Manuel Montesinos-Rongen

Primary central nervous system lymphomas are derived from germinal-center B cells and show a preferential usage of the V4-34 gene segment

Primary central nervous system lymphomas (PCNSLs) have recently received considerable clinical attention due to their increasing incidence. To clarify the histogenetic origin of these intriguing neoplasms, PCNSLs from 10 HIV-negative patients were analyzed for immunoglobulin (Ig) gene rearrangements. All tumors exhibited clonal IgH gene rearrangements. Of the 10 cases, 5 used the V4-34 gene segment, and all of these lymphomas shared an amino acid exchange from glycine to aspartate due to a mutation in the first codon of the complementarity-determining region 1. No preferential usage of D(H), J(H), V(kappa), J(kappa), V(lambda), or J(lambda) gene segments was observed. All potentially functional rearrangements exhibited somatic mutations. The pattern of somatic mutations indicated selection of the tumor cells (or their precursors) for expression of a functional antibody. Mean mutation frequencies of 13. 2% and 8.3% were detected for the heavy and light chains, respectively, thereby exceeding other lymphoma entities. Cloning experiments of three tumors showed ongoing mutation in at least one case. These data suggest that PCNSLs are derived from highly mutated germinal-center B cells. The frequent usage of the V4-34 gene and the presence of a shared replacement mutation may indicate that the tumor precursors recognized a shared (super) antigen.

A Comprehensive Microarray-Based DNA Methylation Study of 367 Hematological Neoplasms

Background 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. Methodology/Principal Findings Here, we report for the first time a microarray-based DNA methylation study of 767 genes in 367 HNs diagnosed with 16 of the most representative B-cell (n = 203), T-cell (n = 30), and myeloid (n = 134) neoplasias, as well as 37 samples from different cell types of the hematopoietic system. Using appropriate controls of B-, T-, or myeloid cellular origin, we identified a total of 220 genes hypermethylated in at least one HN entity. In general, promoter hypermethylation was more frequent in lymphoid malignancies than in myeloid malignancies, being germinal center mature B-cell lymphomas as well as B and T precursor lymphoid neoplasias those entities with highest frequency of gene-associated DNA hypermethylation. We also observed a significant correlation between the number of hypermethylated and hypomethylated genes in several mature B-cell neoplasias, but not in precursor B- and T-cell leukemias. Most of the genes becoming hypermethylated contained promoters with high CpG content, and a significant fraction of them are targets of the polycomb repressor complex. Interestingly, T-cell prolymphocytic leukemias show low levels of DNA hypermethylation and a comparatively large number of hypomethylated genes, many of them showing an increased gene expression. Conclusions/Significance We have characterized the DNA methylation profile of a wide range of different HNs entities. As well as identifying genes showing aberrant DNA methylation in certain HN subtypes, we also detected six genes—DBC1, DIO3, FZD9, HS3ST2, MOS, and MYOD1—that were significantly hypermethylated in B-cell, T-cell, and myeloid malignancies. These might therefore play an important role in the development of different HNs.

Activating L265P mutations of the MYD88 gene are common in primary central nervous system lymphoma.

Primary central nervous system lymphoma (PCNSL) is a special lymphoma entity. Although being a rare disease, the incidence of PCNSL has significantly raised in the last decades [3, 4], however, a specific standard therapeutic regimen is still a matter of debate [2]. Despite the fact that PCNSL histopathologically resemble diffuse large B cell lymphoma (DLBCL) [3, 4], they are characterized by unique clinical and molecular features [6], including their exclusive manifestation in the unique microenvironment of the immunologically privileged CNS. Activation of the nuclear factor jB (NF-jB) pathway is a hallmark of PCNSL [1, 5]. Various mechanisms of NF-jB activation have been identified in PCNSL. These include gains in chromosome 18q21 being present in 37% of PCNSL and activating mutations of the CARD11 gene being present in 16% of PCNSL [5, 6]. Moreover, NF-jB activation might be triggered by stimulation of either the B cell receptor pathway, the tumor necrosis factor or the toll-like receptor (TLR) pathway. Here, we provide evidence for deregulation of the TLR pathway in the pathogenesis of PCNSL through mutation of the myeloid differentiation primary response gene 88 (MYD88). Analysis of the MYD88 gene, the central integrator of the TLR pathway, in a series of 14 PCNSL by a biphased PCR approach followed by sequencing of all the exons revealed mutations in seven (50%) of the tumors (for details see supplementary information). Interestingly, in five of these seven (71%) PCNSL, i.e. 36% of all tumors analyzed, mutations were identified as a leucine to proline exchange at position 265 (L265P), which is an oncogenically activating mutation and has recently been shown to be of somatic origin [7]. In the remaining two PCNSL, MYD88 mutations resided at positions 103 and 143, respectively. In one tumor a nucleotide exchange corresponded to a silent mutation (L103L). In the other PCNSL the mutation resulted in an amino acid exchange (Q143E), which has not been reported before and the functional impact of which remains to be elucidated. Till date, MYD88 mutations have been described only in 9% of gastric mucosa-associated lymphoid tissue lymphoma, in 3% of chronic lymphocytic leukemia, in 5% of Burkitt’s lymphoma, and in systemic DLBCL, affecting 39% of the activated B cell like (ABC)-DLBCL subtype and 6% of the germinal center B cell like subgroup, respectively [7, 8]. Interestingly, 29% of systemic ABCDLBCL harbored the MYD88 L265P mutation [7]. Two of the five PCNSL (40%) with the recurrent MYD88 L265P mutation concomitantly harbored a CARD11 mutation, which results in the constitutive activation of the CARD11 protein [5]. While either the MYD88 L265P or the CARD11 mutation activates the NF-jB pathway, the combined presence of these mutations may act synergistically; thus, further enhancing NFjB activation [1, 5]. In addition to a direct effect on the NF-jB pathway, MYD88 mutations may alter the Electronic supplementary material The online version of this article (doi:10.1007/s00401-011-0891-2) contains supplementary material, which is available to authorized users.

