Citations

Abstract Abstract 3088 Background: Despite the use of modern immunochemotherapy (R-CHOP) regimens, almost 50% of patients with diffuse large-B-cell lymphoma (DLBCL) will relapse. Current prognostic models, most notably the International Prognostic Index, are comprised of patient and tumor characteristics and are unable to identify patients with less than a 50% chance of long-term survival. However, recent observations demonstrate that factors related to host adaptive immunity and the tumor microenvironment are powerful prognostic variables in non-Hodgkin lymphoma Methods: We retrospectively examined the absolute neutrophil count (ANC), monocyte count (AMC) and lymphocyte count (ALC), obtained from an automated complete blood count with differential, as prognostic variables in a cohort of 255 consecutive DLBCL patients that were uniformly treated with R-CHOP between 2000 and 2007 at a single institution. The primary study objective was to assess if ANC, AMC, and ALC at diagnosis were predictors of overall survival (OS) in DLBCL. Results: At diagnosis, the median ANC was 4720/uL (range 1190–17690), the median AMC was 610/uL (range 30–4040), and the median ALC was 1220/uL (range 140–5410). The median follow-up for these patients was 48 months. In the univariate analysis, each of these variables predicted OS as continuous variables. As dichotomized variables, an elevated ANC (≥5500/μL; hazard ratio 1.75, 95% confidence interval 1.14–2.60, p=0.01) and AMC (≥610/μL; hazard ratio 3.36, 95% confidence interval 2.10–5.59, p<0.0001) were each associated with inferior OS. In contrast, the presence of lymphopenia, defined as an ALC ≤1000/uL, was associated with inferior OS (hazard ratio 2.21, 95% confidence interval 1.43–3.39, p=0.0004). When components of the IPI were included on multivariate analysis only the AMC and ALC were independently significant prognostic factors for OS, with hazard ratios of 3.37 (95% confidence interval 2.05–5.74, p<0.0001) and 2.19 (95% confidence interval 1.38–3.44, p=0.0009), respectively. The dichotomized AMC and ALC generated the AMC/ALC prognostic index (PI) and stratified patients into 3 risk groups: very good (AMC <610/uL and ALC >1000/uL), good (AMC ≥610/uL or ALC ≤1000/uL), and poor-risk (AMC ≥610/uL and ALC ≤1000/uL) populations. For both the very good (n=79) and good-risk (n=134) groups median OS has not been reached with estimated 5-year overall survival of 88% and 69%, respectively. Median OS for poor-risk (n=42) patients was 1.7 years (95% confidence interval 1.1–2.7 years) with an estimated 5-year overall survival of 28% (p<0.0001). By comparison, the R-IPI was unable to identify a group of patients with a median survival less than 8 years. The estimated 5-year OS was 93%, 71% and 53% for very good, good and poor-risk patients, respectively. We sought to determine whether the AMC/ALC PI may provide additional prognostic information when combined with the R-IPI. To test this possibility, the 171 very good/good risk and 84 poor risk patients identified by the R-IPI were subsequently risk stratified using the AMC/ALC PI. Among R-IPI very good/good risk patients a subset of poor risk patients (n=21) with a median OS of 2.2 years (95% confidence interval 1.1–6.6 years) and 35% 5-year OS could be identified with the AMC/ALC PI. In contrast, 5-year OS ranged from 75%-88% among very good and good risk patients. Similarly, stratification of R-IPI poor risk patients by the AMC/ALC PI identified subsets of very good (n=19) and good risk (n=44) patients with median OS that had not been reached and 86% and 55% 5-year OS, respectively. High risk (n=21) patients had a median OS of 1.4 years (95% confidence interval 0.9–2.2 years) and an estimated 5-year OS of less than 25%. Conclusions: Measurement of AMC and ALC at diagnosis is widely applicable, cost effective, predicts OS, and identifies high-risk patients with DLBCL. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2802 Background: Redundancy of pro-survival signaling pathways promotes survival and drug resistance in lymphoid and plasma cell malignancies. In particular, the PI3K-Akt and the MEK-ERK pathways have been shown to play an important role in the proliferation and survival of these malignant cells induced by various cytokines in the tumor microenvironment. Sorafenib, a Raf kinase and VEGF receptor inhibitor, and everolimus, an mTOR inhibitor, have synergistic cytotoxicity in myeloma and lymphoma cells due to inhibition of multiple signaling pathways. Methods: We designed a Phase 1/2 clinical trial to identify the maximum tolerated doses of the two drugs used in combination and the efficacy of the combination. Patients (Pts) with relapsed myeloma or lymphoma were eligible for enrollment. Pts were required to have an absolute neutrophil count ≥1500 × 10(6)/L, a platelet count ≥75,000, and a serum creatinine 21.5 mg/dL. The study utilized the classic 3+3 design. Extensive pharmacokinetic studies were performed to better delineate potential drug interactions. Results: Twenty-six pts were accrued from August 2007 to February 2009. Four pts discontinued sorafenib during cycle 1 for various reasons (2 patient refusal, 1 unrelated medical condition and 1 physician discretion) and were excluded from MTD determination. An additional pt did not have measurable disease and was ineligible, leaving 19 pts with lymphoma (including 6 with Hodgkin lymphoma) and 2 with myeloma for phase I analysis. The pts had a median age of 56 years (range, 22, 69) and were heavily pretreated with a median of 4 prior therapies (range, 1–10). Eighteen (86%) had received a prior stem cell transplant. Four dose limiting toxicities were seen across all dose levels (Table). These included grade 3 vomiting (level 1), grade 4 thrombocytopenia (level 2 and 3, one each) and grade 2 hand and foot rash leading to treatment delay (level 3). Overall, 13 pts experienced a grade 3 or 4 hematologic toxicity. Grade 3 or 4 anemia, neutropenia, and thrombocytopenia occurred in 19%, 43%, and 38% of pts, respectively. Four pts have experienced a grade 3 non-hematologic toxicity; no grade 4 non-hematologic toxicities were seen. Grade 3 non-hematologic toxicities included hypokalemia, weight loss, vomiting, hand-foot skin reaction, fatigue, and elevated alkaline phosphatase. Dose level 1 (sorafenib 200 mg and everolimus 5 mg daily) was best tolerated and was selected for phase 2 evaluation. The ORR was 33% (7/21;95% CI: 15–57%, Table) with 3 pts at dose level 0 (2 PR, 1 CR), one at level 2 (1 PR) and three at level 3 (2 PR, 1 CR) responding. The responders included 5 pts with Hodgkin's disease and one each with an NK cell and T-cell lymphoma. Pts have received a median of 6 cycles (range: 1–19) of treatment. 16 pts have discontinued treatment due to disease progression (13 pts), non-resolution of cytopenias (1 pt), physician discretion (1 pt), and death on study due to lymphoma (1 pt). Disease progression has been seen in 16 pts; 9 pts have died. Median follow-up for pts still alive is 18.7 months (range: 11.5–29.4). 6 pts died from disease progression, one each due to sepsis unlikely related to treatment, cholecystitis, and unknown causes. Sorafenib is metabolized by the cytochrome P450 CYP3A enzyme and RAD-001 mainly by the CYP3A4 system in the liver, hence there is a potential for interactions. The detailed PK analyses performed as part of this trial showed a decrease in the RAD001 levels following initiation of sorafenib on day 8 of cycle 1 (Figure). Conclusion: The combination of sorafenib and everolimus is safe at a recommended phase 2 dose of sorafenib 200 mg and everolimus 5 mg daily. There is no significant drug interaction seen. Activity has been observed, especially in the setting of Hodgkins Disease. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding. Off Label Use: Lenalidomide for treatment of newly diagnosed myeloma. Witzig:Novartis and Celgene: Patents & Royalties, Research Funding, Served on advisory boards with Novartis and Celgene – both uncompensated with compensation to Mayo Clinic.

