Therapy For Mantle Cell Lymphoma Research Articles

Synergistic anti-tumor activity of acadesine (AICAR) in combination with the anti-CD20 monoclonal antibody rituximab in in vivo and in vitro models of mantle cell lymphoma

Mantle cell lymphoma (MCL) is considered one of the most challenging lymphoma, with limited responses to current therapies. Acadesine, a nucleoside analogue has shown antitumoral effects in different preclinical cancer models as well as in a recent phase I/II clinical trial conducted in patients with chronic lymphocytic leukemia. Here we observed that acadesine exerted a selective antitumoral activity in the majority of MCL cell lines and primary MCL samples, independently of adverse cytogenetic factors. Moreover, acadesine was highly synergistic, both in vitro and in vivo, with the anti-CD20 monoclonal antibody rituximab, commonly used in combination therapy for MCL. Gene expression profiling analysis in harvested tumors suggested that acadesine modulates immune response, actin cytoskeleton organization and metal binding, pointing out a substantial impact on metabolic processes by the nucleoside analog. Rituximab also induced changes on metal binding and immune responses.The combination of both drugs enhanced the gene signature corresponding to each single agent, showing an enrichment of genes involved in inflammation, metabolic stress, apoptosis and proliferation. These effects could be important as aberrant apoptotic and proinflammatory pathways play a significant role in the pathogenesis of MCL. In summary, our results suggest that acadesine exerts a cytotoxic effect in MCL in combination with rituximab, by decreasing the proliferative and survival signatures of the disease, thus supporting the clinical examination of this strategy in MCL patients.

Is Early Hematopoietic Stem-Cell Transplantation Necessary in Mantle-Cell Lymphoma?

Is Early Hematopoietic Stem-Cell Transplantation Necessary in Mantle-Cell Lymphoma?

Long-Term Follow-Up of R-CHOP With Bevacizumab as Initial Therapy for Mantle Cell Lymphoma: Clinical and Correlative Results

Emerging evidence indicates that MCL has increased angiogenesis within the tumor microenvironment. We initiated a phase II trial to determine if the addition of bevacizumab to the standard R-CHOP regimen could enhance antitumor effects in patients with previously untreated MCL. Eleven patients with previously untreated MCL received bevacizumab at 15 mg/kg on day 1, and standard CHOP-21 (CHOP given every 21 days per cycle) with rituximab (375 mg/m(2) per cycle) on day 3 of each cycle for a total of 6 cycles. Planned study end points included safety and efficacy assessment, and exploratory analysis of angiogenic profiles. The study was suspended in August of 2010 based on safety findings in DLBCL (diffuse large B-cell lymphoma) of increased cardiovascular events with the regimen. Beyond the standard R-CHOP safety profile, Grade 3 left ventricular dysfunction developed in 2 patients (18%), Grade 1/2 hypertension, proteinuria, and bleeding each developed in 1 patient (9%). The overall response rate was 82% with 36% complete response (CR)/complete response unconfirmed (CRu). The median progression-free survival (n = 11) was 18 months (95% confidence interval, 3-not reached), and 3-year overall survival rate was 82%. Correlative studies showed increased vascular endothelial growth factor receptor 1 expression in tumor cells at baseline, and elevated levels of plasma vascular endothelial growth factor (VEGF) throughout treatment. The addition of bevacizumab to the standard R-CHOP regimen did not appear to significantly improve efficacy beyond that observed from previous studies using R-CHOP alone. Therapeutic strategies that provide sustained inhibition on VEGF-related and VEGF-independent targets within the tumor microenvironment might further improve antiangiogenic effects and warrant further exploration in MCL.

Cyclin D1 Maintains Mantle Cell Lymphoma Through CDK4-Independent Regulation Of DNA Replicative Checkpoints

Mantle cell lymphoma (MCL) is rarely curable and therapy resistance often leaves few viable treatment options for patients. Previous studies have identified the importance of cyclin D1 (CCND1) translocation and overexpression in MCL pathogenesis, which leads to increased cyclin-dependent kinase 4 (CDK4) activity and accelerated cell cycle progression. However, targeting this abnormal cell cycle control, mainly through CDK4 inhibition causes only G1-phase growth arrest without significant cell death (Marzec et al. 2006). In contrast, prolonged inhibition of CCND1 with RNA interference induces apoptosis in MCL cell lines (Weinstein et al. 2012), suggesting an essential function of CCND1 independent of CDK4 activity. The mechanism of this non-catalytic role of CCND1 in maintaining MCL cell survival is largely unknown.To clarify the cell cycle role of CCND1 in addition to its CDK4-dependent function, we compared the effects of CCND1 and CDK4 silencing on MCL cell survival. MCL cell lines co-expressing GFP and doxycycline-inducible shRNA targeting CCND1 or CDK4 were generated. Cells with similar GFP expression levels were FACS sorted to normalize for shRNA expression. Both CCND1 and CDK4 silencing resulted in G1-phase arrest, but only CCND1-silenced cells demonstrated a marked increase in apoptosis. Investigation of the potential cause of apoptosis revealed significant accumulation of DNA double-strand breaks following CCND1 ablation, as measured by nuclear gamma-H2AX focus formation. Interestingly, CCND1-silenced cells exhibited a significant increase in 53BP1+ nuclear bodies in G1-phase, reminiscent of 53BP1 foci observed by Lukas and colleagues in cells undergoing aphidicolin-induced replication stress (Lukas et al. 2011). Analysis of replication fork movement in CCND1-depleted cells showed substantially reduced fork speed and increased frequency of origin firing, both of which are indicative of replication stress. In contrast, knockdown of CDK4 did not result in slower forks or increase in the frequency of origin firing. Genomic instability associated with replication stress was also apparent in CCND1-silenced cells, including increased micronucleus formation and recurrent chromatid gaps or breaks detected by cytokinesis-block assay and karyotyping, respectively.Analysis of DNA replicative and damage checkpoints revealed that both ATR-CHEK1 and ATM-CHEK2 pathways were activated by phosphorylation following CCND1 silencing in MCL cell lines, a xenograft animal model, and primary tumor samples, but not in non-MCL tumors. Interestingly, this activation (with the exception of ATM phosphorylation) was unsustainable over time and did not cause down-regulation of the downstream targets CDC25 and CDK1/2 but, instead, we observed an increase in CDC25A/B protein levels and CDK1/2 activity, indicating defective cell cycle checkpoints. Exposing CCND1-silenced cells to replication stress-inducing or DNA-damaging agents such hydroxyurea, aphidicolin, etoposide or ionizing radiation further amplified the checkpoint defects seen in unperturbed cells. We did not observe any significant difference in this checkpoint signaling in control and CDK4 knockdown cells under these conditions. Furthermore, CCND1-deficient cells were more sensitive to pharmacological inhibition of ATR and CHEK1 but not ATM, confirming a constitutive role of CCND1 in the ATR-CHEK1 pathway.In conclusion, these studies revealed an unexpected CDK4-independent role of CCND1 in maintaining DNA replicative checkpoints to prevent replication stress and genome instability in MCL cells. As most cancer treatments rely on agents that create DNA replication stress, targeting this function of CCND1 could provide a rational approach to overcome resistance to conventional therapies in MCL. Disclosures:No relevant conflicts of interest to declare.