Endogenous interleukin-10 is required for prevention of a hyperinflammatory intracerebral immune response in Listeria monocytogenes meningoencephalitis

ABSTRACT To analyze the role of interleukin-10 (IL-10) in bacterial cerebral infections, we studied cerebral listeriosis in IL-10-deficient (IL-10−/−) and wild-type (WT) mice, the latter of which express high levels of IL-10 in both primary and secondary cerebral listeriosis. IL-10−/− mice succumbed to primary as well as secondary listeriosis, whereas WT mice were significantly protected from secondary listeriosis by prior intraperitoneal immunization withListeria monocytogenes. Meningoencephalitis developed in both strains; however, in IL-10−/− mice the inflammation was more severe and associated with increased brain edema and multiple intracerebral hemorrhages. IL-10−/− mice recruited significantly increased numbers of leukocytes, in particular granulocytes, to the brain, and the intracerebral cytokine (tumor necrosis factor, IL-1, IL-12, gamma interferon, and inducible nitric oxide synthase) and chemokine (crg2/IP-10, RANTES, MuMig, macrophage inflammatory protein 1α [MIP-1α], and MIP-1β) transcription was enhanced compared to that in WT mice. Despite this prominent hyperinflammation, the frequencies of intracerebral L. monocytogenes-specific CD8+ T cells were reduced and the intracerebral bacterial load was not reduced in IL-10−/− mice compared to WT mice. Following intraperitoneal infection, IL-10−/− mice exhibited hepatic hyperinflammation without better bacterial clearance; however, in contrast to the mice with cerebral listeriosis, they did not succumb, illustrating that intrinsic factors of the target organ have a strong impact on the course and outcome of the infection.

Mutations of CARD11 but not TNFAIP3 may activate the NF-κB pathway in primary CNS lymphoma

Primary CNS lymphoma (PCNSL), the intracerebral subgroup of diffuse large B cell lymphoma (DLBCL), shows evidence for aberrant activation of the nuclear factor (NF)-κB pathway. In order to identify potential activators of the NF-κB complex, we analyzed the CARD11 and TNFAIP3 genes for the presence of somatic mutations and TNFAIP3 for aberrant promoter methylation in PCNSL. We also compared PCNSL to spinal DLBCL, because CARD11 and TNFAIP3 mutations have been described in systemic DLBCL. CARD11 mutations, located in the coiled-coil region, which may activate NF-κB, were detected in 16% (5/32) of PCNSL, while TNFAIP3 mutations were detected in 3% (1/32) of PCNSL. In PCNSL, all CARD11 mutations were heterozygous, in-frame, induced amino acid exchanges, and presumably led to activation of this oncogene. Spinal DLBCL harbored mutations of CARD11 and TNFAIP3 in 10% (1/10) and 20% (2/10) of cases, respectively. In both PCNSL and spinal DLBCL, mutations in CARD11 and TNFAIP3 were mutually exclusive. TNFAIP3 was unmethylated in all PCNSLs (30/30) and spinal DLBCLs (10/10). We conclude that mutations of the oncogene CARD11 may contribute to NF-κB activation and thereby play a role in the pathogenesis of PCNSL, while, in contrast to systemic DLBCL, inactivation of TNFAIP3 either by mutation or methylation seems to be of minor significance.

Primary lymphoma of the central nervous system: just DLBCL or not?

