. Additionally, we generated RNA sequencing data on the same EATL tumors. Corresponding clinical and outcome data was collected on the same cohort.
Results
We found that both type 1 and type 2 EATLs had overlapping patterns of mutations and similar overall survival. The most commonly mutated genes were chromatin modifier genes (34%) including ATRX and ARID1B. We also identified recurrent somatic mutations in signal transduction genes, including JAK1 and BCL9L. TP53 mutations were also recurrent (12%). Copy number amplifications in 9q, 1q, and 8q occurred most frequently and were present in both subtypes. We further compared the mutational profiles to peripheral T cell lymphoma, angioimmunoblastic T cell lymphoma, cutaneous T cell lymphoma, natural killer/T cell lymphoma, diffuse large B cell lymphoma, and Burkitt lymphoma. These comparisons identify EATL as a genetically distinct disease with a very different pattern of mutations. RNAseq identified the gene expression patterns that are unique to EATL and also identified gene expression signatures that distinguish the two types of EATL. The DQ2 or DQ8 HLA genotype is present in the majority of type 1 cases (73%) while occurring infrequently in type 2 cases (27%).
Conclusions
Our study defines the genetic landscape of enteropathy associated T cell lymphoma and highlights the genetic and clinical overlap between the two types. While the two types have differences in mutations and gene expression patterns, they have more in common with each other compared to other lymphoma types. Our data may inform future decisions regarding the potential separation of the two EATL types as distinct entities.
No relevant conflicts of interest to declare.
Diffuse large B cell lymphoma (DLBCL) is the most common hematologic malignancy and is characterized by a striking degree of clinical and molecular heterogeneity. Gene expression profiling has long ...been used to define subtypes and understand the heterogeneity within DLBCL. Microarray-based gene expression studies have identified cell of origin subgroups (Alizadeh et al., 2000), as well as gene expression signatures derived from host inflammatory response (Monti et al., 2005), stromal tissues (Lenz et al., 2008) as well as other biological processes. However, the connections between these gene expression signatures and genetic alterations is largely unknown. In this study, we sought to define connections between gene expression signatures and genetic alterations to better understand the biology of DLBCL.
We enrolled a total of 1001 DLBCL patients and comprehensively defined the landscape of genetic mutations, copy number alterations and expression through whole-exome and transcriptome sequencing. We identified 150 genetic drivers of DLBCL including many novel, clinically relevant genes (e.g. SPEN, SETD1B and KLHL14). We began the gene expression analysis with a comprehensive universe of nearly 9500 annotated gene sets from widely used gene set databases (Kegg, Reactome, MSigDB), several lymphoma-specific gene signature sources (Lenz et al. 2008, Monti et al. 2005, and Shaffer et al.) and other published sources. From this large universe, we identified 1228 gene sets that comprised genes with high intra gene set correlation. The vast majority of these 1228 gene sets also exhibited high inter-gene set correlation indicating a high degree of redundancy between these gene sets. We developed an approach to singular value decomposition to define 31 exemplar gene sets that essentially replicated the patterns of the 1228 described gene sets. Our results recapitulated a number of described patterns underlying DLBCL gene expression including distinct gene sets reflecting cell of origin, host response, BCR and Oxidative Phosphorylation (Monti et al., 2005), as well as the “Stromal 1” and “Stromal 2” signatures (Lenz et al., 2008) signatures. There was no association between the expression of stromal signatures and overall mutational burden (p=0.4). Our data identified the genetic underpinnings of each of these distinct gene sets that provide a better understanding of the underlying biology affected by different genetic mutations.
Interestingly, genetic alterations in RHOA were strongly associated with the proliferation gene signature and poor survival in DLBCL. We sought to better define the role of RHOA mutations in DLBCL which are mostly unknown. We knocked out the expression of RHOA in vitro through the CRISPR-Cas9 approach and found that RHOA deletion was lethal in both ABC DLBCL (LY3, TMD8, HBL1) and GCB DLBCLs (Pfeiffer and SUDHL4), results were validated with shRNAs. Conversely, cells overexpressing R5Q mutant proliferated 30% faster than vector control cells and more than 10% faster than wildtype overexpressing cells, directly implicating these mutations and RHOA overexpression in the growth of DLBCL cells.
