T-cell help in the tumor microenvironment enhances rituximab-mediated NK-cell ADCC.

ABSTRACT: Rituximab (RTX) and other monoclonal antibodies (mAbs) that bind directly to malignant cells are of great clinical value but are not effective for all patients. A major mechanism of action of RTX is antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells. Prior in vitro studies in our laboratory demonstrated that T cells contribute to maintaining the viability and cytotoxic potential of NK cells activated by anti-CD20-coated target B cells. Here, we conducted studies using a novel mouse model and clinical correlative analysis to assess whether T-cell help contribute to RTX-mediated NK-cell ADCC in the tumor microenvironment (TME) in vivo. A humanized mouse model was developed using Raji lymphoma cells and normal donor peripheral blood mononuclear cells that allows for control of T-cell numbers in the lymphoma TME. In this model, NK-cell viability and CD16 and CD25 expression dropped after RTX in the absence of T cells but increased in the presence of T cells. RTX therapy was more effective when T cells were present and was ineffective when NK cells were depleted. In patients with indolent lymphoma, fine needle aspirates were obtained before and ∼1 week after treatment with a RTX-containing regimen. There was a strong correlation between CD4+ T cells as well as total T cells in the pretherapy TME and an increase in NK-cell CD16 and CD25 expression after RTX. We conclude that T-cell help in the TME enhances RTX-mediated NK-cell viability and ADCC.

The Lymphoma Epidemiology of Outcomes cohort study: Design, baseline characteristics, and early outcomes.

To address the current and long-term unmet health needs of the growing population of non-Hodgkin lymphoma (NHL) patients, we established the Lymphoma Epidemiology of Outcomes (LEO) cohort study (NCT02736357; https://leocohort.org/). A total of 7735 newly diagnosed patients aged 18 years and older with NHL were prospectively enrolled from 7/1/2015 to 5/31/2020 at 8 academic centers in the United States. The median age at diagnosis was 62 years (range, 18-99). Participants came from 49 US states and included 538 Black/African-Americans (AA), 822 Hispanics (regardless of race), 3386 women, 716 age <40 years, and 1513 rural residents. At study baseline, we abstracted clinical, pathology, and treatment data; banked serum/plasma (N = 5883, 76.0%) and germline DNA (N = 5465, 70.7%); constructed tissue microarrays for four major NHL subtypes (N = 1189); and collected quality of life (N = 5281, 68.3%) and epidemiologic risk factor (N = 4489, 58.0%) data. Through August 2022, there were 1492 deaths. Compared to population-based SEER data (2015-2019), LEO participants had a similar distribution of gender, AA race, Hispanic ethnicity, and NHL subtype, while LEO was underrepresented for patients who were Asian and aged 80 years and above. Observed overall survival rates for LEO at 1 and 2 years were similar to population-based SEER rates for indolent B-cell (follicular and marginal zone) and T-cell lymphomas, but were 10%-15% higher than SEER rates for aggressive B-cell subtypes (diffuse large B-cell and mantle cell). The LEO cohort is a robust and comprehensive national resource to address the role of clinical, tumor, host genetic, epidemiologic, and other biologic factors in NHL prognosis and survivorship.

T cells, particularly activated CD4+ cells, maintain anti-CD20-mediated NK cell viability and antibody dependent cellular cytotoxicity.

Anti-CD20 monoclonal antibody (mAb) therapy is a mainstay of therapy for B cell malignancies, however many patients fail to respond or eventually develop resistance. The current understanding of mechanisms responsible for this resistance is limited. When peripheral blood mononuclear cells of healthy donors were cultured with Raji cells for 7 days, rituximab (RTX) induced NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC), enhanced NK cell viability and increased or maintained NK expression of CD56, CD16, CD57 and KIR. T cells, mainly CD4+, mediated these changes in a contact-dependent manner, with local T cell production of IL2 playing a central role. Similar findings were found when autologous B cells were used as target cells demonstrating the need for T cell help was not due to allogenic reaction. Results with other anti-CD20 and anti-EGFR antibodies were consistent. Small numbers of T cells activated by anti-CD3/CD28 beads or bispecific antibody enhanced RTX-mediated NK cell ADCC, viability and phenotypical changes. Pathway analysis of bulk NK cell mRNA sequencing after activation by RTX with and without T cells was consistent with T cells maintaining the viability of the activated NK cells. These findings suggest T cell help, mediated in large part by local production of IL2, contributes to NK cell ADCC and viability, and that activating T cells in the tumor microenvironment, such as through the use of anti-CD3 based bispecific antibodies, could enhance the efficacy of anti-CD20 and other mAb therapies where NK-mediated ADCC is a primary mechanism of action.

