Conditional expression of HGAL leads to the development of diffuse large B-cell lymphoma in mice

Diffuse large B-cell lymphomas (DLBCLs) are clinically and genetically heterogeneous tumors. Deregulation of diverse biological processes specific to B cells, such as B-cell receptor (BCR) signaling and motility regulation, contribute to lymphomagenesis. Human germinal center associated lymphoma (HGAL) is a B-cell–specific adaptor protein controlling BCR signaling and B lymphocyte motility. In normal B cells, it is expressed in germinal center (GC) B lymphocytes and promptly downregulated upon further differentiation. The majority of DLBCL tumors, primarily GC B-cell types, but also activated types, express HGAL. To investigate the consequences of constitutive expression of HGAL in vivo, we generated mice that conditionally express human HGAL at different stages of hematopoietic development using 3 restricted Cre-mediated approaches to initiate expression of HGAL in hematopoietic stem cells, pro-B cells, or GC B cells. Following immune stimulation, we observed larger GCs in mice in which HGAL expression was initiated in GC B cells. All 3 mouse strains developed DLBCL at a frequency of 12% to 30% starting at age 13 months, leading to shorter survival. Immunohistochemical studies showed that all analyzed tumors were of the GC B-cell type. Exon sequencing revealed mutations reported in human DLBCL. Our data demonstrate that constitutive enforced expression of HGAL leads to DLBCL development

Prognostic Significance of MYC Rearrangement and Translocation Partner in Diffuse Large B-Cell Lymphoma : A Study by the Lunenburg Lymphoma Biomarker Consortium

PURPOSE: MYC rearrangement (MYC-R) occurs in approximately 10% of diffuse large B-cell lymphomas (DLBCLs) and has been associated with poor prognosis in many studies. The impact of MYC-R on prognosis may be influenced by the MYC partner gene (immunoglobulin [IG] or a non-IG gene). We evaluated a large cohort of patients through the Lunenburg Lymphoma Biomarker Consortium to validate the prognostic significance of MYC-R (single-, double-, and triple-hit status) in DLBCL within the context of the MYC partner gene. METHODS: The study cohort included patients with histologically confirmed DLBCL morphology derived from large prospective trials and patient registries in Europe and North America who were uniformly treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone therapy or the like. Fluorescence in situ hybridization for the MYC, BCL2, BCL6, and IG heavy and light chain loci was used, and results were correlated with clinical outcomes. RESULTS: A total of 5,117 patients were identified of whom 2,383 (47%) had biopsy material available to assess for MYC-R. MYC-R was present in 264 (11%) of 2,383 patients and was associated with a significantly shorter progression-free and overall survival, with a strong time-dependent effect within the first 24 months after diagnosis. The adverse prognostic impact of MYC-R was only evident in patients with a concurrent rearrangement of BCL2 and/or BCL6 and an IG partner (hazard ratio, 2.4; 95% CI, 1.6 to 3.6; P < .001). CONCLUSION: The negative prognostic impact of MYC-R in DLBCL is largely observed in patients with MYC double hit/triple-hit disease in which MYC is translocated to an IG partner, and this effect is restricted to the first 2 years after diagnosis. Our results suggest that diagnostic strategies should be adopted to identify this high-risk cohort, and risk-adjusted therapeutic approaches should be refined further

Gray zones around diffuse large B cell lymphoma. Conclusions based on the workshop of the XIV meeting of the European Association for Hematopathology and the Society of Hematopathology in Bordeaux, France