We further investigated the effects of RHOA knockout in vivo using two complementary mouse models. We bred Rhoa conditional knockout mice with Mb1-Cre transgenic mice, in which Cre is restricted to the B cell lineage as well as AID-Cre transgenic mice in which Cre is restricted to germinal center (GC) B cells. While B cells represented the vast majority of cells in the lymphoid organs of Rhoa wildtype mice, we found a dramatic reduction in B cell population and a resulting increase in T cell percentages in Rhoa deleted mice (p<0.001), indicating a critical role of RHOA for B cell development. We also observed a significant reduction in GC B cells as a proportion of total B cells (p=0.01) and a significant disruption of the normally regulated ratio of dark zone and light zone germinal center B cells (p<0.001). Rhoa knockout in GC B cells had significantly reduced filamentous actin (p=0.03) which is associated with cytoskeletal modeling and cell migration.
Collectively, our data define the genetic basis for observed changes in gene expression in DLBCL. Our experimental data define RHOA as an oncogene with roles in B cell development, germinal center maintenance, and DLCBL growth.
Leppa:Roche: Consultancy, Honoraria, Research Funding; Bayer: Research Funding; Merck: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding; Janssen Cilag: Consultancy, Research Funding. Flowers:Abbvie: Consultancy, Research Funding; Infinity: Research Funding; Clinical Care Options: Research Funding; National Cancer Institute: Research Funding; Spectrum: Consultancy; Onyx: Research Funding; OptumRx: Consultancy; Prime Oncology: Research Funding; Janssen Pharmaceutical: Research Funding; Acerta: Research Funding; Gilead: Consultancy; Seattle Genetics: Consultancy; Eastern Cooperative Oncology Group: Research Funding; Research to Practice: Research Funding; Burroughs Welcome Fund: Research Funding; Celgene: Consultancy, Research Funding; V Foundation: Research Funding; Bayer: Consultancy; Educational Concepts: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Genentech/Roche: Consultancy, Research Funding; Millennium/Takeda: Research Funding; National Institutes Of Health: Research Funding; TG Therapeutics: Research Funding. Gribben:TG Therapeutics: Honoraria; Kite: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Pharmacyclics: Honoraria; Abbvie: Honoraria; Acerta: Honoraria; Karyopharm: Honoraria; Genentech/Roche: Honoraria. Hsi:Cellerant Therapeutics: Research Funding; Abbvie: Research Funding; Seattle Genetics: Consultancy, Honoraria, Speakers Bureau; Eli Lilly and Co.: Research Funding. Evens:Merck: Consultancy; Amgen: Consultancy; Seattle Genetics: Consultancy; Spectrum Pharmaceuticals: Consultancy; Novartis: Consultancy; Millennium: Consultancy; AbbVie: Consultancy; Affimed: Consultancy; Pharmacyclics: Consultancy; Celgene: Consultancy; Kite Pharma: Consultancy. Reddy:Abbvie: Consultancy; BMS: Consultancy; Celgene: Consultancy; Gilead: Speakers Bureau. Gordon:Janssen: Other: Data Monitoring Committee.
Introduction
Diffuse large B cell lymphoma (DLBCL), the most common lymphoma world-wide, is strikingly heterogeneous. This heterogeneity creates a daunting challenge for conducting well-powered ...studies connecting molecular features to clinical outcome. Not only is the association of genetic mutations with clinical outcome in DLBCL mostly unknown, the relative importance of other well-described features, such as MYC and BCL2 translocation/expression and cell of origin based subsets (ABC and GCB DLBCL), is difficult to interpret due to conflicting reports.
We sought to comprehensively define the spectrum of genetic mutations and their association with clinical outcome in DLBCL. Our calculations indicated that 500 tumor-normal pairs would provide 95% power to define mutations occurring in at least 5% of patients, and that 800 cases would be required to define the clinical correlations with cross-validation.
Methods
We enrolled 1001 de novo DLBCL patients, with complete IPI and survival data, who were treated uniformly with standard rituximab and anthracycline containing regimens. All tumors were subjected to whole exome and transcriptome sequencing (RNAseq), as well as SNP arrays to confirm genetic alterations. ABC (38%) and GCB DLBCL (36%) subtypes were defined using microarrays and RNAseq in these patients to examine subgroup-based differences in mutations and outcome. MYC and BCL2 expression were quantified separately.
