Rituximab In Chronic Lymphocytic Leukemia Research Articles

A single bout of vigorous intensity exercise enhances the efficacy of rituximab against human chronic lymphocytic leukaemia B-cells ex vivo

Chronic lymphocytic leukaemia (CLL) is characterised by the clonal proliferation and accumulation of mature B-cells and is often treated with rituximab, an anti-CD20 monoclonal antibody immunotherapy. Rituximab often fails to induce stringent disease eradication, due in part to failure of antibody-dependent cellular cytotoxicity (ADCC) which relies on natural killer (NK)-cells binding to rituximab-bound CD20 on B-cells. CLL cells are diffusely spread across lymphoid and other bodily tissues, and ADCC resistance in survival niches may be due to several factors including low NK-cell frequency and a suppressive stromal environment that promotes CLL cell survival. It is well established that exercise bouts induce a transient relocation of NK-cells and B-cells into peripheral blood, which could be harnessed to enhance the efficacy of rituximab in CLL by relocating both target and effector cells together with rituximab in blood. In this pilot study, n = 20 patients with treatment-naïve CLL completed a bout of cycling 15 % above anaerobic threshold for ∼ 30-minutes, with blood samples collected pre-, immediately post-, and 1-hour post-exercise. Flow cytometry revealed that exercise evoked a 254 % increase in effector (CD3−CD56+CD16+) NK-cells in blood, and a 67 % increase in CD5+CD19+CD20+ CLL cells in blood (all p < 0.005). NK-cells were isolated from blood samples pre-, and immediately post-exercise and incubated with primary isolated CLL cells with or without the presence of rituximab to determine specific lysis using a calcein-release assay. Rituximab-mediated cell lysis increased by 129 % following exercise (p < 0.001). Direct NK-cell lysis of CLL cells – independent of rituximab – was unchanged following exercise (p = 0.25). We conclude that exercise improved the efficacy of rituximab-mediated ADCC against autologous CLL cells ex vivo and propose that exercise should be explored as a means of enhancing clinical responses in patients receiving anti-CD20 immunotherapy.

A Single Bout of Exercise Enhances the Efficacy of Rituximab Against Autologous Chronic Lymphocytic Leukaemia B Cells Ex Vivo By Transiently Increasing Natural Killer Cell Frequency in Blood, and Simultaneously Mobilises CD5 +CD19 +CD20 + B Cells into Blood

Chronic Lymphocytic Leukaemia (CLL) is characterised by the proliferation and accumulation of clonal B cells (B-CLL cells) and is often treated with anti-CD20 monoclonal antibody immunotherapies, including rituximab. One of the mechanism-of-action of rituximab is antibody-dependent cellular cytotoxicity (ADCC) which occurs when natural killer (NK) cells detect rituximab bound to CD20 + target cells. Rituximab often fails to induce stringent disease eradication, due in part to the diffuse distribution of clonal cells across multiple lymphoid and non-lymphoid tissues where NK cell frequency can be low. It is well established that an individual bout of aerobic exercise induces a transient relocation of lymphocytes - including NK cells and B cells - into peripheral blood. We hypothesised that this exercise-induced lymphocytosis could be harnessed to enhance the efficacy of rituximab in CLL by relocating both target and effector cells together with rituximab into blood. In this pilot study n = 20 treatment naïve patients with CLL (mean ± SD: age = 62 ± 10 years; height = 174.0 ± 7.5 cm; body mass = 83.3 ± 16.8 kg; body fat = 31.7 ± 9.8 %; blood leukocytes = 30.70 ± 22.21 ×10 9/L; anaerobic threshold = 14.1 ± 2.9 mL.kg -1.min -1) participated. Participants cycled at a moderate intensity (15% above their anaerobic threshold) for ~30-minutes, with blood samples collected pre-, post-, and 1-hour post-exercise. Given the importance of CD16 + NK cells in evoking ADCC, we enumerated NK cell subsets in blood samples collected pre, post-, and 1-hour post-exercise by flow cytometry. As expected, exercise induced a preferential increase of CD56 +CD16 + (+255%, p &amp;lt; 0.001) and mature, cytotoxic CD56 +CD57 +CD16 + NK cells (+322%, p &amp;lt; 0.001) pre- to post-exercise. Next, using immunomagnetic negative separation, NK cells and primary B-CLL cells were isolated from blood pre- and post-exercise and incubated together with heat inactivated foetal calf serum, with or without the presence of rituximab to determine specific lysis using a calcein-release assay. Rituximab mediated cell lysis increased by +129% following exercise ( p &amp;lt; 0.001), with no change in antibody independent NK cell lysis of B-CLL cells - independent of rituximab - following exercise ( p = 0.25). Blocking CD16 on NK cells - vital for ADCC - blunted the effects of exercise on B-CLL cell lysis ( p = 0.84). In a subset of patients ( n = 9) we also explored the effects of autologous time-point matched plasma (instead of heat inactivated foetal calf serum) on rituximab mediated ADCC, which evoked a +92% increase in lysis pre- to post-exercise ( p = 0.038). Collectively, our results suggest that augmented efficacy of rituximab mediated ADCC was driven by an increase in CD16 + NK cells. We posited that the enhancement to rituximab mediated ADCC would have greater, clinically relevant implications if there was also a concomitant exercise-induced mobilisation of B-CLL cells expressing CD20 into blood from different body tissues. Flow cytometry revealed a +63% increase in CD5 +CD19 +CD20 + B-CLL cells ( p = 0.002) in blood after exercise. Further analyses revealed that CD5 +CD19 +CD20 + B-CLL cells with a phenotype consistent with recent egress from lymphoid tissue (CD5 brightCXCR4 dim; 70%, p = 0.004) and B-CLL cells with a propensity to migrate to peripheral tissues (CD5 dimCXCR4 bright; 67%, p = 0.002) were mobilised, with no change to overall CD20 surface antigen density ( p = 1.0) - determined by median fluorescence intensity. Furthermore, exercise evoked a +69% ( p = 0.022) increase in CD5 +CD19 +CD20 + B-CLL cells expressing CD49d, which is considered one of the strongest predictors of CLL prognosis. Taken together, these data demonstrate that exercise increased the frequency of CD20 + B-CLL cells with lymphoid origins and prognostic relevance into the blood, therefore rendering them susceptible to rituximab mediated ADCC. Our results show that individual bouts of moderate intensity aerobic exercise temporarily increased the number of cytotoxic CD16 + NK cells, and CD20 + B-CLL cells in blood. Additionally, our ex vivo investigations demonstrated enhanced rituximab mediated ADCC following exercise. Thus, exercise could be explored as a means of improving clinical responses in patients receiving rituximab, and/or other anti-CD20 monoclonal antibodies such as, Obinutuzumab.

