Witzig, Thomas E.

Abstract Abstract 281 Elevated serum levels of soluble interleukin-2 receptor alpha (sIL-2Ra) have been shown to be a poor prognostic factor in various malignancies and sIL-2Ra levels have generally correlated with tumor bulk and advanced stage of disease. In a clinical trial of single agent rituximab in previously untreated follicular lymphoma (FL) patients, we found that sIL-2Ra levels were elevated compared to controls and increased sIL-2Ra levels prior to treatment were associated with a poor outcome. The mean sIL-2Ra level (+/− standard deviation) in untreated FL patients (n=32) was 2.13 ng/ml (+/− 1.81) and 1.09 ng/ml (+/− 1.53) for normal controls (n=16). The time to progression for previously untreated FL patients after 4 doses of rituximab was 12 months for patients with sIL-2Ra levels above the mean compared to 40 months for patients with low sIL-2Ra levels (p=0.003) confirming that elevated sIL-2Ra is a poor prognostic factor in FL. To explore the mechanism by which sIL-2Ra may contribute to a poor prognosis, we determined the source of sIL-2Ra and whether sIL-2Ra facilitates interleukin-2 (IL-2) signaling. Based on our previous work showing that IL-2 induces Foxp3 expression, we particularly evaluated whether sIL-2Ra may bind IL-2 and contribute to impaired anti-tumor immunity by promoting Treg cell development. We found that sIL-2Ra could be detected by ELISA in the culture medium of IL-2Ra-expressing cell lines such as SuDHL1 and Karpas299 and that activation of these cells with PMA/Ionomycin significantly increased sIL-2Ra levels in the culture medium. Production of sIL-2Ra was associated with an attenuation of surface IL-2Ra expression on SuDHL1 and Karpas299 cells suggesting that surface IL-2Ra is the source of sIL-2Ra. Next, using histo-tagged sIL-2R, we tested whether the complex between sIL-2Ra and IL-2 was able to bind to IL-2R on the malignant cell. We found that sIL-2Ra bound IL-2 and that the complex could be detected on the surface of IL-2R-expressing cells. We then compared the signaling of the sIL-2Ra/IL-2 complex to that of IL-2 alone. As expected, we found that IL-2 stimulated cell proliferation, enhanced STAT5 phosphorylation and upregulated Foxp3 expression in CD4+ T cells in a dose-dependent manner. Administration of an anti-IL-2 antibody attenuated these effects. In contrast, sIL-2Ra alone had little effect on cell proliferation, STAT5 phosphorylation and Foxp3 expression. However, the addition of sIL-2Ra to IL-2 prior to incubation with IL-2R-expressing cells enhanced IL-2-mediated phosphorylation of STAT5 and Foxp3 expression in CD4+ T cells. Compared to IL-2 alone, treatment with sIL-2Ra (25ng/ml) and IL-2 (25ng/ml) increases the number of CD4+ T cells with phosphorylation of STAT5 from 7.9% to 17.4%. Taken together, these results indicate that sIL-2R plays an active biologic role in FL by binding IL-2 and promoting IL-2 signaling rather than depleting IL-2 and blocking its function. Given the findings that IL-2 strongly induces Foxp3 expression and promotes Treg cell development and that Treg cells suppress anti-tumor immunity, we conclude that elevated serum levels of sIL-2Ra facilitate IL-2 signaling and thereby contribute to a poor prognosis in FL. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2868 Poster Board II-844 Background and Objective: In newly diagnosed multiple myeloma (MM), the combination of lenalidomide plus high-dose dexamethasone (RD) is superior to high-dose dexamethasone (Zonder JA et al, Blood 2007;110:77). Preliminary results show that lenalidomide plus low-dose dexamethasone (Rd) has better 2-year overall survival (OS) compared with RD (Rajkumar SV et al, J Clin Oncol 2008;26:8504). The addition of clarithromycin (Biaxin) to lenalidomide and low-dose dexamethasone (BiRd) has been investigated in a phase II study, demonstrating a high response rate and 2-year OS (Niesvizky R at al, Blood 2008;111:1101-1109). However, the additive value of clarithromycin is not known. No randomized trials have compared Rd versus BiRd, none are planned. The objective of this case–matched study was to compare the efficacy and the toxicity of BiRd vs Rd as initial therapy for newly diagnosed MM. Patients and methods: Data from 72 newly diagnosed MM patients treated at the New