Safety and efficacy of ibrutinib in combination with rituximab and lenalidomide in previously untreated follicular and marginal zone lymphoma: An open label, phase 2 study.

BACKGROUND: Follicular lymphoma (FL) and marginal zone lymphoma (MZL) are indolent non-Hodgkin lymphomas (iNHL). Median survival for iNHL is approximately 20 years. Because standard treatments are not curative, patients often receive multiple lines of therapy with associated toxicity-rationally designed, combination therapies with curative potential are needed. The immunomodulatory drug lenalidomide was evaluated in combination with rituximab for the frontline treatment of FL in the phase 3 RELEVANCE study. Ibrutinib, an oral Bruton tyrosine kinase inhibitor, is active in NHL and was evaluated in combination with lenalidomide, rituximab, and ibrutinib (IRR) in a phase 1 study. METHODS: The authors conducted an open-label, phase 2 clinical trial of IRR for previously untreated FL and MZL. The primary end point was progression-free survival (PFS) at 24 months. RESULTS: This study included 48 participants with previously untreated FL grade 1-3a (N = 38), or MZL (N = 10). Participants received 12, 28-day cycles of lenalidomide (15 mg, days 1-21 cycle 1; 20 mg, cycles 2-12), rituximab (375 mg/m2 weekly in cycle 1; day 1 cycles 2-12), and ibrutinib 560 mg daily. With a median follow-up of 65.3 months, the estimated PFS at 24 months was 78.8% (95% confidence interval [CI], 68.0%-91.4%) and 60-month PFS was 59.7% (95% CI, 46.6%-76.4%). One death occurred unrelated to disease progression. Grade 3-4 adverse events were observed in 64.6%, including 50% with grade 3-4 rash. CONCLUSIONS: IRR is highly active as frontline therapy for FL and MZL. Compared to historical results with lenalidomide and rituximab, PFS is similar with higher grade 3-4 toxicity, particularly rash. The study was registered with ClinicalTrials.gov (NCT02532257).

Subtype-specific and co-occurring genetic alterations in B-cell non-Hodgkin lymphoma.

B-cell non-Hodgkin lymphoma (B-NHL) encompasses multiple clinically and phenotypically distinct subtypes of malignancy with unique molecular etiologies. Common subtypes of B-NHL, such as diffuse large B-cell lymphoma, have been comprehensively interrogated at the genomic level, but rarer subtypes, such as mantle cell lymphoma, remain less extensively characterized. Furthermore, multiple B-NHL subtypes have thus far not been comprehensively compared using the same methodology to identify conserved or subtype-specific patterns of genomic alterations. Here, we employed a large targeted hybrid-capture sequencing approach encompassing 380 genes to interrogate the genomic landscapes of 685 B-NHL tumors at high depth, including diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, and Burkitt lymphoma. We identified conserved hallmarks of B-NHL that were deregulated in the majority of tumors from each subtype, including frequent genetic deregulation of the ubiquitin proteasome system. In addition, we identified subtype-specific patterns of genetic alterations, including clusters of co-occurring mutations and DNA copy number alterations. The cumulative burden of mutations within a single cluster were more discriminatory of B-NHL subtypes than individual mutations, implicating likely patterns of genetic cooperation that contribute to disease etiology. We therefore provide the first cross-sectional analysis of mutations and DNA copy number alterations across major B-NHL subtypes and a framework of co-occurring genetic alterations that deregulate genetic hallmarks and likely cooperate in lymphomagenesis.

Ubiquitin-activating enzyme inhibition induces an unfolded protein response and overcomes drug resistance in myeloma.

