Deregulation of ETS1 and FLI1 contributes to the pathogenesis of diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common form of human lymphoma. DLBCL is a heterogeneous disease characterized by different genetic lesions. We herein report the functional characterization of a recurrent gain mapping on chromosome 11q24.3, found in 23% of 166 DLBCL cases analyzed. The transcription factors ETS1 and FLI1, located within the 11q24.3 region, had significantly higher expression in clinical samples carrying the gain. Functional studies on cell lines showed that ETS1 and FLI1 cooperate in sustaining DLBCL proliferation and viability and regulate genes involved in germinal center differentiation. Taken together, these data identify the 11q24.3 gain as a recurrent lesion in DLBCL leading to ETS1 and FLI1 deregulated expression, which can contribute to the pathogenesis of this disease.

Mutations of multiple genes cause deregulation of NF-kappaB in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL), the most common form of lymphoma in adulthood, comprises multiple biologically and clinically distinct subtypes including germinal centre B-cell-like (GCB) and activated B-cell-like (ABC) DLBCL. Gene expression profile studies have shown that its most aggressive subtype, ABC-DLBCL, is associated with constitutive activation of the NF-kappaB transcription complex. However, except for a small fraction of cases, it remains unclear whether NF-kappaB activation in these tumours represents an intrinsic program of the tumour cell of origin or a pathogenetic event. Here we show that >50% of ABC-DLBCL and a smaller fraction of GCB-DLBCL carry somatic mutations in multiple genes, including negative (TNFAIP3, also called A20) and positive (CARD11, TRAF2, TRAF5, MAP3K7 (TAK1) and TNFRSF11A (RANK)) regulators of NF-kappaB. Of these, the A20 gene, which encodes a ubiquitin-modifying enzyme involved in termination of NF-kappaB responses, is most commonly affected, with approximately 30% of patients displaying biallelic inactivation by mutations and/or deletions. When reintroduced in cell lines carrying biallelic inactivation of the gene, A20 induced apoptosis and cell growth arrest, indicating a tumour suppressor role. Less frequently, missense mutations of TRAF2 and CARD11 produce molecules with significantly enhanced ability to activate NF-kappaB. Thus, our results demonstrate that NF-kappaB activation in DLBCL is caused by genetic lesions affecting multiple genes, the loss or activation of which may promote lymphomagenesis by leading to abnormally prolonged NF-kappaB responses.

Diffuse large B-cell lymphoma with concordant bone marrow involvement has peculiar genomic profile and poor clinical outcome.

Bone marrow (BM) involvement in diffuse large B-cell lymphoma (DLBCL) can be morphologically discordant from the primary tumor. Concordant BM infiltration has been shown associated with a poorer outcome in patients treated with CHOP. In order to evaluate tumor-related factors leading to BM involvement in DLBCL, we performed an integrated analysis of i) genomic profiles obtained with a high-density genome wide SNP-based arrays ii) immunomorphological and iii) clinical data from 133 patients uniformly treated with R-CHOP. BM infiltration was found in 27 of 133 (20%) cases; and it was concordant in 18/27 (67%) cases. Concordant infiltration, but not discordant, influenced negatively OS, PFS and DFS and was associated with higher serum LDH, lower CR and higher PD rates. No association with cell of origin was found between BM+ and BM- DLBCL. As compared with BM- cases, BM+ DLBCL showed absence of 7q gain.

Expression of proinflammatory and regulatory cytokines via NF-κB and MAPK-dependent and IFN regulatory factor-3-independent mechanisms in human primary monocytes infected by Mycobacterium tuberculosis.

Knowledge of the molecular events regulating the innate response to Mycobacterium tuberculosis (Mtb) is critical for understanding immunological pathogenesis and protection from tuberculosis. To this aim, the regulation and the expression of regulatory and proinflammatory cytokines were investigated in human primary monocytes upon Mtb infection. We found that Mtb-infected monocytes preferentially express a proinflammatory cytokine profile, including IL-6, TNF-α, and IL-1ß. Conversely, among the regulatory cytokines, Mtb elicited IL-10 and IL-23 release while no expression of IL-12p70, IL-27, and IFN-ß was observed. The analysis of the signalling pathways leading to this selective cytokine expression showed that in monocytes Mtb activates MAPK and NF-κB but is unable to stimulate IRF-3 phosphorylation, a transcription factor required for IL-12p35 and IFN-ß gene expression. Thus, by inducing a specific cytokine profile, Mtb can influence the immunoregulatory properties of monocytes, which represent important target of novel vaccinal strategies against Mtb infection.

Single nucleotide polymorphism-arrays provide new insights in the pathogenesis of post-transplant diffuse large B-cell lymphoma.

