Grote, Deanna M.

AbstractAlthough 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.

AbstractElevated 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 Treg cells rather than TH1 or TH17 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.

Abstract 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.

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.

AbstractWaldenström macroglobulinemia (WM) is a serious and frequently fatal B-cell malignancy associated with an elevated monoclonal IgM protein in the serum. Many of the mechanisms leading to this disease are not yet known. B-lymphocyte stimulator (BLyS) is a TNF family member that is critical for maintenance of normal B-cell development and homeostasis. BLyS is overexpressed in a variety of B-cell malignancies and has been shown to inhibit apoptosis in malignant B cells. It also regulates immunoglobulin secretion by normal B cells. To determine the relevance of BLyS in WM, we examined the role of BLyS in WM patient samples. Malignant B cells were found to bind soluble BLyS and variably express the receptors BAFF-R, TACI, and BCMA. We also found expression of BLyS in bone marrow specimens by immunohistochemistry and elevated serum BLyS levels in patients with WM. BLyS, alone or in combination with cytokines that induce immunoglobulin production, was found to increase IgM secretion by malignant B cells. Furthermore, BLyS was found to increase the viability and proliferation of malignant B cells from WM patients. Due to the role of BLyS in WM, strategies to inhibit BLyS may potentially have therapeutic efficacy in these patients.

Abstract BLyS, recently shown to be critical for survival of normal B cells, has been found to be elevated in a number of immune disease models. A role for BLyS in the survival of malignant B cells has also been revealed and we therefore sought to identify a role for BLyS and its receptors in non-Hodgkin lymphoma (NHL). We found that tumor cells from all NHL histologic subtypes expressed one or more of 3 known receptors (BCMA, TACI, and BAFF-R) for BLyS; however, the pattern of expression was variable. We provide evidence that BLyS is expressed in tumors from patients with NHL and that BLyS levels increase as tumors transform to a more aggressive phenotype. Additionally, we provide evidence that serum BLyS levels are elevated in a subgroup of patients with NHL. In patients with de novo large B-cell lymphoma, a high BLyS level correlated with a poorer median overall survival, the presence of constitutional symptoms, and elevated values of lactic dehydrogenase. When BLyS levels were correlated with response to therapy in all patients, responding patients had a significantly lower BLyS level than those with progressive disease. In summary, we found that BLyS and its receptors represent a potentially important therapeutic target in B-cell lymphoma.

Derivatives of the Edmonston-B strain of measles virus (MV-Ed) are safe, live attenuated measles virus (MV) vaccines that have been used worldwide for more than 30 years. The cytoreductive potential of MV-Ed has been investigated in murine models of both aggressive and indolent B-cell lymphoma in severe combined immunodeficient (SCID) mice. The rationale for these studies was generated by experience with viral fusogenic membrane glycoproteins as cytotoxic genes and the recognition of the potential of replicating viruses in the treatment of human malignancy. Intratumoral injection of both unmodified MV-Ed and a strain of MV-Ed genetically modified by the addition of a β-galactosidase reporter gene (MVlacZ) induced regression of large established human lymphoma xenografts, in contrast to control therapy with UV-inactivated virus, in which all tumors progressed. The antitumor effect still occurred in the presence of passively transferred anti-MV antibody. Intravenous administration of MV also resulted in considerable slowing of tumor progression. Analysis of sections of residual tumor confirmed replication of MV within the tumors. Thus, the vaccine strain of MV mediates regression of large, established human B-cell lymphoma xenografts in SCID mice, and proof of principle is established that MV is oncolytic for lymphomas in vivo. Attenuated MVs may have value as a novel replicating-virus therapy for this group of disorders.