Michelle K. Manske

Elevated Serum B-Lymphocyte Stimulator Levels in Patients With Familial Lymphoproliferative Disorders

PURPOSE Serum B-lymphocyte stimulator (BLyS) levels have been found to be elevated in a number of immune disease models. Therefore, we sought to establish whether BLyS levels were elevated in patients with B-cell lymphoproliferative disorders and to determine whether elevated BLyS levels correlated with clinical characteristics of the disease. PATIENTS AND METHODS Specimens were collected from the peripheral blood of individuals diagnosed with B-cell chronic lymphocytic leukemia (B-CLL; n = 70) or from age- and sex-matched patients seen at the same institution (n = 41). Serum BLyS levels were determined by enzyme-linked immunosorbent assay, and sequencing of the BLyS promoter was performed by conventional methods and confirmed by restriction fragment length polymorphism analysis. RESULTS We found that elevated BLyS levels were more common in patients with familial B-CLL than individuals with sporadic B-CLL or normal controls. Because of this association, we sequenced the BLyS promoter in patients with B-CLL and normal controls and identified a polymorphic site, -871 C/T. We found that the wild-type sequence was significantly underrepresented in patients with familial B-CLL (4%) compared with patients with sporadic B-CLL (30%; P = .01) or controls (24%; P = .04). Furthermore, using a luciferase reporter under control of the BLyS promoter containing either a C or a T at position -871, we found that the reporter construct containing a T at -871 had a 2.6-fold increase in activity (P = .004). CONCLUSION Our data suggest serum BLyS levels are elevated in patients with familial B-CLL and that elevated BLyS levels correlate with the presence of a T at -871 in the BLyS promoter.

Soluble IL-2Rα facilitates IL-2–mediated immune responses and predicts reduced survival in follicular B-cell non-Hodgkin lymphoma

Elevated serum levels of the soluble form of IL-2 receptor α (sIL-2Rα) have been correlated with a poor prognosis in a variety of different types of cancers. However, its biologic relevance remains unclear and controversial. In patients with follicular B-cell non-Hodgkin lymphoma (FL), we observed that serum sIL-2Rα levels were elevated compared with controls and that elevated sIL-2Rα levels before treatment were associated with a poor outcome. To explore the mechanism by which sIL-2Rα may contribute to a poor prognosis in FL, we determined the effects of sIL-2Rα on IL-2 signaling and found that the sIL-2Rα-IL-2 complex promoted T-cell differentiation toward to inhibitory T(reg) cells rather than T(H)1 or T(H)17 cells. Shed by activated T cells that express membrane-bound IL-2Rα, sIL-2Rα further enhanced IL-2-mediated phosphorylation of Stat5 thereby significantly up-regulating Foxp3 expression in CD4(+) T cells. We found that CD4(+) T cells treated with either IL-2 or sIL-2Rα-IL-2 complex, but not with sIL-2Rα alone, inhibited the function of CD8(+) T cells. Taken together, these results indicate that sIL-2Rα actually plays an active biologic role in FL by binding IL-2 and promoting IL-2 signaling rather than depleting IL-2 and blocking its function.

A BAFF-R mutation associated with non-Hodgkin lymphoma alters TRAF recruitment and reveals new insights into BAFF-R signaling.

A BAFF receptor mutation associated with non-Hodgkin lymphoma provides new insight into the proximal players of normal BAFF-R signaling.

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.

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.

t(X;14)(p11;q32) in MALT lymphoma involving GPR34 reveals a role for GPR34 in tumor cell growth

Genetic aberrations, including trisomies 3 and 18, and well-defined IGH translocations, have been described in marginal zone lymphomas (MZLs); however, these known genetic events are present in only a subset of cases. Here, we report the cloning of an IGH translocation partner on chromosome X, t(X;14)(p11.4;q32) that deregulates expression of an poorly characterized orphan G-protein-coupled receptor, GPR34. Elevated GPR34 gene expression was detected independent of the translocation in multiple subtypes of non-Hodgkin lymphoma and distinguished a unique molecular subtype of MZL. Increased expression of GPR34 was also detected in tissue from brain tumors and surface expression of GPR34 was detected on human MZL tumor cells and normal immune cells. Overexpression of GPR34 in lymphoma and HeLa cells resulted in phosphorylation of ERK, PKC, and CREB; induced CRE, AP1, and NF-κB-mediated gene transcription; and increased cell proliferation. In summary, these results are the first to identify a role for a GPR34 in lymphoma cell growth, provide insight into GPR34-mediated signaling, identify a genetically unique subset of MZLs that express high levels of GPR34, and suggest that MEK inhibitors may be useful for treatment of GPR34-expressing tumors.

