Citations

Abstract Abstract 3115 Background. Rituximab significantly improves the outcome of patients with B-cell non-Hodgkin lymphomas, but it does not completely eradicate residual B-cell populations in the bone marrow (BM) and lymph nodes (LN). The architecture and gene expression profile of LN stromal cells in diffuse large cell lymphoma correlates with outcome following treatment with R-CHOP (Lenz et al. 2008). Interestingly, multiple studies have demonstrated the importance of B-cells adhesion and pro-survival signaling mediated by integrin alfa-4-beta-1 (VLA-4/CD49d), which is constitutively expressed on malignant B-cells. Although several studies have demonstrated that adhesion to stromal cell cultures or ligand coated surfaces can protect malignant B-cells from apoptosis induced by chemotherapy drugs (cell adhesion-mediated drug resistance (CAM-DR); Dalton, 2002), a similar mechanism of resistance to rituximab has not, to our knowledge, been described. Hypothesis. In this study we tested the hypothesis that interactions with the microenvironment protect malignant B-cells from rituximab induced apoptosis, and that targeting these interactions with natalizumab, a humanized monoclonal anti-alfa-4 antibody approved for treatment of multiple sclerosis and Crohn's disease, can overcome this protection. Methods. Lymphoma B-cell lines were cultured on either a plastic surface, confluent HS-5 cells or a fibronectin (alfa-4-beta-1 ligand) coated surface. HS-5 is an immortalized human bone marrow stromal cell line and a well characterized model for a component of the BM microenvironment (Roecklein and Torok-Storb, 1995). Cells were treated with rituximab (10 ug/mL; Genentech), natalizumab (10ug/mL; Biogen IDEC), control IgG (10ug/mL, Sigma-Aldrich). Cell viability was measured by flow cytometry using Annexin V-FITC, propidium iodide in malignant B-cell population (anti-CD19 antibody, BD PharMingen). Results. We studied ten CD20-positive B-cell lymphoma cell lines and the CD20-positive MEC-1 cell line (derived from prolymfocytoid transformation of CLL) for rituximab induced apoptosis. The four most rituximab sensitive cell lines were used for further experiments (Karpas-422, Raji, DOHH2, SUDHL-4). Cell lines were co-cultured for 24hrs with confluent HS-5 stromal cells and subsequently treated for 24hrs with rituximab. The percentage of apoptotic cells was significantly lower (17-23% vs. 30–42%, p<0.05) for cells co-cultured with HS-5 cells compared to control cells cultivated under the same conditions on the plastic surface for all four cell lines. Similar results were obtained for several primary CLL samples (3-5% vs. 13–18%, p<0.05). We next examined if natalizumab could disrupt cell adhesion to fibronectin and overcome the stromal cell-mediated resistance to rituximab. Natalizumab reduced the number of lymphoma cells that adhered to fibronectin by 75–95% (p<0.05). Moreover, adherent cells cultivated on fibronectin coated surface completely lost the adhesion morphology after the addition of natalizumab to the cultivation media. The combination of natalizumab and rituximab versus rituximab alone increased by 26–32% (p<0.05) the number of apoptotic B-cells in co-culture with HS-5 for three of four rituximab-responding cell lines. Conclusion. We have shown that human bone marrow stromal cells (HS-5) protect lymphoma B cells from rituximab induced apoptosis suggesting existence of stromal cell adhesion-mediated antibody resistance (CAM-AR) analogous to CAM-DR. Targeting integrin alfa-4-beta-1 with natalizumab partially overcomes this cell adhesion-mediated resistance to rituximab. Research supported by P50CA97274-8 LYMPHOMA SPORE, Genentech, MSMT-MSM0021622430 and IGAMZCR NT11218-3/2010. Disclosures: Zent: Genentech: Research Funding; Genzyme: Research Funding; Novartis: Research Funding.