Antibody Opsonization of a TLR9 Agonist-Containing Virus-like Particle Enhances In Situ Immunization.

The immunologic and therapeutic effects of intratumoral (IT) delivery of a novel virus-like particle as a lymphoma immunotherapy were evaluated in preclinical studies with human cells and a murine model. CMP-001 is a virus-like particle composed of the Qß bacteriophage capsid protein encapsulating an immunostimulatory CpG-A oligodeoxynucleotide TLR9 agonist. In vitro, CMP-001 induced cytokine production, including IFN-α from plasmacytoid dendritic cells, but only in the presence of anti-Qß Ab. In vivo, IT CMP-001 treatment of murine A20 lymphoma enhanced survival and reduced growth of both injected and contralateral noninjected tumors in a manner dependent on both the ability of mice to generate anti-Qß Ab and the presence of T cells. The combination of IT CMP-001 with systemic anti-PD-1 enhanced antitumor responses in both injected and noninjected tumors. IT CMP-001 alone or combined with anti-PD-1 augmented T cell infiltration in tumor-draining lymph nodes. We conclude IT CMP-001 induces a robust antitumor T cell response in an anti-Qß Ab-dependent manner and results in systemic antitumor T cell effects that are enhanced by anti-PD-1 in a mouse model of B cell lymphoma. Early-phase clinical evaluation of CMP-001 and anti-PD-1 combination therapy in lymphoma will begin shortly, based in part on these results.

Intraperitoneal CMP-001: A Novel Immunotherapy for Treating Peritoneal Carcinomatosis of Gastrointestinal and Pancreaticobiliary Cancer.

BACKGROUND: The treatment options for patients with peritoneal carcinomatosis (PC) of gastrointestinal and pancreaticobiliary origins are limited. The virus-like particle, CMP-001, composed of the Qß bacteriophage capsid protein encapsulating a CpG-A oligodeoxynucleotide, activates plasmacytoid dendritic cells (pDCs) and triggers interferon alpha (IFNα) release, leading to a cascade of anti-tumor immune effects. METHODS: To evaluate the ability of CMP-001 to trigger an immune response in patients with PC, peritoneal cells were isolated and stimulated ex vivo with CMP-001. Both IFNα release and percentage of pDC were quantified using enzyme-linked immunosorbent assay (ELISA) and flow cytometry, respectively. To evaluate the anti-tumor response in vivo, murine PC models were generated using mouse cancer cell lines (Panc02 and MC38) in immunocompetent mice treated with intraperitoneal CMP-001 or saline control. Survival was followed, and the immunophenotype of cells in the peritoneal tumor microenvironment was evaluated. RESULTS: The pDCs accounted for 1% (range 0.1-3.9%; n = 17) of the isolated peritoneal cells. Ex vivo CMP-001 stimulation of the peritoneal cells released an average of 0.77 ng/ml of IFNα (range, 0-4700 pg/ml; n = 14). The IFNα concentration was proportional to the percentage of pDCs present in the peritoneal cell mixture (r = 0.6; p = 0.037). In murine PC models, intraperitoneal CMP-001 treatment elicited an anti-tumor immune response including an increase in chemokines (RANTES and MIP-1ß), pro-inflammatory cytokines (IFNγ, interleukin 6 [IL-6], and IL-12), and peritoneal/tumor immune infiltration (CD4+/CD8+ T and natural killer [NK] cells). The CMP-001 treatment improved survival in both the Panc02 (median, 35 vs 28 days) and the MC38 (median: 57 vs 35 days) PC models (p < 0.05). CONCLUSIONS: As a novel immunotherapeutic agent, CMP-001 may be effective for treating patients with PC.