York Presbyterian Hospital–Cornell Medical Center, from December 2004 to December 2006, with BiRD, were analyzed. For comparison, an equal number of pair mates were selected among newly diagnosed patients seen at the Mayo Clinic who received Rd, from March 2005 to December 2008. Case matching was performed with respect to age, gender, and transplant status. Patients treated with BiRd received oral lenalidomide (25 mg/day, days 3-21 of cycle 1; days 1-21 of subsequent cycles); dexamethasone (40 mg days 1, 2, 3, 8, 15, 22 of cycle 1; days 1, 8, 15, and 22 of each subsequent cycle); clarithromycin (500 mg twice daily, from day 2 of cycle 1). Patients treated with Rd received oral lenalidomide (25 mg/day, days 1-21) plus low-dose dexamethasone (40 mg days 1, 8, 15, 22). In both groups patients were allowed to discontinue treatment to pursue transplant, but treatment until progression, relapse or unacceptable toxicity was permitted at the physician's discretion. Outcome was analyzed on an intention-to-treat basis. The Chi-square or the rank sum tests were used to compare variables. Time-to-event analysis was performed using the Kaplan-Meier method; comparisons were determined by the log-rank test and the Cox proportional hazards model. Results: Median duration of treatment was 11.8 months in the BiRd group vs 6 months in the Rd group. On intention-to-treat analysis, complete response was significantly higher with BiRd compared to Rd (45.8% vs 13.9%, respectively, p<0.001); similarly very good partial response (VGPR) or better was higher with BiRd (73.6% vs 33.3%, p<0.001). Rates of VGPR or better, after 6 months and 1 year of therapy, were significantly higher in BiRd patients. Both time-to-progression (TTP) (median 48.3 months vs 27.5 months; HR 0.51; 95% CI 0.25-1.06; p=0.071) and PFS (median 48.3 months vs 27.5 months, HR 0.50; 95% CI 0.25-0.98; p=0.044) were higher with BiRd. Median time to next treatment (TTNT) was not reached in BiRd group compared to 29.9 months in Rd patients (HR 0.37 95% CI 0.20-0.66, p<0.001). There was a trend toward better OS with BiRd, (3-year OS: 89.7% vs 73.0%), but the difference was not statistically significant (HR 0.48; 95% CI 0.17-1.37; p=0.170). Thirty-two patients in each group received transplant; median time to transplant was longer in BiRd group (13.5 vs 5.9 months). Results of survival, for patients who received transplant and patients who did not, were similar to those of the total population. On subset analysis, among patients presenting with International Staging System (ISS) stage I/II at diagnosis, TTP, PFS and TTNT were significantly longer in BiRd patients; no significant differences were found among patients with ISS stage III. Main grade 3-4 toxicities of BiRd were hematological, in particular thrombocytopenia (23.6% vs 8.3%, p=0.012); rate of neutropenia was similar between the 2 groups (19.4% vs 16.7%, p=0.665). Infections (16.7% vs 9.7%, p=0.218) and dermatological toxicity (12.5% vs 4.2%, p=0.129) were higher in patients who received Rd. Rate of venous thromboembolism was similar in the 2 groups (9.7% vs 12.5%, respectively in Rd and BiRd patients, p=0.596). Conclusion: Results of this case-control analysis suggest superiority of BiRd in terms of response rate and survival, compared with Rd, and suggest that there may be a significant additive value when clarithromycin is added to Rd. These data indicate the need for randomized prospective phase III studies to evaluate BiRd versus Rd in the treatment of MM. Disclosures: Off Label Use: research drug in combination to standard of care. Kumar:celgene: Research Funding; millenium: Research Funding; bayer: Research Funding; novartis: Research Funding; genzyme: Research Funding. Mark:celgene: Research Funding. Dispenzieri:celgene: Research Funding. Gertz:celgene: Honoraria; genzime: Honoraria; millenium: Honoraria; amgen: Honoraria. Leonard:celgene: Consultancy. Lacy:celgene: Research Funding. Witzig:celgene: Research Funding. Fonseca:medtronic: Consultancy; genzyme: Consultancy; celgene: Consultancy; amgen: Consultancy; BMS: Consultancy; otsuka: Consultancy. Greipp:celgene: Research Funding. Niesvizky:celgene: Research Funding, Speakers Bureau; Millenium: Research Funding, Speakers Bureau; Proteolix: Research Funding, data monitoring committee.