Three proteasome inhibitors have garnered regulatory approvals in various multiple myeloma settings; but drug resistance is an emerging challenge, prompting interest in blocking upstream components of the ubiquitin-proteasome pathway. One such attractive target is the E1 ubiquitin-activating enzyme (UAE); we therefore evaluated the activity of TAK-243, a novel and specific UAE inhibitor. TAK-243 potently suppressed myeloma cell line growth, induced apoptosis, and activated caspases while decreasing the abundance of ubiquitin-protein conjugates. This was accompanied by stabilization of many short-lived proteins, including p53, myeloid cell leukemia 1 (MCL-1), and c-MYC, and activation of the activating transcription factor 6 (ATF-6), inositol-requiring enzyme 1 (IRE-1), and protein kinase RNA-like endoplasmic reticulum (ER) kinase (PERK) arms of the ER stress response pathway, as well as oxidative stress. UAE inhibition showed comparable activity against otherwise isogenic cell lines with wild-type (WT) or deleted p53 despite induction of TP53 signaling in WT cells. Notably, TAK-243 overcame resistance to conventional drugs and novel agents in cell-line models, including bortezomib and carfilzomib resistance, and showed activity against primary cells from relapsed/refractory myeloma patients. In addition, TAK-243 showed strong synergy with a number of antimyeloma agents, including doxorubicin, melphalan, and panobinostat as measured by low combination indices. Finally, TAK-243 was active against a number of in vivo myeloma models in association with activation of ER stress. Taken together, the data support the conclusion that UAE inhibition could be an attractive strategy to move forward to the clinic for patients with relapsed and/or refractory multiple myeloma.

Ibrutinib inhibits pre-BCR+ B-cell acute lymphoblastic leukemia progression by targeting BTK and BLK.

Targeting B-cell receptor (BCR) signaling is a successful therapeutic strategy in mature B-cell malignancies. Precursor BCR (pre-BCR) signaling, which is critical during normal B lymphopoiesis, also plays an important role in pre-BCR+ B cell acute lymphoblastic leukemia (B-ALL). Here, we investigated the activity and mechanism of action of the BTK inhibitor ibrutinib in preclinical models of B-ALL. Pre-BCR+ ALL cells were exquisitely sensitive to ibrutinib at therapeutically relevant drug concentrations. In pre-BCR+ ALL, ibrutinib thwarted autonomous and induced pre-BCR signaling, resulting in deactivation of PI3K/Akt signaling. Ibrutinib modulated the expression of pre-BCR regulators (PTPN6, CD22, CD72, and PKCß) and substantially reduced BCL6 levels. Ibrutinib inhibited ALL cell migration toward CXCL12 and beneath marrow stromal cells and reduced CD44 expression. CRISPR-Cas9 gene editing revealed that both BTK and B lymphocyte kinase (BLK) are relevant targets of ibrutinib in pre-BCR+ ALL. Consequently, in mouse xenograft models of pre-BCR+ ALL, ibrutinib treatment significantly prolonged survival. Combination treatment of ibrutinib with dexamethasone or vincristine demonstrated synergistic activity against pre-BCR+ ALL. These data corroborate ibrutinib as a promising targeted agent for pre-BCR+ ALL and highlight the importance of ibrutinib effects on alternative kinase targets.

Tonic B-cell receptor signaling in diffuse large B-cell lymphoma.

We used clustered regularly interspaced short palindromic repeats/Cas9-mediated genomic modification to investigate B-cell receptor (BCR) signaling in cell lines of diffuse large B-cell lymphoma (DLBCL). Three manipulations that altered BCR genes without affecting surface BCR levels showed that BCR signaling differs between the germinal center B-cell (GCB) subtype, which is insensitive to Bruton tyrosine kinase inhibition by ibrutinib, and the activated B-cell (ABC) subtype. Replacing antigen-binding BCR regions had no effect on BCR signaling in GCB-DLBCL lines, reflecting this subtype's exclusive use of tonic BCR signaling. Conversely, Y188F mutation in the immunoreceptor tyrosine-based activation motif of CD79A inhibited tonic BCR signaling in GCB-DLBCL lines but did not affect their calcium flux after BCR cross-linking or the proliferation of otherwise-unmodified ABC-DLBCL lines. CD79A-GFP fusion showed BCR clustering or diffuse distribution, respectively, in lines of ABC and GCB subtypes. Tonic BCR signaling acts principally to activate AKT, and forced activation of AKT rescued GCB-DLBCL lines from knockout (KO) of the BCR or 2 mediators of tonic BCR signaling, SYK and CD19. The magnitude and importance of tonic BCR signaling to proliferation and size of GCB-DLBCL lines, shown by the effect of BCR KO, was highly variable; in contrast, pan-AKT KO was uniformly toxic. This discrepancy was explained by finding that BCR KO-induced changes in AKT activity (measured by gene expression, CXCR4 level, and a fluorescent reporter) correlated with changes in proliferation and with baseline BCR surface density. PTEN protein expression and BCR surface density may influence clinical response to therapeutic inhibition of tonic BCR signaling in DLBCL.