Prospective correlation between the patient microbiome with response to and development of immune-mediated adverse effects to immunotherapy in lung cancer.

BACKGROUND: Though the gut microbiome has been associated with efficacy of immunotherapy (ICI) in certain cancers, similar findings have not been identified for microbiomes from other body sites and their correlation to treatment response and immune related adverse events (irAEs) in lung cancer (LC) patients receiving ICIs. METHODS: We designed a prospective cohort study conducted from 2018 to 2020 at a single-center academic institution to assess for correlations between the microbiome in various body sites with treatment response and development of irAEs in LC patients treated with ICIs. Patients must have had measurable disease, ECOG 0-2, and good organ function to be included. Data was collected for analysis from January 2019 to October 2020. Patients with histopathologically confirmed, advanced/metastatic LC planned to undergo immunotherapy-based treatment were enrolled between September 2018 and June 2019. Nasal, buccal and gut microbiome samples were obtained prior to initiation of immunotherapy +/- chemotherapy, at development of adverse events (irAEs), and at improvement of irAEs to grade 1 or less. RESULTS: Thirty-seven patients were enrolled, and 34 patients were evaluable for this report. 32 healthy controls (HC) from the same geographic region were included to compare baseline gut microbiota. Compared to HC, LC gut microbiota exhibited significantly lower α-diversity. The gut microbiome of patients who did not suffer irAEs were found to have relative enrichment of Bifidobacterium (p = 0.001) and Desulfovibrio (p = 0.0002). Responders to combined chemoimmunotherapy exhibited increased Clostridiales (p = 0.018) but reduced Rikenellaceae (p = 0.016). In responders to chemoimmunotherapy we also observed enrichment of Finegoldia in nasal microbiome, and increased Megasphaera but reduced Actinobacillus in buccal samples. Longitudinal samples exhibited a trend of α-diversity and certain microbial changes during the development and resolution of irAEs. CONCLUSIONS: This pilot study identifies significant differences in the gut microbiome between HC and LC patients, and their correlation to treatment response and irAEs in LC. In addition, it suggests potential predictive utility in nasal and buccal microbiomes, warranting further validation with a larger cohort and mechanistic dissection using preclinical models. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03688347 . Retrospectively registered 09/28/2018.

Status of Cancer Care at Network Sites of the Nation's Academic Cancer Centers.

BACKGROUND: Cancer care coordination across major academic medical centers and their networks is evolving rapidly, but the spectrum of organizational efforts has not been described. We conducted a mixed-methods survey of leading cancer centers and their networks to document care coordination and identify opportunities to improve geographically dispersed care. METHODS: A mixed-methods survey was sent to 91 cancer centers in the United States and Canada. We analyzed the number and locations of network sites; access to electronic medical records (EMRs); clinical research support and participation at networks; use of patient navigators, care paths, and quality measures; and physician workforce. Responses were collected via Qualtrics software between September 2017 and December 2018. RESULTS: Of the 69 responding cancer centers, 74% were NCI-designated. Eighty-seven percent of respondents were part of a matrix health system, and 13% were freestanding. Fifty-six reported having network sites. Forty-three respondents use navigators for disease-specific populations, and 24 use them for all patients. Thirty-five respondents use ≥1 types of care path. Fifty-seven percent of networks had complete, integrated access to their main center's EMRs. Thirty-nine respondents said the main center provides funding for clinical research at networks, with 22 reporting the main center provides all funding. Thirty-five said the main center provided pharmacy support at the networks, with 15 indicating the main center provides 100% pharmacy support. Certification program participation varied extensively across networks. CONCLUSIONS: The data show academic cancer centers have extensive involvement in network cancer care, often extending into rural communities. Coordinating care through improved clinical trial access and greater use of patient navigation, care paths, coordinated EMRs, and quality measures is likely to improve patient outcomes. Although it is premature to draw firm conclusions, the survey results are appropriate for mapping next steps and data queries.