Abstract Abstract 2634 The peripheral blood absolute lymphocyte/monocyte ratio (ALC/AMC) at diagnosis has been recently reported [Porrata et al, Hematologica 2012; 97(2):262-9] and confirmed [Koh et al, Oncologist 2012; 17(6):871-80] to be a predictor of clinical outcomes in classical Hodgkin lymphoma (HL). In addition, an inversed correlation has been reported between the ALC/AMC ratio and tumor associated macrophages in classical HL suggesting an association between the host biological response in the macro-environment and micro-environment. However, a limitation of the ALC/AMC ratio at diagnosis, the International Prognostic Score at diagnosis, the Positron-Emission Tomography (PET-scan) at diagnosis, or interim PET-scan during treatment is their inability to sequentially assess the host/tumor interaction during treatment. Therefore, we studied the ALC/AMC ratio recovery, as a surrogate marker of host immunity (i.e., ALC) and tumor micro-environment (i.e., AMC), at each treatment cycle phase to assess its predictive ability for clinical outcomes in classical HL. To participate in the study, patients were required to be diagnosed, treated, and followed at Mayo Clinic, Rochester, Minnesota and treated with adriamycin, bleomycin, vinblastine, and dacarbazine (ABVD) with or without radiation therapy. An ALC/AMC ratio ≥ 1.1 cut-off was used in the study based on our previous publication [Porrata et al, Hematologica 2012; 97(2):262-9]. From 1990 until 2008, 190 classical HL patients qualified for the study. The cohort was evenly split with 50% males and 50% females. Fifty-eight percent of the patients had an interim PET-scan performed. The median age at diagnosis was 36 years (range: 18–83 years). Seventeen patients received 2 cycles; 5 patients 3 cycles; 51 patients 4 cycles; 5 patients 5 cycles and 112 patients 6 cycles. The median follow-up was 3.7 years (range: 0.2–20 years). The ALC/AMC ratio ≥ 1.1 at each treatment cycle phase of ABVD was a predictor for overall survival (OS), lymphoma-specific survival (LSS), progression-free survival (PFS) and time to progression (TTP). Patients were stratified based on how many cycles the ALC/AMC ratio did not reach the cut-off of 1.1. The more cycles with an ALC/AMC < 1.1 the worse survival was observed. The 23 patients who did not achieve an ALC/AMC ≥ 1.1 during the treatment cycles phase experienced the worst survival rates: 5 year OS of 33%; 5 year LSS of 43%; 5 year PFS of 16%; and 5 year TTP of 27%. Patients were then dichotomized into patients that did not achieve an ALC/AMC ratio ≥ 1.1 for all the cycles versus patients with an ALC/AMC ratio ≥ 1.1 in any cycle during treatment. Patients with an ALC/AMC ratio ≥ 1.1 in any cycle (N=167) experienced better survival compared with patients that in all cycles the ALC/AMC ratio was < 1.1 [ OS, the hazard ratio(HR) was 0.09, 95% CI (0.04–0.19), p < 0.0001; for LSS, HR =0.07, 95% CI (0.02–0.17), p <0.0001; for PFS, HR = 0.08, 95%CI (0.04–0.14), p < 0.0001; and for TTP, HR = 0.05, 95% CI (0.02–0.11), p < 0.0001]. In the multivariate analysis, an ALC/AMC ratio ≥ 1.1 in any cycle versus none was an independent predictor for clinical outcome when compared to ALC/AMC ratio at diagnsosis, PET-scan, International Prognostic Score, limited verus advanced stage, and radiation therapy [OS, HR = 0.14, 95% CI (0.04–0.40), p< 0.0002; for LSS, HR = 0.09, 95% CI (0.01–0.37), p <0.0006); for PFS, HR = 0.19, 95% CI (0.05–0.82), p < 0.03); and for TTP, HR = 0.15, 95% CI (0.02–0.89), p < 0.02]. In conclusion, the ALC/AMC ratio during ABVD treatment cycles in classical HL is a predictor for survival and provides an opportunity to develop therapeutic modalities to manipulate the ALC/AMC ratio during ABVD chemotherapy to improve clinical outcomes in classical HL. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 287 Purpose: Farnesyltransferases are key cellular enzymes involved in the prenylation of proteins. Prenylated proteins are important for malignant cell growth. The purpose of this phase II study (NCT00082888) was to assess tumor response and toxicity associated with the oral farnesyltransferase inhibitor tipifarnib (R115777, Johnson and Johnson) in patients with relapsed aggressive, indolent, or uncommon lymphoma. The uncommon group included low incidence lymphoma types such as Hodgkin lymphoma (HL) and T-cell non-Hodgkin lymphoma- cutaneous T-cell lymphoma (CTCL) and peripheral T-cell lymphoma (PTCL). Patients and Methods: Patients with relapsed or refractory non-Hodgkin (NHL) or Hodgkin (HL) were eligible if they had measurable disease, performance status ≤2, platelet count ≥75,000 × 10(6)/L, an absolute neutrophil count ≥1000 × 10(6)/L and a creatinine ≤2 × upper limit of normal. Patients initially received tipifarnib 300 mg twice daily, days 1–21 every 28 days. The trial was conducted by the University of Iowa/Mayo Clinic Lymphoma SPORE. Results: Ninety-three patients with lymphoma (42 aggressive, 15 indolent, and 36 uncommon) enrolled in the trial between March 2004 and November 2008. The median age was 62 years (range, 18–91). The median number of prior therapies was 5 (range, 1–17). The overall response rate (ORR) was 20% (19/93) with 6% (6/93) complete responses (CR) and 14% (13/93) partial responses (PR). The ORR was 17%, 7%, and 31% for the aggressive, indolent, and uncommon groups, respectively (Table). Among the 19 responders were 7 diffuse large B-cell lymphoma (DLBCL), 7 T-cell NHL, 1 follicular grade II, and 4 HL. The overall median response duration for the 19 responders was 7.5 months (mean, 12.8; range, 1.8 – 48.1 months). The median response duration was 11.3 months (mean, 10.1; range, 1.8 – 18.5 months), 2 months, and 7.5 months (mean, 15.5; range, 1.9 – 48.1 months) for the aggressive (7), indolent (1), and uncommon (11) groups, respectively. Five patients in the uncommon group are still receiving treatment with a range of treatment duration between 25 – 60+ months. Among the 5 still receiving treatment 4 responded and 1 is stable. The range of response duration is 15.4, 17.7, 43.1, and 48.1 months. These five patients (2 with HL, 1 anaplastic large cell cutaneous, and 2 PTCL) are currently receiving doses of 400 (1), 200 (2), and 100 (2) mg twice daily. The median time to progression for all patients was 3.6 months (95% CI: 2.1 – 4.5 months). The grade 3,4 toxicities observed were primarily fatigue (10%) and reversible myelosuppression with 11% anemia, 37% neutropenia, and 32% thrombocytopenia. Correlative studies demonstrated tipifarnib-induced upregulation of Bim, a marker of tipifarnib action in lymphoma cell lines, in 7 of 9 paired lymphoma biopsies. Conclusions: Tipifarnib has activity in lymphoma, with responses in patients with heavily pretreated DLBCL, HL and T-cell types, but little activity in follicular NHL. This agent has an excellent toxicity profile and targets a novel pathway. Further studies in combination with other agents are warranted for patients with DLBCL, HL or T-cell NHL. Disclosures: No relevant conflicts of interest to declare.

