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Ronald Levy

Publications and source records attributed to Ronald Levy.

At least 19 recordsLinked to original sources
PubMed2006

Cell-free production of scFv fusion proteins: an efficient approach for personalized lymphoma vaccines.

The unique immunoglobulin (Ig) idiotype on the surface of each B-cell lymphoma represents an ideal tumor-specific antigen for use as a therapeutic vaccine. We have used an Escherichia coli-based, cell-free protein-expression system to produce a vaccine within hours of cloning the Ig genes from a B-cell tumor. We demonstrated that a fusion protein consisting of an idiotypic single chain Fv antibody fragment (scFv) linked to a cytokine (GM-CSF) or to an immunostimulatory peptide was an effective lymphoma vaccine. These vaccines elicited humoral immune responses against the native Ig protein displayed on the surface of a tumor and protected mice against tumor challenge with efficacy equal to that of the conventional Ig produced in a mammalian cell and chemically coupled to keyhole limpet hemocyanin. The cell-free E coli system offers a platform for rapidly generating individualized vaccines, thereby allowing much more efficient application in the clinic.

Animals↗
PubMed2006

The oncoprotein LMO2 is expressed in normal germinal-center B cells and in human B-cell lymphomas.

We previously developed a multivariate model based on the RNA expression of 6 genes (LMO2, BCL6, FN1, CCND2, SCYA3, and BCL2) that predicts survival in diffuse large B-cell lymphoma (DLBCL) patients. Since LMO2 emerged as the strongest predictor of superior outcome, we generated a monoclonal anti-LMO2 antibody in order to study its tissue expression pattern. Immunohistologic analysis of over 1200 normal and neoplastic tissue and cell lines showed that LMO2 protein is expressed as a nuclear marker in normal germinal-center (GC) B cells and GC-derived B-cell lines and in a subset of GC-derived B-cell lymphomas. LMO2 was also expressed in erythroid and myeloid precursors and in megakaryocytes and also in lymphoblastic and acute myeloid leukemias. It was rarely expressed in mature T, natural killer (NK), and plasma cell neoplasms and was absent from nonhematolymphoid tissues except for endothelial cells. Hierarchical cluster analysis of immunohistologic data in DLBCL demonstrated that the expression profile of the LMO2 protein was similar to that of other GC-associated proteins (HGAL, BCL6, and CD10) but different from that of non-GC proteins (MUM1/IRF4 and BCL2). Our results warrant inclusion of LMO2 in multivariate analyses to construct a clinically applicable immunohistologic algorithm for predicting survival in patients with DLBCL.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Humoral immune response and immunoglobulin G Fc receptor genotype are associated with better clinical outcome following idiotype vaccination in follicular lymphoma patients regardless of their response to induction chemotherapy.

We have reported that anti-idiotype antibody response and FcgammaRIIIa 158 valine/valine (V/V) genotype both correlate with better outcome in a group of 136 follicular lymphoma patients receiving idiotype vaccination after induction chemotherapy. Here, we examined whether this correlation is related in any way to the chemotherapy response. In patients with complete response (CR), the 5-year progression-free survival (PFS) was 69% for patients with antibody response and/or V/V genotype, while the PFS was only 40% for patients with neither; the median time to progression (TTP) was 10.47 versus 3.46 years (P=.012). In patients with partial response (PR), the 5-year PFS was 57% for patients with antibody response and/or V/V genotype, and 17% for patients with neither; the median TTP was not reached versus 1.31 years (P=.001). This study further confirms the strong association of clinical outcome with antibody response and with the functionally more active form of the Fc receptor in patients receiving idiotype vaccination regardless of their response to induction chemotherapy.

Antibodies, Anti-Idiotypic↗
PubMed2006

Expression of the human germinal center-associated lymphoma (HGAL) protein identifies a subset of classic Hodgkin lymphoma of germinal center derivation and improved survival.

The human germinal-center-associated lymphoma (HGAL) gene and its cognate protein are expressed in a germinal center (GC)-specific manner. Its expression in classic Hodgkin lymphoma (cHL) prompted us to address whether HGAL expression could distinguish biologically distinct subgroups of cHL. Tissue microarrays from 145 patients treated with curative intent showed HGAL staining in 75% and was closely correlated with MUM1/IRF4 (92%) expression. BCL6 (26%), CD10 (0%), BCL2 (31%), Blimp1 (0.02%), and Epstein-Barr virus (EBV) (20%) showed no specific correlation; neither did phospho-STAT6, a key mediator of IL-4 and IL-13 signaling that induces HGAL and is implicated in cHL pathogenesis. In our study cohort, the 5-year overall survival (OS) correlated with young age (less than 45 years, P < .001), low stage (stage I and II, P = .04), and low International Prognostic Score (P = .002). In univariate analysis, HGAL expression was associated with improved OS (P = .01) and failure-free survival (FFS) (P = .05) but was not independent of other factors in multivariate analysis of OS or FFS. The expression of the GC-specific marker HGAL in a subset of cHL suggests that these cHLs retain characteristics of GC-derived lymphomas. The association with improved OS in univariate but not multivariate analysis suggests that HGAL expression is related to known clinical parameters of improved survival.