Results
Gene discovery analysis of somatic mutations and copy number alterations in exome sequencing data from 502 tumor-normal pairs of DLBCL identified 197 recurrently mutated genes, including 155 genes previously identified to be mutated in DLBCLs. In addition, our study uncovered 42 novel driver genes in DLBCL (e.g. BTK, SPEN, CD70). Exome sequencing results were validated by Sanger sequencing of 1120 variants with over 90% concordance. We also identified copy number alterations in these genes, with strong agreement (90%) of amplifications and/or deletions to those detected on Illumina high resolution SNP microarrays.
These 197 genes were found to comprise 15 functionally related subnetworks, including those related to histone modification, NFkB, B cell receptor, PI3K and cell cycle (Figure 1). Within each subnetwork, the vast majority of the gene alterations occurred in a mutually exclusive (P<10-3) fashion in patterns consistent with their described functions within the subnetworks. For instance, among genes comprising the NFkB subnetwork, positive regulators of the pathway such as MYD88 and CARD11 showed activating patterns (copy number gains and recurrent hotspot mutations), whereas negative regulators such as TNFAIP3, NFKBIE, and NFKBIA were inactivated through genetic deletions or frequent nonsense or frameshift mutations.
We examined the associations between the mutations and clinical outcome in all 1001 patients. All survival analyses were conducted using nearly equally split training and validation sets, corrected for multiple comparisons with significance of P<0.01 in the validation set (Figure 2). The cell of origin classification was strongly associated with survival in our cases and was independent of MYC and BCL2 co-expression, which was separately associated with survival (Figure 2A). Figure 2B shows hazard ratios for select genes, as well as associated Kaplan-Meier survival curves for a subset of those genes. We further identified combinations of different genetic and expression features that point to context dependence for survival associations (Figure 2C). For instance, mutations in KLHL14 were associated with a particularly poor prognosis in ABC DLBCL, while CREBBP mutations in ABC DLBCL patients were associated with better prognosis than average GCB DLBCLs. Mutations in EZH2 and CD70 were associated with a highly favorable prognosis within the GCB DLBCL subgroup. TP53 mutations were found to be prognostic only in the presence of MLL2 mutations and high BCL2 expression. Importantly, these risk groups are mutually exclusive and inform clinical outcome significantly better than existing metrics.
Conclusion
To our knowledge, this is the largest whole exome sequencing study in any single cancer. Our study answers many long-standing questions in the disease, informing a comprehensive understanding of genetic drivers of DLBCL, their organization into pathways, and their relationship to clinical outcome.
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Leppä:Roche: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Takeda Pharmaceuticals: Consultancy, Honoraria, Other: Travel expenses; Janssen: Consultancy, Research Funding; CTI Bio Pharma: Consultancy; Mundipharma: Research Funding; Bayer: Other: Travel expenses, Research Funding. Flowers:ECOG: Research Funding; Gilead: Consultancy, Research Funding; Millenium/Takeda: Research Funding; Pharmacyclics, LLC, an AbbVie Company: Research Funding; TG Therapeutics: Research Funding; Mayo Clinic: Research Funding; NIH: Research Funding; Infinity: Research Funding; AbbVie: Research Funding; Genentech: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Acerta: Research Funding. Hsi:Seattle Genetics: Honoraria, Speakers Bureau; HTG Molecular Diagnostics: Consultancy, Honoraria; Eli Lilly: Research Funding; Cellerant Therapeutics: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Evens:Takeda: Other: Advisory board. Reddy:GILEAD: Membership on an entity’s Board of Directors or advisory committees; INFINITY: Membership on an entity’s Board of Directors or advisory committees; celgene: Membership on an entity’s Board of Directors or advisory committees; KITE: Membership on an entity’s Board of Directors or advisory committees.
Differential gene expression between sexes is the main contributor of the morphological and behavioral differences observed between them. Studying the signatures of these differences at the genetic ...level will help us understand the forces acting on them. The existence of androdioecious and gonochoristic species in the genus Caenorhabditis makes it suitable for sex-biased gene expression studies. In this thesis, I have assembled the transcriptome of C. sp. 9 and C. sp. 5 using de novo and reference-based techniques. Evolutionary analysis of the assembled contigs showed that genes with male-biased expression evolve faster than those with a female bias, as observed in other taxa. Furthermore, I found a positive correlation between gene expression and codon usage bias.
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