Abstract Introduction Relapse in the central nervous system is an uncommon complication of diffuse large B cell lymphoma (DLBCL) associated with a poor prognosis. The addition of rituximab to chemotherapy has improved outcomes in patients with DLBCL but its effect on the outcome of patients with CNS relapse is not well characterized. Here we present the natural history of patients with CNS relapse in a large cohort of patients with DLBCL who were treated in the immunochemotherapy (IC) era. Methods Newly diagnosed patients with DLBCL or primary mediastinal B-cell lymphoma (PMBCL) and treated with primary anthracycline based IC were prospectively enrolled on the University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource (MER) within 9 months of diagnosis and followed for relapse, retreatment, and death. Clinical management at diagnosis and subsequent therapies were per treating physician. Patients with documented CNS involvement at diagnosis, primary CNS lymphoma, or post-transplant lymphoproliferative disorder at diagnosis were excluded. All CNS relapse and retreatment details were verified by medical record review. Results 1017 patients with newly diagnosed DLBCL or PMBCL, no documented CNS disease at diagnosis, and treated with IC were enrolled in the MER between 2002 and 2012. At a median follow-up of 59 months (range 1-148), 36 patients had CNS relapse. The cumulative incidence of CNS relapse was 3.1% (95% CI: 2.2%-4.4%) at 2 years and 3.7% (95% CI: 2.7%-5.0%) at 5 years after diagnosis. CNS involvement was identified after first line IC in 25 patients, and after salvage therapy in 11 patients. 22 patients had an isolated CNS relapse, while 14 had both systemic and CNS disease at relapse. The incidence of isolated CNS relapse at two years from diagnosis was 1.9% (95% CI: 1.2%-3.0%, figure 1). CNS involvement was parenchymal in 22 patients, leptomeningeal in 11, and parenchymal and leptomeningeal in 3 patients. At diagnosis, this subset of 36 patients had a median age of 61 years (range 20-86); 25 (69%) were male. IPI was 0-1 in 6 patients, 2 in 13 patients, 3 in 11 patients and 4-5 in 6 patients. Cell of origin per Hans algorithm was available in 17 patients, 8 were GCB and 9 were non-GCB. Extranodal sites of disease at diagnosis included testicular (2), renal (5), bone (10) and bone marrow (6). 7 patients had no sites of extranodal disease. LDH was elevated in 71%. The German High-Grade Non-Hodgkin Lymphoma Study Group CNS risk score was low risk in 6 patients, intermediate risk in 22 patients, and high risk in 8 patients. 6 patients received CNS prophylaxis with their initial immunochemotherapy, including 3 of the 21 patients who subsequently developed isolated CNS disease. First line therapy after CNS relapse included high dose methotrexate based chemotherapy in 22 patients and other systemic regimens in 6, with intrathecal (IT) chemotherapy in 5. Overall survival in patients after CNS relapse was poor with a median survival of 6.3 months (95% CI: 2.9-15.5) and 12-month survival rate of 29% (95% CI: 17%-49%). Patients who had CNS relapse following initial IC had better subsequent survival (median OS = 7.6 months) compared to patients with first presentation of CNS relapse following salvage therapy (OS = 2.1 months, p=0.005, see figure 2). 10 patients (28%) proceeded to SCT after CNS relapse, of which 6 remain alive at a median follow-up of 39 months. There were no differences in post-CNS relapse survival by age, sex, CNS risk score, cell of origin, extranodal site involvement at diagnosis, or site of CNS relapse (all p>0.10), though power was limited due to small numbers. Conclusions The incidence of CNS relapse in DLBCL is approximately 4% in the immunochemotherapy era. The vast majority of CNS relapses occur within 2 years of diagnosis. Isolated relapse in patients without CNS involvement at diagnosis is a relatively rare event with an incidence of approximately 2% at 2 years of diagnosis. Outcomes remain poor, and standard clinical variables do not predict survival after CNS relapse. Novel therapeutic approaches are needed in this population, with consideration to autologous SCT, which produces durable remission in a subset of patients. Disclosures Maurer: Kite Pharma: Research Funding. Farooq:Kite Pharma: Research Funding. Thompson:Kite Pharma: Research Funding. Ansell:Bristol-Myers Squibb: Research Funding; Celldex: Research Funding. Cerhan:Kite Pharma: Research Funding. Link:Genentech: Consultancy, Research Funding; Kite Pharma: Research Funding.