Helminth-Induced Production of TGF-ß and Suppression of Graft-versus-Host Disease Is Dependent on IL-4 Production by Host Cells.

Helminths stimulate the secretion of Th2 cytokines, like IL-4, and suppress lethal graft-versus-host disease (GVHD) after bone marrow transplantation. This suppression depends on the production of immune-modulatory TGF-ß and is associated with TGF-ß-dependent in vivo expansion of Foxp3+ regulatory T cells (Treg). In vivo expansion of Tregs is under investigation for its potential as a therapy for GVHD. Nonetheless, the mechanism of induced and TGF-ß-dependent in vivo expansion of Tregs, in a Th2 polarized environment after helminth infection, is unknown. In this study, we show that helminth-induced IL-4 production by host cells is critical to the induction and maintenance of TGF-ß secretion, TGF-ß-dependent expansion of Foxp3+ Tregs, and the suppression of GVHD. In mice with GVHD, the expanding donor Tregs express the Th2-driving transcription factor, GATA3, which is required for helminth-induced production of IL-4 and TGF-ß. In contrast, TGF-ß is not necessary for GATA3 expression by Foxp3+ Tregs or by Foxp3- CD4 T cells. Various cell types of innate or adaptive immune compartments produce high quantities of IL-4 after helminth infection. As a result, IL-4-mediated suppression of GVHD does not require invariant NKT cells of the host, a cell type known to produce IL-4 and suppress GVHD in other models. Thus, TGF-ß generation, in a manner dependent on IL-4 secretion by host cells and GATA3 expression, constitutes a critical effector arm of helminthic immune modulation that promotes the in vivo expansion of Tregs and suppresses GVHD.

Outcomes among North American patients with diffuse large B-cell lymphoma are independent of tumor Epstein-Barr virus positivity or immunosuppression.

The prevalence, presenting clinical and pathological characteristics, and outcomes for patients with diffuse large B-cell lymphoma that is Epstein-Barr virus positive remain uncertain as does the impact of congenital or iatrogenic immunosuppression. Patients with newly diagnosed diffuse large B-cell lymphoma with available tissue arrays were identified from the University of Iowa/Mayo Clinic Molecular Epidemiology Resource. Patients infected with human immunodeficiency virus or who had undergone a prior organ transplant were excluded. Epstein-Barr virus-associated ribonucleic acid testing was performed on all tissue arrays. A history of significant congenital or iatrogenic immunosuppression was determined for all patients. At enrollment, 16 of the 362 (4.4%) biopsies were positive for Epstein-Barr virus. Thirty-nine (10.8%) patients had a significant history of immunosuppression. Patients with Epstein-Barr-positive diffuse large B-cell lymphoma had no unique clinical characteristics but on pathology exhibited a higher frequency of CD30 positivity (25.0% versus 8.1%, respectively; P<0.01), and non-germinal-center subtype (62.5% versus 34.1%, respectively; P<0.01). No baseline clinical characteristics were associated with a history of immunosuppression. With a median follow up of 59 months, and after adjustment for International Prognostic Index, there was no association of Epstein-Barr virus positivity or immunosuppression with event-free survival at 24 months (odds ratio=0.49; 95% confidence interval: 0.13-1.84 and odds ratio=0.81; 95% confidence interval: 0.37-1.77) or overall survival (hazard ratio=0.86; 95% confidence interval: 0.38-1.97 and hazard ratio=1.00; 95% confidence interval: 0.57-1.74). In contrast to non-Western populations, our North American population had a low prevalence of Epstein-Barr virus-positive diffuse large B-cell lymphoma that did not convey an adverse prognosis. A history of immunosuppression, while known to be a risk factor for the development of diffuse large B-cell lymphoma, did not affect subsequent prognosis.

Academic Cancer Center Phase I Program Development.

