Citations

Foxp3 expression was initially thought to be restricted to the CD4+CD25+ regulatory T-cell population. However, recent studies suggest that forkhead box P3 (Foxp3) is expressed in CD4+CD25− T cells in aged mice. In the present study in B-cell non-Hodgkin lymphoma (NHL), we found that a subset of intratumoral but not peripheral blood CD4+CD25− T cells, comprising about 15% of intratumoral CD4+ T cells, express Foxp3 and are capable of suppressing the proliferation of autologous infiltrating CD8+ T cells. In vitro activation with OKT3/anti-CD28 antibody (Ab) or dendritic cells (DCs) induced Foxp3 expression in a subset of these CD4+CD25−Foxp3− T cells. We found that the presence of lymphoma B cells during activation augmented activation-induced Foxp3 expression in CD4+CD25− T cells. We also found that CD70+ lymphoma B cells significantly contributed to the activation-induced Foxp3 expression in intratumoral CD4+CD25− T cells. Furthermore, the blockade of CD27-CD70 interaction by anti-CD70 Ab abrogated lymphoma B-cell–mediated induction of Foxp3 expression in intratumoral CD4+CD25− T cells. Taken together, these studies reveal a novel role for NHL B cells in the development of intratumoral regulatory T cells.

Abstract To better understand the molecular changes that occur in Waldenstrom macroglobulinemia (WM), we employed antibody-based protein microarrays to compare patterns of protein expression between untreated WM and normal bone marrow controls. Protein expression was defined as a >2-fold or 1.3-fold change in at least 67% of the tumor samples. Proteins up-regulated by >2-fold included Ras family proteins, such as Rab-4 and p62DOK, and Rho family proteins, such as CDC42GAP and ROKα. Other proteins up-regulated by >1.3-fold included cyclin-dependent kinases, apoptosis regulators, and histone deacetylases (HDAC). We then compared the samples of patients with symptomatic and asymptomatic WM and showed similar protein expression signatures, indicating that the dysregulation of signaling pathways occurs early in the disease course. Three proteins were different by >2-fold in symptomatic versus asymptomatic, including the heat shock protein HSP90. Elevated protein expression was confirmed by immunohistochemistry and immunoblotting. Functional significance was validated by the induction of apoptosis and inhibition of proliferation using specific HDAC and HSP90 inhibitors. This study, therefore, identifies, for the first time, multiple novel proteins that are dysregulated in WM, which both enhance our understanding of disease pathogenesis and represent targets of novel therapeutics. [Cancer Res 2007;67(8):3777–8]

Abstract The mechanisms by which multiple myeloma (MM) cells migrate and home to the bone marrow are not well understood. In this study, we sought to determine the effect of the chemokine SDF-1 (CXCL12) and its receptor CXCR4 on the migration and homing of MM cells. We demonstrated that CXCR4 is differentially expressed at high levels in the peripheral blood and is down-regulated in the bone marrow in response to high levels of SDF-1. SDF-1 induced motility, internalization, and cytoskeletal rearrangement in MM cells evidenced by confocal microscopy. The specific CXCR4 inhibitor AMD3100 and the anti-CXCR4 antibody MAB171 inhibited the migration of MM cells in vitro. CXCR4 knockdown experiments demonstrated that SDF-1–dependent migration was regulated by the PI3K and ERK/MAPK pathways but not by p38 MAPK. In addition, we demonstrated that AMD3100 inhibited the homing of MM cells to the bone marrow niches using in vivo flow cytometry, in vivo confocal microscopy, and whole body bioluminescence imaging. This study, therefore, demonstrates that SDF-1/CXCR4 is a critical regulator of MM homing and that it provides the framework for inhibitors of this pathway to be used in future clinical trials to abrogate MM trafficking.

Abstract Purpose: Rituximab is a chimeric antibody that induces B-cell apoptosis and recruits immune effector cells to mediate cell lysis. Interleukin-12 (IL-12) facilitates cytolytic responses by T cells and natural killer cells. This phase II study was done to determine the efficacy and toxicity of IL-12 in combination with rituximab in patients with B-cell non-Hodgkin's lymphoma (NHL). Experimental Design: Fifty-eight patients with histologically confirmed relapsed B-cell NHL were randomized to receive concurrent treatment with rituximab and IL-12 (arm A) or rituximab with subsequent treatment with IL-12 after documented nonresponse or progression after rituximab (arm B). Treatment consisted of 375 mg/m2 rituximab on days 1, 8, 15, and 22 and 300 ng/kg IL-12 given s.c. twice weekly starting on day 2 for arm A or upon progression for arm B. Results: The overall response rate was 37% (11 of 30) in arm A and 52% (13 of 25) in arm B. All of the responses seen in arm B occurred while patients received rituximab, and no responses occurred during treatment with subsequent IL-12. The median duration of response was 16 months for arm A and 12 months for arm B. Biopsy specimens were serially obtained in a subset of patients and showed that changes in gene expression were different when cells from the peripheral blood were compared with cells from lymph node biopsies. Conclusions: The concomitant use of IL-12 and rituximab had modest disease activity in patients with B-cell NHL, but the sequential administration of IL-12 after rituximab did not result in additional clinical responses.