Abstract Abstract 2862 Follicular lymphoma (FL) is a B-cell malignancy that exhibits significant intratumoral infiltration by non-malignant T lymphocytes. The pathophysiological significance of infiltrating T cells is poorly understood but recent studies have suggested that CD4+CD25+ regulatory T (Treg) cells are highly represented in lymph nodes involved by FL. These Treg cells display the ability to suppress the proliferation and cytokine production of other tumor-infiltrating T cells and migrate to areas of B-cell lymphoma in response to chemotactic signals provided by the malignant B-cells. Denileukin diftitox, a chimeric immunotoxin composed of the modified cytotoxic domain of diphtheria toxin and human interleukin-2 (IL-2) protein, targets cells expressing CD25 and has proven efficacy in patients with relapsed B-cell lymphoma. In this study, we combined denileukin diftitox with rituximab in a cohort of previously untreated, advanced-stage follicular lymphoma patients. Our hypothesis was that denileukin diftitox would deplete the Treg cells, thereby removing the inhibition of the immune response, and rituximab would deplete the B-cells thereby preventing further recruitment of Treg cells to the areas of lymphoma. Between August 2008 and March 2010, twenty-four patients with stage III and IV follicular grade 1 or 2 non-Hodgkin lymphoma were accrued to the study. One patient died before treatment was given and is not included in the analysis. The median age was 60 years (range: 27 – 79), 12 (52%) of the patients were male, 19 (83%) had a PS of 0 and 4 (17%) had a PS of 1. Based on the Follicular Lymphoma International Prognostic Index (FLIPI), 3 (13%) were low risk, 14 (61%) were intermediate risk and 6 (26%) were high risk. Patients received rituximab 375 mg/m2 on days 1, 8, 15 and 22 and denileukin diftitox 18 mcg/kg/day on days 1–5 every 3 weeks for 4 cycles. A median of 4 cycles of therapy was given (range: 1 – 4). Thirteen patients completed treatment per protocol (57%), however 5 patients discontinued treatment due to adverse events (22%), 2 refused further treatment (9%) and 1 discontinued due to disease progression (4%). Nine of the 23 patients (39%; 95% CI: 21–61%) responded to treatment, 3 (13%) had a complete response and 6 (26%) had a partial response. Twenty-one patients (91%) are alive with a median follow-up of 8.7 months (range: 3.4–19.5). Seven (30%) patients have progressed and two (8.7%) has died. The median time to progression is 13.4 months (95% CI: 10.4 – NA). The combination, however, was associated with significant toxicity. Thirteen patients (57%) experienced grade 3 or greater adverse events. Six patients (26%) had symptoms of capillary leak syndrome, 1 of whom died. In correlative studies performed on the peripheral blood, the number of CD25+ T-cells decreased after treatment when compared to pretreatment numbers (median 24%; range: 8–44%). We conclude that while the addition of denileukin diftitox to rituximab decreased the numbers of CD25+ T-cells, denileukin diftitox contributed significantly to the toxicity of the combination. Furthermore, the overall response rate and time to progression in this study were no better than what would be expected in follicular lymphoma patients treated with rituximab alone. Disclosures: No relevant conflicts of interest to declare.

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 Genetic analysis has an increasing role in the diagnosis, classification, and management of patients with CLL. Although translocations involving the IgH locus are well characterized in B-cell malignancies such as follicular cell lymphoma, their frequency and significance in CLL are less well defined. We studied a series of patients with a clinical diagnosis of CLL using a FISH probe set including IgH. Methods: FISH was performed on blood using a DNA probe set designed to detect common chromosome anomalies associated with CLL and translocations involving IgH. Patients with an IgH abnormality were further studied using FISH probes sets for IgH and cyclin D1, BCL-2, BCL-3, BCL-6, BCL-11a, c-MYC, MALT-1, c-MAF, FGFR-3 or PAX-5. The clinical presentation, pathology, and flow cytometric immunophenotype was reviewed in patients with various translocations involving IgH. Results: Between 1/1/03 and 