Post-transplant lymphoproliferative disorders (PTLD) are complications of solid organ transplantation associated with severe morbidity and mortality. Diffuse large B-cell lymphoma (DLBCL) represents the most common form of monomorphic PTLD. We studied 44 cases of post-transplant DLBCL (PT-DLBCL) with high-density genome wide single nucleotide polymorphism-based arrays, and compared them with 105 cases of immunocompetent DLBCL (IC-DLBCL) and 28 cases of Human Immunodeficiency Virus-associated DLBCL (HIV-DLBCL). PT-DLBCL showed a genomic profile with specific features, although their genomic complexity was overall similar to that observed in IC- and HIV-DLBCL. Among the loci more frequently deleted in PT-DLBCL there were small interstitial deletions targeting known fragile sites, such as FRA1B, FRA2E and FRA3B. Deletions at 2p16.1 (FRA2E) were the most common lesions in PT-DLBCL, occurring at a frequency that was significantly higher than in IC-DLBCL. Genetic lesions that characterized post-germinal center IC-DLBCL were under-represented in our series of PT-DLBCL. Two other differences between IC-DLBCL and PT-DLBCL were the lack of del(13q14.3) (MIR15/MIR16) and of copy neutral LOH affecting 6p [major histocompatibility complex (MHC) locus] in the latter group. In conclusion, PT-DLBCL presented unique features when compared with IC-DLBCL. Changes in PT-DLBCL were partially different to those in HIV-DLBCL, suggesting different pathogenetic mechanisms in the two conditions linked to immunodeficiency.

Genomic lesions associated with a different clinical outcome in diffuse large B-Cell lymphoma treated with R-CHOP-21.

Despite recent therapeutic improvements, the clinical course of diffuse large B-cell lymphoma (DLBCL) still differs considerably among patients. We conducted this retrospective multi-centre study to evaluate the impact of genomic aberrations detected using a high-density genome wide-single nucleotide polymorphism-based array on clinical outcome in a population of DLBCL patients treated with R-CHOP-21 (rituximab, cyclophosphamide, doxorubicine, vincristine and prednisone repeated every 21 d). 166 DNA samples were analysed using the GeneChip Human Mapping 250K NspI. Genomic anomalies were analysed regarding their impact on the clinical course of 124 patients treated with R-CHOP-21. Unsupervised clustering was performed to identify genetically related subgroups of patients with different clinical outcomes. Twenty recurrent genetic lesions showed an impact on the clinical course. Loss of genomic material at 8p23.1 showed the strongest statistical significance and was associated with additional aberrations, such as 17p- and 15q-. Unsupervised clustering identified five DLBCL clusters with distinct genetic profiles, clinical characteristics and outcomes. Genetic features and clusters, associated with a different outcome in patients treated with R-CHOP, have been identified by arrayCGH.

Genome wide DNA-profiling of HIV-related B-cell lymphomas.

Non-Hodgkin lymphomas (NHL) represent a frequent complication of human immunodeficiency virus (HIV) infection. To elucidate HIV-NHL pathogenesis, we performed a genome-wide DNA profiling based on a single nucleotide polymorphism-based microarray comparative genomic hybridization in 57 HIV-lymphomas and, for comparison, in 105 immunocompetent diffuse large B-cell lymphomas (IC-DLBCL). Genomic complexity varied across HIV-NHL subtypes. HIV-Burkitt lymphoma showed a significantly lower number of lesions than HIV-DLBCL (P = 0.032), whereas the median number of copy number changes was significantly higher in Epstein-Barr virus negative (EBV-) HIV-DLBCL (42.5, range 8-153) compared to EBV+ cases (22; range 3-41; P = 0.029). Compared to IC-DLBCL, HIV-DLBCL displayed a distinct genomic profile with no gains of 18q and specific genetic lesions. Fragile sites-associated genes, including FHIT (FRA3B), WWOX (FRA16D), DCC (FRA18B) and PARK2 (FRA6E) were frequently inactivated in HIV-NHL by interstitial deletions, and a significantly higher prevalence of FHIT alterations was observed in HIV-DLBCL compared to IC-DLBCL. The same genes involved by fragile site deletions were also frequently affected by aberrant methylation of regulative regions.

Genomic profiling of Richter's syndrome: recurrent lesions and differences with de novo diffuse large B-cell lymphomas.

Richter's syndrome (RS) represents the transformation of chronic lymphocytic leukaemia (CLL) to aggressive lymphoma and is mostly represented by diffuse large B-cell lymphoma (DLBCL), with a post-germinal centre (GC) phenotype, clonally related to the pre-existing CLL. RS has a very poor prognosis and its pathogenetic mechanisms are poorly understood. In order to gain additional hints in RS pathogenesis, we performed a genome-wide DNA profiling study of 13 RS phases and eight matched CLL phases using the Affymetrix Human Mapping 250K NspI SNP arrays. Individual genomic profiles were heterogeneous, with no individual lesions occurring in more than half of the cases. However, several observations suggest that MYC pathway might be involved in RS. The 13q13.3-qter region containing MIRHG1 (MIR-17-92), a cluster of microRNA interacting with c-MYC, was acquired at the time of transformation. The 13q gain was coupled with the gain of c-MYC and loss of TP53. Translocation of c-MYC was acquired at transformation in a fraction of cases and this event appeared mutually exclusive with gain of MIRHG1. MYCN, a c-MYC homologue, was also recurrently gained. By comparing RS with 48 de novo DLBCL, RS presented a significantly lower prevalence of deletions affecting the PRDM1 and TNFAIP3, genes on 6q, known to be associated with a post-GC phenotype. In conclusion, the genomic profile of RS seems to differ from what observed in de novo DLBCL and in other transformed DLBCL. Genomic lesions occurring in RS are heterogeneous suggesting the existence of different RS subsets, possibly due to different transforming mechanisms. A deregulation of MYC pathway might represent one of the main transformation events in the pathogenesis of a subset of RS clonally related to the previous CLL.

