Entirely noninvasive outcome prediction in central nervous system lymphomas using circulating tumor DNA.

ABSTRACT: State-of-the-art response assessment of central nervous system lymphoma (CNSL) by magnetic resonance imaging is challenging and an insufficient predictor of treatment outcomes. Accordingly, the development of novel risk stratification strategies in CNSL is a high unmet medical need. We applied ultrasensitive circulating tumor DNA (ctDNA) sequencing to 146 plasma and cerebrospinal fluid (CSF) samples from 67 patients, aiming to develop an entirely noninvasive dynamic risk model considering clinical and molecular features of CNSL. Our ultrasensitive method allowed for the detection of CNSL-derived mutations in plasma ctDNA with high concordance to CSF and tumor tissue. Undetectable plasma ctDNA at baseline was associated with favorable outcomes. We tracked tumor-specific mutations in plasma-derived ctDNA over time and developed a novel CNSL biomarker based on this information: peripheral residual disease (PRD). Persistence of PRD after treatment was highly predictive of relapse. Integrating established baseline clinical risk factors with assessment of radiographic response and PRD during treatment resulted in the development and independent validation of a novel tool for risk stratification: molecular prognostic index for CNSL (MOP-C). MOP-C proved to be highly predictive of outcomes in patients with CNSL (failure-free survival hazard ratio per risk group of 6.60; 95% confidence interval, 3.12-13.97; P < .0001) and is publicly available at www.mop-c.com. Our results highlight the role of ctDNA sequencing in CNSL. MOP-C has the potential to improve the current standard of clinical risk stratification and radiographic response assessment in patients with CNSL, ultimately paving the way toward individualized treatment.

AMD3100-Mediated CXCR4 Inhibition Impairs Development of Primary Lymphoma of the Central Nervous System.

A hallmark of primary lymphoma of the central nervous system (CNS; PCNSL) is the strong CXCR4 expression of the tumor cells, the function of which is still unknown. In vitro treatment of BAL17CNS lymphoma cells by AMD3100, which inhibits CXCR4-CXCL12 interactions, resulted in the significantly differential expression of 273 genes encoding proteins involved in cell motility, cell-cell signaling and interaction, hematological system development and function, and immunologic disease. Among the genes down-regulated was the one encoding CD200, a regulator of CNS immunologic activity. These data directly translated into the in vivo situation; BAL17CNS CD200 expression was down-regulated by 89% (3% versus 28% CD200+ lymphoma cells) in AMD3100-treated versus untreated mice with BAL17CNS-induced PCNSL. Reduced lymphoma cell CD200 expression may contribute to the markedly increased microglial activation in AMD3100-treated mice. AMD3100 also maintained the structural integrity of blood-brain barrier tight junctions and the outer basal lamina of cerebral blood vessels. Subsequently, lymphoma cell invasion of the brain parenchyma was impaired, and maximal parenchymal tumor size was significantly reduced by 82% in the induction phase. Thus, AMD3100 qualified as a potentially attractive candidate to be included into the therapeutic concept of PCNSL. Beyond therapy, CXCR4-induced suppression of microglial activity is of general neuroimmunologic interest. This study identified CD200 expressed by the lymphoma cells as a novel mechanism of immune escape in PCNSL.

The process of somatic hypermutation increases polyreactivity for central nervous system antigens in primary central nervous system lymphoma.

The immunoglobulin (Ig) heavy and light chain variable gene mutational pattern of the B cell receptor (BCR) in primary central nervous system (CNS) lymphoma (PCNSL) cells suggests antigenic selection to drive pathogenesis and confinement to the CNS. This hypothesis is supported by the observation that the tumor B cell receptor (tBCR) of PCNSL is polyreactive and may be stimulated by CNS proteins. To obtain further insight into the role of the germinal center (GC) reaction on BCR reactivity, we constructed recombinant antibodies (recAb) with Ig heavy and light chain sequences of the corresponding naive BCR (nBCR) by reverting tBCR somatic mutations in 10 PCNSL. Analysis of nBCR-derived recAb reactivity by a protein microarray and immunoprecipitation demonstrated auto- and polyreactivity in all cases. Self-/polyreactivity was not lost during the GC reaction; surprisingly, tBCR significantly increased self-/polyreactivity. In addition to proteins recognized by both the nBCR and tBCR, tBCR gained self-/polyreactivity particularly for proteins expressed in the CNS including proteins of oligodendrocytes/myelin, the S100 protein family, and splicing factors. Thus, in PCNSL pathogenesis, a faulty GC reaction may increase self-/polyreactivity, hereby facilitating BCR signaling via multiple CNS antigens, and may ultimately foster tumor cell survival in the CNS.

