Therapy For non-Hodgkin Lymphoma Research Articles

Cooperation of the proapoptotic receptor agonist rhApo2L/TRAIL with the CD20 antibody rituximab against non-Hodgkin lymphoma xenografts

AbstractRecombinant human rhApo2L/TRAIL selectively stimulates apoptosis in various cancer cells through its receptors DR4 and DR5, and is currently in clinical trials. Preclinical studies have established antitumor activity of rhApo2L/TRAIL in models of epithelial cancers; however, efficacy in non-Hodgkin lymphoma (NHL) models is not well studied. Of 7 NHL cell lines tested in vitro, rhApo2L/TRAIL stimulated apoptosis in BJAB, Ramos RA1, and DoHH-2 cells. Rituximab, a CD20 antibody used to treat certain types of NHL, augmented rhApo2L/TRAIL-induced caspase activation in Ramos RA1 and DoHH2 but not BJAB or SC-1 cells, through modulation of intrinsic rather than extrinsic apoptosis signaling. In vivo, rhApo2L/TRAIL and rituximab cooperated to attenuate or reverse growth of tumor xenografts of all 4 of these cell lines. Depletion of natural killer (NK) cells or serum complement substantially reduced combined efficacy against Ramos RA1 tumors, suggesting involvement of antibodydependent cell- and complement-mediated cytotoxicity. Both agents exhibited greater activity against disseminated than subcutaneous BJAB xenografts, and worked together to inhibit or abolish disseminated tumors and increase survival. Moreover, rhApo2L/TRAIL helped circumvent acquired rituximab resistance of a Ramos variant. These findings provide a strong rationale for clinical investigation of rhApo2L/TRAIL in combination with rituximab as a novel strategy for NHL therapy.

Serum Cytokine Levels Predict Time to Progression after Rituximab as Initial Therapy in Patients with Follicular Grade 1 Non-Hodgkin Lymphoma.

Background: Patients with newly diagnosed, advanced-stage, follicular B-cell non-Hodgkin's lymphomas (NHL) commonly receive rituximab as initial therapy. While antibody dependent cytotoxicity (ADCC) is felt to be the predominant mechanism of action for rituximab, the immunologic effects of rituximab are largely unknown. We have previously shown that genetic polymorphisms in IL-8, IL-2, IL-12 and IL-1 receptor antagonist (IL-1RN) have prognostic significance in follicular lymphoma. The goal of this study was to measure changes in the abovementioned and other serum cytokine levels in patients receiving single agent rituximab as initial therapy for follicular NHL and to identify cytokines that are associated with an increased risk of disease relapse.Methods: Patients with untreated follicular grade 1 NHL, and measurable stage III/IV disease were treated with rituximab 375 mg/m2 intravenous weekly for 4 doses and were then followed for response and time to progression (TTP); no maintenance therapy was provided. Thirty-seven patients were enrolled in this clinical trial between July 1999 and May 2001. As previously reported, the overall response rate in the 36 eligible patients was 72%, with 36% complete remissions. Now, with a median follow up of 5.1 years, the median TTP is 2.2 years (95% CI, 1.2–3.3 years) and the 5-year overall survival is 77% (95% CI: 64–92%). We measured the serum levels of a variety of inflammatory cytokines by ELISA at baseline and 1 month after completing treatment. Serum specimens were available in 30 patients. Thirty two cytokines or receptors including IL-1β, IL-1RN, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 p40/p70, IL-13, IL-15, IL-17, TNF-α, IFN-α, IFN-γ, GM-CSF, MIP-1α, MIP-1β, IP-10, MIG, Eotaxin, RANTES, MCP-1, VEGF, GCSF, EGF, FGF-basic, HGF, BLyS and APRIL were analyzed.Results: A significant increase was seen in the serum levels of BLyS (p=0.0001) after rituximab therapy, while IL-12 (p=0.0002), IL-1RN (p=0.0001), IL-2R (p=0.0001), IL-1β (p=0.02), IL-7 (p=0.02), IL-10 (p=0.02), and MIG (p=0.05) all significantly decreased. All other cytokines/receptors remained unchanged. Higher pretreatment serum levels of IL-2R (p=0.008) and IL-12 (p=0.001) correlated with a shorter TTP. Furthermore, elevated serum IL-2R levels pretreatment correlated with a lack of response to rituximab (p=0.03). Differences in overall survival could not be determined due to the fact that only 8 patients enrolled in the study have died. Because serum IL-2R levels correlated with response and TTP, we measured the cell surface expression of IL-2R in the tumor by flow cytometry. IL-2R was detected on a subpopulation of CD4+ T-cells in all specimens tested (n=14), and on malignant B-cells in 5 cases (36%).Conclusions: Pretreatment serum levels of IL-2R and IL-12 are important prognostic factors for response to therapy or TTP in follicular lymphoma patients treated with rituximab. Furthermore, serum levels of BLyS, IL-1RN, IL-7, IL-10, IL-1β and MIG also change after rituximab treatment. Targeting these cytokines may therefore provide additional clinical benefit to patients treated with rituximab.

Superior Anti-Tumor Activity of the CD19-Directed Immunotoxin, SAR3419 to Rituximab in Non-Hodgkin's Xenograft Animal Models: Preclinical Evaluation.

