Biology Of Mantle Cell Lymphoma Research Articles

CD31/PECAM-1 Is Overexpressed On Mantle-Cell Lymphoma (MCL) Primary Cells and Positively Regulates Engraftment and Growth of MCL Cell Lines in Immunodeficient Mice.

AbstractAbstract 2681Mantle-cell lymphoma (MCL) is a lymphoma subtype with poor prognosis. Recently, new drugs targeting at least partially the angiogenesis cascade have been successfully tested in the therapy of relapsed MCL patients, including temsirolimus, enzastaurin and lenalidomide. To reveal molecular mechanisms that regulate MCL-induced angiogenesis (and that might represent potential new druggable targets), we established and analyzed two mouse models of human MCL. Immunodeficient mice were subcutaneously (s.c.) xenografted with MCL cell lines JEKO-1 and HBL-2, and when tumor diameters reached 3cm in any size, the mice were sacrificed, and the excized tumors subjected to immunohistochemical (IHC) analysis. Alternatively, ex vivo obtained MCL cells were magnetically sorted using CD45 microbeads, and subjected to gene expression and flow cytometry analyses compared to the in vitro growing controls.IHC analysis proved that the tumors were neovascularized. Gene expression profiling by TaqMan Human Angiogenesis Array revealed that the most upregulated gene in both JEKO-1 and HBL-2 in vivo vs. in vitro growing cells was platelet/endothelial cell adhesion molecule CD31/PECAM-1 (fold change 148.9 ± 19.4, and 127.6 ± 10.5, respectively). Recently, Boyd et al. reported upregulation of CD31 in three of five primary MCL samples compared to normal B cells by Western blotting. By flow cytometry we assessed surface expression of CD31 on primary cells obtained from peripheral blood and bone marrow of 25 MCL patients before therapy. The percentage of CD31 positive cells was significantly higher in the subset of CD19+CD5+ peripheral blood MCL cells compared to CD19+CD5+ healthy donor B-cells (78.6± 4.4 vs. 14.7±2.6, p< 0.001), as well as in the subset of CD19+CD5+ bone marrow MCL cells compared to CD19+CD5+ B-cells obtained from the bone marrow of patients with various lymphoma subtypes with no detectable bone marrow involvement (81.9±3.9 vs. 20.5±4.8, p< 0.0001).In addition to surface CD31 we asked if patients with MCL have increased levels of soluble form of CD31 (sCD31) compared to healthy volunteers. We measured concentrations of sCD31 in plasma samples obtained from 17 MCL patients before therapy by ELISA. Despite the fact that MCL patients demonstrated higher variability in sCD31 concentrations (range 10.7–135.6 ng/ml) compared to healthy volunteers (range 43.3– 92.0 ng/ml), the medians were not statistically different (56.4 vs. 53.3 ng/ml). We also measured concentrations of sCD31 in plasma of two patients with MCL before treatment and after three cycles of chemotherapy, and found that the post-chemotherapy levels of sCD31 were lower in both patients (91.8 vs. 60.7 ng/ml; 56.4 vs. 30.4 ng/ml).To investigate the role of CD31 in the biology of MCL we derived JEKO-1 and HBL-2 clones with stable downregulation of CD31 by siRNA approach. In addition, two JEKO-1 subclones with upregulated CD31 were established by limiting dilution from the original cell line (=controls). The limiting dilution approach was not feasible in HBL-2 cells, which completely lack CD31 expression. While HBL-2 clones with downregulated CD31 engrafted constantly, JEKO-1 clones with downregulated CD31 engrafted only in 2 out of 8 mice. Growth of the tumors derived from both HBL-2 and JEKO-1 clones with downregulated CD31 was significantly slower compared to that of control tumors (HBL-2: 2.1±0.4 g versus 4.1±0.2 g, p=0.002; JEKO-1: 0.5±0.1 g versus 2.2±0.2 g, p=0.0019). Xenotransplantation of JEKO-1 clones with upregulated CD31 resulted in accelerated tumor growth compared to controls (3.4±0.2 g versus 2.2±0.2 g, p=0.0027). Importantly, the in vitro proliferation rate between the clones with changed CD31 expression and the original cell lines were not statistically different suggesting that the different growth pattern of tumors was a consequence of altered interaction between the tumor cells and the murine microenvironment.In summary, CD31/PECAM-1 antigen is overexpressed on primary CD19+CD5+ MCL cells obtained from the peripheral blood and bone marrow of MCL patients before therapy compared to control CD19+CD5+ B-cells. The upregulated CD31/PECAM-1 appears to play important role in MCL biology, and might represent potential druggable target.Financial Support: IGA-MZ NT13201-4/2012, GAUK 259211/110709, GAUK 446211, UNCE 204021, PRVOUK P24/LF1/3, PRVOUK 1–5101–280002 PVK, SVV-2012–254260507 Disclosures:No relevant conflicts of interest to declare.