Abstract Abstract 3211 Poster Board III-148 Although autologous stem cell transplantation (ASCT) has become standard of care for many patients with hematologic malignancies, many patients fail to collect a minimum number of CD34 + stem cells to support high dose chemotherapy and ASCT. Recently, plerixafor, a CXCR4 antagonist, was FDA approved for use in combination with G-CSF for autologous peripheral blood stem cell mobilization in patients with NHL or Multiple Myeloma. In February 2009, we commenced a risk adapted approach to the utilization of plerixafor. The study was restricted to patients mobilized with GCSF alone and patients undergoing chemotherapy primed PBSC mobilization were excluded. Peripheral blood stem cell mobilization was commenced with G-CSF at 10 mcg/kg/day. On day 4, a peripheral blood (PB) CD34 count was measured. For patients whose PB CD34 was ≥10/μL, apheresis was commenced the following morning. For patients whose PB CD34 was <10/μL, it was measured again on day 5; if ≥10/μL then apheresis was commenced the following morning. If PB CD34 was <10/μL on day 5, plerixafor (0.24 mg/kg sc) was administered on the evening of day 5 and apheresis was commenced the following day (Group A). In addition, during apheresis, for patients whose collection yield was < 0.5 × 106 CD34/kg, in the absence of instrument failure or problems with the collection procedure, plerixafor was added (Group B). Morning administration of G-CSF and evening dosing of plerixafor continued daily until apheresis was complete. From February to July 2009, 174 mobilization attempts occurred; 27 with chemotherapy and 147 with cytokines alone. The 147 pts who underwent mobilization with cytokines alone are presented here. The underlying diagnosis was as follows: Myeloma 61 pts, NHL 54 pts, Amyloid 17 pts, Hodgkin 10 pts, POEMS 4 pts and 1 pt with a solid tumor. For the entire group the median number of CD34 cells collected was 5.5 × 106 CD34/kg (range 0.1-17). The median number of apheresis was 3 (range 1-12). 67 patients (46%) received plerixafor; 37 patients started plerixafor during mobilization (Group A) and 30 patients during collections due to a poor yield (Group B). 12 pts of the 37 received plerixafor on day 4 because of prior mobilization failure or high risk of mobilization failure and are included in Group A. Table 1 outlines the details of mobilization and collection by groups. By disease category, of the 61 patients with MM, 28 (46%) received plerixafor (8 Group A and 20 Group B). Median apheresis in all the MM pts was 2 (range 1-12) with a total of 6.8 × 106 CD34/kg (2.2-16.7). In the 54 NHL pts, 32 (59%) received plerixafor (24 Group A and 8 Group B). Median apheresis for all pts with NHL was 3 (range 1-7) with a total of 4.6 × 106 CD34/kg (range 0-11.4). Overall, only 7 of 147 (5%) mobilization attempts failed to achieve a minimum of 2 × 106 CD34/kg. This compares to a 22% failure rate prior to institution of this risk adapted approach. In conclusion, implementing this risk adapted approach allows poor mobilizers to be identified promptly and for plerixafor to be initiated during mobilization and collection, thereby reducing the number of mobilization failures. In patients who predictably would not have successful collection based on a PB CD34 <10/μL, addition of plerixafor results in a majority of patients achieving an adequate collection. This risk adapted approach may be more cost effective than reattempting mobilization after a prior failure or utilizing combination G-CSF and plerixafor for upfront mobilization. Table 1. Mobilization and Collection data. All patients N=147 Group A1 N=37 Group B2 N=30 No Plerixafor N=80 PB CD34 day 4 Median 11 0 7 19 Range 0-331 0-7 0-32 0-331 PB CD34 day 5 Median 10 4.5 12 15 Range 0-51 2-9 11-23 9-51 Apheresis Yield Median 5.5 4.4 6.0 6.2 Range 0.1-17 3.1-12.7 3.0-12.2 2-17 Number of Apheresis Median 3 3 5 2 Range 1-12 1-7 4-12 1-8 Days of Plerixafor Median n/a 3 2 n/a Rang 1-7 1-8 1 Group A – Plerixafor initiated prior to apheresis 2 Group B – Plerixafor initiated during apheresis Disclosures No relevant conflicts of interest to declare.