TLR signals license CD8 T cells to destroy oligodendrocytes expressing an antigen shared with a Listeria pathogen.

Increasing evidence suggests a role of CD8 T cells in autoimmune demyelinating CNS disease, which, however, is still controversially discussed. Mice, which express ovalbumin (OVA) as cytosolic self-antigen in oligodendrocytes (ODC-OVA mice), respond to CNS infection induced by OVA-expressing attenuated Listeria with CD8 T cell-mediated inflammatory demyelination. This model is suitable to decipher the contribution of CD8 T cells and the pathogen in autoimmune CNS disease. Here, we show that both antigen and pathogen are required in the CNS for disease induction, though not in a physically linked fashion. Intracerebral challenge with combined toll like receptor (TLR) TLR2 and TLR9 as well as TLR7 and TLR9 agonists substituted for the bacterial stimulus, but not with individual TLR agonists (TLR2, TLR3,TLR5,TLR7, TLR9). Furthermore, MyD88 inactivation rendered ODC-OVA mice resistant to disease induction. Collectively, CD8 T cell-mediated destruction of oligodendrocytes is activated if (i) an antigen shared with an infectious agent is provided in the CNS microenvironment and (ii) innate immune signals inform the CNS microenvironment that pathogen removal warrants an immune attack by CD8 T cells, even at the expense of locally restricted demyelination.

Enteric Murine Ganglionitis Induced by Autoimmune CD8 T Cells Mimics Human Gastrointestinal Dysmotility.

Inflammatory bowel diseases frequently cause gastrointestinal dysmotility, suggesting that they may also affect the enteric nervous system. So far, the precise mechanisms that lead to gastrointestinal dysmotility in inflammatory bowel diseases have not been elucidated. To determine the effect of CD8 T cells on gastrointestinal motility, transgenic mice expressing ovalbumin on enteric neurons were generated. In these mice, adoptive transfer of ovalbumin-specific OT-I CD8 T cells induced severe enteric ganglionitis. CD8 T cells homed to submucosal and myenteric plexus neurons, 60% of which were lost, clinically resulting in severely impaired gastrointestinal transition. Anti-interferon-γ treatment rescued neurons by preventing their up-regulation of major histocompatibility complex class I antigen, thus preserving gut motility. These preclinical murine data translated well into human gastrointestinal dysmotility. In a series of 30 colonic biopsy specimens from patients with gastrointestinal dysmotility, CD8 T cell-mediated ganglionitis was detected that was followed by severe loss of enteric neurons (74.8%). Together, the preclinical and clinical data support the concept that autoimmune CD8 T cells play an important pathogenetic role in gastrointestinal dysmotility and may destroy enteric neurons.

Array-based profiling of the lymphoma cell DNA methylome does not unequivocally distinguish primary lymphomas of the central nervous system from non-CNS diffuse large B-cell lymphomas.

Primary lymphomas of the central nervous system (PCNSL) are diffuse large B-cell lymphomas (DLBCLs) confined to the central nervous system (CNS). We here performed array-based DNA methylation analyses of 26 PCNSL and 78 DLBCL and validated our findings in an independent dataset. We identified 2847 CpGs differentially methylated between PCNSL and non-CNS-DLBCL. Neither a supervised analysis using these CpGs nor application of 3 CpG classifiers selected for class separation unambiguously separated PCNSL from non-CNS-DLBCL. Remarkably, 6/78 non-CNS-DLBCL consistently segregated with PCNSL, which displayed molecular features typical for PCNSL. Our findings suggest that a subset of non-CNS-DLBCL exists which molecularly resembles PCNSL.

