Lucy S. Hodge

Comprehensive analysis of tumor microenvironment cytokines in Waldenstrom macroglobulinemia identifies CCL5 as a novel modulator of IL-6 activity

Although proinflammatory and chemotactic cytokines can profoundly affect the tumor microenvironment, and many of them have been shown to have therapeutic efficacy in preclinical models, the role of these molecules in Waldenström macroglobulinemia (WM) remains poorly understood. In this study, simultaneous analysis of WM patient sera and bone marrow biopsies identified a set of dysregulated cytokines including CCL5, G-CSF, and soluble IL-2 receptor, that were significantly elevated in WM patients whereas IL-8 and EGF levels were significantly lower in these patients compared with healthy controls. Interestingly, CCL5 levels positively correlated with features of disease aggressiveness such as elevated IgM levels and bone marrow involvement. Functional analysis of tumor microenvironment revealed a functional correlation between CCL5 levels and IL-6 levels, a proinflammatory cytokine with an important role in normal and malignant B-cell biology. Furthermore, CCL5 stimulated IL-6 secretion in WM stromal cells resulting in increased IgM secretion by WM malignant cells via the JAK/STAT signaling pathway. Thus, together these results define a novel signaling network in the WM tumor microenvironment controlling IgM secretion and suggest CCL5 as a potential target for the treatment of this disease.

Activation of TAK1 by MYD88 L265P drives malignant B-cell Growth in non-Hodgkin lymphoma

Massively parallel sequencing analyses have revealed a common mutation within the MYD88 gene (MYD88L265P) occurring at high frequencies in many non-Hodgkin lymphomas (NHLs) including the rare lymphoplasmacytic lymphoma, Waldenströms macroglobulinemia (WM). Using whole-exome sequencing, Sanger sequencing and allele-specific PCR, we validate the initial studies and detect the MYD88L265P mutation in the tumor genome of 97% of WM patients analyzed (n=39). Due to the high frequency of MYD88 mutation in WM and other NHL, and its known effects on malignant B-cell survival, therapeutic targeting of MYD88 signaling pathways may be clinically useful. However, we are lacking a thorough characterization of the role of intermediary signaling proteins on the biology of MYD88L265P-expressing B cells. We report here that MYD88L265P signaling is constitutively active in both WM and diffuse large B-cell lymphoma cells leading to heightened MYD88L265P, IRAK and TRAF6 oligomerization and NF-κB activation. Furthermore, we have identified the signaling protein, TAK1, to be an essential mediator of MYD88L265P-driven signaling, cellular proliferation and cytokine secretion in malignant B cells. Our studies highlight the biological significance of MYD88L265P in NHL and reveal TAK1 inhibition to be a potential therapeutic strategy for the treatment of WM and other diseases characterized by MYD88L265P.

TGF-Β upregulates CD70 expression and induces exhaustion of effector memory T cells in B-cell non-Hodgkin's lymphoma

Transforming growth factor beta (TGF-β) has an important role in mediating T-cell suppression in B-cell non-Hodgkin lymphoma (NHL). However, the underlying mechanism responsible for TGF-β-mediated inhibition of effector memory T (Tm) cells is largely unknown. As reported here, we show that exhaustion is a major mechanism by which TGF-β inhibits Tm cells, and TGF-β mediated exhaustion is associated with upregulation of CD70. We found that TGF-β upregulates CD70 expression on effector Tm cells while it preferentially induces Foxp3 expression in naive T cells. CD70 induction by TGF-β is Smad3-dependent and involves IL-2/Stat5 signaling. CD70+ T cells account for TGF-β-induced exhaustion of effector Tm cells. Both TGF-β-induced and preexisting intratumoral CD70+ effector Tm cells from B-cell NHL have an exhausted phenotype and express higher levels of PD-1 and TIM-3 compared with CD70− T cells. Signaling transduction, proliferation and cytokine production are profoundly decreased in these cells, and they are highly susceptible to apoptosis. Clinically, intratumoral CD70-expressing T cells are prevalent in follicular B-cell lymphoma (FL) biopsy specimens, and increased numbers of intratumoral CD70+ T cells correlate with an inferior patient outcome. These findings confirm TGF-β-mediated effector Tm cell exhaustion as an important mechanism of immune suppression in B-cell NHL.

