John J. Densmore

Rituximab Infusion Promotes Rapid Complement Depletion and Acute CD20 Loss in Chronic Lymphocytic Leukemia

Complement plays an important role in the immunotherapeutic action of the anti-CD20 mAb rituximab, and therefore we investigated whether complement might be the limiting factor in rituximab therapy. Our in vitro studies indicate that at high cell densities, binding of rituximab to human CD20+ cells leads to loss of complement activity and consumption of component C2. Infusion of rituximab in chronic lymphocytic leukemia patients also depletes complement; sera of treated patients have reduced capacity to C3b opsonize and kill CD20+ cells unless supplemented with normal serum or component C2. Initiation of rituximab infusion in chronic lymphocytic leukemia patients leads to rapid clearance of CD20+ cells. However, substantial numbers of B cells, with significantly reduced levels of CD20, return to the bloodstream immediately after rituximab infusion. In addition, a mAb specific for the Fc region of rituximab does not bind to these recirculating cells, suggesting that the rituximab-opsonized cells were temporarily sequestered by the mononuclear phagocytic system, and then released back into the circulation after the rituximab-CD20 complexes were removed by phagocytic cells. Western blots provide additional evidence for this escape mechanism that appears to occur as a consequence of CD20 loss. Treatment paradigms to prevent this escape, such as use of engineered or alternative anti-CD20 mAbs, may allow for more effective immunotherapy of chronic lymphocytic leukemia.

Thrice-Weekly Low-Dose Rituximab Decreases CD20 Loss via Shaving and Promotes Enhanced Targeting in Chronic Lymphocytic Leukemia

Treatment of chronic lymphocytic leukemia (CLL) patients with standard dose infusion of rituximab (RTX), 375 mg/m2, induces clearance of malignant cells from peripheral blood after infusion of 30 mg of RTX. After completion of the full RTX infusion, substantial recrudescence of CLL cells occurs, and these cells have lost >90% of CD20. To gain insight into mechanism(s) of CD20 loss, we investigated the hypothesis that thrice-weekly low-dose RTX (20 or 60 mg/m2) treatment for CLL over 4 wk would preserve CD20 and enhance leukemic cell clearance. During initial infusions in all 12 patients, the first 30 mg of RTX promoted clearance of >75% leukemic cells. Four of six patients receiving 20 mg/m2 RTX retained ≥50% CD20, and additional RTX infusions promoted further cell clearance. However, four of six patients receiving 60 mg/m2 had CD20 levels <20% baseline 2 days after initial infusions, and additional RTX infusions were less effective, presumably due to epitope loss. Our results suggest that when a threshold RTX dose is exceeded, recrudesced RTX-opsonized cells are not cleared, due to saturation of the mononuclear phagocytic system, but instead are shaved of RTX-CD20 complexes by acceptor cells. Thrice-weekly low-dose RTX may promote enhanced clearance of circulating CLL cells by preserving CD20.

Perifosine Plus Bortezomib and Dexamethasone in Patients With Relapsed/Refractory Multiple Myeloma Previously Treated With Bortezomib: Results of a Multicenter Phase I/II Trial

PURPOSE Novel agents have improved patient outcome in relapsed or relapsed/refractory multiple myeloma (MM). Preclinical data show that the novel signal transduction modulator, perifosine, enhances the cytotoxicity of dexamethasone and bortezomib. Clinical data suggest that perifosine in combination with dexamethasone has activity in relapsed or relapsed/refractory MM. PATIENTS AND METHODS In a phase I/II study, perifosine in combination with bortezomib with or without dexamethasone was prospectively evaluated in 84 patients with relapsed or relapsed/refractory MM. All were heavily pretreated and bortezomib exposed; 73% were refractory to bortezomib, and 51% were refractory to bortezomib and dexamethasone. The dose selected for the phase II study was perifosine 50 mg/d plus bortezomib 1.3 mg/m(2), with the addition of low-dose dexamethasone at 20 mg if progression occurred on perifosine plus bortezomib alone. RESULTS An overall response rate (ORR; defined as minimal response or better) of 41% was demonstrated with this combination in 73 evaluable patients, including an ORR of 65% in bortezomib-relapsed patients and 32% in bortezomib-refractory patients. Therapy was generally well tolerated; toxicities, including gastrointestinal adverse effects and fatigue, proved manageable. No treatment-related mortality was seen. Median progression-free survival was 6.4 months, with a median overall survival of 25 months (22.5 months in bortezomib-refractory patients). CONCLUSION Perifosine-bortezomib ± dexamethasone demonstrated encouraging activity in heavily pretreated bortezomib-exposed patients with advanced MM. A phase III trial is underway comparing perifosine-bortezomib plus dexamethasone with bortezomib-dexamethasone in patients with relapsed/refractory MM previously treated with bortezomib.