Abstract Background Immune editing is a major mechanism used by tumors to promote their survival. We have previously reported the presence of immunosuppressive monocytes (CD14+HLA-DR low/neg) in a number of cancers. Increased presence of these cells was associated with decreased treatment response and OS. Here we report an in vitro model to examine tumor monocyte cross talk and demonstrate that these monocytes may directly promote tumor cells’ resistance to chemotherapy. Method Monocytes from healthy donors were co-cultured with lymphoma cell lines (OCI-Ly1, OCI-Ly3, OCI-Ly7, OCI-Ly10, Su-DHL2, Su-DHL6, Jeko-1, Granta-519) with or without doxorubicin (DOX). Cultured cells were assessed for phenotype, viability, proliferation, and induced protein expression. Changes in vitro were confirmed in primary tissue by gene expression profile from 48 primary diffuse large B cell (DLBCL) lymphoma tumors. Results Monocytes converted CD14+HLA-DR+ to the CD14+HLA-DRlow/neg phenotype by co-culture with 5 of the 8 B cell lymphoma cell lines. The HLA-DR suppression was not due to the known regulatory effects of IL-10 nor was the effect seen in co-culture using B cells from healthy donors. Thus, the magnitude of DR loss is mediated by a yet undetermined mechanism. DOX incubation induced apoptosis in all of the cell lines. Co-culture with monocytes improved lymphoma cell survival in 3 of the 4 lymphoma cell lines. For example, untreated OCI-Ly3 had a 1.3±0.2 fold expansion that was reduced to 0.6±0.1 when treated with DOX (p<0.0001, n=13). However, when the cells were treated with DOX and co-cultured with monocytes the survival improved to 0.8±0.2 (p<0.0001, n=13). Having previously shown that CD14+HLA-DRlow/neg were capable of suppressing patient immunity, partially through arginase I, we determined if co-culture of monocytes with tumor cells increased arginase I expression in either monocytes or tumor cells. Not only did we see arginase I expression increases during co-culture(n=10, p<0.01), but surprisingly, the greatest increases were seen during co-culture and DOX treatment (n=10, p=0.01). To determine if our in vitro observations reflect human tumors, we used gene expression data obtained from 48 DLBCL tumors and found that increased CD14 gene expression was positively correlated with increased arginase I expression (n=48, p=0.02). Conclusions We have demonstrated that certain tumor cells induced phenotype changes in monocytes in an IL-10 independent and a tumor specific way. In those cells where tumors factors mediate monocyte phenotype, the monocytes can promote tumor cell resistance to cytotoxic killing by DOX, thus demonstrating a cross-talk between monocyte and tumor function. We found that in an environment of monocyte/tumor cross talk, arginase I expression is further increased in a cytotoxic environment. Together, this data demonstrates an active cross talk between monocytes and tumors resulting in multiple mechanisms of tumor resistance to chemotherapy. Disclosures: No relevant conflicts of interest to declare.