Adult↗
PubMed2006

Kinetics of B cell receptor signaling in human B cell subsets mapped by phosphospecific flow cytometry.

Differences in BCR signaling may govern outcomes as diverse as proliferation and cell death. We profiled BCR signaling kinetics in subsets of primary human B cells using flow cytometry. In the predominant population expressing IgM, BCR cross-linking led to a quick burst of Syk, ERK1/2, and p38 signaling. In contrast, IgG B cells sustained higher per-cell ERK1/2 phosphorylation over time. This dichotomy suggested a mechanism for dampening signals transmitted by IgM. Regulatory phosphatase activity in IgM B cells was BCR-mediated and initiated more slowly than kinase activity. This BCR-mediated phosphatase activity was sensitive to inhibition by H(2)O(2) and required to attenuate IgM BCR signaling. These results provide the first kinetic maps of BCR signaling in primary human B cell subsets and enable new studies of signaling in B cell disorders, such as autoimmunity and cancer.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2006

Altered B-cell receptor signaling kinetics distinguish human follicular lymphoma B cells from tumor-infiltrating nonmalignant B cells.

The B-cell receptor (BCR) transmits life and death signals throughout B-cell development, and altered BCR signaling may be required for survival of B-lymphoma cells. We used single-cell signaling profiles to compare follicular lymphoma (FL) B cells and nonmalignant host B cells within individual patient biopsies and identified BCR-mediated signaling events specific to lymphoma B cells. Expression of CD20, Bcl-2, and BCR light chain isotype (kappa or lambda) distinguished FL tumor B-cell and nontumor host B-cell subsets within FL patient biopsies. BCR-mediated signaling via phosphorylation of Btk, Syk, Erk1/2, and p38 occurred more rapidly in tumor B cells from FL samples than in infiltrating nontumor B cells, achieved greater levels of per-cell signaling, and sustained this level of signaling for hours longer than nontumor B cells. The timing and magnitude of BCR-mediated signaling in nontumor B cells within an FL sample instead resembled that observed in mature B cells from the peripheral blood of healthy subjects. BCR signaling pathways that are potentiated specifically in lymphoma cells should provide new targets for therapeutic attention.

B-Lymphocytes↗
PubMed2005

Therapeutic vaccination against murine lymphoma by intratumoral injection of naive dendritic cells.

Dendritic cells are potent antigen-presenting cells that can induce both immune responses and tolerance depending on their state of activation. Immunologic tolerance to established tumors is a major impediment for the development of effective cancer immunotherapy. Dendritic cells may be deficient in number or in function at the tumor site. To address this problem, we evaluated the ability of immature naïve dendritic cells to induce an antitumor immune response when injected directly into a murine B-cell lymphoma. Mice with advanced transplanted syngeneic tumor were given intratumoral injections of bone marrow-derived dendritic cells. Intratumoral dendritic cell injection alone had no antitumor effect. Systemic chemotherapy alone resulted in only transient tumor regression. However, the intratumoral injection of dendritic cells after chemotherapy led to complete, long-term tumor regression in the majority of treated mice. This dendritic cell-mediated antitumor effect was systemic, resulting in simultaneous elimination of the tumor at second uninjected sites. In addition, it resulted in long-term memory with resistance to tumor rechallenge. Both CD4+ and CD8+ T cells are necessary for the antitumor effect. Furthermore, tumors that occasionally recurred in mice with initial complete tumor regression could be retreated by the same combined chemoimmunotherapy approach. These results show that immunotherapy can succeed in the setting of advanced lymphoma if dendritic cells are restored and loaded with tumor antigens in situ at a single tumor site.

Animals↗
PubMed2005

Rapid expression of vaccine proteins for B-cell lymphoma in a cell-free system.