Multiple factors critical to the effectiveness of academic phase I cancer programs were assessed among 16 academic centers in the U.S. Successful cancer centers were defined as having broad phase I and I/II clinical trial portfolios, multiple investigator-initiated studies, and correlative science. The most significant elements were institutional philanthropic support, experienced clinical research managers, robust institutional basic research, institutional administrative efforts to reduce bureaucratic regulatory delays, phase I navigators to inform patients and physicians of new studies, and a large cancer center patient base. New programs may benefit from a separate stand-alone operation, but mature phase I programs work well when many of the activities are transferred to disease-oriented teams. The metrics may be useful as a rubric for new and established academic phase I programs. The Oncologist 2017;22:369-374.

Intestinal helminths regulate lethal acute graft-versus-host disease and preserve the graft-versus-tumor effect in mice.

Donor T lymphocyte transfer with hematopoietic stem cells suppresses residual tumor growth (graft-versus-tumor [GVT]) in cancer patients undergoing bone marrow transplantation (BMT). However, donor T cell reactivity to host organs causes severe and potentially lethal inflammation called graft-versus-host disease (GVHD). High-dose steroids or other immunosuppressive drugs are used to treat GVHD that have limited ability to control the inflammation while incurring long-term toxicity. Novel strategies are needed to modulate GVHD, preserve GVT, and improve the outcome of BMT. Regulatory T cells (Tregs) control alloantigen-sensitized inflammation of GVHD, sustain GVT, and prevent mortality in BMT. Helminths colonizing the alimentary tract dramatically increase the Treg activity, thereby modulating intestinal or systemic inflammatory responses. These observations led us to hypothesize that helminths can regulate GVHD and maintain GVT in mice. Acute GVHD was induced in helminth (Heligmosomoides polygyrus)-infected or uninfected BALB/c recipients of C57BL/6 donor grafts. Helminth infection suppressed donor T cell inflammatory cytokine generation and reduced GVHD-related mortality, but maintained GVT. H. polygyrus colonization promoted the survival of TGF-ß-generating recipient Tregs after a conditioning regimen with total body irradiation and led to a TGF-ß-dependent in vivo expansion/maturation of donor Tregs after BMT. Helminths did not control GVHD when T cells unresponsive to TGF-ß-mediated immune regulation were used as donor T lymphocytes. These results suggest that helminths suppress acute GVHD using Tregs and TGF-ß-dependent pathways in mice. Helminthic regulation of GVHD and GVT through intestinal immune conditioning may improve the outcome of BMT.

Early event status informs subsequent outcome in newly diagnosed follicular lymphoma.

Recent advances in follicular lymphoma (FL) have resulted in prolongation of overall survival (OS). Here we assessed if early events as defined by event-free survival (EFS) at 12 and 24 months from diagnosis (EFS12/EFS24) can inform subsequent OS in FL. 920 newly diagnosed grade 1-3A FL patients enrolled on the University of Iowa/Mayo Clinic Lymphoma SPORE Molecular Epidemiology Resource (MER) from 2002-2012 were initially evaluated. EFS was defined as time from diagnosis to progression, relapse, re-treatment, or death due to any cause. OS was compared to age-and-sex-matched survival in the general US population using standardized mortality ratios (SMR) and 95% confidence intervals (CI). We used a cohort of 412 FL patients from two Lyon, France hospital registries for independent replication. Patients who failed to achieve EFS12 had poor subsequent OS (MER SMR = 3.72, 95%CI: 2.78-4.88; Lyon SMR = 8.74, 95%CI: 5.41-13.36). Conversely, patients achieving EFS12 had no added mortality beyond the background population (MER SMR = 0.73, 95%CI: 0.56-0.94, Lyon SMR = 1.02, 95%CI: 0.58-1.65). Patients with early events after immunochemotherapy had especially poor outcomes (EFS12 failure: MER SMR = 17.63, 95%CI:11.97-25.02, Lyon SMR = 19.10, 95%CI:9.86-33.36; EFS24 failure: MER SMR = 13.02, 95%CI:9.31-17.74, Lyon SMR = 7.22, 95%CI:4.13-11.74). In a combined dataset of all patients from both cohorts, baseline FLIPI was no longer informative in EFS12 achievers. Reassessment of patient status at 12 months from diagnosis in follicular lymphoma patients, or at 24 months in patients treated with immunochemotherapy, is a strong predictor of subsequent overall survival in FL. Early event status provides a simple, clinically relevant endpoint for studies assessing outcome in FL. Am. J. Hematol. 91:1096-1101, 2016. © 2016 Wiley Periodicals, Inc.