It is estimated that 63,000 cases of non-Hodgkin's lymphoma (NHL) will be diagnosed in the United States alone in 2007, of which 19,000 will die of their disease. The selectively over-expressed, tumor-associated, lineage specific CD19 antigen is one of the hallmarks of B-cell NHL. Therefore, the design of high affinity-CD19-based modality epitomize an important step towards improving treatment outcome. In this report, we tested the efficacy of SAR3419, a novel humanized anti-CD19 antibody, i.e., huB4, conjugated to the cytotoxic maytansine derivative, DM4. The in vivo anti-tumor activity of SAR3419 was assessed as a single agent and in comparison with conventional therapies using our established CD19-positive human lymphoma xenograft models, the diffuse large cell lymphoma (WSU-DLCL2) and the transformed follicular small cleaved cell lymphoma (WSU-FSCCL), both EBV-negative. In all, we followed 112 tumor-engrafted SCID mice, 56 bearing bilateral WSU-DLCL2 tumors (subcutaneous) and 56 bearing the WSU-FSCCL (systemic disease). Starting 7 days after xenograft establishment (100 – 200 mg tumor size), SAR3419 (7.5, 15 and 30mg/kg), free DM4 (0.6mg/kg), naked huB4 (30mg/kg), CHOP (at maximum tolerated dose in SCID mice as previously defined) or rituximab (40mg/kg) were administered iv on Days 1 and 4 (except CHOP given on day 1 only). Animals were monitored 3 times weekly for tumor measurements and toxicity and were euthanized when tumor burden reached 10% of body weight (WSU-DLCL2) or became moribund (WSU-FSCCL). Our results showed that WSU-DLCL2-bearing animals treated with SAR3419 dosage of 15 mg/kg had no measurable tumor up to Day 155 (end of experiment) and considered cured (7/7 tumor-free survivors, TFS). In the higher dose of SAR3419 (30mg/kg), all animals (7/7) were tumor-free at time of death. However, 4/7 animals died of unrelated causes before the end of the experiment. At the dosage of 7.5 mg/kg, SAR3419 induced a T-C of 64 days and Log10 kill of 4.6 (2/7 TFS). Treatment with rituximab resulted in high anti-tumor activity (T-C of 72 days; Log10 kill of 5.19 and 4/7 TFS). Animals treated with huB4 or CHOP had T-C of 5 and 13 days, and Log10 kill 0.15 and 0.82, respectively. In the WSU-FSCCL model, by the end of the experiment (Day 150), SAR3419 induced 100%, 4/7 and 2/7 survivors, at 30, 15 and 7.5 mg/kg, respectively. The number of survivors in the other arms of the study was 2/7 for rituximab, 3/7 for CHOP and 2/7 for huB4. All animals in the no treatment (control arm) had died by Day 75. Necropsy and H&E analysis revealed that all deaths featured leptomeningeal lymphoma in the control and treated groups. Overall, SAR3419 is a very active immunoconjugate that produced 100% cures in the two models tested. SAR3419 was found better than either rituximab or CHOP at optimal dosages. We conclude that SAR3419 holds promise as novel and well tolerated immunoconjugate therapy in B-cell NHL.

Durable Complete Responses Following Therapy with Epratuzumab Plus Rituximab: Final Efficacy Results of a Multicenter Study in Recurrent Indolent Non-Hodgkin's Lymphoma (NHL).

BACKGROUND: Epratuzumab, an anti-CD22 humanized monoclonal antibody, has shown clinical activity as a single-agent (Leonard et al, J Clin Oncol. 2003; 21:3051–3059) and in combination with rituximab in relapsed/refractory B-cell NHL (Leonard et al, J Clin Oncol. 2005; 23:5044–5051). To evaluate this combination regimen in a larger cohort of patients and to evaluate long-term efficacy, we conducted an international multicenter, open-label, single-arm study in patients with recurrent indolent NHL.METHODS: Forty-nine patients (23F/26M, median age: 61, elevated LDH: 25%, bone marrow involvement: 49%) with chemotherapy-relapsed or refractory follicular NHL (n=41) or small lymphocytic lymphoma (n=7) (as well as one enrolled patient with histological evidence of follicular and diffuse large B-cell lymphoma) were evaluated. Thirty-five (71%) were rituximab-naive, with the remainder having previously received and responded to then relapsed from rituximab (single agent or with chemotherapy). Patients received 360 mg/m2 IV of epratuzumab, followed by 375 mg/m2 IV of rituximab, weekly for 4 consecutive weeks.RESULTS: As preliminarily reported (Emmanouilides et al, Blood 2003; 102/11:69a), the combination therapy was well tolerated, without notable additive toxicity over that expected with single-agent rituximab. Twenty-two of the 41 (54%, 95% CI: 37% – 69%) follicular NHL (FL) patients achieved an objective response (OR) using IWG response criteria, including 10 (24%) patients with complete responses (CR), half of whom remained in remission at the last evaluation with a median duration of follow-up of 44.3 months (range: 18.2 – 52.4 months). The median duration of response (DR) was 13.4 months (95% CI: 8.4 – 28.2 months) and the median progression-free survival (PFS) was 10.2 months (95% CI: 6.3 – 13.6 months), with a Kaplan-Meier estimated median DR of 33.4 months (range: 11.2 – 47.9 months) and an estimated median PFS of 35.1 months (range: 12.9 – 52.4 months) for the 10 patients who achieved CR. Importantly, 4/7 SLL patients (57%) experienced OR, including 2 CR, 1 CRu and 1 PR. Furthermore, while the rituximab-naive FL and SLL patients (n=34) showed an OR of 50% (26% CR/CRu, 24% PR), those patients who had responded to prior rituximab (n=14) had an OR of 64% (29% CR, 36% PR).CONCLUSIONS: Overall, this study confirms that the combination of epratuzumab and rituximab, in addition to being well tolerated, demonstrates promising anti-lymphoma activity for indolent NHL, and can result in durable complete remissions in a subset of patients. Further evaluation of this combination regimen as initial therapy for indolent NHL is planned (CALGB 50701), and in diffuse large B cell lymphoma (CHOP + epratuzumab + rituximab) is ongoing (NCCTG N0489).