Combination Biologic Therapy Without Chemotherapy As Initial Treatment For Mantle Cell Lymphoma: Multi-Center Phase II Study Of Lenalidomide Plus Rituximab

BackgroundInitial treatment for mantle cell lymphoma (MCL) is not standardized. Current conventional upfront chemoimmunotherapies are generally not curative and can be deferred in some patients. This presents an opportunity to evaluate novel therapeutic approaches in the first line setting. Lenalidomide, an immunomodulatory compound which targets both the tumor cells directly and the tumor microenvironment, has shown clinical efficacy either alone or in combination with rituximab in relapsed MCL. We report findings of the first study of a chemotherapy-free approach as initial treatment for MCL, using lenalidomide and rituximab as a combination biologic doublet. MethodsThe study protocol includes both an induction phase and a maintenance phase. During the induction phase, lenalidomide is administered at 20 mg daily on days 1-21 of a 28-day cycle for a total of 12 cycles, with dose escalation to 25 mg daily if tolerated. Standard dose rituximab is administered weekly x 4 during cycle 1, then once every other cycle, for a total of 9 doses. During the maintenance phase which starts with cycle 13, lenalidomide is administered at 15 mg daily on days 1-21 of a 28-day cycle, with rituximab maintenance once every other cycle until progression of disease. The primary objective was to evaluate overall response rate (ORR). Secondary objectives included safety analysis, progression-free survival, overall survival, and QOL assessment. Based on a Simon two-stage design comparing an ORR of ≥60% with treatment to an unacceptable ORR of ≤40% (alpha=10%, power=80%), 15 or more overall responses out of 28 enrolled patients were required to declare the treatment effective and worthy of further testing. ResultsFrom 7/2011 to 2/2013, 31 subjects with previously untreated MCL were enrolled at 4 centers, and the study met its accrual. At study entry, median age was 65 years (range 42-86), and the M:F ratio was 3:1. All patients had stage III/IV disease, 12 (39%) had elevated LDH, and 27 (87%) had bone marrow involvement. MIPI scores were evenly distributed between low-, intermediate-, and high-risk (36%, 32%, and 32% respectively). Ki67 index was <30% in 23 (74%) subjects. Treatment was generally well tolerated with expected side effects. Grade 3-4 hematologic toxicities included neutropenia (39%), thrombocytopenia (13%) and anemia (7%). Grade 3-4 non-hematologic toxicities included rash (23%), tumor flare (7%) and serum sickness associated with rituximab (7%). Grade 1-2 infections included URI (29%), UTI (10%), pneumonia (10%) and sinusitis (7%). One incidence each of DVT and PE were observed and resolved with treatment. As of July 2013 at a median follow-up of 12 months (range 5-23 months), 27 (87%) patients remain on study without evidence of disease progression, including 18 who have completed induction and now in the maintenance phase. Four patients went off study – one withdrew consent, two had progression of disease, and one could not tolerate tumor flare associated side effects. Thirty patients are evaluable for efficacy with at least one response assessment. The preliminary ORR for evaluable patients is 77% (95% CI = 57% to 89%) with 40% CR/CRu (95% CI = 23% to 59%), and may further improve with additional follow-up on continued treatment. Median time to objective response was 2.8 months, with CR typically confirmed between 6-12 months. Four patients (13%) have stable disease with ongoing clinical benefit at 5+, 6+, 12+ and 13+ months. Median progression-free survival and duration of response have not been reached. Neither MIPI score nor Ki67 index correlated with response. All patients have maintained or improved quality of life parameters during treatment by FACT-Lym analysis. ConclusionsThis study provides the first demonstration that a chemotherapy-free, combination biologic approach is feasible as initial therapy for mantle cell lymphoma. Lenalidomide up to 25 mg daily given 21 out of 28 days can be safely combined with rituximab as frontline therapy for MCL. Preliminary efficacy data on response rates are encouraging. More precise assessment of response rate and durability will require more follow-up with additional subjects. However, these data justify further evaluation of the lenalidomide + rituximab regimen both alone and as a platform for the integration of novel agents in combination approaches in MCL both in the upfront and relapsed settings. Disclosures:Ruan:Celgene: Consultancy, Research Funding, Speakers Bureau. Off Label Use: Lenalidomide in the frontline treatment of mantle cell lymphoma. Martin:Seattle Genetics: Consultancy, Speakers Bureau; Millennium: Research Funding; Genentech: Speakers Bureau; Celgene: Consultancy, Research Funding; Teva: Consultancy, Research Funding. Shah:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Schuster:Celgene: Research Funding. Smith:Micromet: Consultancy; Seattle Genetics: Consultancy; Celgene: Consultancy; Allos: Consultancy; Genentech: Consultancy; Onyx: Consultancy. Furman:Celgene: Research Funding. Coleman:Celgene: Consultancy. Leonard:Celgene: Consultancy; Genentech: Consultancy.