Assessment of rituximab-abbs, a biosimilar, and rituximab outcomes in patients with CLL or NHL: A real-world UK study

BackgroundRituximab (chimeric anti-CD20 monoclonal antibody) treatment is approved for chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL). Rituximab-abbs (first biosimilar approved in 2017) is expected to significantly reduce healthcare economic burden due to lower acquisition costs. This non-interventional, non-comparative study assessed real-world effectiveness and tolerability of rituximab-abbs and rituximab in treatment-naive patients with CLL or NHL. Materials and methodsVia an online physician survey, 46 UK-registered hematologists and oncologists retrospectively reported on randomly selected patients aged ≥18 years with CLL or NHL with rituximab-abbs or rituximab as first-line immunotherapy. Overall, 201 patient charts were examined across 4 cohorts: rituximab-abbs in CLL, rituximab-abbs in NHL, rituximab in CLL, rituximab in NHL. ResultsDemographic profiles across cohorts were similar. Most patients (94 %–100 %) received combination therapy (rituximab-abbs or rituximab mainly with chemotherapy). For both treatments, overall response rate (94 %–98 %) and 1-year overall survival (98 %–100 %) were very high for patients with CLL or NHL. Most common serious adverse events were neutropenia, fatigue, anemia and infusion reactions. The majority of patients (54 %–66 %) did not experience a grade ≥3 adverse event. Healthcare resource utilization was similarly high across cohorts, driven by diagnostic testing, oncologist office visits, and day-case hospital admissions; many patients required supportive medical therapies. Mean annual savings of ∼£1000/patient driven by acquisition costs occurred with rituximab-abbs versus rituximab, administration costs were similar. ConclusionRituximab-abbs and rituximab demonstrated similar effectiveness and tolerability in treating CLL and NHL in routine UK clinical practice and demonstrate the utility of the biosimilar as a cost-saving alternative treatment.

Prognostic role of CD4 T-cell depletion after frontline fludarabine, cyclophosphamide and rituximab in chronic lymphocytic leukaemia

BackgroundEradication of minimal residual disease (MRD), at the end of Fludarabine-Cyclophosphamide-Rituximab (FCR) treatment, is a validated surrogate marker for progression-free and overall survival in chronic lymphocytic leukaemia. But such deep responses are also associated with severe immuno-depletion, leading to infections and the development of secondary cancers.MethodsWe assessed, blood MRD and normal immune cell levels at the end of treatment, in 162 first-line FCR patients, and analysed survival and adverse event.ResultsMultivariate Landmark analysis 3 months after FCR completion identified unmutated IGHV status (HR, 2.03, p = 0.043), the level of MRD reached (intermediate versus low, HR, 2.43, p = 0.002; high versus low, HR, 4.56, p = 0.002) and CD4 > 200/mm3 (HR, 3.30, p < 0.001) as factors independently associated with progression-free survival (PFS); neither CD8 nor NK counts were associated with PFS. The CD4 count was associated with PFS irrespective of IGHV mutational status, but only in patients with detectable MRD (HR, 3.51, p = 0.0004, whereas it had no prognostic impact in MRD < 10− 4 patients: p = 0.6998). We next used a competitive risk model to investigate whether immune cell subsets could be associated with the risk of infection and found no association between CD4, CD8 and NK cells and infection.ConclusionsConsolidation/maintenance trials based on detectable MRD after FCR should investigate CD4 T-cell numbers both as a selection and a response criterion, and consolidation treatments should target B-cell/T-cell interactions.