B-CLL cells acquire APC- and CTL-like phenotypic characteristics after stimulation with CpG ODN and IL-21.

CpG oligodeoxynucleotides (CpG) and IL-21 are two promising agents for the treatment of B-cell chronic lymphocytic leukemia (B-CLL). Recently, we reported that the combination of CpG and IL-21 (CpG/IL-21) can induce granzyme B (GrB)-dependent apoptosis in B-CLL cells. Here, we demonstrate that treatment of B-CLL cells with CpG and IL-21 results in the development of antigen-presenting cell (APC)-like cells with cytotoxic features. These properties eventually give rise to B-CLL cell apoptosis, independently of their cytogenetic phenotype, whereas normal B-cell survival is not negatively affected by CpG/IL-21. APC- and CTL-typical molecules found to be up-regulated in CpG/IL-21-stimulated B-CLL cells include GrB, perforin, T-bet, monokine-induced by IFN-γ and IFN-γ-inducible protein 10 (IP-10), as well as molecules important for cell adhesion, antigen cross-presentation and costimulation. Also induced are molecules involved in GrB induction, trafficking and processing, whereas the GrB inhibitor Serpin B9 [formerly proteinase inhibitor-9 (PI-9)] is down-modulated by CpG/IL-21. In conclusion, CpG/IL-21-stimulated B-CLL cells acquire features that are reminiscent of killer dendritic cells, and which result in enhanced immunogenicity, cytotoxicity and apoptosis. Our results provide novel insights into the aberrant immune state of B-CLL cells and may establish a basis for the development of an innovative cellular vaccination approach in B-CLL.

Anti-Lymphoma Monoclonal Antibodies: Making Better Antibodies and Making Antibodies Better.

Monoclonal antibodies (mAbs) are highly effective anti-cancer agents; however, there is still considerable room for improving their therapeutic impact. Understanding the mechanisms of action of anti-cancer mAbs, and modifying mAb structure based on this understanding, is leading to the design and testing of more effective mAbs.

Elevated soluble IL-2Rα, IL-8, and MIP-1ß levels are associated with inferior outcome and are independent of MIPI score in patients with mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a unique type of lymphoma with a prognosis intermediate between indolent and aggressive types. The purpose of this study was to study blood cytokine levels in newly diagnosed and relapsed MCL patients with respect to patterns of abnormalities and relationship to the MCL International Prognostic Index (MIPI) score. We analyzed blood levels of 30 cytokines using a multiplex ELISA in 88 patients with newly diagnosed MCL (pre-treatment levels) and 20 with relapsed MCL and compared them with controls without known lymphoma. Elevated cytokine levels were compared with clinical outcome and the MIPI score. In the 88 newly diagnosed MCL patients, we found significantly elevated levels compared with controls of IL-12, IP-10, sIL-2Rα, MIG, IL-1RA, IL-8, MIP-1α, and MIP-1ß (all P < 0.05). Of these elevated cytokines, sIL-2Rα, IL-8, MIG, MIP-1α, and MIP-1ß were predictive of inferior event-free survival, and sIL-2Rα (HR = 1.94; P = 0.038), IL-8 (HR = 2.17; P = 0.015), and MIP-1ß (HR = 2.10; P = 0.016) were independent of MIPI score; only sIL-2Rα (HR = 2.35; P = 0.041) was associated with overall survival after adjustment for MIPI. In the relapsed MCL patient group, the only significantly elevated plasma cytokines that predicted EFS were sIL-2Rα (HR = 2.90; P = 0.04) and IL-8 (HR = 3.75; P = 0.02). Elevated blood levels of sIL-2Rα and the pro-inflammatory cytokines IL-8 and MIP-1ß are poor prognostic factors in MCL patients and independent of MIPI score. These factors, if validated, will provide important additions to the MIPI and guide the development of new therapies for patients with elevated levels of these cytokines.