Atg7 suppression enhances chemotherapeutic agent sensitivity and overcomes stroma-mediated chemoresistance in acute myeloid leukemia.

Autophagy is a cellular adaptive mechanism to stress, including that induced by chemotherapeutic agents. Reverse phase protein array suggested that high expression of the essential autophagy-related protein, Atg7, was associated with shorter remission in newly diagnosed acute myeloid leukemia (AML) patient samples, indicating a role in chemoresistance. Knockdown of Atg7 in AML cells using short hairpin RNA markedly increased apoptosis and DNA damage following treatment with cytarabine and idarubicin. Interestingly, coculture of AML cells with stromal cells increased autophagy and chemoresistance in the AML cells exposed to chemotherapeutic agents, and this was reversed following Atg7 knockdown. This effect was further enhanced by concomitant knockdown of Atg7 in both AML and stromal cells. These findings strongly suggest that Atg7, and likely microenvironment autophagy in general, plays an important role in AML chemoresistance. Mechanistic studies revealed that Atg7 knockdown induced a proapoptotic phenotype in AML cells, which was manifested by an increased NOXA expression at the transcriptional level. Finally, in a mouse model of human leukemia, Atg7 knockdown extended overall survival after chemotherapy. Thus, the inhibition of Atg7 appears to be a valid strategy to enhance chemosensitivity, and it could indeed improve outcomes in AML therapy.

Combination of galectin inhibitor GCS-100 and BH3 mimetics eliminates both p53 wild type and p53 null AML cells.

Galectin 3 (LGALS3) expression is prognostic for poor survival in acute myeloid leukemia (AML) patients. GCS-100 is a novel galectin inhibitor that may prove useful for AML therapy. In this study, we found that GCS-100 induced apoptosis in AML cells. The agent reduced MCL-1 expression suggesting that GCS-100 could be more effective when combined with a BH3 mimetic. Indeed, potent synergistic cytotoxicity was achieved when GCS-100 was combined with ABT-737 or ABT-199. Furthermore, the GCS-100/ABT-199 combination was effective against primary AML blast cells from patients with FLT3 ITD mutations, which is another prognostic factor for poor outcome in AML. This activity may involve wild-type p53 as shRNA knockdown of LGALS3 or galectin 1 (LGALS1) sensitized wild-type p53 OCI-AML3 cells to GCS-100/ABT-737-induced apoptosis to a much greater extent than p53 null THP-1 cells. Suppression of LGALS3 by shRNA inhibited MCL-1 expression in OCI-AML3 cells, but not THP-1 cells, suggesting the induced sensitivity to ABT-737 may involve a MCL-1 mediated mechanism. OCI-AML3 cells with LGALS1 shRNA were also sensitized to ABT-737. However, these cells exhibited increased MCL-1 expression, so MCL-1 reduction is apparently not required in this process. A role for p53 appears important as GCS-100 induces p53 expression and shRNA knockdown of p53 protected OCI-AML3 cells from the cytotoxic effects of the GCS-100/ABT-737 treatment combination. Our results suggest that galectins regulate a survival axis in AML cells, which may be targeted via combined inhibition with drugs such as GCS-100 and ABT-199.

Antileukemia activity of the novel peptidic CXCR4 antagonist LY2510924 as monotherapy and in combination with chemotherapy.

Targeting the stromal cell-derived factor 1α (SDF-1α)/C-X-C chemokine receptor type 4 (CXCR4) axis has been shown to be a promising therapeutic approach to overcome chemoresistance in acute myeloid leukemia (AML). We investigated the antileukemia efficacy of a novel peptidic CXCR4 antagonist, LY2510924, in preclinical models of AML. LY2510924 rapidly and durably blocked surface CXCR4 and inhibited stromal cell-derived factor 1 (SDF-1)α-induced chemotaxis and prosurvival signals of AML cells at nanomolar concentrations more effectively than the small-molecule CXCR4 antagonist AMD3100. In vitro, LY2510924 chiefly inhibited the proliferation of AML cells with little induction of cell death and reduced protection against chemotherapy by stromal cells. In mice with established AML, LY2510924 caused initial mobilization of leukemic cells into the circulation followed by reduction in total tumor burden. LY2510924 had antileukemia effects as monotherapy as well as in combination with chemotherapy. Gene expression profiling of AML cells isolated from LY2510924-treated mice demonstrated changes consistent with loss of SDF-1α/CXCR4 signaling and suggested reduced proliferation and induction of differentiation, which was proved by showing the attenuation of multiple prosurvival pathways such as PI3K/AKT, MAPK, and ß-catenin and myeloid differentiation in vivo. Effective disruption of the SDF-1α/CXCR4 axis by LY2510924 may translate into effective antileukemia therapy in future clinical applications.