6/15/04, samples from 1032 patients with a clinical diagnosis of CLL were analyzed by FISH. Of these, 76 patients (7%) had translocations involving the IgH locus. Sixty one patients had a single translocation partner gene (cyclin D1 45%, BCL-2 24%, BCL-3 8%, c-MYC 2.5%, or BCL-11a 1.3%). Two patients had two independent clones (cyclin D1 and BCL-2; BCL-3 and BCL-11a). In 13 pts (17%) we could not identify the translocation partner. Thirty five (46%) of the 76 patients with IgH translocations had been seen at Mayo Clinic Rochester and had clinical and laboratory data for detailed review. Among these 35 patients, 16 (45%) had fusion of IgH and cyclin D1 and were diagnosed with mantle cell lymphoma (MCL). The immunophenotype of the monoclonal B cells was typical of MCL (CD5+, CD23-) in 6 patients and typical of CLL (CD5+, CD23+) in 4 patients. Six MCL patients had non-diagnostic immunophenotypes: CD5-, CD 23+ (n = 3) and CD5-, CD23- (n = 3). Twelve (34%) of the 35 patients had the IGH/BCL-2 fusion gene. Of these, 3 patients had leukemic phase follicular lymphoma, one had both CLL and MCL, and 8 had the diagnostic immunophenotype of CLL. The remaining 7 patients (19%) had a variety of translocation partners: BCL-3 (n = 1), BCL-3 and BCL-11a (n = 1), c-MYC (n = 2), and unknown (n = 3). The patients with a translocation involving BCL-3 had a typical CLL immunophenotype. Discussion: The IgH FISH probe detected a molecular defect in 7% of patients with a circulating monoclonal B cell population. In patients with a CLL immunophenotype, use of the IgH probe identified the IgH/cyclin D1 translocation characteristic of MCL in 4 patients, a finding of major clinical significance. Interestingly, after exclusion of MCL patients, more than 4% patients with CLL had other translocations involving IgH, a proportion comparable with other better defined chromosomal abnormalities in CLL. Although IgH/BCL-2 translocation is usually associated with follicular lymphoma, we identified 8 patients with CLL and this translocation. Translocations involving BCL-3 had been described before in CLL with an “atypical” immunophenotype. In contrast both of our patients with BCL-3 had typical phenotypes. Translocations involving IgH and BCL-11a have not been previously described. We conclude that FISH studies using the IgH probe improve the precision of diagnosis in CLL and could also have prognostic value.

Abstract Background: We have previously shown that CLL B cells secrete vascular endothelial growth factor (VEGF) and express VEGF receptors: VEGFR-1 and VEGFR-2. Secreted VEGF protects CLL B cells from spontaneous and drug induced apoptosis via increased levels of Mcl-1 and XIAP; however, the exact mechanism of this process is unknown. In solid tumors, there is increasing evidence that signaling through VEGF receptor known as neuropillin-1 (NRP-1) is critical for VEGF induced resistance to apoptosis. Hypothesis: NRP-1 is expressed by CLL B cells and is critical for VEGF mediated protection from apoptosis. Methods: To demonstrate the presence of NRP-1 on CLL B cells, we conducted flow cytometry and immunoblotting. We then evaluated the ability of NRP-1 blocking antibodies to induce apoptosis of primary CLL B cells. To do this, circulating CLL B cells were isolated by density gradient centrifugation. Patient samples with greater than 80% of CD5+ CD19+ cells in a mononuclear cell population, as assessed by flow cytometry, were cultured in AIM-V media in 24-well plates at 1.5 x 106 cells/mL. To occupy NRP-1, we added NRP-1 blocking antibodies (Calbiochem, Darmstadt, Germany) at increasing concentrations (0.5 μg/mL -10 μg/mL) to cultured CLL B cells. Cell death was assessed using an annexin and propidium iodide flow assay after 24 h of in vitro culture. CLL cells cultured without antibodies and isotype nonspecific monoclonal antibodies were used as controls. Results: CLL B cells were found to express NRP-1 but not uniformly. Most patients had flow positive evidence for NRP-1 but a distinct percentage (35%) was very low (≤5%) or negative. However, immunoblot analysis revealed moderate to low levels of NRP-1 protein with evidence of tyrosine phosphorylation in all tested CLL patients (N=9). NRP-1 blocking antibodies, but not VEGF-R1 and -R2 blocking antibodies, induced apoptosis