Waldenström macroglobulinemia (WM) is a B cell disorder with a highly variable clinical outcome, where some patients remain asymptomatic, while others have significant symptoms and require therapeutic intervention. Clinical symptoms include infiltration of lymphoplasmacytic cells into the bone marrow, production of a monoclonal IgM protein, anemia, lymphadenopathy, and serum hyperviscosity. Despite the introduction of multiple chemotherapeutic regimens over the past several decades, WM remains an incurable disease. 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) and its methyl ester derivative (CDDO-Me) and imidazolide derivative (CDDO-Im) are synthetic triterpenoids derived from oleanolic acid. These compounds have been shown to induce apoptosis of several tumor cell types including breast cancer, lung cancer, ovarian cancer, melanoma, osteosarcoma, leukemia, and multiple myeloma cells. The goal of this study was to evaluate the potential role of synthetic triterpenoids in WM. Preliminary studies on malignant B cells indicated that CDDO-Im induced the greatest amount of cell death and we therefore used this derivative of CDDO for our studies. CD19+ CD138+ cells from bone marrow biopsy specimens obtained from WM patients were isolated by positive selection and were treated with varying concentrations of CDDO-Im (62.5 nM to 750 nM ) and cell viability was determined after 24 hours (n=3). Compared to the nil control 47% of the malignant cells remained viable at a CDDO-Im concentration of 62.5 nM and only 11% remained viable at 125 nM CDDO-Im. To determine if CDDO-Im had specific toxic effects on non-malignant cells, we cultured CD19- CD138- cells from WM patient bone marrows with CDDO-Im and found that non-malignant cells were less sensitive to the drug, 80% being viable at 62.5 nM and 65% being viable at 125 nM. Similarly, we found that normal peripheral blood B cells and CD19+ CD138+ bone marrow B cells from healthy donors were less sensitive to CDDO-Im. Compared to the nil control 93% of the CD19+ CD138+ bone marrow B cells and 70% of the peripheral blood B cells remained viable at a CDDO-Im concentration of 62.5 nM and 95% and 68% remained viable at 125 nM CDDO-Im respectively. We next examined the effect of CDDO-Im on WM cell proliferation and found that CDDO-Im inhibited cell proliferation in a dose-dependent manner. Similar to the viability assays, there was a differential effect of CDDO-Im on malignant and non-malignant cells. Compared to the nil control, at 125 nM, there was a complete inhibition of malignant cell growth, while approximately 40% of the non-malignant cells remained proliferative. To determine the mechanism of cell death, CD19+ CD138+ cells were cultured in the presence or absence of various doses of CDDO-Im for 6 hours and cell lysates were examined for cleavage of PARP. There was evidence of PARP cleavage in a dose-dependent manner, suggesting that CDDO-Im induced malignant cell death occurs through a caspase-dependent mechanism. In summary, the synthetic triterpenoid CDDO-Im decreased the viability of WM B cells in a dose-dependent manner, and CDDO-Im had a greater effect on the viability of the malignant cells compared to non-malignant cells from the same WM patients. CDDO-Im also inhibited malignant cell growth in a dose-dependent manner and the mechanism of CDDO-Im mediated cell death appears to be a caspase-mediated event. Overall, our data indicate that CDDO-Im may have potential efficacy in WM patients.