Current and Emerging Therapies in Mantle Cell Lymphoma

Mantle Cell Lymphoma, characterized by the t(11;14)(q13; q32) chromosomal translocation and cyclin D1 expression, remains one of the most challenging lymphoma subtypes to treat. Therapy can be divided into treatment modalities for younger, stem cell transplant (SCT)-eligible patients vs older, SCT-ineligible patients. For clinically fit patients younger than 60-65 years of age we recommend cytarabine-containing induction and conditioning regimens such as Rituximab (R)-CHOP alternating with R-DHAP followed by autologous SCT consolidation. Elderly patients benefit from R-bendamustine or R-CHOP with maintenance rituximab following induction therapy, especially after R-CHOP. While standard chemoimmunotherapy provides high overall response rates, the responses are not durable and sequential therapies are thus necessary. MCL is proving to be sensitive to novel therapies that may in the near future become useful adjuncts to standard regimens. For example, bortezomib, lenalidomide, and temsirolimus each have single-agent efficacy in relapsed and refractory disease. Several targeted agents are emerging that likewise may transform management of MCL. The B-cell receptor pathway appears to be critical in the pathogenesis of MCL, and novel agents such as ibrutinib and idelalisib that target this signaling pathway are highly active in relapsed and refractory MCL. Similarly, cell cycle inhibitors targeting cyclin dependent kinases as well as HDAC inhibitors have shown promise in early studies.

Activity and complexes of mTOR in diffuse large B-cell lymphomas—a tissue microarray study

Diffuse large B-cell lymphoma is a heterogeneous group of diseases with different responses to therapy. Targeting mTOR (mammalian target of rapamycin) offers a new approach to improve the treatment. mTOR inhibitors are being developed and are in clinical use in mantle cell lymphoma therapy and clinical trials are ongoing in other high-grade lymphomas as well. However, there is limited data about mTOR activity and the expression of its different complexes in diffuse large B-cell lymphomas. Tissue microarray blocks were constructed from paraffin-embedded biopsy specimens. More than 700 immunohistochemical stainings (mTOR signaling-related proteins and phosphoproteins, markers for lymphoma classification) were evaluated from 68 diffuse large B-cell lymphoma biopsies from conventionally treated and followed patients. Approximately 30% of cases were characterized as germinal center-derived diffuse large B-cell lymphomas, which showed virtually no mTOR activity, as determined by phospho-ribosomal S6 expression, the most sensitive marker of mTOR activity. In about 80% of non-germinal center-derived diffuse large B-cell lymphoma cases, positivity of mTOR-related phosphoproteins was observed, denoting mTOR activity. Moreover, Rictor (a characteristic protein of the mTOR complex2) was overexpressed in 43% of all diffuse large B-cell lymphomas and in 63% of mTOR-active non-germinal center diffuse large B-cell lymphoma samples. Rictor overexpression with mTOR activity indicated significantly worse survival for patients than mTOR inactivity or mTOR activity with low Rictor expression. These results suggest that mTOR activity is characteristic in most non-germinal center-derived diffuse large B-cell lymphomas with potentially variable mTOR-inhibitor sensitivity. Taken together, mTOR inhibitors may be useful in addition to regular therapy in diffuse large B-cell lymphomas, however, patient and inhibitor selection criteria must be carefully considered.

A Synthetic Lethal RNA Interference Screen Identifies Components of Genotoxic Stress Response As Targets for Anti-CCND1 Therapy in Mantle Cell Lymphoma.