Obinutuzumab for the treatment of chronic lymphocytic leukemia and other B-cell lymphoproliferative disorders

ABSTRACTIntroduction: Chemoimmunotherapeutic regimens using the anti-CD20 antibody rituximab improved significantly the survival rates in various B-cell lymphoproliferative disorders (LPDs), including chronic lymphocytic leukemia (CLL). The next-generation CD20 antibody obinutuzumab represents an addition to the drug armamentarium used for the therapeutic management of patients with LPDs.Areas covered: Herein, the authors discuss the biochemical and conformational engineering of obinutuzumab to increase antibody-dependent cell-mediated cytotoxicity and direct cell death. They also describe the available preclinical data on obinutuzumab’s role in B-cell LPDs. Furthermore, the authors summarize the Phase I and II clinical trials of obinutuzumab, focusing on the main pharmacokinetic/pharmacodynamic characteristics, the most common clinically significant adverse events, dose optimization, and clinical outcomes of patients with CLL and other B-cell LPDs treated with obinutuzumab as monotherapy or in combination with other agents. To put these data in perspective, the use of obinutuzumab is compared with that of rituximab in CLL and other B-cell LPDs.Expert opinion: Clinical trials have demonstrated that obinutuzumab is well tolerated. The novel mechanism of action of obinutuzumab is associated with significant efficacy in CLL and other B-cell LPDs. Ongoing clinical trials are expected to determine the optimal use of obinutuzumab in these diseases.

Outcomes of patients with chronic lymphocytic leukemia treated with first-line idelalisib plus rituximab after cessation of treatment for toxicity.

More active therapies are needed for older and unfit patients with chronic lymphocytic leukemia (CLL) who are not eligible for chemoimmunotherapy with fludarabine, cyclophosphamide, and rituximab. The phosphyotidylinositol-3-kinase δ inhibitor idelalisib is effective in patients with treatment-naive and relapsed/refractory CLL as monotherapy and in combination with rituximab, but it can be associated with treatment-limiting adverse events, particularly diarrhea/colitis. The outcomes for patients who cease treatment for adverse events have not been previously described. The authors analyzed long-term follow-up data from 40 treatment-naïve patients aged ≥65 years who received treatment at The University of Texas MD Anderson Cancer Center on a phase 2 study of idelalisib plus rituximab for CLL. In patients who permanently ceased treatment because of toxicity, the time to subsequent disease progression was analyzed according to baseline characteristics. Fifteen patients permanently ceased therapy (PCT) because of toxicity (PCTTOX ), most commonly diarrhea/colitis (n = 7), at a median of 11 months after commencing treatment. PCTTOX was associated with a higher risk of subsequent disease progression (hazard ratio, 6.61; 95% confidence interval, 1.77-16.15) relative to that observed in patients who remained on therapy. Ten patients subsequently progressed, and 7 required salvage therapy; 5 patients remained progression-free at a median of 23.3 months (range, 8.5-28.6 months). Patients who were positive for ζ-associated protein-70 had more rapid disease progression after treatment cessation (P = .048). There were no CLL-related deaths. PCTTOX is the major determinant of PFS in patients who receive first-line idelalisib-based treatment. However, a subgroup of patients with favorable biologic characteristics has prolonged PFS, even after PCTTOX . The absence of CLL-related deaths indicates that salvage treatment is generally successful after PCTTOX . Cancer 2016;122:2505-11. © 2016 American Cancer Society.

A Phase 2 Study of Alemtuzumab-Ofatumumab (A+O) Combination in Patients with Previously Untreated Chronic Lymphocytic Leukemia (CLL)

