T-cell prolymphocytic leukemia (T-PLL) is an aggressive post-thymic T-cell malignancy characterized by the recurrent inv(14)(q11q32)/t(14;14)(q11;q32) or t(X;14)(q28;q11) leading to activation of either the TCL1 or MTCP1 gene, respectively. However, these primary genetic events are insufficient to drive leukemogenesis. Recently, activating mutations in JAK3 have been identified in other T-cell malignancies. Since JAK3 is essential for T-cell maturation, we analyzed a cohort of 32 T-PLL patients for mutational hot spots in the JAK3 gene using a step-wise screening approach. We identified 14 mutations in 11 of 32 patients (34%). The most frequently detected mutation in our cohort was M511I (seen in 57% of cases) previously described as an activating change in other T-cell malignancies. Three patients carried two mutations in JAK3. In two patients M511I and R657Q were simultaneously detected and in another patient V674F and V678L. In the latter case we could demonstrate that the mutations were on the same allele in cis. Protein modeling and homology analyses of mutations present in other members of the JAK family suggested that these mutations likely activate JAK3, possibly by disrupting the activation loop and the interface between N and C lobes, increasing the accessibility of the catalytic loop. In addition, four of the 21 patients lacking a JAK3 point mutation presented an aberrant karyotype involving the chromosomal band 19p13 harboring the JAK3 locus. The finding of recurrent activating JAK3 mutations in patients with T-PLL could enable the use of JAK3 inhibitors to treat patients with this unfavorable malignancy who otherwise have a very poor prognosis.
Tumor cell survival critically depends on heterotypic communication with benign cells in the microenvironment. Here, we describe a survival signaling pathway activated in stromal cells by contact to B cells from patients with chronic lymphocytic leukemia (CLL). The expression of protein kinase C (PKC)-?II and the subsequent activation of NF-?B in bone marrow stromal cells are prerequisites to support the survival of malignant B cells. PKC-? knockout mice are insusceptible to CLL transplantations, underscoring the in vivo significance of the PKC-?II-NF-?B signaling pathway in the tumor microenvironment. Upregulated stromal PKC-?II in biopsies from patients with CLL, acute lymphoblastic leukemia, and mantle cell lymphoma suggests that this pathway may commonly be activated in a variety of hematological malignancies.
Immunoglobulin (Ig) gene remodeling by V(D)J recombination plays a central role in the generation of normal B cells, and somatic hypermutation and class switching of Ig genes are key processes during antigen-driven B cell differentiation. However, errors of these processes are involved in the development of B cell lymphomas. Ig locus-associated translocations of proto-oncogenes are a hallmark of many B cell malignancies. Additional transforming events include inactivating mutations in various tumor suppressor genes, and also latent infection of B cells with viruses, such as Epstein-Barr virus. Many B cell lymphomas require B cell antigen receptor expression, and in several instances chronic antigenic stimulation plays a role in sustaining tumor growth. Often, survival and proliferation signals provided by other cells in the microenvironment are a further critical factor in lymphoma development and pathophysiology. Many B cell malignancies derive from germinal center B cells, most likely because of the high proliferation rate of these cells and the high activity of mutagenic processes.
In the present study, telomere length, telomerase activity, the mutation load of immunoglobulin variable heavy chain (IGHV) genes, and established prognostic factors were investigated in 78 patients with chronic lymphocytic leukaemia (CLL) to determine the impact of telomere biology on the pathogenesis of CLL. Telomere length was measured by an automated multi-colour flow-FISH, and an age-independent delta telomere length (?TL) was calculated. CLL with unmutated IGHV genes was associated with shorter telomeres (p = 0.002). Furthermore, we observed a linear correlation between the frequency of IGHV gene mutations and elongation of telomeres (r = 0.509, p < 0.001). With respect to prognosis, a threshold ?TL of -4.2 kb was the best predictor for progression-free and overall survival. ?TL was not significantly altered over time or with therapy. The correlation between the mutational load in IGHV genes and the ?TL in CLL might reflect the initial telomere length of the putative cell of origin (pre- versus post-germinal center B cells). In conclusion, the ?TL is a reliable prognostic marker for patients with CLL. Short telomeres and high telomerase activity as occurs in some patients with CLL with a worse prognosis might be an ideal target for treatment with telomerase inhibitors.
The origin of IgM(+)CD27(+) B lymphocytes with mutated IgV genes, which account for approximately 20% of human peripheral blood (PB) B cells, is controversially discussed. A generation in a primary diversification pathway, in T cell-independent immune responses, or in T cell-dependent germinal center (GC) reactions has been proposed. We show here that IgM(+)IgD(+)CD27(+) and IgM(+)IgD(-/low)CD27(+) B cell subsets carry, like class-switched memory B cells, mutations in the Bcl6 gene as a genetic trait of a GC experience. Moreover, the identification of PB IgM(+)IgD(+)CD27(+) B cells clonally related to GC-derived IgG(+) memory B cells with shared and distinct IgV gene mutations demonstrates the GC origin also of the former subset. These findings provide genetic evidence for a GC derivation of somatically mutated IgM(+) B cells and indicate that adult humans harbor a large population of IgM(+)IgD(+) post-GC memory B cells. Furthermore, the analysis revealed that a highly diverse and often very large population of memory B cells is generated from a given GC B cell clone, and that (preferentially IgM) memory B cells are generated already early in the GC reaction. This provides novel insights into the dynamics of GC reactions and the generation of a memory B cell repertoire.