AbstractAbstract 2430 Background.Almost all cases of mantle cell lymphoma (MCL) harbor the t(11:14) chromosomal translocation resulting in overexpression of the cell cycle regulatory protein cyclin D1 (CCND1), which promotes cell proliferation and poor survival. Targeting CCND1 in vitro and clinically, however, is not sufficient to cause tumor cell death, suggesting that additional mechanisms compensate for MCL growth and survival. Unraveling these additional signals will help identify novel targets for rational combination therapies in MCL. Previously, we developed a novel functional genomics tool using an inducible RNA interference (RNAi) library, which can simultaneously assess the role of thousands of genes in cell viability in tumor cell lines. Here we apply the inducible RNAi screen to identify synthetic lethal interactions with CCND1 in MCL. The screen uncovered several components of the DNA damage response as potential new combination targets for anti-CCND1 therapy in mantle cell lymphoma. Methods.The RNAi library was previously constructed using a retroviral vector that inducibly expresses small-hairpin RNA (shRNA). Each shRNA vector contains a unique 60-mer bar code that can hybridize to a corresponding complementary sequence spotted on a custom Agilent oligonucleotide microarray. To perform a synthetic lethal RNAi screen for CCND1, we first established a stable MCL line (UPN-1) that express an inducible CCND1 or control shRNA, transduced these lines with the pooled shRNA library, selected for transduced cells with puromycin, and induced shRNA expression for eight days. Genomic DNA containing bar code sequences was then amplified by PCR, fluorescently labeled, and hybridized onto microarrays. Each screen was repeated four times to enable statistical analysis. Candidate shRNAs obtained from the screen were validated for synergistic killing of MCL cells when combined with CCND1 knockdown. We evaluated genotoxic stress response triggered by DNA damage following CCND1 inactivation in MCL lines by Western blots. DNA damage and repair were assessed by comet assays and immuno-fluorescent staining of DNA repair proteins including phospho-H2AX, RAD51 and 53BP1. Results.The RNAi screen uncovers multiple shRNAs targeting RIPK1, RIPK3, NEMO, and TAK1, which sensitize MCL cells to CCND1 inhibition. RIPK1, NEMO, and TAK1 have been shown to play an essential role in cells undergoing genotoxic stress by linking DNA damage-induced ATM activation and NF-kB activity. We demonstrated that silencing CCND1 in the MCL cell lines UPN-1, JEKO-1, Z138, and Granta-519 up-regulates RIPK1 mRNA and protein expression, in addition to increased phosphorylation of DNA damage response proteins such as ATM, CHEK1/2 and H2AX. We observed a two-fold increase of DNA damage levels in CCND1 knockdown cells as assessed by comet assays. We also detected cell cycle-independent increase of DNA double strand break (DSB) foci in CCND1 knockdown cells by staining with fluorescently labeled anti-phospho-H2AX antibody.Knockdown of RIPK1 in MCL lines (UPN-1 and JEKO-1) resulted in apoptotic cell death and these RIPK1 shRNA-transduced cells are hypersensitive to irradiation and DNA damaging agents, indicating RIPK1 plays a protective role against DNA damage-induced apoptosis. The survival role of RIPK1 in MCL cells may correlate with the DNA repair function as demonstrated by the inability of RIPK1 knockdown cells to efficiently resolve etoposide-induced DNA DSB foci over time. Furthermore, we also found that RIPK1 knockdown cells failed to down-regulate the G2/M cell cycle checkpoint protein CDC25B and to up-regulate ATM phosphorylation and Ku86 protein expression in response to genotoxic stress. Blocking these RIPK1-dependent responses could sensitize MCL cells to CCND1 knockdown-induced DNA damage. Similar analyses of the other hits from the RNAi screen are on going. Conclusions.There are few viable treatment options for mantle cell lymphoma. We have identified the receptor interacting protein kinase 1 (RIPK1), of the DNA damage response pathway, as a potential therapeutic target whose downregulation sensitizes MCL cells to anti-CCND1 treatment, possibly by promoting insurmountable genotoxic stress. Successful implementation of our functional genetic screens for genes that sensitize MCL cells to anti-CCND1 treatment could define novel targets suitable for effective combination therapies. Disclosures:No relevant conflicts of interest to declare.

SOX11 Directly Represses Wnt/β-Catenin Signaling and Identifies a Subgroup of Mantle Cell Lymphoma Patients with Improved Survival with Intensive Treatment

AbstractAbstract 895The SOX11 (Sex determining region Y)-box 11 transcription factor is aberrantly expressed in the majority (78-93%) of Mantle Cell Lymphoma (MCL) patients. Functionally, SOX11 represses MCL cell proliferation in vitro. We identify SOX11 mediated repression of Wnt signaling as contributory to the proliferation block in vitro and determine the clinical consequences of SOX11 overexpression in a meta-analysis of 3 large international patient cohorts.We have previously identified SOX11 direct binding target genes using chromatin immunoprecipitation coupled with high resolution, next generation sequencing (ChIP-Seq). Members of the Wnt signaling pathway, which is known to promote proliferation in MCL, were significantly enriched in our ChIP analysis in MCL patients and cell lines. Using a luciferase reporter assay for nuclear beta-catenin activity, we find that overexpression of SOX11 represses Wnt signaling (p<0.005) in vitro in Z138 and JEKO-1 cells. Conversely, depletion of SOX11 using siRNA increased Wnt signaling (p<0.05) in the same cell lines. The overexpression phenotype is characterized by G2/M arrest and proliferation block in five MCL cell lines studied, irrespective of p53 status or endogenous SOX11 levels.The relationship between SOX11 and proliferation was validated in primary patient samples. Interrogation of an eighty-two patient gene-expression dataset demonstrates that SOX11 mRNA expression is significantly (p<0.05) and inversely proportional to Ki-67, a marker of cell proliferation. Similarly, expression of SOX11 is inversely proportional to beta-catenin (p<0.05) supporting our in vitro data showing crosstalk between SOX11 and Wnt pathways in SOX11 mediated repression of cell proliferation in MCL.Prior studies examining the impact of SOX11 expression on patient outcomes using immunohistochemistry have been hampered by small numbers and heterogeneity in treatment. To overcome patient selection and treatment biases, we analyzed SOX11 expression in a total of 386 patients from three independent well-annotated tissue microarrays from University of Wisconsin, Karolinska Institute and British Columbia Cancer Agency. Higher SOX11 expression was associated with improved overall survival (p<0.03) in patients treated with intensive regimens such as R-Hyper-CVAD or VCR-CVAD, independent of IPI/MIPI or p53 overexpression in these patients and similar to reports in epithelial malignancies. Conversely, there was no significant impact of SOX11 expression on survival in R-CHOP treated patients.In conclusion, building from our previous ChIP-seq data, we further validated the inverse association between SOX11 and proliferation in primary patient samples. Our functional data showing direct repression of Wnt signaling represents one pathway through which SOX11 can exert its anti-proliferative effect. Our meta-analysis indicates that higher SOX11 expression is associated with improved survival in MCL patients treated with intensive chemotherapy. This supports prospective validation of SOX11 as a bioassay to select patients more likely to benefit from intensive therapy in MCL. Transcriptional regulation of Wnt and other biological pathways affected by SOX11 target genes help explain the impact of SOX11 expression on patient outcomes. Disclosures:Goy:Milennium: Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity’s Board of Directors or advisory committees; Seattle Genetics: Membership on an entity’s Board of Directors or advisory committees; Pharmacyclics: Membership on an entity’s Board of Directors or advisory committees; J & J: Membership on an entity’s Board of Directors or advisory committees; Pfizer: Membership on an entity’s Board of Directors or advisory committees.