Intravenous, Intraperitoneal, and Subcutaneous Routes of Administering 64Cu-DOTA-HB22.7 Display Equivalent Tumor Targeting Ability.

BACKGROUND: Chemotherapy for Non-Hodgkin's Lymphoma (NHL) is initially effective, but often limited by toxicity and resistance. New agents for the treatment of NHL are needed. HB22.7 is a monoclonal antibody that binds CD22 and has previously been shown to reduce human lymphoma xenograft volume in nude mice.OBJECTIVES: (1) Develop a 64Cu-DOTA-HB22.7 antibody, for in vivo imaging and potential therapy of NHL. (2) Determine if tumor targeting of 64Cu-DOTA-HB22.7 is equivalent between intravenous (IV), intraperitoneal (IP), and subcutaneous (SQ) routes of administration.METHODS: DOTA-HB22.7's ability to bind CD22+ cells was assessed by flow cytometry. 64Cu-DOTA-HB22.7 was injected IV, IP, or SQ into xenograft-bearing nude mice and tumor targeting assessed by positron emission tomography (PET) and biodistribution assay. Pharmacokinetics were determined by measuring radioactivity of blood samples. Serum was analyzed by radio-TLC.RESULTS: DOTA conjugation does not affect HB22.7's ability to bind CD22. 64Cu-DOTA-HB22.7 demonstrates specific tumor targeting at 24 and 48 hrs. Targeting is equivalent regardless of route of administration. Pharmacokinetics demonstrate that 64Cu-DOTA-HB22.7 can access the bloodstream (and thus, target tumor) in IP or SQ injections. By 48 hrs, blood 64Cu levels are (a) equivalent, regardless of injection route and (b) below peak levels, indicating clearance from the circulation. Serum analysis shows that right-shifted TLC peaks, possibly 64Cu-DOTA-HB22.7 metabolites, are present only in the IV injected group at 48 hrs.CONCLUSION: These findings establish 64Cu-DOTA-HB22.7 as an NHL-specific imaging agent and indicate its potential for use in radioimmunotherapy. These findings provide evidence that more accessible routes of administration can achieve equivalent targeting results, and may lead to more efficient and accurate administration of antibody-based therapeutics in mice.

Biokinetics and Dosimetry of 188Re-anti-CD20 in Patients with Non-Hodgkin's Lymphoma: Preliminary Experience

Radioimmunotherapy is a molecular targeting treatment for high-risk leukemia and lymphoma. Rhenium-188-labeled anti-CD66 monoclonal antibody has been used successfully in patients with high-risk acute myeloid leukemia or myelodysplastic syndrome. Our aim was to establish the biokinetics of (188)Re-anti-CD20 in patients and to evaluate its dosimetry as a target-specific radiopharmaceutical for non-Hodgkin's lymphoma (NHL) radioimmunotherapy. Whole-body images were acquired at various times after administration of (188)Re-anti-CD20, obtained from instant freeze-dried kit formulations with radiochemical purity >95%. Regions of interest (ROIs) were drawn around source organs in each time frame. The cpm of each ROI was converted to activity using the conjugate view counting method. The image sequence was used to extrapolate time-activity curves in each organ to calculate the total number of disintegrations (N) that occurred in the source regions. N data were the input for the OLINDA/EXM code to calculate internal radiation dose estimates. Dosimetric studies indicated that after administration of 4.87-8.72 GBq of (188)Re-anti-CD20, the absorbed dose to total body would be 0.75 Gy, which corresponds with the recommended dose for NHL therapies. The calculated absorbed doses of (188)Re-anti-CD20 indicate that it may be used in radioimmunotherapy. Therefore, these preliminary data justify a full assessment of the safety, toxicity, and efficacy of (188)Re-anti-CD20 in a clinical study.

Biokinetics and dosimetry of target-specific radiopharmaceuticals for molecular imaging and therapy

Molecular imaging techniques directly or indirectly monitor and record the spatiotemporal distribution of molecular or cellular processes for biochemical, biologic, diagnostic or therapeutic applications. 99mTc-HYNIC-TOC has shown high stability both in vitro and in vivo and rapid detection of somatostatin receptor-positive tumors. Therapies using radiolabeled anti-CD20 have demonstrated their efficacy in patients with B-cell non-Hodgkin's lymphoma (NHL). The aim of this study was to establish biokinetic models for 99mTc-HYNIC-TOC and 188Re-anti-CD20 and to evaluate their dosimetry as target-specific radiopharmaceuticals. The OLINDA/EXM code was used to calculate patient-specific internal radiation dose estimates. 99mTc-HYNIC-TOC images showed an average tumor/blood ratio of 4.3±0.7 in receptor-positive tumors with an average effective dose of 4.4 mSv. Dosimetric studies indicated that after administration of 5.8 to 7.5 GBq of 188Re-anti-CD20 the absorbed dose to total body would be 0.75 Gy which corresponds to the recommended dose for NHL therapies.

