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Abstract Introduction. The peripheral blood absolute lymphocyte (ALC)/monocyte (AMC) ratio (ALC/AMC), as a surrogate of host immunity (i.e. ALC) and tumor microenvironment (i.e. AMC), is a predictive biomarker for clinical outcomes in diffuse large B-cell (DLBCL) patients. An ALC/AMC ratio ≥ 1.1 has been shown to be predictive of better survival both at baseline and during each therapy cycle in patients treated with R-CHOP [Porrata et al, 2014]. Lenalidomide is an immunomodulatory drug, effective in DLBCL patients, with various mechanisms of actions on the tumor microenvironment and the host immune response. In this study we analyze the prognostic value of ALC/AMC ratio in a cohort of newly diagnosed DLBCL patients treated with Lenalidomide plus R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone, R2-CHOP) in a phase II trial (MC078E trial, [Nowakowski et al, 2015]). Methods. All patients with de-novo DLBCL enrolled in the phase II trial MC078E and treated at diagnosis with R2-CHOP regimen were included in the analysis. We investigated the ALC/AMC ratio at baseline and each R2-CHOP cycle as predictor of progression-free survival (PFS) and overall survival (OS). The ALC/AMC ratio was obtained by dividing the ALC by the AMC from the automated white blood cell differential obtained from the complete blood cell count on day 0 of each treatment cycle (range from day -3 to day 0 of each course). Patients were then divided in 4 groups on the basis of pattern of ALC/AMC ratio during treatment: group A: patients with ALC/AMC ratio ≥ 1.1 throughout all cycles; group B: patients with ALC/AMC ratio ≥ 1.1 at baseline, but then obtained an ALC/AMC < 1.1 during treatment; group C: patients with ALC/AMC ratio < 1.1 at baseline, but then gained an ALC/AMC ≥ 1.1 during treatment; group D: patients with ALC/AMC ratio < 1.1 throughout all cycles. PFS and OS were analyzed in the different groups. We separately conducted the same analysis in a matched control cohort of 94 DLBCL patients from the Mayo Clinic Lymphoma Database treated with standard R-CHOP. Results. A total of 63 patients with de-novo DLBCL treated with R2CHOP regimen at diagnosis were included in the analysis. Clinical characteristics were: median age 67 years (22-87), male sex 61.9%, III-IV advance stage 87.3%, elevated LDH serum levels 66.7%, intermediate-high/high International Prognostic index (IPI) score in 54% cases. No differences in 3y-PFS and 3y-OS in patients with ALC/AMC ratio ≥ 1.1 vs ALC/AMC < 1.1 at baseline and at each R2-CHOP course have been observed. In a landmark analysis from day 0 of the last cycle, 3y-PFS in group A vs B vs C vs D was: 71.4% vs 67.1% vs 66.7% vs 85.7%, respectively (p 0.80); 3y-OS in group A vs B vs C vs D was: 85.7% vs 86.5% vs 66.7% vs 100%, respectively (p 0.74). No differences in both 3y-PFS and 3y-OS were observed comparing group A vs B, in group C vs D, and in group A vs D. Unlike what was observed in the R-CHOP treated DLBCL patients, in a univariate analysis ALC/AMC < 1.1 during all cycles of R2CHOP was not a predictor for inferior outcomes (Figure 1.a). In the matched cohort of patients treated with standard R-CHOP, ALC/AMC < 1.1 at baseline and at each course demonstrated to be predictive of worse outcome (p <0.01),survival was significantly different in the 4 groups identified according to ALC/AMC ratio during treatment and ALC/AMC < 1.1 maintained during all cycles of R2CHOP was predictive of worse PFS and OS (Figure 1.b). Conclusions. Lenalidomide in combination with standard R-CHOP (R2-CHOP), in previously untreated DLBCL patients, appears to overcome the negative prognostic value of ALC/AMC ratio observed in patients treated with R-CHOP alone. This could reflect the additional effect of lenalidomide to standard therapy on tumor microenvironment and on host immunity response. Figure 1: Landmark Analysis for Progression Free Survival and Overall Survival by ALC/AMC groups within R2CHOP (1.A) and R-CHOP (1.B). Group A: ALC/AMC >=1.1 for all cycles; Group B: ALC/AMC >=1.1 at baseline and obtained <1.1 during treatment; Group C: ALC/AMC <1.1 at baseline and obtained >=1.1 during treatment; Group D: ALC/AMC <1.1 for all cycles. Disclosures Witzig: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract Introduction: The addition of Rituximab to chemotherapy has significantly improved the outcome of patients with newly diagnosed diffuse large B-cell lymphoma (DLBCL). Patients treated with immunochemotherapy for DLBCL who achieve EFS24 (event-free for 2 years after diagnosis) have an overall