B-cell-specific conditional expression of Myd88p.L252P leads to the development of diffuse large B-cell lymphoma in mice.

The adaptor protein MYD88 is critical for relaying activation of Toll-like receptor signaling to NF-κB activation. MYD88 mutations, particularly the p.L265P mutation, have been described in numerous distinct B-cell malignancies, including diffuse large B-cell lymphoma (DLBCL). Twenty-nine percent of activated B-cell-type DLBCL (ABC-DLBCL), which is characterized by constitutive activation of the NF-κB pathway, carry the p.L265P mutation. In addition, ABC-DLBCL frequently displays focal copy number gains affecting BCL2 Here, we generated a novel mouse model in which Cre-mediated recombination, specifically in B cells, leads to the conditional expression of Myd88(p.L252P) (the orthologous position of the human MYD88(p.L265P) mutation) from the endogenous locus. These mice develop a lymphoproliferative disease and occasional transformation into clonal lymphomas. The clonal disease displays the morphologic and immunophenotypical characteristics of ABC-DLBCL. Lymphomagenesis can be accelerated by crossing in a further novel allele, which mediates conditional overexpression of BCL2 Cross-validation experiments in human DLBCL samples revealed that both MYD88 and CD79B mutations are substantially enriched in ABC-DLBCL compared with germinal center B-cell DLBCL. Furthermore, analyses of human DLBCL genome sequencing data confirmed that BCL2 amplifications frequently co-occurred with MYD88 mutations, further validating our approach. Finally, in silico experiments revealed that MYD88-mutant ABC-DLBCL cells in particular display an actionable addiction to BCL2. Altogether, we generated a novel autochthonous mouse model of ABC-DLBCL that could be used as a preclinical platform for the development and validation of novel therapeutic approaches for the treatment of ABC-DLBCL.

Primary Central Nervous System (CNS) Lymphoma B Cell Receptors Recognize CNS Proteins.

Primary lymphoma of the CNS (PCNSL) is a diffuse large B cell lymphoma confined to the CNS. To elucidate its peculiar organ tropism, we generated recombinant Abs (recAbs) identical to the BCR of 23 PCNSLs from immunocompetent patients. Although none of the recAbs showed self-reactivity upon testing with common autoantigens, they recognized 1547 proteins present on a large-scale protein microarray, indicating polyreactivity. Interestingly, proteins (GRINL1A, centaurin-α, BAIAP2) recognized by the recAbs are physiologically expressed by CNS neurons. Furthermore, 87% (20/23) of the recAbs, including all Abs derived from IGHV4-34 using PCNSL, recognized galectin-3, which was upregulated on microglia/macrophages, astrocytes, and cerebral endothelial cells upon CNS invasion by PCNSL. Thus, PCNSL Ig may recognize CNS proteins as self-Ags. Their interaction may contribute to BCR signaling with sustained NF-κB activation and, ultimately, may foster tumor cell proliferation and survival. These data may also explain, at least in part, the affinity of PCNSL cells for the CNS.

CD44 regulates the apoptotic response and promotes disease development in chronic lymphocytic leukemia.

The cell-surface glycoprotein CD44 is expressed in chronic lymphocytic leukemia (CLL), but its functional role in this disease is poorly characterized. We therefore investigated the contribution of CD44 to CLL in a murine disease model, the Eµ-TCL1 transgenic mouse, and in CLL patients. Surface CD44 increased during murine CLL development. CD44 expression in human CLL was induced by stimulation with interleukin 4/soluble CD40 ligand and by stroma cell contact. Engagement of CD44 by its natural ligands, hyaluronic acid or chondroitin sulfate, protected CLL cells from apoptosis, while anti-CD44 small interfering RNAs impaired tumor cell viability. Deletion of CD44 during TCL1-driven murine leukemogenesis reduced the tumor burden in peripheral blood and spleen and led to a prolonged overall survival. The leukemic cells from these CD44 knockout animals revealed lower levels of antiapoptotic MCL1, a higher propensity to apoptosis, and a diminished B-cell receptor kinase response. The inhibitory anti-CD44 antibodies IM7 and A3D8 impaired the viability of CLL cells in suspension cultures, in stroma contact models, and in vivo via MCL1 reduction and by effector caspase activation. Taken together, CD44 expression in CLL is mediated by the tumor microenvironment. As a coreceptor, CD44 promotes leukemogenesis by regulating stimuli of MCL1 expression. Moreover, CD44 can be addressed therapeutically in CLL by specific antibodies.