Management of Mantle Cell Lymphoma: Key Challenges and Next Steps

Mantle cell lymphoma (MCL) is regarded as an aggressive lymphoid malignancy that exhibits varied clinical behavior and prognoses, reflecting the biologic heterogeneity of the disease. In most cases, patients with MCL achieve a shorter median survival compared with more common B-cell lymphomas, such as follicular lymphoma, and are less likely to achieve a durable response with chemotherapy. Currently, there is no defined standard of care for patients with MCL. Rituximab-containing immunochemotherapy strategies are commonly used, but the addition of rituximab to conventional induction chemotherapy has produced suboptimal responses that are relatively short-lived and have not resulted in a survival advantage. Further intensification of the chemotherapy component, including autologous stem cell transplantation, has increased response and survival rates but has not proven to be curative while being associated with higher toxicity. Clearly, there is a need for developing novel agents and strategies that will improve clinical outcomes for patients with MCL. Targeted therapies and new cytotoxic agents are showing great promise and may have a role in maintenance and/or initial therapy. This summary highlights current challenges in the management of MCL, and outlines expert perspectives, key questions, and future directions. For the third consecutive year, a panel of global experts in MCL assembled to deliberate on topical issues in MCL including advances in pathobiology, strategies for risk-adapted therapy, front-line treatment options, consolidation approaches, and novel therapeutic strategies. The proceedings of this workshop, held December 3, 2009 in New Orleans, LA, are summarized here. It must be emphasized that this synopsis is not meant to serve as an exhaustive review of MCL biology and management, but is a distillation of the expert discussions, highlighting key questions and future directions identified.

Mantle cell lymphoma in relapse: the role of emerging new drugs

Despite current advances in the therapy for newly diagnosed mantle cell lymphoma (MCL), relapsed MCL continues to have a poor prognosis. Advances in our understanding of the molecular pathogenesis of MCL are yielding many promising novel therapies. This article reviews the unique biology of MCL and describes how our understanding of its cell cycle dysregulation, and impaired apoptotic pathways is yielding many potential therapeutic targets including cyclin D1 and the cell cycle regulatory proteins, inhibitors of mammalian target of rapamycin, the proteasome, and proapoptotic family members. Recent preclinical and clinical data with cdk inhibitors, histone deacetylase inhibitors, the proteasome inhibitor bortezomib, mammalian target of rapamycin inhibitors, and other experimental strategies such as immunotherapy and microRNA are discussed. Understanding these targeted therapies in the context of the biology of MCL, has the potential to develop novel therapeutic platforms for the treatment of relapsed MCL, and will hopefully change the outcome for patients with this challenging clinical condition.

Current treatment strategy and new agents in mantle cell lymphoma

Mantle cell lymphoma (MCL) is a well-recognized lymphoma subtype that accounts for about 5% of all patients with non-Hodgkin lymphoma. The clinical course of MCL ranges from an indolent disease to a rapidly progressive malignancy, with a poor prognosis and a median overall survival (OS) of about 3-5 years reported in earlier data sets. Knowledge of its biology has increased in the last few years. Unfortunately, this progress has not yet brought any major improvements in therapeutic approaches, which still remain highly unsatisfactory. Recent improvement has been achieved by the successful introduction of monoclonal antibodies and dose-intensified approaches including autologous stem cell transplantation strategies. However, with the exception of allogeneic hematopoietic stem cell transplantation, current treatment approaches are non-curative, and the corresponding survival curves are characterized by a delayed but continuous decline and a median survival of 4-6 years. In recent years, new insights into the biology of MCL have been obtained which have provided the rationale for the development of novel therapeutic strategies. Emerging new drugs such as bendamustine, proteasome inhibitors, antibodies, mTOR inhibitors, and immunomodulatory drugs and others are based on the dysregulated control of cell cycle machinery and impaired apoptotic pathways. The efficacy of these agents as monotherapy was demonstrated to be comparable to conventional chemotherapy in relapsed MCL, and combination strategies are currently being investigated in clinical trials.