Abstract Background: Peripheral T-cell non-Hodgkin’s lymphomas (PTCL) account for approximately 10% of all non-Hodgkin’s lymphomas in the U.S. and Europe. PTCL, unspecified (PTCL-U), as classified by the WHO, is the most common subtype. The ability of high-dose therapy (HDT) and stem cell transplantation (SCT) to improve patient outcome was evaluated in the current study. Methods: All patients with PTCL-U evaluated at our institution who had tissue specimens available for review from January, 1994 to December, 2005 were identified and both clinical characteristics and treatment course obtained from the medical record. Results: 89 patients with PTCL-U were identified, with a median follow up of 13 months (range 1 month–12 years). The median age at diagnosis was 56 years (range 24–90). The IPI was low, intermediate or high in 34%, 44% and 22% of patients, respectively. On univariate analysis, age >60 (p<.0001), ECOG performance status >1 (p<.0001), LDH greater than normal (p=.004), Ann Arbor Stage III/IV (p=.01) and the presence of B symptoms (p=.0004) were associated with a poorer overall survival, as was an intermediate or high IPI (p<.0001). In contrast, on multivariate analysis only age >60 (p=.0006) and ECOG performance status >1 (p=.007) were independently associated with poorer overall survival. Fifty-seven percent of patients were treated initially with an anthracycline-based regimen, most commonly CHOP (53%). Patients with a low (0–1), intermediate (2–3) or high (4–5) IPI experienced a 61%, 29% and 18% 5-year overall survival, respectively. Fourteen patients (16%) received HDT and autologous (n=12) or allogeneic (n=2) SCT, either at the time of a first partial or complete response (n=11) or at the time of first relapse (n=3). The median survival in those patients treated initially with an anthracycline-based regimen alone was 11 months (95% CI 0.6–1.6 years). Improvement in both 5-year overall (75% vs 24%, HR=5.6, 95% CI 2.0–23.4, p=.0004) and disease-free (60% vs 26%, HR=3.2, 95% CI 1.3–9.3, p=.008) survival was observed in patients who received HDT/SCT, as compared to patients treated with anthracycline-based therapy alone. Furthermore, improvement in both 5-year overall (80% vs 26%, HR=5.8, 95% CI 1.7–35.7, p=.002) and disease free (72% vs 26%, HR=3.8, 95% CI 1.3–15.8, p=.009) survival was observed in patients who received HDT/SCT at the time of a first partial or complete response when compared to patients who received anthracycline-based therapy. When patients who failed to achieve a first partial or complete response were excluded from the analysis, improved 5-year overall (80% vs 57%, HR=0.5, 95% CI 0.1–2.1, p=.4) and disease-free (72% vs 53%, HR=0.7, 95% CI 0.1–2.3, p=.5) survival were observed in patients who received upfront HDT/SCT, although this failed to reach statistical significance. The improved overall and disease free survival observed in patients receiving HDT/SCT, either at the time of a first response or at the time of relapse, remained significant when accounting for differences in the IPI. In conclusion, selected patients with PTCL-U may benefit from treatment with HDT/SCT following a first response to conventional anthracycline-based chemotherapy.