survival equivalent to that of the age- and sex-matched general population. Relapses after achieving EFS24 have been considered to be unusual but have been understudied. We sought to define the rate, clinical characteristics, treatment pattern, and outcomes of such relapses. Methods: 1448 patients with newly diagnosed DLBCL from March 2002 to June 2015 were included. Patients were enrolled in the Molecular Epidemiology Resource (MER) of the University of Iowa/Mayo Clinic Lymphoma SPORE, treated per physician choice (predominantly R-CHOP immunochemotherapy) and followed prospectively. An event was defined as progression or relapse, unplanned re-treatment after initial therapy, or death from any cause. Cumulative incidence of relapse and non-relapse mortality after achieving EFS24 were analyzed as competing events using Gray's test in the EZR software. Post-relapse survival was defined as time from relapse to death from any cause and analyzed using Kaplan-Meier method in SPSS (V22). Results: Among the 1448 patients, 1260 (87%) had DLBCL alone at diagnosis, and 188 (13%) had concurrent indolent lymphoma (follicular lymphoma 115, marginal zone lymphoma 18, chronic lymphocytic leukemia 14, lymphoplasmacytic lymphoma 4, unspecified 37) at diagnosis. After a median follow-up of 83.9 months, 896 patients achieved EFS24. For all 896 patients who achieved EFS24, the cumulative incidence of relapse (CIR) was 5.7%, 9.3% and 13.2%, respectively, at 2, 5 and 10 years after achieving EFS24. Patients with concurrent indolent lymphoma at diagnosis had a higher CIR compared to those with DLBCL alone at diagnosis (10.2 vs 4.8% at 2 years, 15.7 vs 8.0% at 5 years, 28.8 vs 9.7% at 10 years, P<0.001; Figure 1). There were a total of 84 patients who relapsed after achieving EFS24. The median age at initial diagnosis was 66 years (range 35-92), and 48 (57%) were male. At diagnosis, 11 (13%) had ECOG PS >1, 37 (50%) had LDH elevation, 62 (74%) were stage III-IV, 14 (17%) had more than 1 extranodal site, and 26 (31%) were poor risk by R-IPI score. There were 58 patients with DLBCL alone at diagnosis who relapsed after achieving EFS24, and 38 (75%) relapsed with DLBCL, 13 (25%) relapsed with indolent lymphoma (predominantly follicular lymphoma), and pathology was unknown in 7 patients. In contrast, there were 26 patients with concurrent indolent lymphoma at diagnosis who relapsed after achieving EFS24, and 9 (41%) relapsed with DLBCL, 13 (59%) relapsed with indolent lymphoma, and pathology was unknown in 4 patients. In the 47 patients who relapsed with DLBCL after achieving EFS24, 45% received intensive salvage chemotherapy, 19% received regular intensity chemotherapy, 9% received CNS directed chemotherapy, and 36% went on to receive autologous stem cell transplant (ASCT). In the 26 patients who relapsed with indolent lymphoma after achieving EFS24, 27% were initially observed, 54% received regular intensity chemotherapy, 4% received intensive salvage chemotherapy, and 19% received ASCT after subsequent progression. The median post-relapse survival (PRS) for all patients with a relapse after achieving EFS24 was 38.0 months (95% CI 27.5-48.5). The median PRS for patients who relapsed with DLBCL and indolent lymphoma after achieving EFS24 were 29.9 (19.9-39.9) and 89.9 (NR-NR) months, respectively (P=0.002; Figure 2). Conclusions: Relapses after achieving EFS24 in patients with DLBCL were uncommon in the rituximab era. Patient with DLBCL alone at diagnosis can relapse with either DLBCL or indolent lymphoma (3:1 ratio). Patients with concurrent DLBCL and indolent lymphoma at diagnosis had a significantly higher CIR, and relapses with DLBCL and indolent lymphoma were similar (2:3 ratio). Even with high intensity salvage chemotherapy and consolidative ASCT, patients who relapsed with DLBCL had a significantly worse survival compared to those who relapsed with indolent lymphoma. Late relapses with DLBCL remain clinically challenging, with a median survival of 2.5 years after relapse. Figure 1. Figure 1. Disclosures Maurer: Celgene: Research Funding; Nanostring: Research Funding; Morphosys: Research Funding. Witzig:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Ansell:Takeda: Research Funding; Pfizer: Research Funding; Affimed: Research Funding; Regeneron: Research Funding; Seattle Genetics: Research Funding; Celldex: Research Funding; LAM Therapeutics: Research Funding; Trillium: Research Funding; Merck & Co: Research Funding; Bristol-Myers Squibb: Research Funding. Cerhan:Celgene: Research Funding; Jannsen: Other: Scientific Advisory Board; Nanostring: Research Funding.