Number of Lymphoma-Associated-Macrophages (LAM) Is An Independent Predictor of Survival in Patients with Mantle Cell Lymphoma (MCL).

Abstract 3944Poster Board III-880 BackgroundMCL is a clinically aggressive lymphoma characterized by frequent relapses and short survival. Clinical behavior and response to therapy vary considerably and are often unpredictable based on clinical factors. Prior gene and protein expression studies have demonstrated proliferation and TP53 expression as biological mechanisms that strongly influence survival (Rosenwald Cancer Cell 2003 & Katzenberger Blood 2006 and Louie Blood 1995). Recent studies have highlighted the role of the microenvironment in follicular lymphoma (FL) including numbers of lymphoma-associated-macrophages (LAM), as important prognostic factors (Dave NEJM 2004 & Farinha Blood 2005). Within most tumors, macrophages display an M2 phenotype that promotes tumor growth and angiogenesis. In MCL, the clinical impact of LAM is unknown. Our aim was to assess the prognostic impact of LAM in MCL patients from a single institution experience using tissue microarrays (TMA), immunohistochemistry and clinical correlates. MethodsA TMA block was built with duplicate 0.6mm cores of paraffin-embedded formalin-fixed diagnostic biopsies from 185 patients treated at the BC Cancer Agency (1983-2004). A subset of cases was also analyzed for the presence of the t(11;14) by FISH. Primary and subsequent therapy included multiple regimens: single or multi-agent chemotherapy with or without rituximab, stem cell transplant, radiation and therapeutic splenectomy. Standard immunohistochemistry was performed with CD20, CCND1, CD68, CD163 (an M2 marker), CD34, TP53 and Ki67. TP53 and Ki67 IHC were scored using image analysis (VIAS). Univariate and multivariate correlates of overall survival (OS) were determined using SPSS software. ResultsThe median follow-up of living patients was 5.75 years. The IPI predicted OS (p=0.0013). Numbers of cases with interpretable staining varied for the different biomarkers (range 157 to 180). All 185 cases were either CCND1 positive and/or t(11;14) FISH positive. Both proliferation (quartiles of Ki67+ cells) and TP53 (dichotomized based on cut-off >40%) were significant predictors of OS independent of IPI (OS, p=0.0002). Cases showing more than 5% infiltrating CD68+ cells (18/185) had a 4-y OS of 5% vs. 35% (p=0.0081). These CD68+ cases significantly correlated with 12 cases showing more than 5% infiltrating CD163+ cells (x2=0.001), which also significantly influenced OS (p=0.008). 17 cases had increased microvessel density (MVD), defined by CD34+ cells, which correlated with inferior OS (p=0.02). Interestingly, cases with increased MVD often had >5% CD163+ cells (x2=0.05), but not increased CD68+ cells. Multivariate analysis including IPI, TP53, Ki67, CD68, CD163 and CD34, showed IPI (RR=1.8, 95%CI=1.0-3.1, p=0.047), TP53 (RR=2.3, 95%CI=1.3-4.3, p=0.006), Ki67 (RR=2.2, 95%CI=1.2-4.2, p=0.012) and CD68 (RR=2.1, 95%CI=1.1-3.8, p=0.022) to be independent predictors of OS. Finally, in the 27 patients who received rituximab at some time during their disease course, increased CD68+ cells did not significantly affect OS (p=0.1), suggesting that, similar to FL, the prognostic effect of LAM may be abolished by rituximab. ConclusionsThe number of LAMs is an important prognostic factor in MCL, independent of clinical parameters, proliferation and TP53 expression. Expression of CD163, described as an M2 polarized macrophages, also predicts survival and correlates with increased angiogenesis. These preliminary data suggest that the use of rituximab may abrogate the prognostic impact of LAM in MCL. In summary, similar to other lymphoma subtypes, the microenvironment is important in MCL biology and prognosis and may be an important new target for therapy in this aggressive disease. [Display omitted] Disclosures:Connors:Roche Canada (F Hoffmann-La Roche): Research Funding. Gascoyne:Roche Canada, Genentech, Lilly, Millennium : Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Frontline therapy in mantle cell lymphoma: The role of high-dose therapy and integration of new agents