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.

Systems biology of primary CNS lymphoma: from genetic aberrations to modeling in mice.

Primary lymphoma of the central nervous system (CNS, PCNSL) is a specific diffuse large B cell lymphoma entity arising in and confined to the CNS. Despite extensive research since many decades, the pathogenetic mechanisms underlying the remarkable tropism of this peculiar malignant hematopoietic tumor remain still to be elucidated. In the present review, we summarize the present knowledge on the genotypic and phenotypic characteristics of the tumor cells of PCNSL, give an overview over deregulated molecular pathways in PCNSL and present recent progress in the field of preclinical modeling of PCNSL in mice. With regard to the phenotype, PCNSL cells resemble late germinal center exit IgM+IgD+ B cells with blocked terminal B cell differentiation. They show continued BCL6 activity in line with ongoing activity of the germinal center program. This together with the pathways deregulated by genetic alterations may foster B cell activation and brisk proliferation, which correlated with the simultaneous MYC and BCL2 overexpression characteristic for PCNSL. On the genetic level, PCNSL are characterized by ongoing aberrant somatic hypermutation that, besides the IG locus, targets the PAX5, TTF, MYC, and PIM1 genes. Moreover, PCNSL cells show impaired IG class switch due to sµ region deletions, and PRDM1 mutations. Several important pathways, i.e., the B cell receptor (BCR), the toll-like receptor, and the nuclear factor-κB pathway, are activated frequently due to genetic changes affecting genes like CD79B, SHIP, CBL, BLNK, CARD11, MALT1, BCL2, and MYD88. These changes likely foster tumor cell survival. Nevertheless, many of these features are also present in subsets of systemic DLBLC and might not be the only reasons for the peculiar tropism of PCNSL. Here, preclinical animal models that closely mimic the clinical course and neuropathology of human PCNSL may provide further insight and we discuss recent advances in this field. Such models enable us to understand the pathogenetic interaction between the malignant B cells, resident cell populations of the CNS, and the associated inflammatory infiltrate. Indeed, the immunophenotype of the CNS as well as tumor cell characteristics and intracerebral interactions may create a micromilieu particularly conducive to PCNSL that may foster aggressiveness of tumor cells and accelerate the fatal course of disease. Suitable animal models may also serve as a well-defined preclinical system and may provide a useful tool for developing new specific therapeutic strategies.

Primary lymphoma of the central nervous system--a diagnostic challenge.

Primary lymphoma of the central nervous system is a distinct diffuse large B-cell lymphoma confined to the nervous system. Whereas classical cases can be classified easily, differential diagnosis can be a challenge in particular in patients who had received treatment prior to biopsy. In the differential diagnosis, other tumours and inflammatory diseases of autoimmune and infectious aetiology need to be considered.

Expression of Cas9 in a Syngeneic Model of Primary Central Nervous System Lymphoma Induces Intracerebral NK and CD8 T Cell-Mediated Lymphoma Cell Lysis Via Perforin.