Abstract Background An association has been identified between the diagnosis-to-treatment interval (DTI) and prognostic clinical factors and outcomes in patients with previously untreated diffuse large B-cell lymphoma (DLBCL) (Maurer et al, JCO 2018). This association can result in inadvertent selection bias in clinical trials to exclude patients with aggressive disease due to the inability to delay treatment long enough to fulfill enrollment criteria, compromising the validity of clinical trial study results to the general population. A similar concern exists in the relapsed/refractory (r/r) DLBCL setting regarding patient selection bias against patients with aggressive disease requiring immediate treatment following relapse or progression. Here we examine the time from progression after immunochemotherapy (IC) to the initiation of first salvage chemotherapy and its association with outcome. Patients and Methods: Newly diagnosed patients were prospectively enrolled within 9 months of diagnosis in the University of Iowa/Mayo Clinic Lymphoma SPORE Molecular Epidemiology Resource (MER), now a subcohort of the Lymphoma Epidemiology of Outcomes (LEO) Cohort Study, and followed for progression/relapse, retreatment, and death. This analysis includes patients with their first r/r DLBCL following frontline IC who initiated aggressive salvage chemotherapy as identified and included in a previous publication (Farooq et al, BJH 2017). The progression-to-treatment interval (PTI) was defined as the time in days from date of progression from IC to initiation of salvage therapy. The date of progression was defined as either a) the date biopsy was obtained for patients who had a biopsy to confirm progressive disease or b) the date of the scan or clinical examination indicating progression in patients who did not have a biopsy performed. Event-free survival (EFS) was defined as time from initiation of first salvage therapy to progression or relapse, initiation of new anti-lymphoma therapy, or death due to any cause; overall survival (OS) was defined as time from initiation of salvage therapy until death due to any cause. Results: 162 patients with r/r DLBCL enrolled in the MER from 2002-2012 who initiated aggressive salvage chemotherapy with intent to transplant and had confirmed dates for both progression after IC and start of salvage therapy were evaluated. Median age at first progression for these patients was 64 years (range 36-76) and 104 (64%) were male. Median time from diagnosis to first progression on IC was 6.7 months (IQR: 4.6-12.7). Initial salvage therapy was R-ICE (80%), R-DHAP (8%), rituximab, oxaliplatin, cytosine arabinoside, and dexamethasone (ROAD) (6%), and other (7%). At a median follow-up of 49 months from initiation of salvage therapy (IQR: 33-74), 116 patients had died (72%). Median PTI was 6 days (IQR: 2-13). 110 patients (68%) had biopsy confirmation of disease prior to initiating salvage therapy; median PTI was 2 days (IQR: 1-7) for patients who had biopsy confirmation vs. 7 days (IQR: 3-16) in patients without biopsy confirmation, Wilcoxon p=<0.0001. There was no difference in OS from initial salvage therapy between patients who did not have biopsy confirmation (HR=1.09, 95% CI: 0.74-1.60, p=0.68) compared to patients with biopsy confirmation. Patients with short PTI (0-6 days) had significantly worse overall survival from initiation of salvage therapy (median OS = 7.6 months, HR=2.10 (95% CI: 1.43-3.08) compared to patients who initiated therapy 7 or more days from progression (median OS = 29.7 months), logrank p<0.0001. This association remained consistent (HR=2.20, 95% CI: 1.48-3.26, p<0.0001) after adjusting for age and biopsy confirmation of progression. Short PTI was also associated with inferior response rate to initial salvage therapy (51% vs. 65%, p=0.058), lower rate of proceeding to transplant after initial salvage therapy (33% vs. 55%, p=0.0063), lower rate of being event-free 24 months from initial salvage therapy (16% vs 33%, p=0.011) and lower rate of ever proceeding to transplant (44% vs. 59%, p=0.057). Conclusions: A short progression-to-treatment interval is strongly associated with inability to proceed to transplant and inferior overall survival in r/r DLBCL. These results have implications for the design and interpretation of clinical trials in the relapsed/refractory setting. Figure. Figure. Disclosures Maurer: Celgene: Research Funding; Nanostring: Research Funding; Morphosys: Research Funding. Ansell:Trillium: Research Funding; Merck & Co: Research Funding; Affimed: Research Funding; Bristol-Myers Squibb: Research Funding; Seattle Genetics: Research Funding; Regeneron: Research Funding; LAM Therapeutics: Research Funding; Pfizer: Research Funding; Celldex: Research Funding; Takeda: Research Funding. Witzig:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Cerhan:Nanostring: Research Funding; Celgene: Research Funding; Jannsen: Other: Scientific Advisory Board.