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 2705 Background: Due to similar cytomorphological features and treatment strategies, T lineage lymphomas (T-LBL) and lymphoblastic leukemias (T-ALL) are usually grouped together as a single entity. Traditionally, the difference between T-LBL and T-ALL has been based on percentage of blasts found in the bone marrow (BM): >25% BM blasts and/or presence of peripheral blood (PB) blasts consistent with T-ALL. Recent studies, based on immunophenotypic, cytogenetic and molecular differences, emphasize the clinical uniqueness between T-LBL and T-ALL and argue against grouping them as a single entity. Objectives: To perform a clinicopathologic and cytogenetic comparison between patients with T-LBL and T-ALL in order to identify differences between these disease groups. Methods: We utilized the Mayo Clinic Lymphoma database to identify consecutive adult patients (>18 years) diagnosed with T-cell leukemia and lymphoma from 1980 to 2010. The patients were sub-divided into T-LBL and T-ALL based on BM and PB blasts (absence of BM and PB blasts consistent with T-LBL). Clinicopathological and cytogenetic characteristics were retrospectively reviewed. Result: 50 patients met the criteria for T-cell leukemia/lymphoma (29-T-ALL, 21-T-LBL). 50% of patients in each group were females with median age at diagnosis of 32 years for T-LBL and 29 years for T-ALL. Characteristics associated with a statistically significant survival difference for the cohort included: white blood cell count at diagnosis (>100,000/cmm), platelet count (<50,000/cmm), LDH (>500 U/L), presence of BM blasts and an adverse karyotype (hypodiploidy and MLL rearrangements). ECOG performance status, number of lymph node groups involved, extranodal disease, CNS involvement and constitutional symptoms had no impact on survival. Furthermore, no overall survival difference between patients with T-LBL and T-ALL was identified (figure 1). Among patients with T-LBL, 11 (51%) patients with extranodal disease had shortened survival (p=0.02). CNS involvement was seen in 4 (19%) of T-LBL patients and 5 (17%) of T-ALL patients, and did not affect overall survival (p=0.8). 4 (19%) T-LBL patients and 1 (3%) T-ALL patient had pleural involvement at diagnosis. There were no morphologic, flow cytometric, or immunohistochemical differences noted between the two groups. Conclusion: In a large study from a single institution, we confirm no survival difference between T-LBL and T-ALL, which further provides ongoing support that from a therapeutic standpoint these groups can be viewed as a single entity. However, we hope to further clarify clinical differences by performing microarray and chemokine analysis on available tissue specimens. Disclosures: No relevant conflicts of interest to declare.

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 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 Abstract 1222 Poster Board I-244 Background Survival for patients with primary CNS lymphoma (PCNSL), in general, is poor with patients requiring frequent chemotherapy treatments or receiving whole-brain radiation therapy, which can potentially result in significant neurologic decline and dementia. Because of the improved survival of high risk patients with aggressive lymphoma undergoing autologous stem cell transplant (ASCT), we began ASCT for patients with PCNSL in first or later remission with chemotherapy sensitive disease. We now update on outcomes of patients who have had at least 100 day follow up post ASCT. Baseline characteristics Between June, 2000 and January, 2009, 22 patients underwent ASCT for PCNSL. Median age at transplant was 50 years old (range 26-67). Median number of prior treatments 1 (range 1-3). Median time from diagnosis to transplant was 7.2 months (range 2.9 to 75.8). Median International Extranodal Working Study Group Prognostic Score: 2 (range 0-3). Disease status at transplant: First CR 10 patients, later CR or PR 12 patients. Results Twenty-two patients underwent ASCT for PCNSL and have a minimum of 100 days follow-up. All patients received BEAM conditioning. Median follow up post-transplant was 30 months (range 3-107 months). Eight patients have relapsed at a median of 217 days (range 40-1349). Of the patients who relapsed, four have died of disease progression and the remaining four are alive after additional therapy. Median overall survival from diagnosis or transplant has not been reached. Median progression free survival from transplant was 70 months. Conclusions Although limited by patient selection and retrospective biases, this review suggests that ASCT for PCNSL demonstrates improved overall survival when compared to historical controls with similar PCNSL Prognostic Scores (2 year survival for patients from diagnosis with PS 2-3 was 48% in a prior published study). ASCT in first remission in patients with PCNSL appears promising and may limit the need for additional therapy which can be myelosuppressive or result in neurologic decline secondary to radiation therapy in patients who are appropriate candidates. Disclosures No relevant conflicts of interest to declare.