Phase II Trial of Idiotype Vaccination in Previously Treated Patients With Indolent Non-Hodgkin’s Lymphoma Resulting in Durable Clinical Responses

PURPOSE To evaluate idiotype (Id) vaccination as a single agent in previously treated patients with indolent non-Hodgkins lymphoma. PATIENTS AND METHODS Patients underwent biopsy for determination of their lymphoma-specific Id sequence. Recombinant Id protein was manufactured and covalently linked with keyhole limpet hemocyanin (KLH) to generate Id/KLH. Patients received Id/KLH 1 mg on day 1 subcutaneously, with granulocyte-macrophage colony-stimulating factor 250 mug on days 1 to 4, monthly for 6 months. Booster injections were administered until progression. Both clinical and immune responses were evaluated. RESULTS Thirty-two previously treated patients received at least one injection of Id/KLH, and 31 were assessed for efficacy. Responses were observed in four patients (one complete response and three partial responses). Median time to onset of response was 5.9 months (range, 2.3 to 14.1 months). Median duration of response has not been reached but should be at least 19.4 months (range, 10.4 to 27.2+ months). Median time to progression is 13.5 months. The most common adverse events were mild to moderate injection site reactions. Six (67%) of nine patients tested demonstrated a cellular immune response, and four (20%) of 20 patients demonstrated an antibody response against their Id. CONCLUSION This trial demonstrates that Id/KLH alone can induce tumor regression and durable objective responses. Further study of Id/KLH is recommended in other settings where efficacy may be further enhanced as in first-line therapy or after cytoreductive therapy.

Biology and therapy of mantle cell lymphoma.

Purpose of review Mantle cell lymphoma is an aggressive non-Hodgkins lymphoma characterized by the t(11;14) chromosomal translocation and overexpression of cyclin D1. Constituting approximately 5 to 8% of all non-Hodgkins lymphomas, it carries the poorest prognosis among non-Hodgkins lymphoma subtypes. Standard and effective treatment approaches have yet to be established. Recent findings Several recent insights regarding the molecular pathogenesis and prognostic biomarkers have been realized by way of comparative genomic hybridization, cDNA microarray, and proteomic analysis. Clinical trials using chemotherapy plus rituximab have shown improved response rates, including complete remissions, but without cure in most cases, indicating a clear need for new treatment approaches. Novel therapies for relapsed disease using the proteasome inhibitor bortezomib, thalidomide plus rituximab, the cyclin inhibitor flavopiridol, or inhibitors of the mammalian target of rapamycin (mTOR) have shown encouraging clinical responses. Stem cell transplantation, including nonmyeloablative approaches, are being incorporated into therapeutic regimens and show promise in both the front-line and relapsed/refractory settings. Summary This review summarizes recent advances in the biology, pathogenesis, and therapy of mantle cell lymphoma and identifies ongoing areas of clinical investigation and new treatment approaches.

A voltage-gated calcium channel is linked to the antigen receptor in Jurkat T lymphocytes

Activation of T lymphocytes results in an increase in intracellular Ca2+ due in large part to influx of extracellular Ca2+. Using the patch clamp technique, an inward current in Jurkat T lymphocytes was observed upon depolarization from a holding potential of −90 mV but not from −60 mV. This whole‐cell current was insensitive to tetrodotoxin, carried by Ba2+, and blocked by Ni2+. Occupancy of the T lymphocyte antigen receptor increased the currents magnitude. These data suggest that antigen receptor‐induced Ca2+ entry in T lymphocytes may be mediated by a voltage‐regulated Ca channel.

Treatment strategies for relapsed and refractory aggressive non-Hodgkin's lymphoma

The aggressive non-Hodgkins lymphomas (NHL) are a clinically heterogeneous group of lymphomas with disparate responses to standard chemotherapy regimens. Aggressive NHL includes diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL) and peripheral T-cell lymphomas (PTCL), among others. Significant advances have been made in the last decade in the initial treatment of DLBCL and MCL, but the treatment of relapsed or refractory disease remains difficult. The addition of rituximab to the treatment of DLBCL and MCL has improved clinical outcomes and is now a critical component of initial therapy and treatment of relapsed disease. The PTCLs, not having a similar agent to significantly change the treatment approach to these diseases, remain a difficult therapeutic problem. This review examines recent advances in the treatment of relapsed or refractory aggressive NHL and discusses novel approaches currently under investigation.

Calmodulin regulation of Ca2+ entry in Jurkat T cells

We have previously proposed a role for calmodulin (CaM) in the regulation of initiation of Ca2+ entry in Jurkat T cells, as well as in the regulation of the current that mediates Ca2+ entry, IT. In this report, we provide evidence for the mechanism of CaM action. We have previously shown that activation-induced Ca2+ entry into Jurkat T cells is mediated by a current we have called IT. In the whole cell variation, but not the perforated patch variation, of the patch clamp technique, this current is short-lived (under 6 min) suggesting that the current is under the control of a diffusible component of the cytosol. Addition of CaM to the whole cell recording pipette solution maintained IT for up to 20 min, suggesting that CaM may be this diffusible component. Pharmacological inhibitors of CaM blocked the augmentation of IT normally induced by an activating stimulus. Cells electroporated in the presence of anti-CaM antibodies had reduced influx of extracellular Ca2+, with no change in release of Ca2+ from the internal stores. These observations suggest that T cell receptor engagement initiates Ca2+ influx by a pathway that likely includes CaM, which may in turn regulate IT. Influx of extracellular Ca2+ is required for cellular proliferation, and inhibition of CaM by pharmacological inhibitors reduced cellular proliferation. This same inhibition of proliferation was seen in cells electroporated with anti-CaM antibodies. This suggests that inhibition of CaM and/or IT may be a target for therapeutic inhibition of inappropriate T cell proliferation.