The term “gray-zone” lymphoma has been used to denote a group of lymphomas with overlapping histological, biological, and clinical features between various types of lymphomas. It has been used in the context of Hodgkin lymphomas (HL) and non-Hodgkin lymphomas (NHL), including classical HL (CHL), and primary mediastinal large B cell lymphoma, cases with overlapping features between nodular lymphocyte predominant Hodgkin lymphoma and T-cell/histiocyte-rich large B cell lymphoma, CHL, and Epstein–Barr-virus-positive lymphoproliferative disorders, and peripheral T cell lymphomas simulating CHL. A second group of gray-zone lymphomas includes B cell NHL with intermediate features between diffuse large B cell lymphoma and classical Burkitt lymphoma. In order to review controversial issues in gray-zone lymphomas, a joint Workshop of the European Association for Hematopathology and the Society for Hematopathology was held in Bordeaux, France, in September 2008. The panel members reviewed and discussed 145 submitted cases and reached consensus diagnoses. This Workshop summary is focused on the most controversial aspects of gray-zone lymphomas and describes the panel’s proposals regarding diagnostic criteria, terminology, and new prognostic and diagnostic parameters

Preclinical Activity of Brentuximab Vedotin (SGN-35) in Primary Effusion Lymphoma (PEL)

Abstract Abstract 3728 Primary effusion lymphoma (PEL) is a distinct and aggressive subtype of non-Hodgkin lymphoma (NHL) commonly presenting with pleural, peritoneal, or pericardial malignant effusions usually without a contiguous tumor mass. PEL is most commonly diagnosed in HIV-positive patients, accounting for 4% of all NHLs in this population, yet may also develop in immunosuppressed HIV-negative individuals. While Human Herpes Virus 8 (HHV8 or Kaposi's sarcoma-associated herpesvirus) is directly implicated in the oncogenesis of this lymphoma, most PEL cases are also associated with Epstein-Barr virus and the combination of the two may facilitate transformation. The tumor cells exhibit plasmablastic features and express CD45, CD38, CD138, HHV8 and CD30. PEL is an aggressive tumor characterized by a short median survival of only 6 months with current therapeutic approaches underscoring the urgent need for development of new therapeutics. Brentuximab vedotin (SGN-35) is an antibody-drug conjugate (ADC) comprised of an anti-CD30 monoclonal antibody cAC10 conjugated by a protease-cleavable dipeptide linker to a potent cell killing agent monomethyl auristatin E (MMAE). Following binding to CD30, brentuximab vedotin is rapidly internalized and is transported to lysosomes, where the peptide linker is selectively cleaved allowing binding of the released MMAE to tubulin and leading to cell cycle arrest and apoptosis. Brentuximab vedotin was recently reported to have promising antitumor activity in CD30 expressing tumors, such as Hodgkin and Anaplastic large cell lymphomas. Since PEL tumors are reported to express CD30, we have hypothesized that brentuximab vedotin might be effective in the treatment of this NHL subtype. Initially, we have confirmed by flow cytometry the expression of CD30 on PEL cell lines (UM-PEL 1, UM-PEL 3, BC-1 and BC-3), and by review of immunohistochemistry and flow cytometry results in patients with previous diagnosis of PEL at our institution. To examine in vitro potency of brentuximab vedotin, UM-PEL 1, UM-PEL 3, BC-1 and BC-3 PEL cell lines were treated with brentuximab vedotin at concentration ranging from 0–100 micrograms/ml. Staining with YO-PRO and Propidium Iodide (PI) demonstrated dose dependent cell apoptosis and death in all the cell lines at 72 hours post treatment. In contrast, control IgG conjugated with MMAE failed to induce apoptosis and cell death of PEL cell lines confirming specific brentuximab vedotin cytotoxicity. Furthermore, brentuximab vedotin decreased proliferation of PEL cells at 48 hours leading to a complete proliferation arrest at 72 hours, as measured by MTS assay. These effects were absent after equivalent doses of control IgG conjugated drug treatment. Supportive to this, labeling of cells with PI to detect active DNA content by flow cytometry showed that bretuximab vedotin induced growth arrest in G2/M phase. To further establish the anti-tumor potential of brentuximab vedotin in vivo, we used the direct xenograft UM-PEL 1 model, established in our laboratory (Sarosiek, PNAS 2010), which mimics human PEL tumors. UM-PEL 1 bearing mice were injected intraperitoneally 3 times a week with brentuximab vedotin or control IgG conjugated MMAE for 4 weeks. Brentuximab vedotin treatment markedly prolonged overall survival of UM-PEL-1 bearing mice compared to controls (p Disclosures: No relevant conflicts of interest to declare