Chemoimmunotherapy for relapsed/refractory and progressive 17p13-deleted chronic lymphocytic leukemia (CLL) combining pentostatin, alemtuzumab, and low-dose rituximab is effective and tolerable and limits loss of CD20 expression by circulating CLL cells.

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) patients with purine analog refractory disease or TP53 dysfunction still have limited treatment options and poor survival. Alemtuzumab-containing chemoimmunotherapy regimens can be effective but frequently cause serious infections. We report a Phase II trial testing the efficacy and tolerability of a short-duration regimen combining pentostatin, alemtuzumab, and low-dose high-frequency rituximab designed to decrease the risk of treatment-associated infections and to limit the loss of CD20 expression by CLL cells. The study enrolled 39 patients with progressive CLL that was either relapsed/refractory (n = 36) or previously untreated with 17p13 deletion (17p13-) (n = 3). Thirteen (33%) patients had both 17p13- and TP53 mutations predicted to be dysfunctional, and eight patients had purine analog refractory CLL without TP53 dysfunction. Twenty-six (67%) patients completed therapy, with only five (13%) patients having treatment-limiting toxicity and no treatment-related deaths. Twenty-two (56%) patients responded to treatment, with 11 (28%) complete responses (four with incomplete bone marrow recovery). Median progression-free survival was 7.2 months, time to next treatment was 9.1 months, and overall survival was 34.1 months. The majority of deaths (82%) were caused by progressive disease, including transformed diffuse large B-cell lymphoma (n = 6). Correlative studies showed that low-dose rituximab activates complement and natural killer cells without a profound and sustained decrease in expression of CD20 by circulating CLL cells. We conclude that pentostatin, alemtuzumab, and low-dose high-frequency rituximab is a tolerable and effective therapy for CLL and that low-dose rituximab therapy can activate innate immune cytotoxic mechanisms without substantially decreasing CD20 expression.

CXCR5 polymorphisms in non-Hodgkin lymphoma risk and prognosis.

CXCR5 [chemokine (C-X-C motif) receptor 5; also known as Burkitt lymphoma receptor 1 (BCR1)] is expressed on mature B-cells, subsets of CD4+ and CD8+ T-cells, and skin-derived migratory dendritic cells. Together with its ligand, CXCL13, CXCR5 is involved in guiding B-cells into the B-cell zones of secondary lymphoid organs as well as T-cell migration. This study evaluated the role of common germline genetic variation in CXCR5 in the risk and prognosis of non-Hodgkin lymphoma (NHL) using a clinic-based study of 1,521 controls and 2,694 NHL cases including 710 chronic lymphocytic leukemia/small lymphocytic lymphoma, 586 diffuse large B-cell lymphoma (DLBCL), 588 follicular lymphoma (FL), 137 mantle cell lymphoma (MCL), 230 marginal zone lymphoma (MZL), and 158 peripheral T-cell lymphoma (PTCL). Of the ten CXCR5 tag SNPs in our study, five were associated with risk of NHL, with rs1790192 having the strongest association (OR 1.19, 95% CI 1.08-1.30; p = 0.0003). This SNP was most strongly associated with the risk of FL (OR 1.44, 95 % CI 1.25-1.66; p = 3.1 × 10(-7)), with a lower degree of association with DLBCL (OR 1.16, 95% CI 1.01-1.33; p = 0.04) and PTCL (OR 1.29, 95 % CI 1.02-1.64; p = 0.04) but no association with the risk of MCL or MZL. For FL patients that were observed as initial disease management, the number of minor alleles of rs1790192 was associated with better event-free survival (HR 0.64; 95% CI 0.47-0.87; p = 0.004). These results provide additional evidence for a role of host genetic variation in CXCR5 in lymphomagenesis, particularly for FL.

Rituximab infusion induces NK activation in lymphoma patients with the high-affinity CD16 polymorphism.

Natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity involving FcγRIIIa (CD16) likely contributes to the clinical efficacy of rituximab. To assess the in vivo effects of CD16 polymorphisms on rituximab-induced NK activation, blood was evaluated before and 4 hours after initiation of the initial dose of rituximab in 21 lymphoma subjects. Rituximab induced NK activation and a drop in circulating NK-cell percentage in subjects with the high-affinity [158(VF/VV)] but not the low-affinity [158(FF)] CD16 polymorphism. There was no correlation between NK-cell activation or NK-cell percentage and polymorphisms in CD32A, C1q, or CH50. We conclude that NK activation occurs within 4 hours of rituximab infusion in subjects with the high-affinity CD16 polymorphism but not those with the low-affinity CD16 polymorphism. This finding may help explain the superior clinical outcome seen in the subset of high-affinity CD16 polymorphism lymphoma patients treated with single-agent rituximab.

Vitamin D insufficiency and prognosis in chronic lymphocytic leukemia.

Vitamin D insufficiency is common globally and low levels are linked to higher cancer incidence. Although vitamin D insufficiency is related to inferior prognosis in some cancers, no data exist for chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). We evaluated the relationship of 25(OH)D serum levels with time-to-treatment (TTT) and overall survival (OS) in newly diagnosed CLL patients participating in a prospective cohort study (discovery cohort) and a separate cohort of previously untreated patients participating in an observational study (confirmation cohort). Of 390 CLL patients in the discovery cohort, 119 (30.5%) were 25(OH)D insufficient. After a median follow-up of 3 years, TTT (hazard ratio[HR] = 1.66; P = .005) and OS (HR = 2.39; P = .01) were shorter for 25(OH)D-insufficient patients. In the validation cohort, 61 of 153 patients (39.9%) were 25(OH)D insufficient. After a median follow-up of 9.9 years, TTT (HR = 1.59; P = .05) and OS (HR 1.63; P = .06) were again shorter for 25(OH)D-insufficient patients. On pooled multivariable analysis of patients in both cohorts adjusting for age, sex, Rai stage, CD38 status, ZAP-70 status, immunoglobulin heavy chain variable (IGHV) gene mutation status, CD49d status, and cytogenetic abnormalities assessed by interphase fluorescent in situ hybridization testing, 25(OH)D insufficiency remained an independent predictor of TTT (HR = 1.47; P = .008), although the association with OS was not significant (HR = 1.47; P = .07). Vitamin D insufficiency is associated with inferior TTT and OS in CLL patients. Whether normalizing vitamin D levels in deficient CLL patients would improve outcome merits clinical testing.

A phase I trial of immunostimulatory CpG 7909 oligodeoxynucleotide and 90 yttrium ibritumomab tiuxetan radioimmunotherapy for relapsed B-cell non-Hodgkin lymphoma.

Radioimmunotherapy (RIT) for relapsed indolent non-Hodgkin lymphoma produces overall response rates (ORR) of 80% with mostly partial remissions. Synthetic CpG oligonucleotides change the phenotype of malignant B-cells, are immunostimulatory, and can produce responses when injected intratumorally and combined with conventional radiation. In this phase I trial, we tested systemic administration of both CpG and RIT. Eligible patients had biopsy-proven previously treated CD20+ B-cell NHL and met criteria for RIT. Patients received rituximab 250 mg/m(2) days 1,8, and 15; (111) In-ibritumomab tiuxetan days 1, 8; CpG 7909 days 6, 13, 20, 27; and 0.4 mCi/kg of (90) Y-ibritumomab tiuxetan day 15. The doses of CpG 7909 tested were 0.08, 0.16, 0.32 (six patients each) and 0.48 mg/kg (12 patients) IV over 2 hr without dose limiting toxicity. The ORR was 93% (28/30) with 63% (19/30) complete remission (CR); median progression free survival of 42.7 months (95% CI 18-NR); and median duration of response (DR) of 35 months (4.6-76+). Correlative studies demonstrated a decrease in IL10 and TNFα, and an increase in IL1ß, in response to therapy. CpG 7909 at a dose of 0.48 mg/kg is safe with standard RIT and produces a high CR rate and long DR; these results warrant confirmation.