The idiotype (Id)-granulocyte-macrophage colony-stimulating factor (GM-CSF) fusion proteins are potential vaccines for immunotherapy of B-cell lymphoma. In this study, four vaccine candidates were constructed by fusing murine GM-CSF to the amino- or carboxy-terminus of the 38C13 murine B-lymphocyte Id scFv with two different arrangements of the variable regions of the heavy chain and light chain (VL-VH and VH-VL). scFv (VH-VL) and GM-CSF/scFv fusion proteins were expressed in an Escherichia coli cell-free protein synthesis system. In order to promote disulfide bond formation during cell-free expression, cell extract was pretreated with iodoacetamide (IAM), and a sulfhydryl redox buffer composed of oxidized and reduced glutathione was added. The E. coli periplasmic disulfide isomerase, DsbC, was also added to rearrange incorrectly formed disulfide linkages. The 38C13 B-lymphocyte Id scFv was expressed with 30% of its soluble yield in active form (43 microg/ml) when tested with an anti-idiotypic mAb, S1C5, as the capture antibody in radioimmunoassay. It was found that the amino-terminal GM-CSF fusion proteins, GM-VL-VH and GM-VH-VL, showed much higher activity than the carboxy-terminal GM-CSF fusion proteins, VL-VH-GM and VH-VL-GM, in stimulating the cell proliferation of a GM-CSF-dependent cell line, NFS-60. Between the two amino-terminal GM-CSF fusion proteins, GM-VL-VH showed a higher total and soluble yield than GM-VH-VL.

Animals↗
PubMed2005

Expression of the human germinal center-associated lymphoma (HGAL) protein, a new marker of germinal center B-cell derivation.

We identified the human germinal center-associated lymphoma (HGAL) in gene-expression profiling studies of diffuse large B-cell lymphoma (DLBCL). The expression of HGAL correlated with survival in patients with DLBCL. The HGAL gene is the human homolog of M17, a mouse gene expressed specifically in normal germinal center (GC) B cells. We generated a monoclonal antibody against the HGAL protein and show that HGAL is expressed in the cytoplasm of GC lymphocytes and in lymphomas of GC derivation. Among 727 lymphomas tested by immunohistochemistry on tissue microarrays, HGAL staining was found in follicular lymphomas (103 of 107), Burkitt lymphomas (40 of 40), mediastinal large B lymphomas (7 of 8), and in DLBCLs (103 of 151). Most marginal zone lymphomas lacked HGAL staining. Lymphocyte-predominant Hodgkin lymphomas (12 of 17) and, surprisingly, classical Hodgkin lymphomas (78 of 107) were found to be positive. Hierarchical clustering of comparative immunohistologic results in DLBCLs demonstrates that the expression of HGAL is similar to 2 other GC-associated proteins, BCL6 and CD10, but different from 2 markers associated with a non-GC phenotype, MUM1/IRF4 and BCL2. The restricted expression and GC specificity of HGAL protein suggest that it may have an important role in the diagnosis of specific lymphomas, and, potentially in the identification of subtypes associated with different prognoses.

Animals↗
PubMed2004

The radioisotope contributes significantly to the activity of radioimmunotherapy.

PURPOSE: A multicenter, randomized study was undertaken to estimate the single agent activity of Tositumomab and to determine the contribution of radioisotope-labeling with (131)I to activity and toxicity by comparing treatment outcomes for Tositumomab and Iodine I 131 Tositumomab (BEXXAR) to an equivalent total dose of unlabeled Tositumomab. EXPERIMENTAL DESIGN: Seventy-eight patients with refractory/relapsed non-Hodgkin's lymphoma were randomized to either unlabeled Tositumomab or Iodine I 131 Tositumomab. Patients progressing after unlabeled Tositumomab could cross over to receive Iodine I 131 Tositumomab. The median follow-up at analysis was 42.6 months (range 1.9 to 71.5 months). RESULTS: Responses in the Iodine I 131 Tositumomab versus unlabeled Tositumomab groups: overall response 55% versus 19% (P = 0.002); complete response 33% versus 8% (P = 0.012); median duration of overall response not reached versus 28.1 months (95% confidence interval: 7.6, not reached); median duration of complete response not reached in either arm; and median TTP 6.3 versus 5.5 months (P = 0.031), respectively. Of the patients who had a complete response after initial Iodine I 131 Tositumomab therapy, 71% (10 of 14) continued in complete response at 29.8 to 71.1 months. Two patients who achieved a complete response after unlabeled Tositumomab had ongoing responses at 48.1 to 56.9 months. Nineteen patients received Iodine I 131 Tositumomab crossover therapy. Responses after crossover versus prior response to unlabeled Tositumomab were as follows: complete response rates of 42% versus 0% (P = 0.008); overall response 68% versus 16% (P = 0.002); median durations of overall response 12.6 versus 7.6 months (P = 0.001); and median TTP 12.4 versus 5.5 months (P = 0.01), respectively. Hematologic toxicity was more severe and nonhematologic adverse events were more frequent after Iodine I 131 Tositumomab than after Tositumomab alone. Elevated thyrotropin occurred in 5% of patients. Seroconversion to human antimurine antibody after Iodine I 131 Tositumomab, unlabeled Tositumomab, and Iodine I 131 Tositumomab-crossover was 27%, 19%, and 0%, respectively. CONCLUSIONS: Unlabeled Tositumomab showed single agent activity, but in this direct comparison, all of the therapeutic outcome measures were significantly enhanced by the conjugation of (131)I to Tositumomab.