Targeted Therapies for Non-Hodgkin Lymphoma: Rationally Designed Combinations

Non-Hodgkin lymphoma is a diverse group of lymphoid malignancies treated historically with cytotoxic chemotherapy. Recent technologic advances have enabled the development of new therapies specifically targeted to inhibit various pathways involved in disease pathophysiology. Led by the anti-CD20 monoclonal antibody rituximab, these agents are expected to revolutionize patient care. Several new drugs might have limited singleagent activity but could combine with other targeted therapies to produce significant effects. Although the current level of understanding makes this sort of rational combination challenging, there is evidence that certain combinations might be very effective. Examples include the combination of rituximab with other monoclonal antibodies, immunomodulatory drugs, Bcl-2 inhibitors, and bortezomib. All of these agents face the challenge of optimal integration with rituximab. The strategies most likely to produce initial success will be those that incorporate rituximab in sensitive patients, as well as those that target patient populations with rituximab-refractory disease. Ideally, these studies will incorporate biologic correlates, such that the mechanisms of action and the patients most likely to benefit can be better defined. We discuss several combinations, their rationale, and the clinical evidence that justifies further evaluation.

Cost-effectiveness of tositumomab and iodine I-131 tositumomab (Bexxar therapeutic regimen (BTR)), in treatment of non- Hodgkin lymphoma (NHL)

8089 Background: BTR has demonstrated efficacy in NHL patients and it has potential to prolong time to progression (TTP) in relapsed/refractory, low grade, follicular, or transformed NHL. This study assessed effectiveness and cost-effectiveness of BTR compared to alternative therapies in first, second, and third line NHL therapy. Methods: Time-to-event models were constructed with 2 events: progression and death. Patient data from 8 BTR clinical trials were combined to fit Weibull models for TTP and overall survival (OS) by including FLIPI covariates. Estimates for BTR were compared with estimates for alternatives from Weibull models fitted to published TTP and BTR OS data by lines of therapy and measured in life-years (LY). Estimated pre-progression costs included drug costs, lab tests, monitoring, and adverse events; post progression costs included NHL costs until death, all valued in 2006 $US and discounted at 3%. Indirect comparisons yielded incremental cost-effectiveness ratios (ICER=Δ cost/Δ LYs) in each line of therapy. Results: As observed in the table , cost of care estimates in BTR were often comparable with alternative therapies, but typically LY gain favored BTR. Mostly in first and third line, a BTR strategy had an ICER less than the cost-effectiveness threshold of $50,000 per LY gained. Conclusion: Overall, a BTR strategy has a favorable cost-effectiveness profile to alternative strategies including rituximab maintenance (RXM) in first, second, and third line NHL therapy. Results imply both a possible survival gain with early BTR use, and the cost-effectiveness of BTR. This modeling approach can aid in clinical decision making regarding the sequence and timing of therapy for patients with follicular NHL. No significant financial relationships to disclose. [Table: see text]

Rituximab and its role as maintenance therapy in non-Hodgkin lymphoma

Since rituximab, a chimeric monoclonal anti-CD20 antibody, was introduced into clinical practice in 1997, data regarding its benefit in terms of response rate, quality of response, progression-free survival and overall survival in B-cell lymphoid malignancies continues to expand. Rituximab has proven to be a relatively well-tolerated drug, with its major side effects being infusion related. Rituximab was approved initially by the US FDA and the European Medicines Agency for relapsed or refractory low-grade or follicular CD20+ B-cell non-Hodgkin lymphomas. Subsequently, its use has been extended to include first-line therapy in low-grade lymphoma as well as the treatment of more aggressive histological subtypes such as diffuse large B-cell lymphoma. In this article, we review the landmark trials that have impacted clinical practice in follicular and diffuse large B-cell lymphomas and the emerging data for use of rituximab as maintenance therapy in non-Hodgkin lymphoma.

Safety and efficacy of external beam radiation therapy for non‐Hodgkin lymphoma in patients with prior 90Y‐ibritumomab tiuxetan radioimmunotherapy

Non-Hodgkin lymphomas (NHL) are radiosensitive tumors. Recent trials have demonstrated the effectiveness of beta irradiation delivered by monoclonal antibodies conjugated to radioisotopes such as yttrium-90 or iodine-131. Many patients eventually relapse after radioimmunotherapy (RIT) and require additional treatment. The goal of this study was to determine the safety and efficacy of external beam radiation therapy (EBRT) delivered after RIT. We reviewed the records of 135 patients with relapsed B-cell NHL who had received RIT with 90Y-ibritumomab tiuxetan to identify patients who subsequently received EBRT. The response rates and radiation-induced toxicity from the EBRT were assessed. Nineteen of 135 (14%) patients received EBRT to a total of 39 tumor sites. Complete radiation therapy records were available for 16 patients (36 tumor sites), which formed the basis for this analysis. The median EBRT dose was 28.5 Gy (range, 10-40 Gy), with a median fraction size of 2 Gy (range, 1-5 Gy). Response data were available for 29 treated sites. The overall response rate was 90% (26/29) with 12 complete responses (41%), 7 complete clinical responses (24%), 7 partial responses (24%), and 3 stable (10%). Toxicities were generally transient, reversible, and corresponded to the anatomic regions irradiated. EBRT can produce tumor responses at sites of NHL that relapse after RIT with acceptable toxicity.