BackgroundCurrent standard immunochemotherapy is highly effective as frontline treatment of CLL but has several limitations including poor tolerability in older patients with comorbidities, potential long-term marrow toxicity, and limited efficacy in high risk CLL. Therapy with monoclonal antibodies alone may offer an alternative, non-stem cell toxic treatment option. Our prior study of alemtuzumab-rituximab in previously untreated CLL showed promising results with good tolerability although lymph node responses were suboptimal (Frankfurt et al., Leuk Lymphoma 2014). Ofatumumab showed improved complement-mediated cytotoxicity over rituximab for CLL in vitro and significant clinical activity in refractory CLL. In this study we aim to test safety and effectiveness of alemtuzumab-ofatumumab (A+O) combination as a frontline treatment for CLL.MethodsThis phase 2 study of A+O first-line treatment for CLL is conducted at Northwestern University in Chicago and Karolinska University Hospital in Stockholm with an accrual goal of 60 patients. Eligibility requires symptomatic, previously untreated CLL, and PS ≤ 2. There was no exclusion for baseline cytopenia or upper age limit. Alemtuzumab (Alem) is given subcutaneously tiw for up to 18 weeks (dose escalation 3mg-10mg-30mg for week 1 and 30mg per dose for subsequent weeks). Starting week 3, ofatumumab (Ofa) is added intravenously q2 weeks up to 8 doses at 300mg for dose 1 and 2000mg for doses 2-8. All patients received anti-herpes, anti-fungal and anti-PCP prophylaxis. Cytomegalovirus (CMV) was monitored by PCR every other week. Bone-marrow biopsy and CT scans are required for response assessment (IWCLL 2008 criteria). Minimal residual disease (MRD) was assessed in bone-marrow using 6-color flow. Hematologic toxicity was assessed using IWCLL 2008 criteria and non-hematologic toxicity graded by CTCAE 4.0.ResultsTo date the study has enrolled 41 patients among whom 31 patients have completed treatment and are reported here in this interim analysis. Median age was 63 (45-77) years; 58% had Rai stage III/IV; 48% had unmutated IgHV, and 7/31 were 17p- and/or had TP53 mutation.During initial safety run-in period to assess dose-limiting toxicity (DLT), one patient after 18 weeks of therapy developed prolonged cytopenia and died of sepsis, and another patient after 14 weeks of therapy developed grade 4 neutropenia which resolved after 16 days. Both patients achieved MRD-negative complete bone-marrow response with hypocellularity. It was speculated that the cause may be continued treatment with Alem beyond the point of MRD-negative CR in bone-marrow. Early stopping rules were implemented using BM biopsy after 9 and 12 weeks of treatment. Alem is stopped if CR in the bone-marrow is achieved, or if cellularity <10%. Ofa is stopped if CR in lymph-nodes on CT after achieving bone-marrow CR. Since this protocol modification after the initial 9 patients, no further DLT was observed and the safety committee approved continued enrollment.The most frequent treatment-emergent AE included neutropenia, anemia, thrombocytopenia, Alem-related local injection-site reaction, Ofa-related infusion-symptoms, pyrexia, and fatigue. Grade 3-4 hematologic AEs included neutropenia (39%), thrombocytopenia (26%). The most frequent Grade 3-4 non-hematologic AE was febrile neutropenia (23%). CMV-reactivation occurred in 16 cases successfully treated with oral valganciclovir or IV ganciclovir.All patients achieved blood lymphocyte response within 4 weeks. Of 31 patients evaluable for response, 11 met early stopping criteria after achieving MRD-negative bone marrow CR (8/11 overall CR) with a median of 10 weeks of Alem and 5 doses of Ofa. The remaining patients (except one with PD) completed all 18 weeks of Alem and 8 doses of Ofa treatment.Overall response rate (ORR) was 97% with 42% CR and 55% PR. MRD by bone marrow flow-cytometry was negative in 12/13 CR patients and 4/17 PR patients. All but one patient remain alive at follow-up and three required subsequent treatment due to progressive disease.ConclusionsThe combination of alemtuzumab-ofatumumab as first-line CLL treatment produced high response rates, including MRD-negative CR, and had acceptable safety. Early stop after achieving CR prevented late-onset cytopenia. DisclosuresMa:GlaxoSmithKline: Research Funding; National Comprehensive Cancer Network (NCCN) Oncology Research Program: Research Funding. Off Label Use: Alemtuzumab and ofatumumab used in combination. Österborg:GlaxoSmithKline: Research Funding. Lundin:GlaxoSmithKline: Research Funding.

Severe infusion-related reactions are uncommon in rituximab-treated CLL patients in clinical practice: Results from a Swedish national observational study

There have been concerns about serious infusion-related adverse drug reactions (ADR) with rituximab in chronic lymphocytic leukemia (CLL). We therefore conducted an observational trial in which CLL patients planned for rituximab-containing therapy were eligible. Ninety-six patients from 19 centers were enrolled. The most common regimen was rituximab, fludarabine and cyclophosphamide. Fifty-six patients experienced ADR during rituximab infusion. Reactions≥grade 3 occurred in five patients and no cases of tumor lysis syndrome were recorded.Despite a high number of circulating tumor cells few severe ADR were noted. Thus, rituximab-containing regimens can be considered safe for CLL patients in general practice.

Modelling the Cost Effectiveness of Rituximab in Chronic Lymphocytic Leukaemia in First-Line Therapy and Following Relapse

The efficacy and safety of adding rituximab to fludarabine and cyclophosphamide (R-FC) for the treatment of chronic lymphocytic leukaemia (CLL) has been demonstrated in two randomised trials: CLL-8 was conducted in previously untreated patients, and REACH was conducted in previously treated patients. In both trials, progression-free survival was increased in the R-FC treatment groups compared with the FC treatment groups. In CLL-8, overall survival was also significantly increased. To develop an economic model to assess the cost effectiveness, from the Australian healthcare perspective, of rituximab when used as a treatment for both previously untreated and relapsed/refractory CLL. A Markov model with three health states (unprogressed, progressed and death) was developed to extrapolate the trial results over a 15-year time horizon. A treatment algorithm was developed with Australian haematologists to inform the treatments to be modelled. The base-case compares up to three courses of six cycles of R-FC ('first-line' treatment) followed by three courses of post-progression salvage ('Salvage') treatment (including rituximab) with three courses of FC followed by three courses of Salvage treatment (excluding rituximab). Subsequent treatments are incorporated into the model by repeating the unprogressed and progressed health states for each treatment. Time-dependent transition probabilities for the model were estimated from an analysis of individual patient data from CLL-8 and REACH. Comparisons of the hazard rates for the CLL-8 and REACH trials enabled an assessment of the impact on the transitions of receiving the same regimen as the first or second treatment, and hence inform assumptions regarding transitions for third and subsequent treatments. Costs applied in the model were based on published Australian prices in 2009. The model predicts patients receive an average of approximately two courses of treatment, and the addition of rituximab results in an incremental gain of 0.94 quality-adjusted life-years (QALYs). The incremental cost associated with the addition of rituximab is A$40,268, and hence the cost per QALY gained (QALYG) is A$42,906. Rituximab, in combination with chemotherapy, when used multiple times throughout the treatment algorithm, appears to be cost effective for CLL from the Australian healthcare perspective, with a cost/QALYG within the range generally accepted as providing value.