Genome-wide DNA analysis identifies recurrent imbalances predicting outcome in chronic lymphocytic leukaemia with 17p deletion.

Deletion of 17p (TP53) identifies a rare subset of chronic lymphocytic leukaemia (17p- CLL) with aggressive behaviour. Genome-wide DNA-profiling was performed to investigate 18 patients with 17p- CLL. All cases had multiple copy-number (CN) changes. Among the several recurrent CN changes identified, 8q24.13-q24.1-gain (MYC), 8p-loss (TNFRSF10A/B, also known as TRAIL1/2) and 2p16.1-p14-gain (REL/BCL11A) appeared frequently represented. 8p-loss and 2p16.1-p14-gain also appeared clinically relevant and predicted significant shorter time from diagnosis to treatment (8p-loss) and overall survival (8p-loss and 2p16.1-p14-gain, P < 0.05). These observations document a highly unstable genome in 17p- CLL and suggest that additional genes outside the TP53 locus may be important for tumour behaviour.

Effect of titanium carbide coating on the osseointegration response in vitro and in vivo.

Titanium has limitations in its clinical performance in dental and orthopaedic applications. This study describes a coating process using pulsed laser deposition (PLD) technology to produce surfaces of titanium carbide (TiC) on titanium substrates and evaluates the biological response both in vitro and in vivo. X-ray photoelectron spectroscopy (XPS) analysis revealed the presence of 18.6-21.5% TiC in the surface layer, accompanied by oxides of titanium 78.5-81.4% in the following concentrations: 11.1-13.0% Ti(2)O(3), 50.8-55.8% TiO(2), 14.5-14.7% TiO. Expression of genes central to osteoblast differentiation (alkaline phosphatase, A2 pro-collagen type 1, osteocalcin, BMP-4, TGFbeta and Cbfa-1) were up-regulated in all cell lines (primary human osteoblasts, hFOB1.19 and ROS.MER#14) grown on TiC compared with uncoated titanium when measured by semiquantitative PCR and real time-PCR, whilst genes involved in modulation of osteoclastogenesis and osteoclast activity (IL-6 and M-CSF) were unchanged. Bone density was shown to be greater around TiC-coated implants after 2 and 4 weeks in sheep and both 4 and 8 weeks in rabbits compared to uncoated titanium. Rapid bone deposition was demonstrated after only 2 weeks in the rabbit model when visualized with intravital staining. It is concluded that coating with TiC will, in comparison to uncoated titanium, improve implant hardness, biocompatibility through surface stability and osseointegration through improved bone growth.

BCL2, BCL6, MYC, MALT 1, and BCL10 rearrangements in nodal diffuse large B-cell lymphomas: a multicenter evaluation of a new set of fluorescent in situ hybridization probes and correlation with clinical outcome.

Diffuse large B-cell lymphoma is the most common type of non-Hodgkin lymphoma. Although it is a curable disease, fewer than half of patients are cured with conventional chemotherapy. The highly variable outcome reflects a heterogeneous group of tumors, with different genetic abnormalities and responses to therapy. We analyzed 74 cases of diffuse large B-cell lymphoma using interphase fluorescent in situ hybridization with commercially available probes for split-signal targeting BCL-2, BCL-6, MYC, BCL-10, and MALT-1. Gene rearrangements were identified in 48 (65%) of 74 cases. BCL-6 was the most rearranged gene (45%), followed by BCL-2 (21%), BCL-10 (18%), and MYC (16%). No MALT-1 rearrangements were found. When diffuse large B-cell lymphoma cases were subdivided into germinal-center B-cell-like and activated B-cell-like groups, an inverse pattern of BCL-2 and BCL-6 rearrangements was observed. Of interest, the presence of chromosome rearrangements was associated with a worse prognosis. The pattern of cytogenetic abnormalities highlighted the fact not only that diffuse large B-cell lymphoma is a heterogeneous entity but also that even individual cases may contain subclones bearing different chromosomal rearrangements. The relevance and the clinical implication of minor clones showing gene rearrangements are poorly understood; however, this first observation suggests that different rearrangements may be involved in the progression of the disease. The fluorescent in situ hybridization analysis with the panel used in this study is useful to detect the heterogeneity of diffuse large B-cell lymphomas and identify alterations with prognostic implications.