Abstract Background: As our healthcare system faces increasing complexity, it is important for all healthcare providers to practice at the full scope of their training. However, there are limited current options for subspecialty training in hematology for advanced practice providers (APPs), a group of health care providers including both nurse practitioners and physician assistants. We are unaware of any existing postgraduate fellowship for APPs focused on education in both malignant and nonmalignant hematologic disorders. We hope that by creation of the first such fellowship, we can better prepare APP graduates with the subspecialty-specific knowledge required to become highly skilled and competent clinicians in hematology. Methods: Using surveyhero.com, we conducted a web-based needs assessment survey which was distributed to 68 APPs currently working in the hematology and blood & marrow transplant (BMT) teams at three Mayo Clinic sites (Rochester, Scottsdale, and Jacksonville). This survey included questions regarding hematology-specific education the respondents had received in their APP schools, as a way to assess participant's perceptions about the readiness to practice in hematology after completion of APP education, and to identify the learning/training needs that are most important for APPs to practice in hematology. Results: Of 68 APPs, 49 (72%; 34 NPs,15 PAs) completed the survey. 57% of respondents were new APP graduates when hired and 35% had no prior work experience in hematology/BMT. All APPs held a Master's or higher degree (doctorate 31%). Thirty-nine (80%) reported <5% of their APP school curriculum was hematology-focused. The majority (92%) felt that the level of subspecialty training they received in APP schools did not adequately prepare them with the full confidence to practice in hematology. Forty-four (90%) APPs believed subspecialty training could help them to become more competent providers for patients with hematologic disorders. Forty-seven (96%) APPs agreed that being confident and knowledgeable in their practice positively impacted their job satisfaction, and most APPs (84%) felt structured hematology training would attract them to stay within the specialty. The educational subjects that APPs identified as most important included variety of malignant and benign hematologic disorders as well as hematopathology, bone marrow transplant, palliative care, transfusion medicine, infectious disease and pharmacology related to hematology practice. The top three effective learning strategies reported by the APP respondents were active learning from direct patient care, cased based teaching, and educational experience during hospital rounds. Conclusions and Discussions: This needs-based assessment educational project revealed to us the significant gaps in subspecialty training of APP education in hematology and confirmed the necessity of this fellowship. The findings of the survey helped us to optimize the fellowship curriculum in order to meet the learning needs of future APP trainees. Development of the subspecialty fellowship program is a step forward in the future training of APPs. We believe our fellowship has the potential to serve as the postgraduate training model for other institutions nationwide. Disclosures Lacy: Celgene: Research Funding.