Decitabine Or 5-Azacitidine Pre-Treatment Does Not Compromise The Novel Fc-Engineered Antibody Mab 33.1-Mediated ADCC Against Primary AML Blasts - A Rationale For Combination Therapy

IntroductionAcute Myeloid leukemia (AML) in patients older than 60 years is a devastating diagnosis with long-term survival rates of 10%. Elderly patients have poor survival both due to chemoresistance and presence of concomitant comorbidities rendering them ineligible for induction chemotherapy. Hence novel treatment options are warranted in this patient population. Promising activity of monoclonal antibodies such as alemtuzumab and rituximab for chronic lymphocytic leukemia (CLL) and rituximab for lymphomas has raised the potential use of antibody therapies in AML. CD33 is expressed on greater than 90% of AML blast cells while absent from all non-hematopoietic tissues. Hence CD33 is a viable target for antibody-based therapeutics in AML. Here, we tested the ex vivo efficacy of the mAb 33.1, a fully human anti-CD33 antibody Fc-engineered for increased binding to Fcγ receptors on AML cell lines and primary AML blasts. The goals of this study are to evaluate 1) the efficacy of mAb33.1 on purified allogeneic and autologous natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) against primary AML Blasts; 2) to evaluate efficacy of mAb 33.1 in combination with azanucleosides (i.e. decitabine, 5-azacitidine) that are currently used in AML therapy on NK cell-mediated ADCC against primary AML blasts; and 3) to correlate the levels of surface expression of CD33 on AML blasts to the mAb 33.1 mediated ADCC. MethodsmAb 33.1 mediated NK cell activation was determined by NK degranulation as determined by CD107a induction, and ADCC was determined by standard 4-hour 51Cr-release assay. An AML cell line HL60 and a total of 15 AML blast samples were used as targets in this study. NK cells enriched from normal donor PBMC (for allogeneic assays) or sorted from AML blast samples (for autologous assays) were used as effector cells. ResultsThe mAb 33.1 induced potent ADCC activity (>40%) compared to control non-Fc engineered antibody at the concentration of 10 μg/ml in the HL60 cell line. For the AML blasts, mAb 33.1 mediated significantly higher ADCC activity when compared to the control antibody (p<0.05). The relative cytotoxicity mediated by mAb 33.1 varied among different patients, ranging from 4.4% to 65.8%. Subsequent quantification of CD33 showed that there is a positive correlation between ADCC activity and the number of surface CD33 molecules on the AML blasts. Induction of CD107a expression was also observed in both allogeneic and autologous NK cells when the blasts were labeled with mAb 33.1. Pre-treatment of the NK cells and/or target blasts with decitabine or 5-azacitidine for 48hrs, did not alter the mAb 33.1 mediated ADCC activity or CD107 induction. ConclusionmAb33.1 mediated potent ADCC activity and NK activation against AML cell lines and primary AML blasts. Both autologous and allogeneic NK cell-mediated ADCC against primary blast cells from AML patients was observed. The level of NK cell-mediated ADCC was positively associated with the levels of the surface CD33 expression on target AML blasts. Pre-treatment of either AML blasts and/or NK effector cells with Decitabine or 5-azacitidine did not compromise mAb 33.1-mediated ADCC. These pre-clinical studies support further clinical development of mAb 33.1 in combination with relevant anti-AML therapies such as decitabine or 5-azacitidine in patients with CD33 expression. Disclosures:Heider:boehringer-ingelheim: Employment.

Bone Marrow May Be More Informative Than Peripheral Blood To Evaluate Minimal Residual Disease During 1st and 2nd Year Follow Up After First-Line Fludarabine, Cyclophosphamide, and Rituximab For Chronic Lymphocytic Leukemia