Updated Analysis of a Prospective Phase II Trial of R-MACLO-IVAM Followed by Maintenance for Mantle Cell Lymphoma

AbstractAbstract 1622 Background:Contemporary therapy for mantle cell lymphoma (MCL) in fit patients usually consists of an induction phase followed by autologous stem cell transplantation (SCT). We previously reported a phase II trial of intensive chemotherapy induction with R-MACLO-IVAM followed by thalidomide maintenance and demonstrated promising progression-free survival (PFS) and overall survival (OS) rates without SCT (Lossos et al, Leuk Lymph 2010). We subsequently modified the protocol to utilize rituximab maintenance instead of thalidomide. Herein we present updated results and follow-up for the patients treated on this trial. Methods:This was a prospective single-arm phase II trial conducted at the University of Miami with IRB approval. Eligible patients were chemotherapy-naïve and had a confirmed diagnosis of MCL using WHO criteria, age 18–75 years, ECOG PS 0–2, adequate organ function and no history of HIV or prior cancer. Pretreatment staging include CT and PET scans, endoscopy, colonoscopy and bone marrow biopsy. Cycle 1 consisted of R-MACLO (rituximab, methotrexate, doxorubicin, cyclophosphamide and vincristine) followed by G-CSF. When the ANC was >1.5×109/L, cycle 2 with R-IVAM (rituximab, ifosfamide, mesna, etoposide and cytarabine) was begun, followed by G-CSF, as previously reported. Fourteen days after ANC recovery from cycle 2, cycles 3 and 4 were given in identical fashion to 1 and 2. Four weeks after ANC recovery from cycle 4, patients were re-staged and responses were assessed by standard criteria. Patients who achieved complete responses (both radiologically and pathologically) were eligible for the maintenance phase. The first 22 patients were treated with thalidomide maintenance (target daily dose of 200mg); the protocol was subsequently modified to utilize rituximab maintenance at a dose of 375 mg/m2 IV weekly × 4 weeks, repeated every 6 months. Maintenance therapy was continued until MCL relapse or intolerable toxicity. OS was calculated from the date of diagnosis to the date of death or last follow up. PFS was calculated from date of diagnosis until the date of pathological evidence of recurrence or death. Data were summarized using descriptive statistics and survival was analyzed using the Kaplan-Meier method. Results:Between June 2004 and June 2012, 32 patients were enrolled, 22 on the first phase and 10 after the amendment to rituximab maintenance. All patients were evaluable for toxicity and 31 were evaluable for response. Median age was 56.5 years (range 39–73). All subjects had at least stage III disease with bone marrow involvement in 84% and gastrointestinal involvement in 38%. Distribution according to MIPI: low 28%; intermediate 38%; high 34%. All patients had diffuse variant except 2 with blastic variant. Twenty-eight patients completed all 4 cycles of therapy; treatment was stopped in 2 after 3 cycles, and in one after 2 cycles, and 1 died during cycle 1. Of the 31 patients completing 2 cycles of chemotherapy, 29 (94%) achieved a complete response (CR) and 2 had a partial response (PR). After a median follow-up of 54.9 months, the 5-year PFS was 69% (95% CI 51% – 82%) and the 5-year OS was 88% (95% CI 72% – 95%) [Fig 1 &2]. Lower MIPI group (low vs intermediate vs high) was associated with longer PFS (log rank p = 0.007) and longer OS (log rank p = 0.036) [Fig 3 &4]. Nine patients relapsed and 5 died; 1 died from sepsis on cycle 1; 1 died in CR at 38 months from non-small cell lung cancer diagnosed 19 months after MCL and the other 3 died from relapsed MCL after 22, 24 and 60 months respectively. Seven of the 9 patients who relapsed were treated with rituximab and bendamustine and one underwent an allogeneic SCT. Toxicities during the chemotherapy phase for the last 10 patients were similar to what was previously published for the first 22 patients, with the exception of lower renal toxicity since the dose of methotrexate was reduced to a total of 4.2 g/m2 instead of 6.7 g/m2 prior to the amendment. The toxicities during the thalidomide maintenance phase were similar to what was previously reported. For the 10 patients who received rituximab maintenance, there were no unexpected toxicities during the maintenance phase. Conclusions:R-MACLO-IVAM results in a high overall response rate (94% CR and 6% PR) and a low relapse rate after over 4.5 years of median follow-up. The median PFS and OS still have not been reached. These results compare favorably with regimens that include upfront SCT. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Immunochemotherapy As Induction (R-CHOP and R-HyperC-VAD/R-MA) in Mantle Cell Lymphoma, a Hungarian Multicenter Open Label Phase II Study (Rituximab [MabThera®] in Mantle Cell Lymphoma, REMENY)