Prognostic Significance of Pretreatment Serum Cytokines in Classical Hodgkin Lymphoma

Purpose: Although the International Prognostic Score (IPS) is the gold standard for risk-stratifying patients with classical Hodgkin lymphoma (cHL), these criteria do not accurately predict outcome. As cytokines are critically involved in driving cHL, we tested whether pretreatment serum cytokine levels could provide additional prognostic information. Experimental Design: Thirty cytokines were measured in pretreatment serum from 140 patients with cHL and compared with 50 nonlymphoma controls. Patients were followed for event-free survival (EFS) and overall survival (OS), and Cox proportional hazards regression models were used to assess the association of individual cytokines and the cytokine profiles with outcome via unadjusted and IPS-adjusted HR. Results: Twelve cytokines (EGF, bFGF, G-CSF, HGF, IL-6, IL-8, IL-12, IL-2R, IP-10, MIG, TNF-α, and VEGF) were significantly (P < 0.05) higher in patients with cHL than controls; elevated levels of HGF, IL-6, IL-2R, IP-10, and MIG were all associated with poorer EFS. Only interleukin-2 receptor (IL-2R; P = 0.002) and interleukin (IL)-6 (P < 0.001) were independently prognostic. Patients with increased IL-6 and IL-2R had a significantly higher risk of early relapse and death, a finding that remained significant even after IPS-based risk stratification. Although elevated IL-6 and IL-2R correlated with the IPS, soluble CD30 (sCD30), and thymus and activation-related chemokine (TARC) levels, the two-cytokine model remained independently predictive of prognosis. Conclusions: Elevated pretreatment serum cytokines are associated with increased disease relapse and inferior survival in cHL. Thus, the pretreatment cytokine profile, particularly serum levels of IL-6 and IL-2R, may be used to identify patients with cHL at high risk for early-disease relapse. Clin Cancer Res; 19(24); 6812–9. ©2013 AACR.

Establishment and characterization of a novel Waldenström macroglobulinemia cell line, MWCL-1

Waldenström macroglobulinemia (WM) is a rare, lymphoplasmacytic lymphoma characterized by hypersecretion of immunoglobulin M (IgM) protein and tumor infiltration into the bone marrow and lymphatic tissue. Our understanding of the mechanisms driving the development and progression of WM is currently by the shortage of representative cell models available for study. We describe here the establishment of a new WM cell line, MWCL-1. Comprehensive genetic analyses have unequivocally confirmed a clonal relationship between this novel cell line and the founding tumor. MWCL-1 cells exhibit an immunophenotype consistent with a diverse, tumor clone composed of both small B lymphocytes and larger lymphoplasmacytic cells and plasma cells: CD3⁻, CD19⁺, CD20⁺, CD27⁺, CD38⁺, CD49D⁺, CD138⁺, cIgM⁺, and κ⁺. Cytogenetic studies identified a monoallelic deletion of 17p13 (TP53) in both the cell line and the primary tumor. Direct DNA resequencing of the remaining copy of TP53 revealed a missense mutation at exon 5 (V143A, GTG>GCG). In accordance with primary WM tumors, MWCL-1 cells retain the ability to secrete high amounts of IgM protein in the absence of an external stimulus. The genetic, immunophenotypic, and biologic data presented here confirm the validity of the MWCL-1 cell line as a representative model of WM.

IL-21 in the bone marrow microenvironment contributes to IgM secretion and proliferation of malignant cells in Waldenstrom macroglobulinemia

Cytokines within the tumor microenvironment play an important role in supporting the growth and survival of B-cell malignancies. One such cytokine, IL-21, promotes the growth of myeloma and Hodgkin lymphoma cells while inducing apoptosis in chronic lymphocytic leukemia. However, the biologic significance of IL-21 has not been examined in Waldenstrom macroglobulinemia (WM), a B-cell lymphoma characterized by elevated serum IgM and a lymphoplasmacytic bone marrow infiltrate. We report here on the presence of IL-21 in the bone marrow of patients with WM and have identified activated T cells as the source of this cytokine. We readily detected the IL-21 receptor on malignant WM B cells and show that IL-21 significantly increases both IgM secretion and cellular proliferation of these cells with no effect on viability. IL-21 rapidly induces phosphorylation of STAT3 in WM cells, and treatment of the WM cell line MWCL-1 with a STAT3 inhibitor abolished the IL-21-mediated increases in cellular proliferation and IgM secretion. IL-21 also increased the expression of known STAT3 targets involved in B-cell differentiation, including BLIMP-1, XBP-1, IL-6, and IL-10. Overall, our data indicate that IL-21 in the bone marrow microenvironment significantly affects the biology of WM tumor cells through a STAT3-dependent mechanism.