Mantle cell lymphoma (MCL) is a unique lymphoma subtype comprising 6% to 8% of new lymphoma diagnoses. It is generally considered to be incurable with standard lymphoma therapies. The median overall survival (OS) is often reported to be 3 to 4 years, but more recent data suggest that the median OS may be longer than 5 years. There is considerable heterogeneity in MCL, with some patients succumbing to their disease in less than a year and others surviving for more than 10 years. The biology of MCL is reasonably well understood, and targeted therapies based on this knowledge are in development. Clinical trials incorporating new agents into standard therapies are under way. The optimal frontline treatment strategy is not defined and is a source of controversy. Intensive therapies administered to younger patients appear to produce better-quality remission than standard treatments, and one randomized clinical trial demonstrates improved OS with an intensive approach. Additional randomized clinical trials defining standard approaches are needed.

Mantle cell lymphoma: Are current therapies changing the course of disease?

Mantle cell lymphoma (MCL) is a rare entity of non-Hodgkin's lymphoma, although it seems to be increasing in incidence and severity. There is no accepted standard therapy; however, one controlled clinical trial demonstrated that intensive induction immunochemotherapy followed by high-dose radiochemotherapy and autologous stem cell transplantation was superior to conventional treatment. Moreover, uncontrolled studies of intensive immunochemotherapy followed by autologous stem cell transplantation now suggest that MCL may be cured. Insight into the biology of MCL is expanding, opening new avenues of treatment with well-defined molecular targets, including CD20, mammalian target of rapamycin, and proteasomes.

Molecular biology of mantle cell lymphoma: From profiling studies to new therapeutic strategies

Mantle cell lymphoma (MCL) is a well-defined lymphoid malignancy characterized by a rapid clinical evolution and poor response to current therapeutic protocols. The hallmark genetic alteration of MCL is the t(11;14)(q13;32) chromosomal translocation that leads to the overexpression of cyclin D1. Recently, new molecular alterations of major importance in the pathogenic mechanisms of this disease have been discovered, and have revealed the biological heterogeneity of MCL. The first section of our review discusses our current understanding of the molecular biology of this entity according to recent information from comparative genomic hybridization (CGH) and expression profiling studies, which are leading to the identification of several druggable targets. In the second section we revise new therapeutic strategies based on new drug families that target key molecular pathways of major relevance in this malignancy. We analyze emerging agents that are already producing significant results in different models of human cancers, including MCL. Based on the current knowledge and recent studies, we suggest that the encouraging results described here should provide a rationale platform for the design of new treatments that may overcome the resistance of this aggressive lymphoma to conventional therapy and improve patient prognosis.

Mantle cell lymphoma: advances in biology and therapy

Mantle cell lymphoma is characterized by dysregulation of cyclin D1, but this is not sufficient for lymphoma development. It is a difficult disease to treat, being incurable with standard chemotherapy and having a median survival of approximately 5 years. The purpose of this review is to update recent advances in mantle cell lymphoma biology with prognostic and potentially therapeutic implications, and mantle cell lymphoma treatment approaches and new agents. Genetic alterations that cooperate with cyclin D1 have been described that alter proliferation, in particular p27Kip and p16INK4, or apoptosis. Biological factors such as high-proliferation signature defined by gene expression profiles, loss of p27 and presence of mutant p53 confer poor prognosis. Proliferative rate also predicts patient outcome. Clinical criteria such as the international prognostic index, follicular lymphoma international prognostic index or a formula using age, performance status, white blood cell count and lactate dehydrogenase, separate prognostic groups. Not all patients require therapy at diagnosis. Although the best reported results have been with rituximab-hyperfractionated cyclophosphamide-vincristine-doxorubicin-dexamethasone-methotrexate/cytarabine, a cooperative group study of this regimen appears not quite as successful. Consolidation of remission after rituximab-cyclophosphamide-doxorubicin-vincristine-prednisone with high-dose therapy/stem-cell support prolongs remission and consolidation with radioimmunotherapy shows promise. Intensifying induction by alternating intensified rituximab-cyclophosphamide-doxorubicin-vincristine-prednisone with rituximab and high-dose cytarabine, followed by high-dose therapy appears quite promising. Novel agents active in relapsed disease include bortezomib, mammalian target of rapamycin inhibitors, immunomodulatory agents, antibodies and cyclin pathway-directed agents such as flavopiridol and cyclin-dependent kinase inhibitors. New insights into mantle cell lymphoma biology may lead to targeted therapy. Meanwhile, combinations of existing therapeutic approaches seem to have improved outcomes.