Abstract Introduction Refractory pleural effusions present a challenging management problem and are associated with a poor prognosis in patients with primary systemic amyloidosis (AL). Vascular endothelial growth factor (VEGF) has been implicated to play a role in both malignant and non-malignant pleural effusions through its ability to regulate vascular permeability. VEGF can induce morphologic changes in vascular endothelial cells, regulate gene expression, stimulate cell proliferation and migration, and inhibit apoptosis. We report a series of four patients with AL who presented with bilateral pleural effusions that were refractory to diuretic therapy. After treatment with bevacizumab, an anti-VEGF antibody, three of the four patients had improvement in their pleural effusions, peripheral edema, and functional status. Methods A retrospective chart review was conducted four patients with AL and refractory pleural effusions who received bevacizumab. Three of the patients had documented renal amyloid by biopsy and two had suspected cardiac involvement. Serum creatinine ranged from 1.3–3.8 mg/dL (mean=2.15 mg/dL), 24 hour urine protein ranged from 190 mg – 12.5 grams per 24 hours (mean=6.1 grams). Bevacizumab 5 mg/kg was administered for 1–4 cycles (mean=2). Results Three of the four patients had demonstrable improvement in their pleural effusions, peripheral edema and functional status after a single dose of bevacizumab. One patient had no objective response to treatment. The three responders experienced a 2–7 kg (mean= 4.6 kg) decrease in weight within two weeks after receiving bevacizumab. This effect was observed in patients who were re-treated. All three patients received concurrent diuretic therapy, however, they had not previously responded to diuretic therapy. Two weeks after the first dose of bevacizumab, the 24 hour urine protein of the patient who received four cycles of bevacizumab increased to 18.2 grams per 24 hours, however it returned to baseline (12.5 grams per 24 hours) one week later, and increased slightly to 13.3 grams per 24 hours after the fourth dose. A second patient also experienced an increase in 24 hour protein excretion (6.9 grams per 24 hours to 9.2 grams per 24 hours) after the first dose of bevacizumab. One patient experienced a deep vein thrombosis after the third cycle of bevacizumab. No hemorrhagic complications were observed. Conclusion Bevacizumab may represent a novel non-invasive management strategy for patients with systemic amyloidosis and diuretic-refractory pleural effusions, however, additional studies are needed to further define the role of bevacizumab in the management of this group of patients.

Abstract Background: Patients (pts) with aggressive NHL who relapse after conventional chemotherapy or stem cell transplant have a poor prognosis. Novel agents are needed for this important subset. Tipifarnib is an orally available methylquinolone derivative that is an inhibitor of the farnesyl transferase enzyme that transfers the farnesyl group from farnesyl pyrophosphate to specific polypeptides including small GTPases of the Ras, Rho-B, and Rheb families, as well as nuclear lamins and kinetochore proteins. Tipifarnib has documented activity in acute leukemia and produces stable disease in relapsed multiple myeloma. The goal of this study was to evaluate the activity of single-agent tipifarnib in patients with relapsed aggressive NHL in the context of a phase II trial. Methods: Pts with relapsed diffuse large, follicular grade III, or mantle cell lymphoma were eligible for this study. Pts were treated with oral tipifarnib 300 mg twice daily d1–21 q 28 days until progression. Pts were restaged after 2 cycles, 6 cycles, and every 3 cycles thereafter. Tumor was sampled prior to tipifarnib and at day 8 in selected patients with accessible tumors. These paired samples were assayed for accumulation of unprocessed HDJ-2 and prelamin A, two polypeptides that require farnesylation for processing. Results: Forty-two pts were accrued between March 2004 and June 2006. Thirty-seven pts had diffuse large cell, 1 follicular grade III, and 4 had mantle cell. Of the 38 patients evaluable for response, 7 (18%) had a partial response and 8 (21%) had stable disease. The 7 responders all had diffuse large cell histology. Median progression-free survival (PFS) for all pts was 2 months (range, 0–25+). The PFS of the 7 responders was noteworthy at 4.2, 4.9+, 6.7, 9.6, 14.9+, 21.6+, and 25.1+ months, respectively. The primary toxicity was myelosuppression and 33% of pts required dose reductions. The median dose received in all pts was 300 mg twice daily (mean 275, range, 100–400). The median dose received by the 7 responders was 200 mg twice daily (range, 100–400). The dose of 300 mg twice daily resulted in accumulation of unfarnesylated forms of one or both markers in all pts studied to date. Conclusion: Oral tipifarnib has antitumor activity as a single agent with an excellent toxicity profile in pts with relapsed/refractory aggressive lymphoma. When given in this dose and schedule, tipifarnib is well-tolerated and can be taken for months without cumulative toxicity. This drug warrants further investigation in combination with conventional chemotherapy and other novel agents for the treatment of this important subset of patients.