AbstractAbstract 4889 Background:Considering the especially poor outcome of mantle cell lymphoma with traditional induction chemotherapies, the Hungarian working group initiated a Hungarian Mantle Cell Lymphoma Study (REMENY) using rituximab (MabThera) based immunochemotherapy. The structure of the trial:National, multicenter, open label trial rituximab based immunochemotherapy induction in mantle cell lymphoma. Two therapies were available according to investigators' choice: R-CHOP-21 (8x) or R-HyperC-VAD/R-MA according to the MD Anderson protocol. High dose therapy + autologous stem cell support (ASCT) was allowed, according to the investigators decision.Diagnosis was based upon standard histology. Initial staging included the standard protocols, imaging techniques, bone marrow evaluation and gastrointestinal endoscopy (not compulsory at initial staging). Recruitment period was planned to be 3 years, the follow-up 2 years.Remission was evaluated according to Cheson (IWC) criteria,CR was qualified only after completion of gastroscopy and colonoscopy, which did not reveal any mantle cell related abnormality. In some cases FDG PET-CT was allowed to substitute for endoscopy.Primary endpoints: OS and PFS, safety Results:Recruitment started in June 2007 and completed unexpectedly early (Nov 2008).48 patients were included, per protocol Patients were 31 (64.58%), follow-up (24 months) was completed for 15 (31.25%) patients. Ann Arbor III-IV B 43 (89.58%) was dominant.Table 1Response to immunochemotherapyR-CHOPR-HyperC-VAD/R-MASignificanceITT N(%)36 (75%)12 (25%)ORR20 (55.6%)7 (58.3 %)p=1.000CR13 (36.1%)6 (32%)p=0.501PR7 (19.4%)1 (8.3%)p=0.659R-CHOPR-HyperC-VAD/R-MASignificancePer protocol N(%)265ORR18 (69.2%)4 (80 %)p=1.000CR11 (42.3%)4 (80%)p=0.172PR7 (26.9%)0 (0%)p=0.562Median PFS for all patients was 30.4 months (SD 5.839, 95% CI: 19–41.9). PFS median for those patients who reached a CR has not been reached while it was 23.7 months in PR (p=0.031). [Display omitted] Safety:One hundred-thirteen adverse events occurred in 31 (64.58%) patients; 53 serious adverse events in 15 (31.25%) patients: 17 cytopenia, including fever with neutropenia 7, transient bronchospasm during rituximab (MabThera) infusion 1, hyperglycemia 1 which may be treatment related. There were 20 death cases, 13 of them due to progression of disease, two due to septic complication, three heart failures and one second malignancy (breast cancer). Significantly more patients on the R-HyperC-VAD/R-MA arm experienced adverse events (11/12 patients, 91.6%) compared to R-CHOP (20/36, 55.5%, p=0.035). Conclusion:This trial further confirmed the value of adding rituximab to standard induction therapies in mantle cell lymphoma. The results are conforming to data published in literature. Higher percentage of patients completed R-CHOP regimen. R-HyperC-VAD/R-MA was more effective in inducing CR, but could be completed only in one third of patients. However, in those patients who adhered to R-HyperC-VAD/R-MA therapy, it resulted in 80% CR, vs. 42.3% in the R-CHOP group. This difference is not statistically significant (p=0.172) possibly due to low case numbers. Those patients who reached CR had significantly longer PFS. PFS obtained in this trial is comparable to international data, and could be further improved by adding immunotherapy maintenance. Planned total number of patients (48) entered the trial during the first 12 months. This was surprising, as the expected number of mantle cell lymphoma based on the international incidence data is cca. 30–35 yearly. The unexpectedly high patient numbers need further explanation. Disclosures:Off Label Use: Rituximab is not authorized for Mantle Cell Lymphoma in Hungary.

Phase I Trial of Rituximab, Cladribine and Temsirolimus (RCT) for Initial Therapy of Mantle Cell Lymphoma

AbstractAbstract 3661 Objective:We conducted this trial to determine the maximum tolerated dose (MTD) and schedule of temsirolimus added to an established regimen comprised of rituximab and cladribine for the initial treatment of mantle cell lymphoma and to generate preliminary information on the toxicity and efficacy of this combination. Methods:A standard phase I cohort of 3 study design was utilized. MTD was defined as the dose level below the lowest dose that induces dose-limiting toxicity (DLT) in at least one-third of patients (at least 2 of a maximum of 6 new patients). DLT was defined as grade 4 ANC (<500) for ≥5 days, grade 4 ANC (<500) associated with fever (>100.5 F) and/or active infection, PLT <25,000, grade 4 infection, or ≥grade 3 non-hematologic toxicity during the first cycle of therapy as per NCI Common Terminology Criteria for Adverse Events v3.0. The fixed doses of rituximab and cladribine were 375 mg/m2 IV day 1 and 5 mg/m2/d IV days 1–5 of a 28 day cycle, respectively, as previously published. There were 5 planned temsirolimus IV dose levels: 15 mg day 1; 25 mg day 1; 25 mg days 1 and 15; 25 mg days 1,8 and 15; and 25 mg days 1,8,15, and 22. The fifth dose level is as previously published in combination with rituximab. Results:A total of 17 patients were treated: 3 each at dose levels 1–4 and 5 at dose level 5 (25 mg temsirolimus days 1,8,15, and 22). The median age was 75 years (52–86). There were 11 males and 6 females. At presentation 88% had stage IV disease, and 94% had extranodal disease. MIPI scores were low in 6% (1 patient), intermediate in 59% (10 patients), and high in 35% (6 patients). There was a single DLT recorded at dose level 3 based on the initial DLT criteria, though this cytokine release syndrome was clearly rituximab related, and occurred prior to the first dose of temsirolimus. Five patients were treated at the highest planned temsirolimus dose level (25 mg days 1,8,15, and 22) with no DLT observed. No further dose escalation was planned, and this level was determined to be tolerated, though higher levels may be tolerable. All patients were evaluable for adverse events. Hematologic toxicity was frequent, with grade 3 anemia in 12% of patients, grade 3 thrombocytopenia in 35%, grade 4 thrombocytopenia in 30%, grade 4 lymphopenia in 47%, grade 3 neutropenia in 24%, and grade 4 neutropenia in 18% of patients. There were 3 thrombotic episodes, 2 of which were attributed to therapy, and 3 episodes of pneumonitis. The overall response rate was 94% with 53% CR and 41% PR. The median progression free survival was 18.7 months. Conclusions:Temsirolimus 25 mg IV weekly may be safely added to rituximab and cladribine at 375 mg/m2 IV day 1 and 5 mg/m2/d IV days 1–5 of a 28 day cycle, respectively. This regimen had promising preliminary activity in an elderly cohort of patients with mantle cell lymphoma. Disclosures:Off Label Use: Temsirolimus for mantle cell lymphoma.