Human Germinal Center-Associated Lymphoma (HGAL) Protein Expression Is Associated with Improved Failure-Free Survival in Brazilian Patients with Classical Hodgkin Lymphoma

Abstract Abstract 4632 BACKGROUND The HGAL gene has prognostic value in diffuse large B-cell lymphoma and expression of its cognate protein is germinal center–specific. A previous study had suggested that HGAL protein expression might also be related to outcome in patients with Hodgkin lymphoma. PATIENTS AND METHODS The aim of the current study was to confirm the prognostic impact of HGAL protein expression in an independent, well-characterized cohort of 232 classic Hodgkin lymphoma patients treated uniformly with the ABVD regimen from 1997 to 2004 in Brazil. The median follow-up was 6.2 years. RESULTS Tissue microarray analysis showed HGAL staining in 188 specimens (81%). The overall survival was better in patients with young age, early-stage, absence of B symptoms, low-risk international prognostic score (IPS) and good performance status. Failure-free survival was superior in patients with early-stage disease, low-risk IPS and HGAL-positive patients. The estimated 5-year failure-free survival for HGAL-positive and HGAL-negative patients was 82% and 67%, respectively (P=0.03). When stage, B symptoms and performance status were included along with HGAL in a multivariate analysis, advanced stage and absence of HGAL staining were independent predictors of a worse failure-free survival. CONCLUSION This study confirms and validates recent findings of a correlation between HGAL expression and outcome in classical Hodgkin lymphoma. Disclosures: No relevant conflicts of interest to declare

MicroRNA Are Useful Biomarkers for Prediction of Response to Therapy and Survival of Patients with Diffuse Large B-Cell Lymphoma

Abstract Abstract 624 Background: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease with a variable clinical outcome even in the current era of rituximab containing anthracycline-based chemotherapy (RCHOP). We have previously reported a 6 gene model which is predictive for outcome in DLBCL (Lossos et al NEJM 2004, 350:1829 and Malumbres et al Blood 2008, 111:5509). Recently, short non-protein coding microRNAs (miRNA) that regulate gene expression by targeting the 3'UTR region of mRNA have been identified and are postulated to play an important role in oncogenesis. We have previously demonstrated that miRNAs exhibit specific expressions at different lymphocyte differentiation stages as well as distinct expression in gene expression-defined DLBCL subtypes (Malumbres et al Blood 2009; 113:3754). Herein, we evaluated whether specific miRNAs differentially expressed across DLBCL cell lines were prognostic biomarkers for prediction of outcome of DLBCL patients (pts). Patients and Methods: RNA was extracted from paraffin-embedded archived specimens (Chen et al Diagn Mol Pathol 2007, 16:61), from pts with newly diagnosed de novo DLBCL treated with R-CHOP at 4 different institutions. Expression of 11 miRNAs, including miR-146a, miR-146b-5p, miR-222, miR-500, miR-574-3p, miR-363, miR-155, and miR-21 that are differentially expressed between the ABC and GCB DLBCL subtypes, as well as expression of miR-18a, miR-140-3p, and miR-181a which has been reported to be variably expressed across DLBCL tumors were analyzed by ABI real-time PCR assays. Expression of the genes comprising the 6 gene survival prediction model were measured as reported previously. Expression was correlated to progression-free survival (PFS) and overall survival (OS). Predictive value of miRNAs was evaluated both as a continuous variable as well as a in a dichotomous model with pts grouped based on miRNA median expression. Results: The study group consisted of 176 pts with a median age 59 years (y) (range 16-92) of which 84 pts (48%) were > 60 y. 54 pts (30%) had an ECOG PS ≥2 and 86 (49%) presented with stage III or IV. Distribution according to the IPI was: 0-1 factor (n=77); 2 factors (n=50); 3 factors (n=30); and ≥4 factors (n=19). There were 41 deaths during a median follow-up of 2.6 years (range 0.04-8.1y). OS correlated with expression of miR-18a as a continuous variable, with higher expression correlating with inferior OS (p=0.038) but was not independent of the IPI in a multivariate model. PFS correlated with expression of miR-181a as a continuous variable (p=0.026; higher expression associated with longer PFS) and with expression of miR-222 as a categorical variable (miR-222cat; p=0.004, lower expression associated with longer PFS). In a multivariable model including IPI, both miR-222cat and miR-181a were IPI independent (p=0.01 and p=0.003, respectively). The mortality-prediction score calculated from the 6-gene model predicted both the OS (p=0.007) and PFS (p=0.004) and was IPI-independent. A multivariate Cox regression analysis that included IPI scores, mortality predictor scores and expression of miR-18a, miR-181a and miR-222cat revealed that all factors except miR-222cat were independent predictors of OS and all factors except miR-18a were independent predictors of PFS. Conclusions: We confirm that in RCHOP treated pts with DLBCL the 6-gene model predicts both OS and PFS independent of the IPI. Expression of miRNAs miR-18a, miR-181a and miR-222 is associated with response to therapy and outcome of pts with DLBCL, independent of the IPI and the mortality-prediction score calculated from the 6-gene model. While the expression of miR-181a and miR-222 may reflect the cell of origin of DLBCL tumors, the expression of miR-18a does not. Further studies are warranted to evaluate the precise role of these miRNAs in B-cell biology and DLBCL pathogenesis and prognosis. Disclosures: Advani: Seattle Genetics, Inc.: Research Funding