The development of clustered regulatory interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR-Cas9)-mediated gene modification has opened an exciting avenue of targeting genes to study the pathogenesis of diseases and to develop novel therapeutic concepts. However, as the effector protein Cas9 is of bacterial origin, unwanted side effects due to a host immune response against Cas9 need to be considered. Here, we used the syngeneic model of BAL17CNS-induced primary lymphoma of the central nervous system (PCNSL, CNS) in BALB/c mice to address this issue. Surprisingly, stable expression of Cas9 in BAL17CNS (BAL17CNS/Cas9) cells rendered them unable to establish PCNSL on intracerebral transplantation. Instead, they induced a prominent intracerebral immune response mediated by CD8 T cells, which lysed BAL17CNS/Cas9 cells via perforin. In addition, B cells contributed to the immune response as evidenced by serum anti-Cas9 antibodies in BALB/c mice as early as day 8 after transplantation of BAL17CNS/Cas9 cells. In athymic BALB/cnu/nu mice, NK cells mounted a vigorous intracerebral immune response with perforin-mediated destruction of BAL17CNS/Cas9 cells. Thus, in the CNS, perforin produced by NK and CD8 T cells was identified as a mediator of cytotoxicity against BAL17CNS/Cas9 cells. These observations should be taken into account when considering therapeutic CRISPR-Cas9-mediated tumor cell manipulation for PCNSL.

COVID-19 Infection Induce miR-371a-3p Upregulation Resulting in Influence on Male Fertility.

In December 2019, the first case of COVID-19 was reported and since then several groups have already published that the virus can be present in the testis. To study the influence of SARS-CoV-2 which cause a dysregulation of the androgen receptor (AR) level, thereby leading to fertility problems and inducing germ cell testicular changes in patients after the infection. Formalin-Fixed-Paraffin-Embedded (FFPE) testicular samples from patients who died with or as a result of COVID-19 (n = 32) with controls (n = 6), inflammatory changes (n = 9), seminoma with/without metastasis (n = 11) compared with healthy biopsy samples (n = 3) were analyzed and compared via qRT-PCR for the expression of miR-371a-3p. An immunohistochemical analysis (IHC) and ELISA were performed in order to highlight the miR-371a-3p targeting the AR. Serum samples of patients with mild or severe COVID-19 symptoms (n = 34) were analyzed for miR-371a-3p expression. In 70% of the analyzed postmortem testicular tissue samples, a significant upregulation of the miR-371a-3p was detected, and 75% of the samples showed a reduced spermatogenesis. In serum samples, the upregulation of the miR-371a-3p was also detectable. The upregulation of the miR-371a-3p is responsible for the downregulation of the AR in SARS-CoV-2-positive patients, resulting in decreased spermatogenesis. Since the dysregulation of the AR is associated with infertility, further studies have to confirm if the identified dysregulation is regressive after a declining infection.

Radiomics for the noninvasive prediction of the BRAF mutation status in patients with melanoma brain metastases.

BACKGROUND: The BRAF V600E mutation is present in approximately 50% of patients with melanoma brain metastases and an important prerequisite for response to targeted therapies, particularly BRAF inhibitors. As heterogeneity in terms of BRAF mutation status may occur in melanoma patients, a wild-type extracranial primary tumor does not necessarily rule out a targetable mutation in brain metastases using BRAF inhibitors. We evaluated the potential of MRI radiomics for a noninvasive prediction of the intracranial BRAF mutation status. METHODS: Fifty-nine patients with melanoma brain metastases from two university brain tumor centers (group 1, 45 patients; group 2, 14 patients) underwent tumor resection with subsequent genetic analysis of the intracranial BRAF mutation status. Preoperative contrast-enhanced MRI was manually segmented and analyzed. Group 1 was used for model training and validation, group 2 for model testing. After radiomics feature extraction, a test-retest analysis was performed to identify robust features prior to feature selection. Finally, the best performing radiomics model was applied to the test data. Diagnostic performances were evaluated using receiver operating characteristic (ROC) analyses. RESULTS: Twenty-two of 45 patients (49%) in group 1, and 8 of 14 patients (57%) in group 2 had an intracranial BRAF V600E mutation. A linear support vector machine classifier using a six-parameter radiomics signature yielded an area under the ROC curve of 0.92 (sensitivity, 83%; specificity, 88%) in the test data. CONCLUSIONS: The developed radiomics classifier allows a noninvasive prediction of the intracranial BRAF V600E mutation status in patients with melanoma brain metastases with high diagnostic performance.

Impact of a Faulty Germinal Center Reaction on the Pathogenesis of Primary Diffuse Large B Cell Lymphoma of the Central Nervous System.