BackgroundMinimal Residual Disease (MRD) status at end of first-line chemoimmunotherapy is an independent prognostic factor for patients (pts) with chronic lymphocytic leukemia (CLL). In the CLL8 trial of the German CLL Study Group, peripheral blood (PB) was monitored for MRD during follow up. Because the microenvironment is important for CLL cell growth and survival and typically it is the last site to eliminate residual disease with chemoimmunotherapy, bone marrow (BM) might be a more reliable site to monitor MRD. MethodsTwo-hundred thirty-seven pts with CLL and an indication for therapy (IWCLL-WG 2008) received first-line fludarabine, cyclophosphamide, and rituximab (FCR) on protocol between 09/2008 and 09/2012. MRD was prospectively assessed in BM and/or PB by flow cytometry using the highly sensitive international standardized approach, 2 months after the last course of treatment (final response assessment) and every 3-6 months thereafter. Kaplan-Meier estimates were compared using the log-rank test. ResultsSixty-one percent of pts were male, 21% were >65 years old, 40% had Rai stage III-IV, 41% had beta2-microglobulin (B2M) ≥4 mg/L, 61% had unmutated IGHV, and 21% had FISH analysis positive for deletion 11q and 7% for deletion 17p. Seventy-five percent of pts received ≥3 total courses of FCR. The complete remission (CR) and overall response (OR) rates were 65 and 97%, respectively. BM MRD negativity was achieved in 59% of pts at final response assessment.For monitoring, BM MRD was assessed in 121 pts during the 1st year and in 30 pts during the 2nd year after completion of treatment with FCR; all samples were serial. PB MRD was assessed in 106 pts during the 1st year and in 57 during the 2nd year of follow up; again all samples were serial. BM MRD negativity was observed in 63 (52%) pts during the 1st year of follow up and in 15 (50%) pts during the 2nd year. PB MRD negativity was observed at the same staging times in 81 (76%) and 29 (51%) pts, respectively. Concurrent BM and PB samples were taken during the 1st year in 51 pts, and in 6 pts during the 2nd year of follow up. We evaluated the association between MRD negativity during the 1st and 2nd year of follow-up and progression-free survival (PFS). BM MRD positive status was associated with shorter PFS when assessed during both the 1st and 2nd year of follow up (p<0.001 and p=0.001, respectively; Figure). In contrast, PB MRD positive status did not correlate with PFS for either time (p=0.15 and p=0.79, respectively; Figure). [Display omitted] ConclusionsAfter first-line FCR for pts with CLL, positive BM MRD may identify pts at higher risk for progression. Based on this finding, BM may be preferred to assess MRD status and pts with positive BM MRD could be considered for maintenance or consolidation strategies. Additional studies confirming these findings are warranted. Disclosures:No relevant conflicts of interest to declare.

Second-line therapies of patients initially treated with fludarabine and cyclophosphamide or fludarabine, cyclophosphamide and rituximab for chronic lymphocytic leukemia within the CLL8 protocol of the German CLL Study Group

Updated results of the CLL8 trial confirm that the addition of rituximab to chemotherapy with fludarabine and cyclophosphamide (FC) leads to a prolongation of progression-free (PFS) and overall survival (OS) in first-line treatment of physically fit patients. After a median observation time of 47 months, median PFS was 57.9 months for patients treated with FC and rituximab (FCR) and 32.9 months for patients treated with FC alone (hazard ratio 0.56, 95% confidence interval 0.465–0.673; p < 0.001). A total of 232 patients were treated for relapse, among them 91 of 408 (22%) initially treated with FCR and 141 of 409 (35%) initially treated with FC. The drugs most frequently used either alone or in combination were rituximab (52% of all second-line therapies), fludarabine (21%), bendamustine (21%) and alemtuzumab (12%). The regimens chosen for second-line treatment after FC or FCR were heterogeneous, which underlines a need for further trials in order to define treatment recommendations for patients with relapsed chronic lymphocytic leukemia.

A phase I study of escalated dose subcutaneous alemtuzumab given weekly with rituximab in relapsed chronic lymphocytic leukemia/small lymphocytic lymphoma

This study assessed the safety and preliminary efficacy of escalated dose subcutaneous alemtuzumab in combination with rituximab in chronic lymphocytic leukemia. Twenty-eight patients with relapsed refractory chronic lymphocytic leukemia were treated on four dosing cohorts of weekly rituximab at 375 mg/m(2) and alemtuzumab doses that started at 30 mg three times per week and escalated to weekly dosing over four weeks, culminating with 90 mg weekly. One dose limiting toxicity of a rituximab infusion reaction was seen in cohort 2, but the regimen was otherwise well tolerated without evidence of differential toxicity by cohort. The overall response rate by National Cancer Institute-Working Group criteria was 61%, and the rate of complete bone marrow response was 43%, most of whom were negative for minimal residual disease. The addition of CT scan evaluation per International Workshop on Chronic Lymphocytic Leukemia 2008 criteria reduced the overall response rate to 14%. Median overall survival was 35 months, with 12 patients able to proceed to stem cell transplantation. Pharmacokinetic studies showed that chronic lymphocytic leukemia involving more than 80% of the bone marrow at study start was associated with lower trough concentrations of alemtuzumab and rituximab, and that higher trough serum concentrations of alemtuzumab were associated with complete bone marrow clearance. We conclude that escalated subcutaneous doses of alemtuzumab given weekly are well tolerated and result in excellent bone marrow clearance of chronic lymphocytic leukemia, helping patients to proceed to stem cell transplantation. This study is registered at ClinicalTrials.gov (Identifier:00330252).