Phase III Intergroup Study of Fludarabine Phosphate Compared With Cyclophosphamide, Vincristine, and Prednisone Chemotherapy in Newly Diagnosed Patients With Stage III and IV Low-Grade Malignant Non-Hodgkin's Lymphoma

To compare the efficacy and safety of fludarabine phosphate with cyclophosphamide, vincristine, and prednisone (CVP) in 381 previously untreated, advanced-stage, low-grade (lg) non-Hodgkin's lymphoma (NHL) patients in a phase III, multicenter study. Between 1993 and 1997, patients were randomly assigned to treatment with either fludarabine (25 mg/m2 intravenously [IV] daily for 5 days every 4 weeks) or CVP (cyclophosphamide 750 mg/m2 IV on day 1; vincristine, 1.4 mg/m2 IV on day 1; and prednisone, 40 mg/m2 orally on days 1 through 5 every 4 weeks). Results Overall response (OR) rates were significantly improved in the fludarabine arm versus the CVP arm, both for the intent-to-treat (ITT) population and assessable patients (P < .001). Complete response (CR) rates in the ITT population were also higher after fludarabine treatment. The CR rate was 38.6% for fludarabine compared with 15.0% for CVP. There were no statistically significant differences in time to progression (TTP), time to treatment failure (TTF), and overall survival (OS) between treatment groups. WHO grades 3 and 4 hematologic adverse events were more common in the fludarabine arm. However, concerning the higher incidence of granulocytopenia, this did not translate to more infections in fludarabine-treated patients. Newly diagnosed lgNHL patients who received fludarabine achieved higher OR and CR rates compared with CVP-treated patients. No differences in TTP, TTF, and OS were noted. Fludarabine is a highly active single agent in lgNHL. Combination therapies incorporating fludarabine are now being further evaluated as first-line therapy in follicular NHL.

Rituximab

Rituximab (MabThera, Rituxan) is an anti-CD20 monoclonal antibody that induces lysis and apoptosis of normal and malignant human B cells, and sensitises malignant B cells to the cytotoxic effect of chemotherapy. In phase III trials in patients with indolent or aggressive B-cell non-Hodgkin's lymphoma (NHL), intravenous rituximab in combination with chemotherapy was more effective as first- or second-line therapy than chemotherapy alone in providing tumour remission and patient survival. Likewise, in patients with chronic lymphocytic leukaemia (CLL), rituximab in combination with chemotherapy appeared more effective than chemotherapy alone as either first- or second-line treatment. In addition, rituximab maintenance therapy was shown to significantly prolong tumour remission and patient survival in patients with indolent B-cell NHL or CLL. The combination of rituximab with cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) was cost effective as first-line therapy for advanced-stage diffuse large B-cell NHL compared with CHOP alone. Rituximab, either alone or in combination with chemotherapy, was generally well tolerated in patients with NHL or CLL. Overall, rituximab in combination with chemotherapy, is a valuable option for first- and second-line therapy in patients with advanced-stage indolent or aggressive B-cell NHL, and possibly those with B-cell CLL, and is included in current treatment guidelines for these indications. The drug is also potentially useful as maintenance therapy in patients with indolent B-cell NHL or CLL.

Spotlight on Rituximab in Non-Hodgkin Lymphoma and Chronic Lymphocytic Leukemia1

Rituximab (MabThera, Rituxan is an anti-CD20 monoclonal antibody that induces lysis and apoptosis of normal and malignant human B cells, and sensitizes malignant B cells to the cytotoxic effect of chemotherapy. In phase III trials in patients with indolent or aggressive B-cell non-Hodgkin lymphoma (NHL), intravenous rituximab in combination with chemotherapy was more effective as first- or second-line therapy than chemotherapy alone in terms of tumor remission and patient survival. Likewise, in patients with chronic lymphocytic leukemia (CLL), rituximab in combination with chemotherapy appeared more effective than chemotherapy alone as either first- or second-line treatment. In addition, rituximab maintenance therapy was shown to significantly prolong tumor remission and patient survival in patients with indolent B-cell NHL or CLL. The combination of rituximab with cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) was cost effective as first-line therapy for advanced-stage diffuse large B-cell NHL compared with CHOP alone. Rituximab, either alone or in combination with chemotherapy, was generally well tolerated in patients with NHL or CLL. Overall, rituximab in combination with chemotherapy, is a valuable option for first- and second-line therapy in patients with advanced-stage indolent or aggressive B-cell NHL, and possibly those with B-cell CLL, and is included in current treatment guidelines for these indications. The drug is also potentially useful as maintenance therapy in patients with indolent B-cell NHL or CLL.

Mechanistic Rationale and Clinical Evidence for the Efficacy of Proteasome Inhibitors against Indolent and Mantle Cell Lymphomas