Novel Treatment for Therapy-Resistant Mantle Cell Lymphoma Targeting NF-κB and mTOR Signaling Pathways in Vitro and in Vivo

AbstractAbstract 63 Background:Mantle cell lymphoma (MCL) is one of the most aggressive B-cell non-Hodgkin lymphomas (NHL) with a median survival of less than five years. Currently, there is no curative therapy available for refractory MCL because of relapse from therapy-resistant tumor cells. It has been well documented that the NF-κB and mTOR pathways are constitutively active in MCL leading to increased survival, proliferation and decreased apoptosis. Therefore, in an effort to improve therapy for refractory MCL, we investigated the antilymphoma activity in vitro and in vivo and associated molecular mechanism of action of 13–197, a quinoxaline analog that specifically perturbs IκB kinase (IKK) β, an upstream kinase of the NF-κB and mTOR pathways. Methods:Established therapy-resistant from Granta 519 (Ahrens and Chaturvedi et al, Leukemia and Lymphoma doi:10.3109/10428194.2012.691481), other MCL cell lines Mino and Rec-1 and primary MCL cells from patients were used in this study. These MCL cells were treated in vitro with varying concentrations of 13–197 for the different time points. Cellular proliferation/viability, cytomorphology, frequency of cells undergoing apoptosis in treated and control cells were evaluated using 3[H]-thymidine uptake, MTT assay, cytomorphology and Annexin-V staining methods respectively. The status of key molecules in the NF-κB and mTOR pathways were examined in therapy-resistant and parental MCL cells following treatment with 13–197 using western blot analyses. The results of these analyses were compared to untreated control cells as appropriate and statistical significance of the results were determined using student‘t' test. In addition, in vivo therapeutic efficacy of 13–197 was investigated using NOD-SCID mouse bearing therapy-resistant MCL. Results:Our results showed that 13–197 significantly decreased the proliferation and induced a ∼four-fold (P<0.005) increase in apoptosis in parental and therapy-resistant MCL cells compared to control cells. At the molecular level, we observed down-regulation of IκBα phosphorylation and inhibition of NF-κB nuclear translocation by the 13–197 in MCL cells. In addition, NF-κB regulated genes such as cyclin D1, Bcl-XL and Mcl-1 were down-regulated in 13–197-treated cells. 13–197 also inhibited the phosphorylation of S6K and 4E-BP1, the downstream molecules of mTOR pathway that are also activated in refractory MCL. Further, to investigate the therapeutic efficacy of 13–197 against therapy-resistant MCL in vivo, we treated NOD-SCID mice bearing therapy-resistant MCL with 13–197; there was significantly reduced tumor burden in the kidney (p>0.05), liver (p>0.01), and lungs (p>0.03) of 13–197 treated mice compared to vehicle treated mice. Indeed, 13–197 treatment significantly increased the survival (p>0.001) of MCL transplanted mice. Taken together, our results suggest that 13–197 targets IKKβ which leads to both the transcriptional (NF-κB) and translational (mTOR) downregulation of gene products (cyclin D1, Bcl-XL and Mcl-1) misregulated in therapy-resistant MCL. Summary/Conclusions:Overall, results suggest that 13–197 perturbs the NF-κB and mTOR pathways leading significant antilymphoma effects in vitro and in vivo thus demonstrates its potentials to be a therapeutic agent for refractory MCL.(This work was supported by the Lymphoma Research Foundation New York, NY) Disclosures:No relevant conflicts of interest to declare.

Pillar-1: Multicenter Phase 2 Study of Everolimus for Patients with Mantle Cell Lymphoma Who Are Refractory or Intolerant to Bortezomib.

Pillar-1: Multicenter Phase 2 Study of Everolimus for Patients with Mantle Cell Lymphoma Who Are Refractory or Intolerant to Bortezomib.

Novel selective inhibitors of nuclear export CRM1 antagonists for therapy in mantle cell lymphoma

Overexpression of the cellular nuclear exportin 1, more commonly called chromosomal region maintenance 1 (CRM1), has been associated with malignant progression and mortality. Therefore, activation of nuclear export can play a significant etiologic role in some forms of human neoplasia and serve as a novel target for the treatment of these cancers. Mantle cell lymphoma (MCL) is an aggressive histotype of B-cell non-Hodgkin lymphoma that remains incurable. The objective of this study was to investigate the functional significance of CRM1 in MCL by evaluating the therapeutic efficacy of CRM1 inhibition in MCL invitro and invivo. Our results showed that CRM1 is highly expressed in MCL cells and is involved in regulating growth and survival mechanisms through the critical nuclear factor-κB survival pathway, which is independent of p53 status. Inhibition of CRM1 by two novel selective inhibitors of nuclear export (SINE), KPT-185 and KPT-276, in MCL cells resulted in significant growth inhibition and apoptosis induction. KPT-185 also induced CRM1 accumulation in the nucleus, resulting in CRM1 degradation by the proteasome. Oral administration of KPT-276 significantly suppressed tumor growth in an MCL-bearing severe combined immunodeficient mouse model, without severe toxicity. Our data suggest that SINE CRM1 antagonists are a potential novel therapy for patients with MCL, particular in relapsed/refractory disease.