Primary lymphoma of the central nervous system (PCNSL, CNS) is a specific diffuse large B cell lymphoma (DLBCL) entity confined to the CNS. Key to its pathogenesis is a failure of B cell differentiation and a lack of appropriate control at differentiation stages before entrance and within the germinal center (GC). Self-/polyreactive B cells rescued from apoptosis by MYD88 and/or CD79B mutations accumulate a high load of somatic mutations in their rearranged immunoglobulin (IG) genes, with ongoing somatic hypermutation (SHM). Furthermore, the targeting of oncogenes by aberrant SHM (e.g., PIM1, PAX5, RHOH, MYC, BTG2, KLHL14, SUSD2), translocations of the IG and BCL6 genes, and genomic instability (e.g., gains of 18q21; losses of 9p21, 8q12, 6q21) occur in these cells in the course of their malignant transformation. Activated Toll-like receptor, B cell receptor (BCR), and NF-κB signaling pathways foster lymphoma cell proliferation. Hence, tumor cells are arrested in a late B cell differentiation stage, corresponding to late GC exit B cells, which are genetically related to IgM+ memory cells. Paradoxically, the GC reaction increases self-/polyreactivity, yielding increased tumor BCR reactivity for multiple CNS proteins, which likely contributes to CNS tropism of the lymphoma. The loss of MHC class I antigen expression supports tumor cell immune escape. Thus, specific and unique interactions of the tumor cells with resident CNS cells determine the hallmarks of PCNSL.

An Autochthonous Mouse Model of Myd88- and BCL2-Driven Diffuse Large B-cell Lymphoma Reveals Actionable Molecular Vulnerabilities.

Based on gene expression profiles, diffuse large B cell lymphoma (DLBCL) is sub-divided into germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL. Two of the most common genomic aberrations in ABC-DLBCL are mutations in MYD88, as well as BCL2 copy number gains. Here, we employ immune phenotyping, RNA-Seq and whole exome sequencing to characterize a Myd88 and Bcl2-driven mouse model of ABC-DLBCL. We show that this model resembles features of human ABC-DLBCL. We further demonstrate an actionable dependence of our murine ABC-DLBCL model on BCL2. This BCL2 dependence was also detectable in human ABC-DLBCL cell lines. Moreover, human ABC-DLBCLs displayed increased PD-L1 expression, compared to GCB-DLBCL. In vivo experiments in our ABC-DLBCL model showed that combined venetoclax and RMP1-14 significantly increased the overall survival of lymphoma bearing animals, indicating that this combination may be a viable option for selected human ABC-DLBCL cases harboring MYD88 and BCL2 aberrations.

Dual role of B cells with accelerated onset but reduced disease activity in P0106₋125-induced experimental autoimmune neuritis of IgH °(/)° mice.

The role of B cells in autoimmune-mediated diseases of the peripheral nervous system was studied in experimental autoimmune neuritis (EAN) in B cell deficient IgH°(/)° C57BL/6J mice having been immunized with P0106₋125 peptide. Compared to coisogenic IgH(+/+) mice, onset of EAN was accelerated [100% disease incidence at day 9 post immunization (p.i.) vs. day 15 p.i.]. At day 9 p.i., numbers of P0106₋125-specific interferon (IFN)-γ-producing CD4(+) T cells were increased, while IL-10 mRNA and production were decreased in IgH°(/)° mice. Beyond day 9 p.i., declining disease activity and a significant reduction of maximal disease activity were correlated with significantly reduced numbers of IFN-γ-producing CD4(+) T cells in IgH(0/0) mice as compared with IgH(+/+) mice. Correspondingly, neuropathology demonstrated only mild axonal damage, while demyelination and dying back axonopathy with spinal cord motor neuron apoptosis were absent. Thus, depending on the stage of EAN, B cells play a dual, i.e. suppressive and enhancing, role during induction and at height of EAN, respectively. The combined interaction of B cells as well as CD4(+) and CD8(+) T cells is required for the development of EAN.

Mutations of CARD11 but not TNFAIP3 may activate the NF-kappaB 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)-kappaB pathway. In order to identify potential activators of the NF-kappaB 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-kappaB, 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-kappaB 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.