Recent advances in understanding the complex biology of the ubiquitin-proteasome pathway have led to the identification of many potentially 'drugable' targets within this pathway. One such inhibitor, bortezomib (formerly known as PS341), has proven to be an effective reversible inhibitor of the chymotryptic protease in the 26S proteasome. Proteasome inhibitors represent a new approach for the treatment of many forms of cancer, especially select hematological malignancies. The proteasome plays an important role in regulating the availability of different intracellular proteins. While only some of the consequences of inhibiting this activity are understood, a growing amount of data suggests that inhibition of the proteasome is associated with a remarkable panoply of different biological effects that include cell cycle arrest, apoptosis, changes in cell surface adhesion markers, and an increased sensitivity to standard chemotherapy and radiation therapy. Bortezomib was recently approved by the US FDA for the treatment of relapsed or refractory multiple myeloma. In addition, bortezomib has also shown encouraging results in the treatment of select types of non-Hodgkin lymphomas (NHLs). Ongoing phase II clinical trials in pretreated patients are exploring bortezomib in different histologies of NHLs and in combination with conventional chemotherapy. Preliminary data have shown interesting activity, especially in patients with follicular, marginal zone, and mantle cell lymphoma; in these populations, durable complete and partial remissions have been reported. The toxicity profile of this drug, coupled with its unusual mechanism of action, make it a potentially important agent warranting further preclinical and clinical attention. However, many unanswered questions remain regarding how best to employ bortezomib in the conventional treatment of lymphoma. The apparent lack of activity in different subtypes of lymphoma, such as small lymphocytic lymphoma/chronic lymphocytic leukemia and diffuse large B-cell lymphoma, as well as a lack of understanding about the best way to combine bortezomib with standard therapies for indolent NHLs, raises important questions regarding the mechanistic basis for its effects. We will undoubtedly need to understand these effects better in order to fully exploit the potential of this new class of drugs.

Survivin—An attractive target for RNAi in non-Hodgkin's lymphoma, Daudi cell line as a model

RNA interference (RNAi) has been widely used in tumor gene therapy, antivirus and gene drug selection. Survivin gene is highly expressed in non-Hodgkin's lymphoma (NHL) tissues and high malignancy Burkitt's lymphoma cell line-Daudi and it is regarded as a potential target of gene therapy for NHL. This study used a vector-based short hairpin RNA (shRNA) technique to explore the effect of RNAi-mediated survivin gene silencing on apoptosis and proliferation of Daudi cells. Recombinant plasmid survivin-shRNA was transfected into Daudi cells transiently and stably. The expression of survivin was detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The apoptosis of Daudi cells after transfection were evaluated by flow cytometry. After transfection of survivin-shRNA, the levels of survivin mRNA were significantly reduced by 64.20% (transient transfection) and 62.32% (stable transfection), respectively; The levels of survivin protein were significantly reduced by 63.50% (transient transfection) and 61.88% (stable transfection); compared with control-shRNA and PBS treated groups. Apoptosis of Daudi cells were significantly higher in the transfection group than in the control group, respectively 21.30 ± 2.96% (transient transfection) and 19.10 ± 2.15% (stable transfection). In conclusion, it was suggested that survivin could be an attractive target for new anti-cancer intervention of NHL and vector-based survivin-shRNA could effectively reduce the expression of survivin and induce cell apoptosis and growth inhibition of NHL cells.

High-Dose Myeloablative Zevalin Radioimmunotherapy with Tandem Stem-Cell Autografting Has Minimal Toxicity and Full Feasibility in an Outpatient Setting.

Background : High dose chemotherapy has an established, albeit limited role, in the management of non-Hodgkin lymphoma (NHL). In fact, because of its toxicity, in particular mucositis, neutropenia and thrombocytopenia, myeloablative regimens with autologous stem cell support can be safely delivered only to clinically fit and younger pts, require prolonged hospitalization, and have a restricted impact in the therapy of NHL. With the primary aim of widening the applicability of myeloablative regimens, we designed a pilot study using high-dose Zevalin with tandem stem-cell support in a prospective cohort of 13 refractory or relapsed NHL patients, unable to safely undergo a BEAM chemotherapy course.Methods : Prior to Zevalin, all pts received one cycle of high-dose cyclophosphamide plus rituximab, and one cycle of high-dose cytarabine and rituximab, at patient-adapted doses. Hematopoietic stem cells were harvested from the peripheral blood during the post-cyclophosphamide and/or the post-cytarabine recovery phase, and tested for MRD. Zevalin was administered at twice the MTD (0.8 mCi/kg), and followed by tandem autografting of a small amount of CD34+ (0.8–2 x 106/kg) on day +7, and an optimal amount (>=5 x 106 CD34+ cells/kg) on day +14, respectively. The former reinfusion (supportive autografting) was done still in the presence of potentially myelotoxic doses of circulating radioactivity, and its aim was solely to achieve an immediate albeit transient hematopoietic recovery. At the time of the second reinfusion (reconstituting autografting) the calculated radiation dose to the reinfused stem cells was less than 5 cGy, in order to ensure a complete and long lasting hematopoietic reconstitution.Results: From July 2004 through March 2005, 13 overall NHL patients entered into the study (DLBCL 5, follicular 3, mantle cell 2, marginal zone nodal 1, small lymphocytic 1, lymphoplasmacytoid 1). Median age was 60 yrs (29–69). The number of prior chemotherapy regimes was 1 (4 pts), 2 (5 pts), 3 (3 pts) and 4 (1 pt), respectively. Neutropenia grade 1 or higher was documented in all but 1 patient, and lasted a median of 7 days (1–15), while grade 4 neutropenia was observed in 8 pts, in which lasted a median of 3.5 days only (1–10). Grade >=1 thrombocytopenia was observed in all patients (median 16 days, range 7–30), while grade 4 thrombocytopenia occurred in 10 pts for a median duration (in the thrombocytopenic pts) of 6 days (1–13). Eight patients required platelet transfusions (median 2, range 1–6), and 9 pts received 1 RBC transfusion each. No extra-hematologic toxicity was observed, and all but 4 patients were cared for as outpatients. The 4 hospital admissions lasted 2 (2 pts) and 4 days (2 pts) respectively, and were required for FUO that rapidly resolved upon antibiotic administration. After a median follow-up of 6 months (1–11), 11 pts are alive, of which 10 in continuous CR.Conclusions : High-dose Zevalin with tandem stem-cell transplantation was minimally toxic in this heavily pretreated patient population, and fully applicable in an outpatient setting. Its administration at 0.8 mCi/kg (and possibly up to 1.2 mCi/kg) as a final consolidation step, warrants prospective comparison with conventional-dose regimens in a minimally selected NHL patient population.