Abstract BACKGROUND: We have demonstrated the green tea extract epigallocatechin-3-gallate (EGCG) has anticancer activity in primary CLL B-cells (Lee, Blood 2004). After dissemination of our in vitro findings by the lay press, many patients with CLL and other low grade non-Hodgkin lymphomas (NHL) began using over the counter green tea extracts as an alternative treatment strategy. We recently reported a case series of 3 patients with CLL and 1 patient with follicular lymphoma who appeared to derive objective clinical benefit from such treatment (Shanafelt, Leukemia Research 2006). Based on these findings EGCG has entered clinical testing for treatment of CLL at Mayo Clinic. Here we explore the in vitro antitumor activity of EGCG against other types of non-Hodgkin lymphoma. METHODS: Five established human B-cell lymphoma cell lines (HT, DOHH2, KARPAS, Ramos, RL) and primary lymphoma cells from 7 patients with various B-cell NHL sub-types [DLBC, FL, SMZ (2), MCL, SLL(2)] were used to evaluate the in vitro sensitivity of human lymphoma cells to EGCG. Freshly isolated primary lymphoma cells harvested in suspension from lymph nodes/spleen were obtained from patients with NHL who provided written informed consent. All patients were untreated at the time of biopsy. Lymphoma cell lines and primary lymphoma cells (n=7) were cultured with increasing doses of purified EGCG (3.12–50 ug/ml) for 24–72 hrs. Viability was assessed by using annexin/PI staining by FACS analysis. RESULTS: EGCG-induced dose dependent cell death in both established human B-cell lymphoma cell lines (average LD50 at 24 hrs between 25–50 ug/mL) and primary NHL cells (average LD50 at 24 hrs between 25–50ug/mL). In contrast, EGCG had minimal effect on purified normal B-cells (n=3) at the highest doses tested (50 ug/mL). By immunoblotting, EGCG-induced death in primary cells and cell lines was associated with PARP cleavage, suggesting the agent induced apoptotic cell death. Despite this finding, EGCG had no effect on levels of MCL-1, XIAP, or Bcl-1 by either immunoblot or FACS analysis. Based on reports that EGCG induces cell death in some cancer cell types through generation of oxidative stress (Furukawa, 2003; Nakazato, 2005), we explored this mechanism in lymphoma cells. To determine whether reactive oxygen species (ROS) generation was necessary for EGCG-induced cell death, lymphoma cell lines were cultured with or without catalase (which catalyzes the conversion of hydrogen peroxide to water and oxygen) for 30 min prior to subsequent 24 hr EGCG exposure (50 and 100 mg/ml). Pre-treatment with catalase (100 U) provided dramatic protection against cell death in both primary NHL cells and NHL cell lines suggesting that EGCG-induced cell death in lymphoma cells is dependent on ROS generation (Fig. 1 shows an example for a patient with mantle cell lymphoma and a patient with splenic marginal zone lymphoma). CONCLUSION: EGCG has in vitro anti-tumor activity against a variety of B-cell NHLs. Given its known favorable toxicity profile in vivo, EGCG is an attractive agent for clinical testing in patients with indolent NHL who otherwise are currently being managed with observation. Figure Figure

Abstract Background: Lenalidomide (Revlimid®), an immunomodulatory drug (IMiD®), was recently approved in the US for treatment of relapsed/refractory multiple myeloma and myelodysplastic syndromes associated with a deletion 5q[31] cytogenetic abnormality. Lenalidomide also has demonstrated activity in chronic lymphocytic leukemia and cutaneous T-cell lymphoma while thalidomide, a less potent IMiD®, has activity in non-Hodgkin’s lymphoma (NHL) as both monotherapy and in combination with rituximab. Aim: To assess the safety and efficacy of lenalidomide monotherapy in subjects with relapsed/refractory indolent NHL. Methods: Subjects with relapsed/refractory indolent NHL following at least 1 prior treatment regimen with measurable disease are eligible. Subjects receive 25 mg lenalidomide orally once daily on Days 1–21 every 28 days and continue therapy for 52 weeks as tolerated or until disease progression. Response and progression are evaluated using the IWLRC methodology. Results: As of July 25, 2006, 23 subjects of a planned 40 have enrolled and received drug. Fifteen subjects are currently evaluable for tumor response. The median age of the 15 response-evaluable subjects is 64 (54–82) and 5 are female. Histology is small lymphocytic lymphoma [SLL] (n=8), follicular center lymphoma grades 1,2 [FL] (n=5) and nodal marginal B-cell lymphoma [NML] (n=2). Median time from diagnosis to lenalidomide monotherapy is 7.6 (3–14) years and median number of prior treatment regimens per subject is 3 (1–17). Two subjects (13%) exhibited an objective partial response (PR) at four months. Both responses, one FL and one NML, occurred in subjects with substantial tumor burden of 61.8 cm2 and 80.3 cm2, respectively. The 4-month time-to-response for these indolent NHL subjects is longer than the 2-month median time-to-response in aggressive NHL subjects (n=7) following lenalidomide monotherapy. Seven patients had stable disease (SD) for a tumor control rate (TCR) of 60% and 6 had progressive disease (PD). Grade 3 or 4 adverse events occurred in 9 (39%) subjects. These were predominantly Grade 3 hematological events (neutropenia, thrombocytopenia) with only 3 subjects (13%) experiencing a Grade 4 adverse reaction (all neutropenia). Conclusion: Early results of lenalidomide monotherapy in indolent NHL show evidence of activity with a safety profile similar to that established previously in other hematological malignancies.

Abstract Background: Atacicept (TACI-Ig) is a recombinant fusion protein that inhibits BLyS (B lymphocyte stimulator) and APRIL (a proliferation-inducing ligand), cytokines that are involved in B-cell homeostasis and immunoglobulin (Ig) expression and are overexpressed in B-cell malignancies. In vitro, atacicept decreases the survival of lymphoma cells and in vivo, atacicept decreases serum immunoglobulin levels. Based on its effects on B cells, atacicept may offer a novel treatment for B-cell malignancies. Methods: A Phase 1, open-label, dose-escalation study of atacicept in patients with relapsed or refractory B-cell lymphoma was performed. Atacicept was administered subcutaneously for 5 weeks in single weekly doses of 2, 4, 7, or 10 mg/kg to sequential patient cohorts. After 8 weeks, patients with responding or stable disease were eligible for treatment on an extension study at the dose previously received for up to 24 weeks or until disease progression. The primary study objective was evaluation of overall safety and the maximum tolerated dose. Pharmacokinetics and biomarkers were also investigated. Results: As of July 2006, 15 patients with relapsed and refractory diffuse large B-cell lymphoma (7), follicular lymphoma (4), small lymphocytic lymphoma/chronic lymphocytic leukemia (2), and mantle-cell lymphoma (2) received 2, 4, or 7 mg/kg atacicept (4 patients per dose cohort) or 10 mg/kg atacicept (3 patients). All patients were heavily pretreated (median number of previous treatments was 5, range 1–10) and 4 patients had previously received a stem cell transplant. All patients completed study treatment (5 doses), except 2 who withdrew due to disease progression after receiving 2 and 4 doses. Atacicept was well tolerated at all doses. The most common adverse events (AEs) that occurred in ≥20% of patients were fatigue (47%) and injection site bruising (20%). Three AEs with ≥ grade 3 severity were reported for 1 patient including pain in jaw, gastrointestinal hemorrhage, and sepsis; all were considered unrelated to atacicept. Four SAEs considered unrelated to atacicept were reported for 2 patients who withdrew due to disease progression. Pharmacokinetic results were nonlinear and consistent with observations in other indications. Five weekly doses produced low to moderate accumulation of free total atacicept and atacicept/BLyS complex in serum. IgA, IgG, and IgM concentrations decreased in a dose-related pattern with a mean decrease of 15–40% from baseline after 4 weeks of atacicept. Peripheral B-cell numbers were variable and were difficult to evaluate due to low B-cell values at baseline in the majority of patients. At the 8-week evaluation, no objective responses were observed. Two patients had stable disease (1 with mantle cell lymphoma and 1 with follicular lymphoma) at 8 weeks, entered the extension study, and received additional doses of atacicept with no safety or tolerability concerns. Both patients however later discontinued treatment due to disease progression. Conclusion: Atacicept at doses of up to 10 mg/kg was well tolerated and demonstrated biological activity by decreasing Ig concentrations in this heavily pretreated cohort of patients with refractory B-cell lymphoma, although tumor responses were not observed.