Paraffin-Based 6-Gene Model Predicts Outcome of Diffuse Large B-Cell Lymphoma Patients Treated with R-CHOP

Abstract Background: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease characterized by highly variable clinical outcomes. It is therefore of paramount importance to be able to predict the outcome of patients at the time of diagnosis. Previously, we constructed a 6 gene model for outcome prediction of DLBCL patients treated with anthracycline-based chemotherapies (Lossos et al NEJM 2004, 350:1829). However, the standard therapy has evolved to rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone (R-CHOP). Subset analyses of clinical trials have suggested that some of the prognostic factors lose their predictive power in R-CHOP treated patients. Consequently, the molecular gold standard for survival prediction in R-CHOP treated patients has not been established. Herein, we evaluated the predictive power of the 6-gene model in R-CHOP treated DLBCL patients. We have employed new methodology that allows quantitation of gene expression from paraffin embedded, fixed tissues. Methods: RNA was extracted from 100 paraffin-embedded specimens (Chen et al Diagn Mol Pathol 2007, 16:61), from patients with DLBCL treated with R-CHOP in British Columbia (73) and at the University of Miami (27). Expression of the 6 genes comprising the model was measured in these samples and the mortality-prediction score was calculated for each patient, as reported previously. Results: The study group consisted of 100 patients with a median age of 58 y (range, 16–92y) that were followed for a median of 2.1 years (range, 0.1–5.6). Distribution according to IPI: 0–1 factor, 42; 2 factors, 24; 3 factors, 19; and ≥4 factors, 14. RNA of sufficient quality and quantity was successfully extracted from all the 100 paraffin embedded specimens tested, some of which had been stored for up to 6 years. The mortality-prediction score derived from the model divided patients into low-risk (50%) and high-risk (50%) subgroups with significantly different overall survival (OS) (p=0.02) and progression free survival (PFS) (p=0.02). Notably the OS was similar between the groups during the 1st year, due to the inclusion of patients with advanced age and poor performance status, but it was markedly different beyond the first 2 years, with the 3 year OS of 80% and 50% in the low risk and high risk groups, respectively. The predictive power of the 6-gene model was independent of the IPI prognostic factors for prediction of OS (P=0.06) and PFS (p=0.01) in these patients. Conclusions: The prognostic value of the 6-gene model remains significant in the era of R-CHOP treatment. Further, using the new RNA extraction methodology, we demonstrate that the model can be applied to routinely available formalin-fixed paraffin blocks from initial diagnostic biopsies, even after long term storage. Following validation in an independent cohort of patients, the six gene model may be practically applied in routine clinical practice