Inhibition of demethylase KDM6B sensitizes diffuse large B-cell lymphoma to chemotherapeutic drugs.

Histone methylation and demethylation regulate B-cell development, and their deregulation correlates with tumor chemoresistance in diffuse large B-cell lymphoma, limiting cure rates. Since histone methylation status correlates with disease aggressiveness and relapse, we investigated the therapeutic potential of inhibiting histone 3 Lys27 demethylase KDM6B, in vitro, using the small molecule inhibitor GSK-J4. KDM6B is overexpressed in the germinal center B-cell subtype of diffuse large B-cell lymphoma, and higher KDM6B levels are associated with worse survival in patients with diffuse large B-cell lymphoma treated with R-CHOP. GSK-J4-induced apoptosis was observed in five (SU-DHL-6, OCI-Ly1, Toledo, OCI-Ly8, SU-DHL-8) out of nine germinal center B-cell diffuse large B-cell lymphoma cell lines. Treatment with GSK-J4 predominantly resulted in downregulation of B-cell receptor signaling and BCL6. Cell lines expressing high BCL6 levels or CREBBP/EP300 mutations were sensitive to GSK-J4. Our results suggest that B-cell receptor-dependent downregulation of BCL6 is responsible for GSK-J4-induced cytotoxicity. Furthermore, GSK-J4-mediated inhibition of KDM6B sensitizes germinal center B-cell diffuse large B-cell lymphoma cells to chemotherapy agents that are currently utilized in treatment regimens for diffuse large B-cell lymphoma.

Reciprocal leukemia-stroma VCAM-1/VLA-4-dependent activation of NF-κB mediates chemoresistance.

Leukemia cells are protected from chemotherapy-induced apoptosis by their interactions with bone marrow mesenchymal stromal cells (BM-MSCs). Yet the underlying mechanisms associated with this protective effect remain unclear. Genome-wide gene expression profiling of BM-MSCs revealed that coculture with leukemia cells upregulated the transcription of genes associated with nuclear factor (NF)-κB signaling. Moreover, primary BM-MSCs from leukemia patients expressed NF-κB target genes at higher levels than their normal BM-MSC counterparts. The blockade of NF-κB activation via chemical agents or the overexpression of the mutant form of inhibitor κB-α (IκBα) in BM-MSCs markedly reduced the stromal-mediated drug resistance in leukemia cells in vitro and in vivo. In particular, our unique in vivo model of human leukemia BM microenvironment illustrated a direct link between NF-κB activation and stromal-associated chemoprotection. Mechanistic in vitro studies revealed that the interaction between vascular cell adhesion molecule 1 (VCAM-1) and very late antigen-4 (VLA-4) played an integral role in the activation of NF-κB in the stromal and tumor cell compartments. Together, these results suggest that reciprocal NF-κB activation in BM-MSCs and leukemia cells is essential for promoting chemoresistance in the transformed cells, and targeting NF-κB or VLA-4/VCAM-1 signaling could be a clinically relevant mechanism to overcome stroma-mediated chemoresistance in BM-resident leukemia cells.

Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma.