Abstract Introduction: Lenalidomide (Len) in association with standard Rituximab-CHOP (R2CHOP21) has been shown to be safe and effective in newly diagnosed Diffuse Large B-cell Lymphomas (DLBCL) [Nowakowski et al. JCO 2014, Vitolo et al. Lancet Oncol 2014]. The addition of lenalidomide appears to benefit primarily patients with non-Germinal Center B-cell (non-GCB) phenotype as determined by immunohistochemistry (IHC). These early results led to currently ongoing randomized trial in ABC subtype of DLBCL, however efficacy of R2CHOP in ABC DLBCL as defined by Gene Expression Prophyling (GEP) has not been reported. In the present combined analysis of two independent phase 2 studies, we report the long-term follow-up (FU) outcome in DLBCL patients treated with R2CHOP, comparing GCB and ABC according to Nanostring Platform. Methods: We included all newly diagnosed histologically-confirmed de-novo DLBCL patients enrolled in two R2CHOP21 phase 2 trials, conducted by Mayo Clinic (MC) and Italian Lymphoma Foundation (FIL). Inclusion criteria in the two trials were similar, main differences included: all age and all International Prognostic Index (IPI) vs age between 60 and 80 years old and IPI >1, in MC vs FIL study, respectively. All pts received R-CHOP21 plus Len at 25 mg/day for 10 days/cycle and 15 mg/day for 14 days/cycle in MC and FIL trial, respectively. Cell of origin (COO) was determined by IHC, according to Hans algorithm, and retrospectively, in patients with available pathological material, by Nanostring, performed according to Scott algorithm and Masque-Soler signature in MC and FIL study, respectively. We analyzed the long-term FU outcome in terms of progression-free survival (PFS), time to progression (TTP), overall survival (OS), comparing between GCB and ABC phenotypes according to GEP. Results: A total of 112 DLBCL pts (63 MC, 49 FIL) were included. Main characteristics were: median age 69 years (y) (range 22-87), male 65 (58%) pts, advanced stage III-IV in 94 (84%), B symptoms in 38 (34%), International Prognostic Index (IPI) intermediate-high/high in 71 (63%). At a median follow-up of 5.1 years (y), 5y-PFS was 63.5%, 5y-TTP 70.1% and 5y-OS 75.4%. A total of 32 relapses were observed, with only 2 cases of Central Nervous System (CNS) relapse. Late relapse occurring beyond 3 years was observed in 4 cases (3 cases with GCB phenotype and one case with missing COO data). In a subgroup analysis by IPI 0-2 vs 3-5 were: 5y-PFS, 5y-TTP and 5y-OS were: 69.0% vs 59.0% (p=0.100), 73.2% vs 67.4% (p=0.285) and 82.3% vs 70.2% (p=0.059), respectively. Regarding for COO defined by IHC, GCB phenotype vs non-GCB were 45 (40%) vs 41 (37%) patients respectively; 26 (23%) patients were not evaluable. 5y-PFS, 5y-TTP and 5y-OS were: 52.8% vs 64.5% (p=0.198), 61.6% vs 69.6% (p=0.444) and 68.6% vs 74.1% (p=0.238) in GCB vs non-GCB, defined by IHC, respectively. Regarding Nanostring analysis, GCB vs ABC vs Unclassified (Uncl) were 31 (46%) vs 22 (32%) vs 15 (22%) respectively; 44 pts were not evaluable. 5y-PFS, 5y-TTP and 5y-OS were: 62.3% vs 70.8% vs 64.2% (p=0.645), 68.1% vs 79.8% vs 64.2% (p=0.662) and 76.0% vs 74.8% vs 79.0% (p=0.658) in GCB vs ABC vs Uncl, defined by Nanostring, respectively (Table1, Fig1). Conclusions: The association of Len with R-CHOP21 appears to overcome the negative prognostic impact of ABC phenotype determined by GEP, with high PFS, TTP and OS rate, maintained at a long-term FU analysis. Figure 1. Figure 1. Disclosures Chiappella: Teva: Other: lecture fees; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: lecture fees; Nanostring: Other: lecture fees; Roche: Other: lecture fees; Amgen: Other: lecture fees; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: lecture fees. Gaidano:Roche: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Morphosys: Honoraria. Witzig:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Vitolo:Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sandoz: Speakers Bureau; Gilead: Speakers Bureau.