Adult↗
PubMed2004

Clinical outcome of lymphoma patients after idiotype vaccination is correlated with humoral immune response and immunoglobulin G Fc receptor genotype.

PURPOSE: The unique immunoglobulin idiotype (Id) expressed by each B-cell lymphoma is a target for immunotherapy. Vaccination with Id induces humoral and/or cellular anti-Id immune responses. However, the clinical impact of these anti-Id immune responses is unknown. We and others have previously reported that immunoglobulin G Fc receptor (FcgammaR) polymorphisms predict the clinical response of lymphoma patients to passive anti-CD20 antibody infusions. In this study, we tested whether anti-Id immune responses or FcgammaR polymorphisms associate with clinical outcome of patients who received Id vaccination. PATIENTS AND METHODS: We analyzed 136 patients with follicular lymphoma who had received Id vaccination. The anti-Id immune responses were measured and FcgammaRIIIa and FcgammaRIIa polymorphisms were determined and correlated with clinical outcome for these patients. RESULTS: Patients who mounted humoral immune responses had a longer progression-free survival (PFS) than those who did not (8.21 v 3.38 years; P = .018). Patients with FcgammaRIIIa 158 valine/valine (V/V) genotype also had a longer PFS than those with valine/phenylalanine (V/F) or phenylalanine/phenylalanine (F/F) genotypes (V/V, 8.21 v V/F, 3.38 years; P = .004; v F/F, 4.47 years; P = .035). Multivariate analysis using the Cox proportional hazards model showed that V/V genotype and humoral immune responses were independent positive predictors for PFS. CONCLUSION: This study is the first to identify the predictive value of FcgammaR polymorphism on clinical outcome in patients who received active immunotherapy with tumor antigen vaccines. Our results imply that the antibodies induced against a tumor antigen are beneficial and that FcgammaR-bearing cells mediate an antitumor effect by killing antibody-coated tumor cells.

Antigens, CD↗
PubMed2004

Prediction of survival in diffuse large-B-cell lymphoma based on the expression of six genes.

BACKGROUND: Several gene-expression signatures can be used to predict the prognosis in diffuse large-B-cell lymphoma, but the lack of practical tests for a genome-scale analysis has restricted the use of this method. METHODS: We studied 36 genes whose expression had been reported to predict survival in diffuse large-B-cell lymphoma. We measured the expression of each of these genes in independent samples of lymphoma from 66 patients by quantitative real-time polymerase-chain-reaction analyses and related the results to overall survival. RESULTS: In a univariate analysis, genes were ranked on the basis of their ability to predict survival. The genes that were the strongest predictors were LMO2, BCL6, FN1, CCND2, SCYA3, and BCL2. We developed a multivariate model that was based on the expression of these six genes, and we validated the model in two independent microarray data sets. The model was independent of the International Prognostic Index and added to its predictive power. CONCLUSIONS: Measurement of the expression of six genes is sufficient to predict overall survival in diffuse large-B-cell lymphoma.

Adolescent↗
PubMed2004

TCR vaccines against a murine T cell lymphoma: a primary role for antibodies of the IgG2c class in tumor protection.

Tumor-associated proteins can act as effective immunotherapeutic targets. Immunization with tumor TCR protein conjugated to the immunogenic protein keyhole limpet hemocyanin (KLH) protects mice from tumor challenge with the murine T cell lymphoma C6VL. The immune mechanisms responsible for this tumor protection are of interest for designing more effective vaccine strategies. Previous studies using depletion experiments had suggested a CD8-mediated component of protection induced by TCR-KLH vaccines. In this study we used CD8alpha knockout, micro MT, and FcgammaR knockout mice to investigate the relative roles of CD8+ T cells and Ab in protective immunity induced by TCR-KLH immunization. We found that CD8+ T cells are not required for tumor protection, although they may contribute to protection. Vaccine-induced Abs are sufficient to mediate protection against this murine T cell lymphoma through an FcR-dependent mechanism. This was confirmed with Ab transfers, which protect challenged mice. Additionally, recombinase-activating gene 1(-/-) splenocytes can mediate Ab-dependent cellular cytotoxicity against this tumor in the presence of bound anti-TCR Abs. IFN-gamma knockout mice demonstrated a requirement for IFN-gamma, probably via generation of IgG2c Abs, in vaccine-induced tumor protection. IFN-gamma knockout mice were not protected by immunization and had a severe impairment in IgG2c Ab production in response to immunization. Although mock-depleted anti-TCR Abs could transfer tumor protection, IgG2c-deficient anti-TCR Abs were unable to transfer tumor protection to wild-type mice. These results suggest that TCR-KLH vaccine-induced tumor protection in the C6VL system is primarily attributable to the induction of IgG2c Abs and humoral immunity.