Prevalence of bortezomib-resistant constitutive NF-kappaB activity in mantle cell lymphoma

BackgroundThe proteasome inhibitor bortezomib can inhibit activation of the transcription factor NF-κB, a mechanism implicated in its anti-neoplastic effects observed in mantle cell lymphoma (MCL). However, NF-κB can be activated through many distinct mechanisms, including proteasome independent pathways. While MCL cells have been shown to harbor constitutive NF-κB activity, what fraction of this activity in primary MCL samples is sensitive or resistant to inhibition by bortezomib remains unclear.ResultsProteasome activity in the EBV-negative MCL cell lines Jeko-1 and Rec-1 is inhibited by greater than 80% after exposure to 20 nM bortezomib for 4 hours. This treatment decreased NF-κB activity in Jeko-1 cells, but failed to do so in Rec-1 cells when assessed by electrophoretic mobility shift assay (EMSA). Concurrently, Rec-1 cells were more resistant to the cytotoxic effects of bortezomib than Jeko-1 cells. Consistent with a proteasome inhibitor resistant pathway of activation described in mouse B-lymphoma cells (WEHI231) and a breast carcinoma cell line (MDA-MB-468), the bortezomib-resistant NF-κB activity in Rec-1 cells is inhibited by calcium chelators, calmodulin inhibitors, and perillyl alcohol, a monoterpene capable of blocking L-type calcium channels. Importantly, the combination of perillyl alcohol and bortezomib is synergistic in eliciting Rec-1 cell cytotoxicity. The relevance of these results is illuminated by the additional finding that a considerable fraction of primary MCL samples (8 out of 10) displayed bortezomib-resistant constitutive NF-κB activity.ConclusionOur findings show that bortezomib-resistant NF-κB activity is frequently observed in MCL samples and suggest that this activity may be relevant to MCL biology as well as serve as a potential therapeutic target.

Bortezomib In Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) represents 6% of non-Hodgkin lymphomas, but is one of the most active fields of clinical investigation. Unfortunately, there is still no standard or curative therapy in MCL. Front-line therapy appears to benefit from intensification either through high-dose therapy with stem cell transplant consolidation or dose-intense chemotherapy with hyperfractionated cyclophosphamide, vincristine, adriamycin/doxorubicin and dexamethasone/rituximab. Most patients still relapse and a multitude of novel agents are currently being tested in this setting, including proteasome inhibitors with bortezomib (the first of its class and the first US FDA-approved drug for MCL), mTOR inhibitors, Bcl-2 inhibitors, antiangiogenesis agents and histone deacetylase inhibitors among others. An obvious effort is needed to enroll patients on clinical trials, the design of which might benefit from pharmacogenomics and a better understanding of MCL biology and its diversity.