A role for B-cell-receptor (BCR) signalling in lymphomagenesis has been inferred by studying immunoglobulin genes in human lymphomas and by engineering mouse models, but genetic and functional evidence for its oncogenic role in human lymphomas is needed. Here we describe a form of 'chronic active' BCR signalling that is required for cell survival in the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). The signalling adaptor CARD11 is required for constitutive NF-kappaB pathway activity and survival in ABC DLBCL. Roughly 10% of ABC DLBCLs have mutant CARD11 isoforms that activate NF-kappaB, but the mechanism that engages wild-type CARD11 in other ABC DLBCLs was unknown. An RNA interference genetic screen revealed that a BCR signalling component, Bruton's tyrosine kinase, is essential for the survival of ABC DLBCLs with wild-type CARD11. In addition, knockdown of proximal BCR subunits (IgM, Ig-kappa, CD79A and CD79B) killed ABC DLBCLs with wild-type CARD11 but not other lymphomas. The BCRs in these ABC DLBCLs formed prominent clusters in the plasma membrane with low diffusion, similarly to BCRs in antigen-stimulated normal B cells. Somatic mutations affecting the immunoreceptor tyrosine-based activation motif (ITAM) signalling modules of CD79B and CD79A were detected frequently in ABC DLBCL biopsy samples but rarely in other DLBCLs and never in Burkitt's lymphoma or mucosa-associated lymphoid tissue lymphoma. In 18% of ABC DLBCLs, one functionally critical residue of CD79B, the first ITAM tyrosine, was mutated. These mutations increased surface BCR expression and attenuated Lyn kinase, a feedback inhibitor of BCR signalling. These findings establish chronic active BCR signalling as a new pathogenetic mechanism in ABC DLBCL, suggesting several therapeutic strategies.

Prognostic impact and targeting of CRM1 in acute myeloid leukemia.

Chromosomal region maintenance 1 (CRM1) is a nuclear export receptor recognizing proteins bearing a leucine-rich nuclear export signal. CRM1 is involved in nuclear export of tumor suppressors such as p53. We investigated the prognostic significance of CRM1 in acute myeloid leukemia (AML) and effects of a novel small-molecule selective inhibitor of CRM1. CRM1 protein expression was determined in 511 newly diagnosed AML patients and was correlated with mouse double minute 2 (MDM2) and p53 levels. High CRM1 expression was associated with short survival of patients and remained an adverse prognostic factor in multivariate analysis. CRM1 inhibitor KPT-185 induced mainly full-length p53 and apoptosis in a p53-dependent manner, whereas inhibition of proliferation was p53 independent. Patient samples with p53 mutations showed low sensitivity to KPT-185. Nuclear retention of p53 induced by CRM1 inhibition synergized with increased levels of p53 induced by MDM2 inhibition in apoptosis induction. KPT-185 and Nutlin-3a, alone and in combination, induced synergistic apoptosis in patient-derived CD34(+)/CD38(-) AML, but not in normal progenitor cells. Data suggest that CRM1 exerts an antiapoptotic function and is highly prognostic in AML. We propose a novel combinatorial approach for the therapy of AML, aimed at maximal activation of p53-mediated apoptosis by concomitant MDM2 and CRM1 inhibition.

Cross talk between follicular Th cells and tumor cells in human follicular lymphoma promotes immune evasion in the tumor microenvironment.

The microenvironment of human follicular lymphoma (FL), an incurable B cell non-Hodgkin's lymphoma, is thought to play a major role in its pathogenesis and course. Microenvironmental cells of likely importance include follicular Th cells (TFH) and regulatory T cells (Tregs), and understanding their interactions with FL tumor cells is necessary to develop novel therapeutic strategies. We found that IL-4 and CD40L are expressed by intratumoral TFH and induce production of CCL17 and CCL22 by FL tumor cells. IL-4 alone induces only CCL17 but enhances stimulation by CD40L of both CCL17 and CCL22. Consistent with our in vitro results, mRNA transcripts of IL-4 correlated with CCL17, but not CCL22, in gene expression profiling studies of FL biopsies, whereas CD40L correlated with both CCL17 and CCL22. Tumor supernatants induced preferential migration of Tregs and IL-4-producing T cells rather than IFN-γ-producing T cells, and Abs to CCR4 significantly abrogated the migration of Tregs. Our results suggest that through two distinct mechanisms, intratumoral TFH induce production of CCL17 and CCL22 by FL tumor cells and facilitate active recruitment of Tregs and IL-4-producing T cells, which, in turn, may stimulate more chemokine production in a feed-forward cycle. Thus, TFH appear to play a major role in generating an immunosuppressive tumor microenvironment that promotes immune escape and tumor survival and growth. Our results provide novel insights into the cross talk among TFH, tumor cells, and Tregs in FL, and offer potential targets for development of therapeutic strategies to overcome immune evasion.

Frequent engagement of the classical and alternative NF-kappaB pathways by diverse genetic abnormalities in multiple myeloma.