Rituximab in the treatment of non-Hodgkin's lymphoma – a critical evaluation of randomized controlled trials

Introduction: The use of rituximab has led to significant improvements in the outcome of both aggressive and indolent Non-Hodgkin's lymphoma (NHL). It is the first targeted therapy to be developed for the treatment of lymphoma which has been widely adopted.Areas covered: This paper discusses the use of rituximab in NHL, mainly concentrating on diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL), with a brief discussion about use in other types of NHL including mantle cell lymphoma (MCL). The use of rituximab in chronic lymphocytic leukemia (CLL) has been recently published in this journal by Robak (Robak T. Rituximab for chronic lymphocytic Leukemia. Expert Opin Biol. Ther. 2012;12(4):503-15). Non hematological indications for rituximab are also not discussed. A Pubmed search was conducted using key words of rituximab, DLBCL, FL, MCL, Burkitt's lymphoma and MALToma. Papers shortlisted for review included randomized control trials and scientific papers discussing CD20.Expert opinion: In conclusion this paper has critically evaluated the use of rituximab in the treatment mainly of DLBCL, FL and MCL both at diagnosis and relapsed disease, and briefly discuses its use in other subtypes of lymphoma. Rituximab has significantly improved the outcome of patients with B cell NHL, in particular those with DLBCL and FL. Patients usually tolerate the treatment well, and studies have shown it to be a cost effective treatment.

Generation and Preclinical Characterization of a Fc-optimized GITR-Ig Fusion Protein for Induction of NK Cell Reactivity Against Leukemia

Natural killer (NK) cells are cytotoxic lymphocytes that largely contribute to the efficacy of therapeutic strategies like allogenic stem cell transplantation in acute myeloid leukemia (AML) and application of Rituximab in chronic lymphocytic leukemia (CLL). The tumor necrosis factor (TNF) family member GITR ligand (GITRL) is frequently expressed on leukemia cells in AML and CLL and impairs the reactivity of NK cells which express GITR and upregulate its expression following activation. We developed a strategy to reinforce NK anti-leukemia reactivity by combining disruption of GITR-GITRL interaction with targeting leukemia cells for NK antibody-dependent cellular cytotoxicity (ADCC) using GITR-Ig fusion proteins with modified Fc moieties. Neutralization of leukemia-expressed GITRL by the GITR domain enhanced cytotoxicity and cytokine production of NK cells depending on activation state with NK reactivity being further largely dependent on the engineered affinity of the fusion proteins to the Fc receptor. Compared with wild-type GITR-Ig, treatment of primary AML and CLL cells with mutants containing a S239D/I332E modification potently increased cytotoxicity, degranulation, and cytokine production of NK cells in a target-antigen-dependent manner with additive effects being observed with CLL cells upon parallel exposure to Rituximab. Fc-optimized GITR-Ig may thus constitute an attractive means for immunotherapy of leukemia that warrants clinical evaluation.

Bendamustine and Rituximab for the Treatment of Chronic Lymphocytic Leukemia: The Moffitt Cancer Center Experience

Abstract 4620 Introduction:Although bendamustine is approved for the treatment of chronic lymphocytic leukemia (CLL), the combination of bendamustine and rituximab (BR) is currently under active investigation in the relapsed/refractory setting as well as for front-line use. Emerging data now suggests BR is acceptable for first-line use and indeed is listed in the NCCN Compendia as a first-line option. Herein, we report the results of a single-center retrospective review of BR use in first-, second- and third-line and beyond and its effects on response rate. Methods:We retrospectively reviewed 21 consecutive patients with CLL that received BR at the Moffitt Cancer Center (MCC) between July 2008 and November 2010. Bendamustine was dosed at 70 mg/m2 IV on Days 1 and 2 every 3–4 weeks; rituximab was dosed at 375 mg/m2 IV once with each cycle along with anti-microbial prophylaxis. Data collected included, but was not limited to age, gender, cytogenetic profile, number of previous therapies, Rai stage at time of treatment and response (based on 1996 NCI-WG definition). The primary objective was to assess response rates. The major secondary objective was to assess the effect of cytogenetics on response rates. All analyses were performed using descriptive statistics. Results:Twenty-one patients received treatment with BR; 7 patients received BR as first-line therapy, 7 received BR as second line therapy and the remaining 7 received BR as third-line therapy or beyond. The median age at time of treatment was 66; 12 of 21 patients were male (57%); All Rai Stages were represented, Stage 0 (n=1), Stage 1 (n=4), Stage 2 (n= 3), Stage 3 (n=5), Stage 4 (n=6). 38% of patients were positive for Del11q, 33% for Del13q, 9.5% for Del17p, and 9.5% for mutated IgVH. In previously untreated patients, 6/7 had a documented complete remission (CR) (71.4%) or partial (PR) (14.3%) response; one patient progressed (PD) on therapy. In second-line therapy, all patients had a documented CR (28.6%) or PR (71.4%). In patients treated with BR as 3rd or 4th line therapy, 3 patients had a CR, 2 patients had stable disease (SD), and 2 had PD. Of the 8 patients with Del11q, 37.5% achieved a CR, 4 had PR and 1 had PD. Of the 7 patients with Del13q, 4 achieved CR and 3 had a PR. Both patients with Del17p had PD. Of the 2 patients with mutated IgVH, one patient achieved a PR while the other had PD. Conclusion:Based on these results, the combination of bendamustine and rituximab in patients with CLL is efficacious and well tolerated in patients with both newly diagnosed and relapsed/refractory disease. Disclosures:Off Label Use: Bendamustine and rituximab for CLL. Ho:Genentech: Honoraria; Cephalon: Consultancy. Cultrera:Genentech: Speakers Bureau. Sotomayor:Genentech: Membership on an entity's Board of Directors or advisory committees. Pinilla-Ibarz:Genentech: Speakers Bureau; Cephalon: Speakers Bureau.