Primary lymphoma of the central nervous system (PCNSL), defined as diffuse large B-cell lymphoma (DLBCL) confined to the central nervous system (CNS), has recently gained attention due to its increased incidence, which is mainly due to the AIDS epidemic and has been observed in some but not all

Delayed development of chronic lymphocytic leukemia in the absence of macrophage migration inhibitory factor

UNLABELLED Survival of chronic lymphocytic leukemia (CLL) cells depends on stimuli provided by a suitable microenvironment. The factors and mechanisms providing this growth support for CLL cells are not fully understood. We found that plasma levels of macrophage migration inhibitory factor (MIF), a proinflammatory and immunoregulatory chemokine, were elevated in CLL patients. Therefore, we characterized the functional role of MIF in a CLL mouse model. For this purpose, we crossed Eμ-TCL1 mice with MIF knockout (MIF-/-) mice. The resulting TCL1+/wtMIF/ mice showed a delayed onset of leukemia, reduced splenomegaly and hepatomegaly, and a longer survival than TCL1+/wtMIFwt/wt controls. Immunohistochemical examination of the lymphoid organs showed that the numbers of macrophages were significantly reduced in the spleen and bone marrow of TCL1+/wtMIF/ mice compared with TCL1+/wtMIFwt/wt controls. Mechanistic studies in vitro revealed that the absence of MIF rendered CLL cells more susceptible to apoptosis. Accordingly, incubation with an anti-MIF antibody reduced the survival of CLL cells on a macrophage feeder layer. In addition, the migratory activity of TCL1+/wtMIF/ macrophages was decreased compared with TCL1+/wtMIFwt/wt macrophages. Taken together, our results provide evidence that MIF supports the development of CLL by enhancing the interaction of CLL cells with macrophages. KEY POINTS Targeted deletion of the gene for macrophage migration inhibitory factor (MIF) delays development of chronic lymphocytic leukemia and prolongs survival in mice. MIF recruits leukemia-associated macrophages to spleen or liver.

Recurrent Inactivation of the PRDM1 Gene in Primary Central Nervous System Lymphoma

Primary lymphomas of the CNS (PCNSLs) show molecular features of the late germinal center exit B-cell phenotype and are impaired in their terminal differentiation as indicated by a lack of immunoglobulin class switching. Because the positive regulatory domain I protein with ZNF domain (PRDM1/BLIMP1) is a master regulator of terminal B-cell differentiation into plasma cells, we investigated a series of 21 PCNSLs for the presence of mutations in the PRDM1 gene and alterations in the expression pattern of the PRDM1 protein. Direct sequencing of all coding exons of the PRDM1 gene identified deleterious mutations associated with abrogation of PRDM1 protein expression in 4 of 21 (19%) PCNSLs. Thus, similar to systemic diffuse large B-cell lymphomas, PRDM1 may be a tumor suppressor in some PCNSL and contribute to lymphomagenesis by impairing terminal differentiation.

Amplification of TCRβ gene rearrangements from micromanipulated single cells: T cells rosetting around Hodgkin and Reed-Sternberg cells in Hodgkin's disease are polyclonal

Despite accounting for only a minor fraction of all cells in Hodgkins lymphoma tissue, the Hodgkin and Reed‐Sternberg (HRS) cells represent the malignant tumor cell clone in Hodgkins disease (HD). By far the most abundant cell type in the tumor tissue are CD4+ T cells. Some of them intimately associate with HRS cells forming rosettes around them. This study addresses the question whether the rosetting phenomenon reflects a specific interaction between T and HRS cells by asking whether the rosettes are composed of T cells expressing a restricted TCR repertoire. Single rosetting T cells were micromanipulated from frozen sections of tumor tissue in two cases of nodular sclerosing HD and one case of lymphocyte predominant HD. TCR Vβ gene rearrangements were amplified from these single cells by PCR. Of 83 potentially functional Vβ gene rearrangements obtained altogether from the three cases, 81 were found to be clonally unrelated. Furthermore, they did not show signs of selection of the receptor chains for recognition of common epitopes: The usage of Vβ and Jβ gene segments as well as the distribution of complementarity‐determining region (CDR) 3 lengths was similar to what was seen in a collection of 60 Vβ gene rearrangements from blood of healthy donors and no recurrent CDR3 amino acid motifs were found. These data suggest that the HRS cells attract CD4+ T cells nonspecifically.

Expression pattern and cellular sources of chemokines in primary central nervous system lymphoma

The expression pattern of a subset of chemokines and their corresponding receptors was investigated in primary central nervous system lymphomas (PCNSL). The tumor cells consistently expressed CXCR4, CXCL12, CXCR5, and CXCL13, both at mRNA and protein levels. Cerebral endothelial cells were positive for CXCL12 and CXCL13, while reactive astrocytes and microglial cells expressed CXCL12, CCR5, and CCR6. Inflammatory T cells in PCNSL were characterized by CCR5 and CCR6 positivity. Taken together, our data indicate a cell type-specific repertoire of chemokine and chemokine receptor expression in PCNSL suggesting that chemokine-mediated interactions facilitate crossing of the blood-brain barrier as well as intracerebral dissemination of PCNSL cells. In addition, chemokines expressed by tumor cells may contribute to induction of reactive glial changes and influence the composition of inflammatory infiltrates in PCNSL. Therefore, cell type specific expression of distinct chemokine profiles likely plays a role in the pathogenesis of PCNSL and may contribute to their characteristic histological appearance.