Sodium Orthovanadate (NaV) Effects CD22 Expression, Intracellular Signaling, and Lymphomacidal Activity of the HB22.7 Anti-CD22 Monoclonal Antibody.

CD22 is a cell-surface molecule on most B-cells and B-cell non-Hodgkin's lymphoma (NHL). A panel of anti-CD22 monoclonal antibodies (mAb) were developed and characterized. Some anti-CD22 mAb bind CD22 but do not stimulate intracellular signaling. However, HB22.7 is an anti-CD22 mAb that binds a unique CD22-epitope, blocks ligand binding, and its binding initiates an intracellular signaling cascade. Sodium orthovanadate (NaV) is a non-specific phosphatase inhibitor whose effect on CD22-mediated signaling in NHL cells was tested with and without HB22.7. Compared to other B-cell receptors, the SHP-1 tyrosine phosphatase is disproportionately associated with the cytoplasmic domain of CD22; NaV inhibits SHP-1. The SHP-1/CD22 interaction presents an opportunity to manipulate CD22-mediated signaling and thus has implications for NHL therapy. Methods: Cultured human NHL cell lines were assessed by trypan blue exclusion for efficacy of treatment with varying concentrations of NaV and HB22.7. Flow cytometry was used to assess how pre-incubation of NHL-cells with NaV before treatment with HB22.7 effected the internalization of CD22 and its cell-surface concentration. Intracellular staining for phospho-p38 and activation of SAP kinase were assessed by flow cytometry. Finally, nude mice bearing Raji (human NHL) xenografts were treated with HB22.7 with and without repeated administrations of oral NaV. Tumor volume, response rate and survival were assessed over the 84-day study period. Results: NaV (1– 100 μM) caused a dose dependent killing of Ramos NHL cells in vitro. When HB22.7 (10 or 30 μg) was given along with NaV cell death was augmented. Flow cytometry of three NHL cell lines (DOHH-2, Karpas, and Ramos) demonstrated that pre-treatment with NaV resulted in less internalization, and more persistence of CD22 on the cell-surface after ligation with HB22.7 than did cells not pretreated with NaV. P38 is a known mediator of CD22 signals. When Ramos cells were pre-treated with NaV a substantial enhancement of phospho-p38 was detected when CD22 was ligated with HB22.7 as compared to either agent alone. Similarly, pretreatment of Ramos cells with NaV resulted in a substantial increase in HB22.7-mediated phosphorylation of SAP kinase as compared to the controls. Pretreatment with NaV did not affect IgM-mediated signals. Studies in nude mice bearing Raji NHL xenografts showed that the in vitro data had applicability to this murine model. Control (untreated) mice and those treated thrice weekly with NaV (150 μg) showed relentless tumor growth and no pattern of response. Mice treated with HB22.7 (2.1 mg, intravenously; 4 doses) had slowing tumor growth by day 21, then regression; there was a 50% complete response rate. Mice treated with the same doses of both NaV and HB22.7 had a more rapid anti-NHL response, less tumor growth, 80% complete responses and the best survival rate. No toxicity could be detected in mice due to NaV treatment. Conclusion: NaV can increase the expression and/or persistence of cell-surface CD22, effect signaling stimulated by HB22.7, and alter or enhance intracellular signaling pathways in ways beneficial to NHL cell killing without observed toxicity in the mouse model. Further experiments will refine the use of these agents in pre-clinical studies of NHL therapy.

Potential Impact of Pathologic Review on Therapy in Non-Hodgkin's Lymphoma (NHL): Analysis from the National Comprehensive Cancer Network (NCCN) NHL Outcomes Project.