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.

Novel Analysis of Clonal Diversification in Blood B Cell and Bone Marrow Plasma Cell Clones in Immunoglobulin Light Chain Amyloidosis

Immunoglobulin light chain amyloidosis (AL) is characterized by a limited clonal expansion of plasma cells and amyloid formation. Here, we report restriction in the diversity of VL gene usage with a dominance of clonally related B cells in the peripheral blood (PB) isotype-specific repertoire of AL patients. A rigorous quantification of lineage trees reveals presence of intraclonal variations in the PB clones compared to the bone marrow (BM) clones, which suggests a common precursor that is still subject to somatic mutation. When compared to normal BM and PB B cells, AL clones showed significant but incomplete impairment of antigenic selection, which could not be detected by conventional R and S mutation analysis. Therefore, graphical analysis of B cell lineage trees and mathematical quantification of tree properties provide novel insights into the process of B cell clonal evolution in AL.

Utility of Doppler Myocardial Imaging, Cardiac Biomarkers, and Clonal Immunoglobulin Genes to Assess Left Ventricular Performance and Stratify Risk Following Peripheral Blood Stem Cell Transplantation in Patients with Systemic Light Chain Amyloidosis (AL)

BACKGROUND Cardiac dysfunction is a well-recognized complication of light chain amyloidosis (AL). Autologous stem cell transplant (auto-SCT) has emerged as a successful treatment modality for AL patients. In this study, we examined the effect of clonal immunoglobulin light chain genes (VL), which encodes the immunoglobulin light chain protein that ultimately forms amyloid, on cardiac function, in the context of auto-SCT and its impact on overall survival. METHODS Longitudinal Doppler myocardial imaging parameters along with cardiac biomarkers were used to assess for cardiac function pre and post auto-SCT. RESULTS VL gene analysis revealed that Vl genes, in particular VlVI, were associated with worse cardiac function parameters than Vk genes. Clonal VL genes appeared to have an impact on left ventricular (LV) function post-transplant and also influenced mortality, with specific VL gene families associated with lower survival. Another key predictor of mortality in this report was change in tricuspid regurgitant flow velocity following auto-SCT. Correlations were also observed between systolic strain rate, systolic strain and VL genes associated with amyloid formation. CONCLUSIONS Clonal VL gene usage influences global cardiac function in AL, with patients having VlVI and VlII-III-associated amyloid more severely affected than those having Vk or VlI amyloid. Pulsed wave tissue Doppler imaging along with immunoglobulin gene analysis offers novel insights into prediction of mortality and cardiac dysfunction in AL after auto-SCT.

B-cell activating factor-receptor specific activation of tumor necrosis factor receptor associated factor 6 and the phosphatidyl inositol 3-kinase pathway in lymphoma B cells

Abstract B-cell activating factor-receptor (BAFF-R) is the primary BAFF receptor that is responsible for promoting B-cell development and survival. Malignant B-cells exploit the BAFF/BAFF-R system, and high serum BAFF levels or genetic alterations in BAFF receptors have been found in B-cell cancers. BAFF signaling impacts pro-survival pathways. However, other than nuclear factor-κB2 (NF-κB2), little is known about the specific pathways activated by individual BAFF receptors. Using a novel BAFF-R expression model we have demonstrated that activation of BAFF-R, independent of transmembrane activator and cytophilin ligand interactor (TACI) and B-cell maturation antigen (BCMA), can induce phosphorylation of Akt and glycogen synthase kinase 3β (GSK3β). Expression of an activated form of BAFF-R also enhanced a pro-survival gene expression pattern, including the novel BAFF-regulated gene Pin1, whose expression was phosphatidyl inositol 3-kinase (PI3K)-dependent. Additionally, we showed that TRAF6 is essential for mediating BAFF-R dependent activation of Akt. Together these data describe a novel role for TRAF6 in BAFF-R-specific activation of the PI3K pathway and provide evidence suggesting a new role for Pin1 in BAFF-R signaling.