Whole-exome analysis reveals novel somatic genomic alterations associated with outcome in immunochemotherapy-treated diffuse large B-cell lymphoma

Lack of remission or early relapse remains a major clinical issue in diffuse large B-cell lymphoma (DLBCL), with 30% of patients failing standard of care. Although clinical factors and molecular signatures can partially predict DLBCL outcome, additional information is needed to identify high-risk patients, particularly biologic factors that might ultimately be amenable to intervention. Using whole-exome sequencing data from 51 newly diagnosed and immunochemotherapy-treated DLBCL patients, we evaluated the association of somatic genomic alterations with patient outcome, defined as failure to achieve event-free survival at 24 months after diagnosis (EFS24). We identified 16 genes with mutations, 374 with copy number gains and 151 with copy number losses that were associated with failure to achieve EFS24 (P<0.05). Except for FOXO1 and CIITA, known driver mutations did not correlate with EFS24. Gene losses were localized to 6q21-6q24.2, and gains to 3q13.12-3q29, 11q23.1-11q23.3 and 19q13.12-19q13.43. Globally, the number of gains was highly associated with poor outcome (P=7.4 × 10−12) and when combined with FOXO1 mutations identified 77% of cases that failed to achieve EFS24. One gene (SLC22A16) at 6q21, a doxorubicin transporter, was lost in 54% of EFS24 failures and our findings suggest it functions as a doxorubicin transporter in DLBCL cells.

Molecular pathogenesis of Waldenström’s macroglobulinemia

Waldenström’s macroglobulinemia is an indolent, lymphoproliferative disease, characterized by a heterogeneous lymphoplasmacytic bone marrow infiltrate and high immunoglobulin M production. While technological advances over the past several decades have dramatically improved the possibilities of studying the molecular basis of Waldenström’s macroglobulinemia, the pathogenesis of the disease remains fragmented. Undoubtedly, research has been successful in uncovering underlying aberrations and deregulated mechanisms in this disease, providing useful information for identifying biomarkers for disease diagnosis, risk stratification and therapeutic intervention, but there is still a long way to go before the pathogenesis of Waldenström’s macroglobulinemia is fully revealed. In addition, the low number of in vitro or in vivo models significantly challenges extensive analysis. In this manuscript, we review the molecular basis of this disease.

Soluble and Membrane-Bound TGF-β-Mediated Regulation of Intratumoral T Cell Differentiation and Function in B-Cell Non-Hodgkin Lymphoma

While the effect of TGF-β on malignant B cells in non-Hodgkin lymphoma (NHL) has been previously evaluated, studies to specifically define the role of TGF-β in tumor immunity in B-cell NHL are limited. We found that soluble TGF-β, secreted by both lymphoma cells and intratumoral T cells, is present in the serum of patients with B-cell NHL. Soluble TGF-β promoted regulatory T (Treg) cells by enhancing expression of Foxp3 in CD4+ T cells and suppressed effector helper T (TH) cells by inhibiting expression of IFN-γ and IL-17. Blockade of the IL-2 signaling pathway diminished the effect of soluble TGF-β on T cell differentiation. Furthermore, we found that membrane-bound TGF-β is expressed specifically on the surface of malignant B cells in B-cell NHL. TGF-β was able to bind to the surface of lymphoma B cells through an interaction with heparan sulfate (HS) but not through the TGF-β receptor. We showed that pretreatment of lymphoma B cells with TGF-β significantly inhibits the proliferation and cytokine production of intratumoral T cells. Taken together, these results suggest that tumor-associated soluble and membrane-bound TGF-β are involved in the regulation of intratumoral T cell differentiation and function in B-cell NHL.

MicroRNA expression in tumor cells from Waldenstrom's macroglobulinemia reflects both their normal and malignant cell counterparts

MicroRNAs (miRNAs) are involved in the regulation of many cellular processes including hematopoiesis, with the aberrant expression of differentiation-stage specific miRNA associated with lymphomagenesis. miRNA profiling has been essential for understanding the underlying biology of many hematological malignancies; however the miRNA signature of the diverse tumor clone associated with Waldenstroms macroglobulinemia (WM), consisting of B lymphocytes, plasmacytes and lymphoplasmacytic cells, has not been characterized. We have investigated the expression of over 13 000 known and candidate miRNAs in both CD19+ and CD138+ WM tumor cells, as well as in their malignant and non-malignant counterparts. Although neither CD19+ nor CD138+ WM cells were defined by a distinct miRNA profile, the combination of all WM cells revealed a unique miRNA transcriptome characterized by the dysregulation of many miRNAs previously identified as crucial for normal B-cell lineage differentiation. Specifically, miRNA-9*/152/182 were underexpressed in WM, whereas the expression of miRNA-21/125b/181a/193b/223/363 were notably increased (analysis of variance; P<0.0001). Future studies focusing on the effects of these dysregulated miRNAs will provide further insight into the mechanisms responsible for the pathogenesis of WM.