Mechanisms of constitutive NF-kappaB signaling in multiple myeloma are unknown. An inhibitor of IkappaB kinase beta (IKKbeta) targeting the classical NF-kappaB pathway was lethal to many myeloma cell lines. Several cell lines had elevated expression of NIK due to genomic alterations or protein stabilization, while others had inactivating mutations of TRAF3; both kinds of abnormality triggered the classical and alternative NF-kappaB pathways. A majority of primary myeloma patient samples and cell lines had elevated NF-kappaB target gene expression, often associated with genetic or epigenetic alteration of NIK, TRAF3, CYLD, BIRC2/BIRC3, CD40, NFKB1, or NFKB2. These data demonstrate that addiction to the NF-kappaB pathway is frequent in myeloma and suggest that IKKbeta inhibitors hold promise for the treatment of this disease.

Casein kinase 1alpha governs antigen-receptor-induced NF-kappaB activation and human lymphoma cell survival.

The transcription factor NF-kappaB is required for lymphocyte activation and proliferation as well as the survival of certain lymphoma types. Antigen receptor stimulation assembles an NF-kappaB activating platform containing the scaffold protein CARMA1 (also called CARD11), the adaptor BCL10 and the paracaspase MALT1 (the CBM complex), linked to the inhibitor of NF-kappaB kinase complex, but signal transduction is not fully understood. We conducted parallel screens involving a mass spectrometry analysis of CARMA1 binding partners and an RNA interference screen for growth inhibition of the CBM-dependent 'activated B-cell-like' (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). Here we report that both screens identified casein kinase 1alpha (CK1alpha) as a bifunctional regulator of NF-kappaB. CK1alpha dynamically associates with the CBM complex on T-cell-receptor (TCR) engagement to participate in cytokine production and lymphocyte proliferation. However, CK1alpha kinase activity has a contrasting role by subsequently promoting the phosphorylation and inactivation of CARMA1. CK1alpha has thus a dual 'gating' function which first promotes and then terminates receptor-induced NF-kappaB. ABC DLBCL cells required CK1alpha for constitutive NF-kappaB activity, indicating that CK1alpha functions as a conditionally essential malignancy gene-a member of a new class of potential cancer therapeutic targets.

Safety and activity of PD1 blockade by pidilizumab in combination with rituximab in patients with relapsed follicular lymphoma: a single group, open-label, phase 2 trial.

BACKGROUND: Endogenous or iatrogenic antitumour immune responses can improve the course of follicular lymphoma, but might be diminished by immune checkpoints in the tumour microenvironment. These checkpoints might include effects of programmed cell death 1 (PD1), a co-inhibitory receptor that impairs T-cell function and is highly expressed on intratumoral T cells. We did this phase 2 trial to investigate the activity of pidilizumab, a humanised anti-PD1 monoclonal antibody, with rituximab in patients with relapsed follicular lymphoma. METHODS: We did this open-label, non-randomised trial at the University of Texas MD Anderson Cancer Center (Houston, TX, USA). Adult (≥18 years) patients with rituximab-sensitive follicular lymphoma relapsing after one to four previous therapies were eligible. Pidilizumab was administered at 3 mg/kg intravenously every 4 weeks for four infusions, plus eight optional infusions every 4 weeks for patients with stable disease or better. Starting 17 days after the first infusion of pidilizumab, rituximab was given at 375 mg/m(2) intravenously weekly for 4 weeks. The primary endpoint was the proportion of patients who achieved an objective response (complete response plus partial response according to Revised Response Criteria for Malignant Lymphoma). Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00904722. FINDINGS: We enrolled 32 patients between Jan 13, 2010, and Jan 20, 2012. Median follow-up was 15.4 months (IQR 10.1-21.0). The combination of pidilizumab and rituximab was well tolerated, with no autoimmune or treatment-related adverse events of grade 3 or 4. The most common adverse events of grade 1 were anaemia (14 patients) and fatigue (13 patients), and the most common adverse event of grade 2 was respiratory infection (five patients). Of the 29 patients evaluable for activity, 19 (66%) achieved an objective response: complete responses were noted in 15 (52%) patients and partial responses in four (14%). INTERPRETATION: The combination of pidilizumab plus rituximab is well tolerated and active in patients with relapsed follicular lymphoma. Our results suggest that immune checkpoint blockade is worthy of further study in follicular lymphoma. FUNDING: National Institutes of Health, Leukemia and Lymphoma Society, Cure Tech, and University of Texas MD Anderson Cancer Center.