AbstractThe underlying mechanisms by which tumor cells are resistant to CTL-mediated apoptosis are not clear. Using a human model of B-cell non-Hodgkin's lymphoma (B-cell NHL), we show that intratumoral Treg cells inhibit the proliferation and granule production of activated autologous infiltrating CD8+ T cells. Our results also show that degranulation and subsequent cytotoxic activity of infiltrating CD8+ T cells exposed to lymphoma B cells is completely attenuated by the presence of intratumoral Treg cells. Furthermore, we show that increased numbers of intratumoral Treg cells correlates with the number of CD8+ T cells in biopsy specimens from patients with B-cell NHL, supporting the in vitro findings that intratumoral Treg cells inhibit proliferation of infiltrating CD8+ T cells. Taken together, these data indicate that human lymphoma B cells are sensitive to autologous CTL-mediated cell death but are protected by the inhibitory function of intratumoral Treg cells. (Cancer Res 2006; 66(20): 10145-52)

AbstractPrimary systemic amyloidosis (AL) is an incurable plasma cell disorder. Lenalidomide, especially in conjunction with dexamethasone, is highly active in patients with multiple myeloma. We studied the toxicity and efficacy of lenalidomide in patients with AL. Patients with symptomatic AL, a measurable plasma cell disorder, and adequate hematologic and renal reserve were eligible. Patients received single-agent lenalidomide. If there was no evidence of progression after 3 months or of hematologic response after 3 cycles, dexamethasone was added. Twenty-three patients were enrolled. Thirteen were previously treated. Organ involvement was cardiac (64%), renal (73%), hepatic (23%), and nerve (14%). Within the first 3 cycles of therapy, 10 patients discontinued treatment: 4 early deaths, 3 adverse events, and 3 other causes. With a median follow-up of 17 months, 10 patients responded to treatment. In these patients, responses included 9 hematologic, 4 renal, 2 cardiac, and 2 hepatic. All but one of the responders had dexamethasone added to their treatment program. The most common grade 3 or 4 adverse events at least possibly attributable to lenalidomide were neutropenia (45%), thrombocytopenia (27%), rash (18%), and fatigue (18%). In AL patients, we saw limited activity of single-agent lenalidomide, but significant activity of the combination with dexamethasone, which warrants further investigation.

Purpose Retrospective studies suggest cytogenetic abnormalities detected by interphase fluorescent in situ hybridization (FISH) can identify patients with chronic lymphocytic leukemia (CLL) who will experience a more aggressive disease course. Other studies suggest that patients may acquire chromosome abnormalities during the course of their disease. There are minimal prospective data on the clinical utility of the widely used hierarchical FISH prognostic categories in patients with newly diagnosed early-stage CLL or the frequency of clonal evolution as determined by interphase FISH. Patients and Methods Between 1994 and 2002, we enrolled 159 patients with previously untreated CLL (83% Rai stage 0/I) on a prospective trial evaluating clonal evolution by FISH. Patients provided baseline and follow-up specimens for FISH testing during 2 to 12 years. Results Chromosomal abnormalities detected by FISH at study entry predicted overall survival. Eighteen patients experienced clonal evolution during follow-up. The rate of clonal evolution increased with duration of follow-up with only one occurrence in the first 2 years (n = 71; 1.4%) but 17 occurrences (n = 63; 27%) among patients tested after 5+ years. Clonal evolution occurred among 10% of ZAP-70–negative and 42% of ZAP-70–positive patients at 5+ years (P = .008). Conclusion This clinical trial confirms prospectively that cytogenetic abnormalities detected by FISH can predict overall survival for CLL patients at the time of diagnosis, but also suggests that many patients acquire new abnormalities during the course of their disease. Patients with higher ZAP-70 expression may be more likely to experience such clonal evolution. These findings have important implications for both clinical management and trials of early treatment for patients with high-risk, early-stage CLL.

Abstract Purpose: Colon cancer cells with high-frequency microsatellite instability (MSI-H) display resistance to 5-fluorouracil (5-FU) that can be reversed by restoring DNA mismatch repair (MMR) proficiency. Given that thymidylate synthase (TS) is inhibited by 5-FU, we studied the relationship between MSI and TS expression, and the prognostic effect of these and other markers (i.e., p53 and 17p allelic imbalance). Experimental Design: Dukes' stage B2 and C colon carcinomas (n = 320) from participants in 5-FU-based adjuvant therapy trials were analyzed for MSI and 17p allelic imbalance. Expression of MMR (hMLH1, hMSH2), TS, and p53 proteins were analyzed by immunohistochemistry. Correlations between markers and associations with overall survival were determined. Results: Of 320 cancers studied, 60 (19%) were MSI-H. TS expression variables were similar in MSI-H and microsatellite stable/low-frequency MSI (MSS/MSI-L) cancers, and unrelated to MMR proteins. MSI-H tumors had lower stage (P = 0.0007), fewer metastatic lymph nodes (P = 0.004), and improved overall survival (P = 0.01). Loss of MMR proteins was also associated with better overall survival (P = 0.006). None of the TS variables were prognostic. Histologic grade (P = 0.0008) and nodal status (P = 0.0002) were associated with overall survival, in contrast to 17p allelic imbalance or p53. Only MSI status or loss of MMR proteins, histologic grade, and tumor stage were independent markers for overall survival. Conclusions: MSI-H tumors show earlier stage at presentation and better stage-adjusted survival rates. MSI status and TS expression were unrelated and TS was not prognostic, suggesting that TS levels cannot explain therapeutic resistance to 5-FU reported in MSI-H colon cancers.

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.