Abstract Background: The clonal plasma cells in multiple myeloma have relatively low rates of apoptosis. We have previously shown that the apoptotic rates of the plasma cells have prognostic value in these patients. However, several methods are used both for targeting the myeloma cells for study as well as for detection of apoptosis in these cells. It is not clear which method is optimal for studying apoptotic rates of plasma cells from primary patient samples. We examined two different ways of studying the plasma cells, namely CD138 magnetic bead based positive selection of myeloma cells (sorted) and gating on the plasma cells in whole bone marrow (unsorted) using CD38/CD45 expression patterns. Additionally, we compared two different methods of identifying apoptotic plasma cells. Methods: Paired samples processed by two different methods were studied. The bone marrow aspirate samples were initially processed using ACK lysis to remove the red cells. The samples were then studied using flow cytometry (unsorted sample) or subjected to a second step of magnetic bead based separation of myeloma cells based on CD138 expression (sorted sample). Each of the samples was studied using Annexin/PI and Apo 2.7 expression to identify the plasma cells undergoing apoptosis. Plasma cells were identified in the unsorted samples by virtue of their CD38/CD45 expression. Samples were studied at collection and after 48 hours of culture. Results: At collection, the median percentages of cells considered apoptotic by annexin/PI (Annexin pos, PI neg) were higher than that detected by the Apo 2.7 method (Apo 2.7 + cells), for both sorted (6.5% vs. 1.5%) and unsorted cells (26% vs. 10%). However, after 48 hours of culture the percentage of apoptotic cells detected using the Apo 2.7 method were higher than that by Annexin/PI for both sorted (22.5% vs. 14.5%) and unsorted cells (43.5% vs. 10%). When the 48 hr study was compared with the initial study, percent apoptotic cells detected by Apo 2.7 had increased at 48 hrs for both sorted and unsorted cells. However, the percentage of apoptotic cells by annexin/PI had decreased in the unsorted cells at 48 hrs compared to initial study. The sorted samples had an overall increase in apoptosis at 48 hours as measured by annexin/PI. We then looked at the percentage of apoptotic cells detected by either method on initial study and compared the sorted cells and unsorted cells. Using either method, the percentage of apoptotic cells was significantly less (P=0.01; paired t-test) among the sorted cells compared to the unsorted cells. Conclusion: Previous studies have shown that CD138 expression is lost from PCs as they enter apoptosis. Here we show in paired samples that the percentage of apoptotic myeloma cells significantly decreases following CD138 sorting. CD138 sorting should not be used when studying apoptosis of myeloma cells in patient samples. Use of unsorted cells also allows for examination of the effects of interventions on other cell populations as well. When serial studies of apoptosis are done with unsorted samples, we recommend using Apo 2.7 rather than Ann/PI, since the percentage of apoptotic cells by Ann/PI expression actually appear to decrease with time in culture, thus making it difficult to assess effects of intervention such as in drug treatment studies. We hypothesize that this is due to an artificially high level of Annexin positivity at the initial study that might be brought on by non-specific damage of the membranes secondary to cell processing.