Safety and activity of lenalidomide and rituximab in untreated indolent lymphoma: an open-label, phase 2 trial.

BACKGROUND: Standard treatments for indolent non-Hodgkin lymphomas are often toxic, and most patients ultimately relapse. Lenalidomide, an immunomodulatory agent, is effective as monotherapy for relapsed indolent non-Hodgkin lymphoma. We assessed the efficacy and safety of lenalidomide plus rituximab in patients with untreated, advanced stage indolent non-Hodgkin lymphoma. METHODS: In this phase 2 trial, undertaken at one instution, patients with follicular lymphoma and marginal zone lymphoma were given lenalidomide, orally, at 20 mg/day on days 1-21 of each 28-day cycle. For patients with small lymphocytic lymphoma, dosing began at 10 mg/day to avoid tumour flare, with an escalation of 5 mg/month to 20 mg/day. Rituximab was given at 375 mg/m(2) as an intravenous infusion on day 1 of each cycle. Patients responding after six cycles could continue therapy for up to 12 cycles. The primary endpoint was overall response, defined as the proportion of patients who achieved a partial or complete response; patients were assessed for response if they had any post-baseline tumour assessment. This trial is registered with ClinicalTrials.gov, number NCT00695786. FINDINGS: 110 patients with follicular lymphoma (n=50), marginal zone lymphoma (n=30), and small lymphocytic lymphoma (n=30) were enrolled from June 30, 2008, until Aug 12, 2011. 93 of 103 evaluable patients had an overall response (90%, 95% CI 83-95). Complete responses occurred in 65 (63%, 95% CI 53-72) and partial responses in 28 patients (27%, 19-37). Of 46 evaluable patients with follicular lymphoma, 40 (87%) patients had a complete response and five (11%) had a partial response. Of 27 evaluable patients with marginal zone lymphoma, 18 (67%) had a complete response and six (22%) had a partial response. Of 30 evaluable patients with small lymphocytic lymphoma, seven (23%) had a complete response and 17 (57%) had a partial response. The most common grade 3 or 4 adverse events were neutropenia (38 [35%] of 110 patients), muscle pain (ten [9%]), rash (eight [7%]), cough, dyspnoea, or other pulmonary symptoms (five [5%]), fatigue (five [5%]), thrombosis (five [5%]), and thrombocytopenia (four [4%]). INTERPRETATION: Lenalidomide plus rituximab is well tolerated and highly active as initial treatment for indolent non-Hodgkin lymphoma. An international phase 3 study (NCT01476787) to compare this regimen with chemotherapy in patients with untreated follicular lymphoma is in progress. FUNDING: Celgene Corporation and Richard Spencer Lewis Memorial Foundation and Cancer Center Support Grant.

Selective targeting of Toll-like receptors and OX40 inhibit regulatory T-cell function in follicular lymphoma.

Immunotherapeutic strategies are promising approaches for the treatment of follicular lymphoma (FL). However, their efficacy may be limited by immunosuppressive elements in the immune system and tumor microenvironment. Therefore, strategies to reverse the effects of the immunosuppressive elements are needed. We observed that regulatory T cells (Tregs) were increased in the peripheral blood at diagnosis and persisted in high numbers after induction of clinical remission with a cyclophosphamide and doxorubicin-containing chemotherapy regimen in FL patients. High levels of peripheral blood Tregs prior to therapy were associated with decreased progression-free survival in FL patients treated with either chemotherapy or combination immunotherapy that targeted CD20 and PD-1 with monoclonal antibodies rituximab and pidilizumab, respectively. Intratumoral and peripheral blood Tregs potently suppressed autologous antitumor effector T cells in FL. However, the effects of FL Tregs could be reversed by triggering Toll-like receptors (TLR) with TLR ligands Pam3 CSK4 (TLR 1/2), flagellin (TLR 5), and CpG-B (TLR 9), and/or OX40. The TLR ligands synergized with each other as well as OX40 signaling to inhibit Tregs. Furthermore, they restored the function of FL tumor-specific effector T cells. Our results suggest that a state of tolerance exists in FL patients at diagnosis and after induction of clinical remission, and agents that activate TLRs 1/2, 5, and 9, and OX40 may serve as adjuvants to enhance the efficacy of antitumor immunotherapeutic strategies and preventive vaccines against infectious diseases in these patients.