STATs are constitutively activated in several malignancies. In primary mediastinal large B-cell lymphoma and Hodgkin lymphoma (HL), inactivating mutations in SOCS1, an inhibitor of JAK/STAT signaling, contribute to deregulated STAT activity. Based on indications that the SOCS1 mutations are caused by the B cell-specific somatic hypermutation (SHM) process, we analyzed B-cell non-HL and normal B cells for mutations in SOCS1. One-fourth of diffuse large B-cell lymphoma and follicular lymphomas carried SOCS1 mutations, which were preferentially targeted to SHM hotspot motifs and frequently obviously inactivating. Rare mutations were observed in Burkitt lymphoma, plasmacytoma, and mantle cell lymphoma but not in tumors of a non-B-cell origin. Mutations in single-sorted germinal center B cells were infrequent relative to other genes mutated as byproducts of normal SHM, indicating that SOCS1 inactivation in primary mediastinal large B-cell lymphoma, HL, diffuse large B-cell lymphoma, and follicular lymphoma is frequently the result of aberrant SHM.
Alterations in the DNA methylation pattern are a hallmark of leukemias and lymphomas. However, most epigenetic studies in hematologic neoplasms (HNs) have focused either on the analysis of few candidate genes or many genes and few HN entities, and comprehensive studies are required.
Human peripheral blood (PB) B cells expressing only IgD and tonsillar IgD-secreting plasma cells carry highly mutated V(H) genes and show preferential Iglambda usage. To further characterize these peculiar cells and gain insight into their generation, we analysed rearranged V(H) and V(L) genes of single IgD-only lambda(+) PB B cells and IgD(+) plasma cells from four individuals each. We demonstrate that the high somatic hypermutation activity in these cells is not restricted to V(H) genes but also present in V(L) genes. Moreover, not only PB IgD-only B cells, as reported earlier, but also IgD-expressing plasma cells often belong to very large clones. Surprisingly, the V(H)3-30 gene segment was used in each PB donor by >30% of IgD-only cells and in 2 tonsils by >50% of IgD plasma cells, whereas it was used less frequent in other B cells. All these features fit to a model in which IgD-only cells develop in superantigen-driven germinal center reactions, in which B cells are activated by binding of antigens to constant parts of Cdelta and often lambda light chains and the V(H)3-30 segment, and are selected for deletion of Cmu. IgD-only B cells may hence represent a unique B lineage subset generated in response to particular antigens.
Lymphomas are assumed to originate at different stages of lymphocyte development through chromosomal aberrations. Thus, different lymphomas resemble lymphocytes at distinct differentiation stages and show characteristic morphologic, genetic, and transcriptional features. Here, we have performed a microarray-based DNA methylation profiling of 83 mature aggressive B-cell non-Hodgkin lymphomas (maB-NHLs) characterized for their morphologic, genetic, and transcriptional features, including molecular Burkitt lymphomas and diffuse large B-cell lymphomas. Hierarchic clustering indicated that methylation patterns in maB-NHLs were not strictly associated with morphologic, genetic, or transcriptional features. By supervised analyses, we identified 56 genes de novo methylated in all lymphoma subtypes studied and 22 methylated in a lymphoma subtype-specific manner. Remarkably, the group of genes de novo methylated in all lymphoma subtypes was significantly enriched for polycomb targets in embryonic stem cells. De novo methylated genes in all maB-NHLs studied were expressed at low levels in lymphomas and normal hematopoietic tissues but not in nonhematopoietic tissues. These findings, especially the enrichment for polycomb targets in stem cells, indicate that maB-NHLs with different morphologic, genetic, and transcriptional background share a similar stem cell-like epigenetic pattern. This suggests that maB-NHLs originate from cells with stem cell features or that stemness was acquired during lymphomagenesis by epigenetic remodeling.
The cellular origin of chronic lymphocytic leukemia (CLL) is still debated, although this information is critical to understanding its pathogenesis. Transcriptome analyses of CLL and the main normal B cell subsets from human blood and spleen revealed that immunoglobulin variable region (IgV) gene unmutated CLL derives from unmutated mature CD5(+) B cells and mutated CLL derives from a distinct, previously unrecognized CD5(+)CD27(+) post-germinal center B cell subset. Stereotyped V gene rearrangements are enriched among CD5(+) B cells, providing independent evidence for a CD5(+) B cell derivation of CLL. Notably, these CD5(+) B cell populations include oligoclonal expansions already found in young healthy adults, putatively representing an early phase in CLL development before the CLL precursor lesion monoclonal B cell lymphocytosis. Finally, we identified deregulated proteins, including EBF1 and KLF transcription factors, that were not detected in previous comparisons of CLL and conventional B cells.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.