Activation of Mammalian Target of Rapamycin Signaling Promotes Cell Cycle Progression and Protects Cells from Apoptosis in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is characterized by the t(11;14) and cyclin D1 overexpression. However, additional molecular events are most likely required for oncogenesis, possibly through cell cycle and apoptosis deregulation. We hypothesized that mammalian target of rapamycin (mTOR) is activated in MCL and contributes to tumor proliferation and survival. In MCL cell lines, pharmacological inhibition of the phosphoinositide 3-kinase/AKT pathway was associated with decreased phosphorylation (activation) of mTOR and its downstream targets phosphorylated (p)-4E-BP1, p-p70S6 kinase, and p-ribosomal protein S6, resulting in apoptosis and cell cycle arrest. These changes were associated with down-regulation of cyclin D1 and the anti-apoptotic proteins cFLIP, BCL-XL, and MCL-1. Furthermore, silencing of mTOR expression using mTOR-specific short interfering RNA decreased phosphorylation of mTOR signaling proteins and induced cell cycle arrest and apoptosis. Silencing of eukaryotic initiation factor (eIF4E), a downstream effector of mTOR, recapitulated these results. We also assessed mTOR signaling in MCL tumors using immunohistochemical methods and a tissue microarray: 10 of 30 (33%) expressed Ser473p-AKT, 13 of 21 (62%) Ser2448p-mTOR, 22 of 22 (100%) p-p70S6K, and 5 of 20 (25%) p-ribosomal protein S6. Total eIF4E binding protein 1 and eukaryotic initiation factor 4E were expressed in 13 of 14 (93%) and 16 of 29 (55%) MCL tumors, respectively. These findings suggest that the mTOR signaling pathway is activated and may contribute to cell cycle progression and tumor cell survival in MCL.

Update on the molecular biology of mantle cell lymphoma

Mantle cell lymphoma (MCL) accounts for 5-10% of non-Hodgkin's lymphomas and it bears the worst prognosis among B cell lymphomas. Even more than in the other lymphomas, new insights in MCL biology are needed. In this review we will discuss the most recent published data. Immunoglobulin sequencing data suggest that the MCL cell of origin is a mature B cell which might have undergone an extra-follicular T-cell independent antigen maturation. The main aberrations that affect cell cycle and DNA repair pathways in MCL are summarized, also discussing the need of the t(11;14)(q13;q32) to make diagnosis of MCL. After the gene expression studies of the last years, now the research focus has moved towards the new opportunities provided by arrayCGH technique for gene discovery and for identification of therapeutic targets. Examples of genes identified because deleted or amplified at DNA level and that might be relevant for MCL pathogenesis are presented.

Mantle cell lymphoma: established therapeutic options and future directions

During the last few years, new insights into the biology of mantle cell lymphoma have been obtained. However, with a median survival of only 3 years, mantle cell lymphoma remains the lymphoma subtype with the poorest prognosis. At initial diagnosis most patients present with advanced Ann Arbor stage III or IV and conventional chemotherapy hardly alters the continuously declining survival curve. Recently, two prospective randomized studies of the German Low Grade Lymphoma Study Group (GLSG) clearly confirmed the superiority of a combined immunochemotherapy. In a randomized study of the European mantle cell lymphoma Network, consolidation with myeloablative radiochemotherapy followed by autologous stem cell transplantation improved the progression-free survival in patients younger than 65 years. However, relapses are still observed at a high frequency. Thus, new therapeutic strategies such as radioactively labeled antibodies or molecular targeting agents (e.g. Bortezomib or flavopiridol) are urgently warranted to further improve the clinical outcome of mantle cell lymphoma.

Proliferative response of mantle cell lymphoma cells stimulated by CD40 ligation and IL-4.

Mantle cell lymphoma (MCL) is a tumor of intermediate-size, IgM+, IgD+ B cells derived from the mantle zone of the germinal center. Little is known about its specific immunologic features or responsiveness to T cell-derived signals. In this work, we evaluated the proliferation and cell cycle properties of freshly isolated MCL cells after CD40 ligation, in the absence and presence of interleukin 4 (IL-4). In each MCL case examined, there was a marked growth-enhancing effect of these two stimuli characterized by improved viability, augmented expression of Ki-67, and induction of the proliferating cell nuclear antigen (PCNA). Cyclin D1 was expressed throughout the cell cycle in MCL cells induced to enter S phase. From these investigations, we conclude that the biology of MCL B lymphocytes is affected by CD154 (CD40 ligand) and IL-4, two signals usually provided by CD4+ T cells. The capacity to manipulate the activation and cell cycle state of MCL cells by these specific immunological stimuli may be exploited to confer susceptibility to chemotherapy agents and develop novel therapies in this disease.