Synergistic anticancer effects of arsenic trioxide with bortezomib in mantle cell lymphoma

Mantle cell lymphoma (MCL) is a subtype of B-cell Non-Hodgkin's Lymphoma (NHL) and accounts for ~6% of all lymphomas. MCL is highly refractory to most chemotherapy including newer antibody-based therapeutic approaches, and high-grade MCL has one of the worst survival rates among NHLs. Therefore, the development of new therapeutic strategies to overcome drug resistance of MCL is important. In this article, we tested the effects of arsenic trioxide (As(2) O(3) , ATO) in bortezomib-resistant MCL. ATO is reported to induce complete remission in the patients with relapsed or refractory acute promyelocytic leukemia. Their effects in MCL, however, have not been explored. In this report, we show that ATO effectively inhibited the growth of MCL cells in vitro. ATO treatment also reduced cyclin D1 expression which is a genetic hallmark of MCL and NF-kB expression which was reported to have a prosurvival role in some MCL cells. The induction of apoptosis in MCL was partially due to reduced levels of cyclin D1 and increased levels of apoptosis-related molecules. The antiproliferative effects of bortezomib on MCL greatly increased when the cells were also treated with ATO, indicating ATO can sensitize MCL to bortezomib. Similar results were noted in bortezomib-resistant cell lines. In conclusion, ATO may be an alternative drug for use in combined adjuvant therapies for MCL, and further clinical testing should be performed.

A new marker, SOX11, aids the diagnosis of mantle cell lymphoma in the prostate: A case report

Mantle cell lymphoma (MCL) of the prostate, either primary or secondary, is a rare entity. This case report examines an 83-year-old male who complained of not only nocturia (2-3 times), but also frequency and urgency of urination. The maximal urinary flow rate was 4.1 ml/sec. A transrectal ultrasound-guided prostate biopsy was advised after a hard enlargement of the prostate was detected; however, it was refused. Therefore, a plasma kinetic transurethral resection of the prostate was performed. Postoperative pathological examinations demonstrated MCL of the prostate. Positive immunohistochemistry for CD5 and cyclin D1 was observed. The diagnosis was confirmed by the introduction of a new diagnostic marker, SOX11. The maximal flow rate achieved was 15 ml/sec following surgery. To the best of our knowledge, this is the first study of MCL being diagnosed using SOX11 as a marker in the prostate. This case should alert clinicians and pathologists to pay close attention to the diagnosis of malignant lymphoma of the prostate. This study provides further insights into the diagnosis and therapy of MCL.

Comparative outcome of initial therapy for younger patients with mantle cell lymphoma: an analysis from the NCCN NHL Database

AbstractFew randomized trials have compared therapies in mantle cell lymphoma (MCL), and the role of aggressive induction is unclear. The National Comprehensive Cancer Network (NCCN) Non-Hodgkin Lymphoma (NHL) Database, a prospective cohort study collecting clinical, treatment, and outcome data at 7 NCCN centers, provides a unique opportunity to compare the effectiveness of initial therapies in MCL. Patients younger than 65 diagnosed between 2000 and 2008 were included if they received RHCVAD (rituximab fractionated cyclophosphamide, vincristine, adriamycin, and dexamethasone), RCHOP+HDT/ASCR (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone + high-dose therapy/autologous stem cell rescue), RHCVAD+HDT/ASCR, or RCHOP. Clinical parameters were similar for patients treated with RHCVAD (n = 83, 50%), RCHOP+HDT/ASCR (n = 34, 20%), RCHOP (n = 29, 17%), or RHCVAD+HDT/ASCR (n = 21, 13%). Overall, 70 (42%) of the 167 patients progressed and 25 (15%) expired with a median follow-up of 33 months. There was no difference in progression-free survival (PFS) between aggressive regimens (P > .57), which all demonstrated superior PFS compared with RCHOP (P < .004). There was no difference in overall survival (OS) between the RHCVAD and RCHOP+HDT/ASCR (P = .98). RCHOP was inferior to RHCVAD and RCHOP+HDT/ASCR, which had similar PFS and OS. Despite aggressive regimens, the median PFS was 3 to 4 years. Future trials should focus on novel agents rather than comparing current approaches.

Novel targeted therapies for mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy characterized by short median survival despite intensive therapies. The clinical behavior of MCL may be due to the complex pathophysiology of the disease which includes its genetic hallmark, the chromosomal translocation t(11;14) resulting in aberrant expression of cyclin D1, alteration in the DNA damage response, and constitutive activation of key anti-apoptotic pathways such as phosphatidyl-inositol 3-kinase (PI3K)/Akt and nuclear factor-kB (NF-kB). Collectively, these changes result in cell cycle dysregulation and give rise to profound genetic instability. Given this complex pathophysiology, the limited number of options for patients with relapsed/refractory MCL, and the difficulty in achieving long-lasting remissions with conventional approaches, it is essential to explore new treatment options targeting the numerous dysregulated pathways that are operable in MCL. We have recently reported that milatuzumab, a fully humanized anti-CD74 monoclonal antibody (mAb), in combination with anti-CD20 mAbs has significant preclinical and clinical activity in MCL. Here we discuss these results, provide additional insights into milatuzumab-mediated MCL cell death, and report preliminary data on the activity of other targeted biologic agents including PCI-32765 and CAL-101 currently undergoing evaluation at our institution and others.

Acute Appendicitis in a Man Undergoing Therapy for Mantle Cell Lymphoma

A 71-year-old man was diagnosed with an aggressive mantle cell lymphoma and was started on six cycles of R-CHOP chemotherapy. Approximately two weeks after starting his first cycle of chemotherapy, he complained of severe right lower quadrant abdominal pain, and an abdominal CT scan demonstrated an enlarged appendix with evidence of contained perforation. The man underwent open appendectomy for acute appendicitis and recovered. The appendectomy specimen was submitted for routine pathological analysis. There was histologic evidence of perforation in association with an inflammatory infiltrate with fibrin adhered to the serosal surface; scattered small lymphoid aggregates were present on the mucosal surface. Although the lymphoid aggregates in the submucosa and lamina propria were rather unremarkable by routine histologic examination, immunohistochemistry revealed the lymphocytes to be predominantly Cyclin D1-overexpressing B cells. To our knowledge, this is the first reported case of acute appendicitis in association with appendiceal involvement by mantle cell lymphoma.