in a dose dependent manner in primary CLL cells (n=5, FIG.) at antibody concentrations starting at 1 μg/mL (p=0.003). The effect of the NRP-1 blockade varied between patients, with a median IC50, 1.5 μg/mL (range 0.5–2 μ/mL). Importantly, concentrations of 5 μg/mL and higher induced apoptosis in more than 90% of the CLL B cells. We also found that the sensitivity of CLL B cells to NRP-1 blocking antibody, in terms of apoptosis induction, was correlated with the number of NRP-1 receptors as assessed by flow cytometry. CLL B cell clones with no detectable NRP-1 had no induction of cell death when exposed to the NRP-1 blocking antibody. Finally, immunoprecipitation and immunoblot assays indicated that NRP-1 physically interacted with VEGF-R2 on CLL B cells. This suggests that NRP-1 could be enhancing VEGF binding affinity on VEGF-R2 to further increase the ability of VEGF to generate signals that lead to apoptosis modulation. Conclusion: We have found that NRP-1 blocking antibodies induce cell death in NRP-1 positive CLL B cells. Similar results using NRP-1 blocking peptides rather than blocking antibodies have been observed in breast cancer (Br J Cancer.2005 Jan 31;92(2):328–33). Our results suggest that NRP-1 represents an attractive therapeutic target in CLL and should be explored further. Figure Figure

Abstract Background: The prognosis of patients with T-cell large granular lymphocyte (LGL) leukemia is diverse, with some patients experiencing rapid progression of the disease while others surviving decades. Despite multiple case reports and a number of case series reported in literature, prognostic factors in this leukemia are unknown and no staging/prognostic system has been developed. Aim: To identify factors predicting the prognosis of patients with newly diagnosed T-cell LGL leukemia. Methods: We reviewed the presentation, laboratory data and the clinical course of patients with newly diagnosed T-cell LGL at seen at the Mayo Clinic, Rochester. T-cell LGL was diagnosed in patients with at least 2 of these criteria: morphological evidence of LGL leukemia on bone marrow biopsy or blood smear; LGL immunophenotype; evidence of clonal T-cell receptor gene rearrangements. Results: 286 patients seen at Mayo Clinic Rochester between 6/1986 and 5/2005 met diagnostic criteria (52% male, median age at presentation 64 years (range 27–88)). Lymphadenopathy, splenomegaly, and hepatomegaly were present in 9%, 4% and 1.5% of the patients respectively. Median hemoglobin was 11.7 g/dL (range 3.5 – 17.1). Median absolute neutrophil count (ANC) was 1.51 × 109/L (range 0.0 – 20 × 109/L) and median absolute lymphocyte count (ALC) was 2.2 × 109/L (range 0.1 – 24). Median LGL count on peripheral smear was 0.81 × 109/L (range 0.06–10.8 × 109/L). Cytogenetic studies were done for 122 patients, of which, only 9 were abnormal. 87 patients (29%) had associated autoimmune conditions: rheumatoid arthritis (n=29), Felty’s syndrome (n=11), other connective tissue disorders (n=9)). Pure red cell aplasia, aplastic anemia, idiopathic thrombocytopenic purpura and autoimmune hemolytic anemia were seen in 17, 5, 8, 1 patients respectively. Median follow up was 50 months (range 1–229) and estimated median survival was 74.3 months. In multivariate analysis, the only independent risk factors were: anemia (hemoglobin<12 g/dl), hazard ratio (HR) 1.75, (95%CI 1.22–2.52, p= 0.0023), severe neutropenia (<0.1 × 109/L), HR 2.11 (95%CI 1.053–4.25) and lymphopenia (ALC< 1 × 109/L), HR 1.93 (95%CI 1.21–3.07). We developed a simple prognostic scoring system based on the above identified independent risk factors, stratifying patients in 3 groups: 0 - no risk factors present, 1 -one risk factor, 2 - two or more risk factors present. The median survival was 105, 55 and 5 months for score of 0, 1 and 2 respectively, p=0.004 (Figure). Conclusion: The prognosis of patients with newly diagnosed T-cell LGL is variable. The presence of anemia, severe neutropenia and lymphopenia are independent poor prognostic factors in T-cell LGL. Combining these factors allows the identification of patients at a high risk of death from the disease. Further studies are needed to prospectively validate these findings and establish if the high risk group would benefit from aggressive therapy. Figure Figure.