Adjuvants, Immunologic↗
PubMed2004

Molecular rescue of tumour-specific T cell receptor idiotype from T cell lymphomas.

The T cell receptor (TCR) idiotype on T cell lymphomas can serve as a vaccine target. To clone the relevant genes, 5' rapid amplification of cDNA ends (RACE) was performed on 13 T cell lymphomas and nine control samples. Two polymerase chain reactions (PCR) were performed for each TCR chain (alpha and beta) and the proportion of the clonal TCR sequence over the total number of TCR sequences was calculated. For alpha, the average proportions were 0.43 vs. 0.05. For beta these were 0.44 and 0.04. The TCR was identified in 10 of 13 lymphoma samples.

Base Sequence↗
PubMed2003

Two immunoglobulin G fragment C receptor polymorphisms independently predict response to rituximab in patients with follicular lymphoma.

PURPOSE: Although rituximab is now routinely used in the treatment of B-cell non-Hodgkin's lymphoma, the mechanism of its antitumor effect is not clear. One potential mechanism of action involves antibody-dependent cellular cytotoxicity (ADCC). Two aspects of ADCC influence the effectiveness of this process: the susceptibility of tumor cells and the activation of effector cells via their immunoglobulin G fragment C receptors (Fc gamma Rs). Several Fc gamma R polymorphisms have been identified that may affect the killing function of natural killer cells and macrophages. PATIENTS AND METHODS: The pretreatment tumor cells from 43 patients with follicular lymphoma were tested for their intrinsic susceptibility to rituximab-mediated ADCC. In addition, the Fc gamma RIIIa (CD16) and Fc gamma RIIa (CD32) polymorphisms were determined in an expanded group of 87 patients. The results were then correlated with clinical outcome of these patients. RESULTS: No difference was found between the susceptibility of tumors from patients who clinically responded to rituximab versus those who did not respond. Conversely, both the Fc gamma RIIIa 158 valine/valine and the Fc gamma RIIa 131 histidine/histidine genotypes were found to be independently associated with the response rate and freedom from progression. CONCLUSION: These data support the hypothesis that ADCC plays an important role in the clinical effect of rituximab at the level of the effector cell. It will be important to include information on Fc receptor polymorphisms in future trials of rituximab therapy.

Antibodies, Monoclonal↗
PubMed2003

BCL6 gene translocation in follicular lymphoma: a harbinger of eventual transformation to diffuse aggressive lymphoma.

Follicular lymphoma (FL) is characterized by a relatively indolent clinical course, but the disease often transforms into a more aggressive large cell lymphoma with a rapidly progressive clinical course. In the present study, we analyzed 41 cases of FL known to have subsequently transformed to aggressive lymphoma and an additional 64 FL samples from patients not subsequently transformed. We studied BCL6 gene rearrangement by the methodology of long-distance inverse polymerase chain reaction (LDI-PCR). Of the 41 cases known to transform, 16 (39.0%) harbored BCL6 translocation or deletion at the time of FL diagnosis. Among 64 cases not known to transform, BCL6 translocation was detected in 9 (14.1%). The prevalence of BCL6 translocation in the group known to transform was significantly higher (P =.0048). Among the transformation cases, the partners of the BCL6 translocation were identified in 13 cases and included IGH, CIITA, U50HG, MBNL, GRHPR, LRMP, EIF4A2, RhoH/TTF, and LOC92656 (similar to NAPA), whereas in the control group the BCL6 partners were IGH, CIITA, SIAT1, and MBNL. In 13 cases paired specimens before and after transformation were available. Among these paired specimens, a loss (3 cases) or a gain (1 case) of BCL6 translocation was observed after the transformation. Analysis of clonality showed that all of these cases represented the evolution of a subclone of the original tumor population. Our study demonstrated that BCL6 translocation is not necessary for transformation but that BCL6 translocation in FL may constitute a subgroup with a higher risk to transform into aggressive lymphoma.

Cell Transformation, Neoplastic↗