The spectrum of use of rituximab in chronic lymphocytic leukemia.

The monoclonal chimeric anti-CD20 antibody, rituximab, has considerably improved therapeutic outcome in B-cell chronic lymphocytic leukemia. Rituximab has limited clinical activity when used as a single agent. The combination of the monoclonal antibody with fludarabine-based regimens clearly demonstrated, in Phase II and randomized trials, an increase in clinical efficacy in previously untreated and pretreated patients. Furthermore the addition of rituximab enabled the eradication of minimal residual disease, which is correlated with the prognosis in a high proportion of patients. Although the combination of rituximab with fludarabine-based regimens increased myelosuppression and immunosuppression, incidence of infections did not increase. The benefit of adding rituximab to other purine analogs or other chemotherapeutic combination regimens has also been explored. Moreover there could be a role for achieving better quality of responses with the combination of different monoclonal antibodies, considering that they target different antigens and exert different mechanism of action. Although the role of rituximab as maintenance therapy in low grade non-Hodgkin’s lymphomas has been determined, the benefit and optimal schedule in chronic lymphocytic leukemia are still under investigation. This review brings together knowledge of the pharmacokinetics, mechanism of action and clinical use of rituximab in chronic lymphocytic leukemia.

Rituximab in Chronic Lymphocytic Leukemia

Rituximab is a class I chimeric anti-CD20 antibody that has shown efficacy in chronic lymphocytic leukemia (CLL), both as a single agent and in combination with traditional chemotherapies. The modest activity demonstrated in early studies evaluating rituximab in relapsed CLL was improved with higher doses or more dose-intensive regimens that overcame the unfavorable pharmacokinetic features commonly found in CLL. These studies led to a variety of combination trials of rituximab with chemotherapy, where both phase II and later phase III studies have shown great promise for the advancement of CLL therapy. Despite the therapeutic success of rituximab in CLL, studies demonstrating the definitive relative mechanism of tumor clearance are still lacking and this requires further investigation. In addition to being used as a therapy for CLL, rituximab is an effective treatment for autoimmune CLL complications such as hemolytic anemia and immune thrombocytopenia (ITP). Patients with CLL may experience early infusion-related side effects that can be diminished with corticosteroid pretreatment and stepped-up dosing. Risk factors for infusion-related toxicity may relate to atypical CLL expressing bright CD20 antigen expression, although several different studies have not clearly implicated elevated white blood cell count as a risk factor. Other adverse events, including delayed cytopenias, reactivation of hepatitis B, and development of progressive multifocal leukoencephalopathy, are rare. Future efforts focusing on novel combination-based strategies will be required to fully appreciate the benefit of this therapy in CLL.

Progressive multifocal leukencephalopathy and cerebral toxoplasmosis in a patient with CLL

A 63-year-old man with a 10 year history of Chronic Lymphocytic Leukemia (CLL) presented with apraxia, forgetfulness, and cognitive impairment. He had been treated with single agent fludarabine at diagnosis and combination therapy with fludarabine, cyclophosphamide, and rituximab 8 months previously. MRI showed high signal in the white matter of right frontal, left frontal, and parietal areas. (see Image 1) Cerebrospinal fluid (CSF) analysis was normal with no organisms seen. Brain biopsy showed white matter replacement with oligodendroglial intranuclear inclusions and adjacent ruptured toxoplasmosis cysts. (see Image 2) Immunohistochemistry was positive for JC virus and PCR of CSF confirmed the presence of the virus as well as toxoplasma. He developed progressive ataxia, dysphagia, and dementia and died 4 weeks after diagnosis. Progressive multifocal leukoencephalopathy is associated with the use of purine analogues and rituximab in CLL [1, 2]. The incidental finding of toxoplasma illustrates how markedly immunosuppressive these regimens are. MRI image. Brain biopsy.

Improvement of Rituximab Efficiency in Chronic Lymphocytic Leukemia by CpG‐Mediated Upregulation of CD20 Expression Independently of PU.1

Lipopolysaccharide (LPS), CpG-containing phosphothioate oligonucleotides (CpG) and various cytokines impact chronic lymphocytic leukemia (CLL) B cells. For example, they influence cell cycle entry, expression of co-receptors, and CD20. Rituximab (RTX), for which CD20 molecule is the target, proved to be less efficient in CLL than in lymphoma. This is accounted for by a lower CD20 level in the former than in the latter B lymphocytes. CD20 transcription is mediated by four transcription factors, of which only purine-rich box-1 (PU.1) is reduced in CLL. We thus examined the effects of LPS, CpG, tumor necrosis factor-alpha, interferon-alpha, interleukin (IL)-3, IL-4, IL-21, granulocyte macrophage-colony stimulating factor (CSF), and granulocyte-CSF on the transcription of PU.1, and the subsequent expression of CD20. It appeared that CpG was unique in that it raised the membrane expression of CD20 on malignant B cells, owing to a PU.1 independent increase in its gene transcription. Moreover, RTX-induced complement-mediated lysis was also ameliorated. Thus, CpG accelerates the transcription of CD20 independently of PU.1, and thereby improves the efficacy of RTX in CLL.