Background: Optimal therapy of NHL is critically dependent upon an accurate pathologic diagnosis. High inter-rater reliability has been demonstrated among expert hematopathologists for common B cell NHLs using the WHO classification system. However, less is known about the accuracy and reliability of pathologic diagnosis of lymphoma in the routine care setting.Methods: We used data from the NCCN NHL Outcomes Project, a prospective cohort study collecting comprehensive clinical, treatment, and outcome data for patients seen at 5 participating centers (City of Hope, Dana-Farber, Fox Chase, MD Anderson, and Roswell Park), to compare pathologic diagnosis assigned at referring and NCCN centers. Newly diagnosed patients presenting between 7/00 and 12/04 with a final diagnosis of one of the following NHL subtypes were included in this analysis: follicular lymphoma (FL, grades 1,2,NOS), FL grade 3, marginal zone (MZL, extranodal, nodal, and splenic), small lymphocytic (SLL), diffuse large B-cell (DLBCL) and mantle cell (MCL). Diagnosis was considered concordant if the NCCN institution assigned the same WHO diagnosis as the referring institution. In order to assess the potential impact of diagnostic reclassification on treatment and outcomes, we defined 6 outside institution treatment-oriented categories: Indolent 1 (FL 1,2,NOS, all types of MZL and SLL), Indolent 2 (FL 3), Aggressive (i.e. DLBCL), MCL, Highly Aggressive (e.g. Burkitt or lymphoblastic lymphoma) and Other Cancer. NCCN diagnoses were classified into 4 treatment-oriented categories: Indolent 1, Indolent 2, DLBCL, and MCL. Treatment category was considered concordant if the diagnosis assigned by the referring institution and the NCCN institution mapped to the same treatment category.Results: Of 928 patients eligible for this analysis, 741 had specimens reviewed at both a referring and NCCN institution. Among these patients, the final diagnosis was discordant for 9% (66/741, 95%CI [7%,11%]). The rates of discordance by histologic type (as assigned by the NCCN center) were: FL 6% (13/218), FL3 16%(5/31), MZL 8% (5/60), SLL 29% (9/31), DLBCL 8% (25/304) and MCL 9% (9/97). In SLL, almost half of discordance was due to use of non-WHO diagnoses (n=4). Of the 66 discordant cases, 51 (7% of total, 95% CI [5%, 9%]) were ultimately diagnosed with a histology that mapped to a discordant treatment category. This included 15 patients (29%) whose final diagnosis was a more aggressive histology (DLBCL), potentially curable if treated with appropriate first-line therapy.Conclusions: We found that the vast majority of patients with common NHL were classified accurately by referring centers. For 9% of patients, however, review by an expert hematopathologist resulted in an alternative diagnosis, and for the majority a change in expected treatment potentially impacting therapeutic outcome.Treatment CategoryNCCNReferring InstitutionIndolent 1 (N=309)Indolent 2 (N=31)DLBCL (N=304)MCL (N=97)Indolent 194%10%4%7%Indolent 21%84%1%0Aggressive1%6%93%1%MCL2%0091%Highly Aggressive1%01%1%Other Cancer1%0<1%0

Secondary Neoplasms Subsequent to Berlin-Frankfurt-Muenster (BFM) Therapy of Non-Hodgkin Lymphoma of Childhood: Significantly Higher Risk for Patients with Lymphoblastic Lymphoma Compared to Other NHL-Subtypes.

We evaluated the rate and type of second malignant neoplasms (SMN) after BFM treatment of children with Non-Hodgkin lymphoma (NHL). Between January 1981 and February 2003 2451 patients (pts) <15 years (y) of age at diagnosis were enrolled into the subsequent trials NHL-BFM 81, 83, 86, 90, and 95. Pts with lymphoblastic lymphoma (LBL) (n=547) or non-anaplastic peripheral T-cell lymphoma (n=97) received acute lymphoblastic leukemia (ALL)-type therapy including cumulative doses of cyclophosphamide (max. 3g/m2), daunorubicine/doxorubicin (max. 280mg/m2), but, except few pts, no etoposide. Prophylactic cranial radiotherapy (CRT) was given in stage III/IV (omitted in NHL-BFM95). Pts with mature B-cell neoplasms (n=1597), or anaplastic large cell lymphoma (n=210) received B-type therapy, consisting of 2–8, 5-day courses including cumulative doses of cyclophosphamide (max. 7g/m2), ifosfamide (max. 8g/m2), doxorubicine max. 150mg/m2 (in trial 81 max. 200mg/m2), and etoposide (max. 1.4g/m2). CRT was omitted since trial NHL-BFM86. With a median follow-up of 6.9 (range 0.2–22.6) years the probability of survival at 15 y was 83+1%. By June 2005, 47 SMN were documented, including 16 acute myeloid leukemias/myelodysplastic syndromes (AML/MDS), 11 NHL, 2 ALL, 1 Hodgkin's lymphoma, 7 brain tumors, and 10 other SMN. All SMN occurred in first remission after a median time of 2.9 (range: 0.4–12.3) years from diagnosis of NHL. The cumulative incidence of SMN at 15 y was 4.0% (95% confidence interval [CI]: 1.9%–6.1%) for the total group. The cumulative incidence of SMN was significantly higher among pts with LBL receiving ALL-type therapy (6.3% at 15 y [95%CI: 2.4%–10.3%] (13 AML/MDS, 2 NHL, 3 brain tumors, and 3 other SMN), as compared to pts with other NHL-entities receiving B-type therapy (3.4% at 15 y [95% CI: 0.5–6.4%] (3 AML/MDS, 9 NHL, 2 ALL, 1 Hodgkin's lymphoma, 4 brain tumors, and 7 other SMN), p=0.002. There was no significant difference of cumulative incidence of SMN in pts who received CRT compared to pts not receiving CRT. However, 5 of 7 pts, who developed brain tumor, received CRT of 12–24 Gy. Also, there was no significant correlation between the incidence of SMN and the cumulative doses of drugs, except for anthracyclines. For pts receiving a cumulative dose of anthracyclines of >160mg/m2 (almost exclusively pts with LBL receiving ALL-type therapy) the cumulative risk for SMN at 15 y was 6.5% (95% CI: 1.5-11.5%), as compared to 2.0% (95% CI: 1.1–2.9%) for pts with lower doses, p=0.007. Exposure to etoposide was not a risk factor for secondary AML/MDS (11 of 16 pts with sec. AML/MDS did not receive etoposide). In a Cox regression analysis only diagnosis of LBL remained a significant risk factor for SMN (RR 2.5, 95% CI 1.4–4.4). Our analysis revealed a cumulative risk for SMN of 4% at 15 y after successful treatment of childhood NHL. The cumulative incidence of SMN was significantly higher in LBL-pts than in other pts. AML/MDS were the most frequent SMN following LBL while second lymphoid malignancies were the most frequent SMN following non-LBL.