Abstract Abstract 85 Background: Follicular lymphoma (FL) is an indolent non-Hodgkin lymphoma (NHL) with a highly variable natural history. Clinical variables describing either patient- or tumor-specific characteristics, many of which are included in the Follicular Lymphoma International Prognostic Index (FLIPI), are used to risk-stratify patients at the time of diagnosis. However, characteristics of non-malignant cells present within the tumor microenvironment provide important prognostic information. For example, both immunohistochemistry and gene expression profiling studies demonstrate that lymphoma-associated macrophages (LAMs) are prognostically important in FL. As LAMs are monocyte-derived, we sought to determine whether the absolute monocyte count (AMC) obtained from a CBC at diagnosis is an adverse prognostic factor in FL. Methods: Consecutive patients with FL who were evaluated and treated at the Mayo Clinic, Rochester between 1998 and 2007 were considered for study participation. Study approval was granted by the Mayo Clinic Institutional Review Board. The primary objective of the study was to determine the significance of the AMC, obtained at the time of diagnosis from a standard complete blood count with differential, in predicting overall survival. The secondary study objective was to determine whether the AMC is an independently significant prognostic factor when compared with the FLIPI. Results: The median follow-up was 4.2 years (range: 2 months to 11 years) for the 355 patients included in this study. At diagnosis, 43%, 34% and approximately 23% of patients were classified as low-, intermediate- or high-risk by the FLIPI. The median AMC at diagnosis was 510/μL, with 8% of patients presenting with an absolute monocytosis. At diagnosis, 45% of patients were initially observed, 42% received combination chemotherapy or immunochemotherapy alone, while the remaining 13% underwent surgery or radiation. The AMC was evaluated as both a continuous and dichotomized variable on univariate analysis. The optimal cut-point for the AMC was determined to be 570/μL by ROC analysis and was used when analyzing the AMC as a dichotomized variable. A relative elevation in the AMC was associated with inferior overall survival, whether analyzed as a continuous or dichotomized variable, with hazard ratios (HR) of 1.002 (HR per unit change in AMC; 95% confidence interval 1.001–1.002; p=0.0002) and 3.4 (95% confidence interval 2.0–6.0; p<0.0001), respectively. The median OS for patients with an AMC <570/μL at diagnosis has not been reached, whereas a median OS of 10.2 years (95% confidence interval 7.8–11.2 years; p<0.0001) was observed for those patients with an AMC ≥570/μL. For comparison, median OS has not been reached for high-risk patients identified by the FLIPI. Among those patients who were initially observed, the median time to progression (TTP) was 31 months (95% confidence interval 25–37 months) for those with an AMC <570/μL at diagnosis. In contrast, patients with an AMC ≥570/μL experienced a significantly shorter TTP following initial observation, with a median TTP of 18 months (95% confidence interval 8–28 months; p=0.03). When adjusting for prognostic factors included in the FLIPI on multivariate analysis, the association between the AMC as a dichotomized variable and survival remained significant with a HR of 2.6 (95% confidence interval 1.5–4.6; p=0.001). When the FLIPI alone (high vs. low/intermediate) was analyzed on multivariate analysis, the AMC remained an independent predictor of survival (p<0.0001). Furthermore, the AMC provided additional prognostic information when superimposed upon the FLIPI. For example, a subset of patients classified as high-risk by the FLIPI were observed to have an OS comparable to low/intermediate risk patients identified by the FLIPI upon further risk-stratification by the AMC, with a 6-year OS of 80%. In contrast, high-risk FLIPI patients with an AMC ≥570/uL were observed to have a median OS of 4.2 years (95% confidence interval 2.7–7.2) and 6-year OS of 47%. Conclusions: The AMC is associated with overall survival in FL and is independent from the FLIPI on multivariate analysis. While the AMC did not appear inferior to the FLIPI in identifying high-risk patients, it may be most helpful when used in conjunction with the FLIPI. These results further support the central role of nonneoplastic myeloid-lineage cells in FL biology. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 880 Pulmonary marginal zone lymphoma is a distinct subgroup of B-cell non-Hodgkin's lymphomas originating from the bronchial-associated lymphoid tissue (BALT) and characterized by an indolent clinical course. Here we present the data of the IELSG-28 study of the International Extranodal Lymphoma Study Group (IELSG) on primary pulmonary lymphoma patients. Clinical records and histological material of 186 patients diagnosed with pulmonary marginal zone lymphoma from December 1986 to June 2009 were collected. The pathology was reviewed centrally and the diagnosis was confirmed in 180 (96%) patients. The median age at diagnosis was 62 (30 – 88). The majority of the patients (149/180) had localized disease at diagnosis with a good performance status (0–1) and low prognostic index (IPI 0–2). Eighty-one patients (45%) received local treatment, including surgery (74%) or radiotherapy (4%). Ninety-nine patients with advanced stage disease or incomplete surgical resection received an additional systemic treatment. Most of these patients (66; 66%) received an alkylating containing regimen, 22 (22%) patients were treated with an anthracycline containing regimen and 29 (29%) received the anti-CD20 monoclonal antibody in combination with chemotherapy. With a median follow up of 67 (range 1–256) months, 47/180 patients (26%) experienced disease progression. (median time to progression 30 months, range 0–109). Progression free survival analysis showed a trend in favour of patients treated with immuno-chemotherapy versus those receiving chemotherapy alone (HR 0.53–95%CI: 0.21, 1.33), while no difference in terms of OS and PFS was observed between patients receiving systemic anthracycline or alkylating containing regimens. In twenty five cases a specific PCR-assay was carried out for detecting the presence of Achromobacter (Alcaligenes) xylosoxidans. The presence of Achromobacter was detected in 50% of patients. Our results confirm that local therapy (surgery or radiotherapy) is the treatment of choice for patients with localized disease. Chemotherapy has to be considered in case of relapse, incomplete surgical excision or advanced disease. In this setting, alkylating containing regimens plus rituximab is the most effective option. Further studies are ongoing to confirm the relevance of infection/colonization with Achromobacter (Alcaligenes) xylosoxidans for pathogenesis of BALT lymphoma. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2836 Monoclonal antibodies (mAb) are an effective but non-curative treatment of CLL. Although the mechanisms of action of mAb in vivo are not fully defined, complement dependent cytotoxicity (CDC) appears to play an important role. We have previously shown that addition of ofatumumab (OFA) significantly increases alemtuzumab (ALM) CDC in vitro and identified a subpopulation of CLL cells that are intrinsically resistant to activated complement. We propose that resistance to CDC could be an important cause of mAb treatment failure in CLL patients. To test the hypothesis that CLL cells treated in vivo with OFA would be resistant to subsequent OFA retreatment in vitro, we studied CLL cells and serum from 10 previously untreated patients with progressive CLL treated with pentostatin, cyclophosphamide, and ofatumumab (PCO). Methods. Patients received 300 mg of OFA followed by 2 mg/m2 of pentostatin and 600 mg/m2 of cyclophosphamide on day 1 of therapy. Samples were taken prior to treatment (S1), immediately after the OFA infusion and prior to chemotherapy (S2), and post-chemotherapy before the second dose of OFA on day 2 (S3). Mononuclear blood cells isolated from EDTA anticoagulated blood by Ficoll-Paque centrifugation were purified to achieve a concentration of > 90% CLL. CDC was assayed at a concentration of 2 × 106/ml CLL cells with 10 mg/ml of ALM (Genzyme), OFA (G.S.K.) or rituximab (RTX)(Genentech) in AIM V medium (Invitrogen, CA) and 10% normal human serum (10%NHS)(Sigma, MO) as a source of complement for 1 hour at 37°C. Absolute viable cell counts were measured by flow cytometry using counting beads (Trucount, BD, CA) and propidium iodide staining (Sigma, MO) with a FACSCalibur (BD, CA) and CellQuest Pro software (BD, CA). Percent CDC was calculated relative to counts for CLL cells treated with only 10%NHS. Binding of mAb and C3b to CLL cells was measured by flow cytometry with mouse anti-human Fc antibody FITC-HB43 and anti-C3b antibody FITC-7C12. C5 deficient serum (C5-serum)(Sigma, MO) was used to examine mAb and C3b binding without CDC. Results were expressed as delta mean fluorescent intensity (dMFI) relative to cells treated without mAb in 10%NHS or C5-serum as appropriate. Complement (CH50) was measured based on lysis of opsonized sheep red cells using standard methods. OFA concentrations in serum samples were determined by measuring the level of binding to Daudi cells compared to standards as previously described. Results: S1 CLL cells were significantly more susceptible to in vitro OFA CDC (median 30%) than S2 and S3 cells (both 0%, p=0.002)(Fig. 1). In contrast, high levels of CDC (median 86 – 88%) were induced in vitro by ALM in all these samples and prior in vivo exposure to OFA did not affect susceptibility to ALM CDC in vitro (p>0.1). CLL cells from S2&S3 had low levels of OFA binding both prior to and post in vitro exposure to OFA (median dMFI 2 and 3 respectively) which was significantly less than OFA binding for S1 (median dMFI 38 p=0.002) likely reflecting in vivo trogocytosis of bound OFA and CD20, as previously described for RTX. In vitro ALM binding was higher than OFA (median dMFI>150) in all specimens. C3b binding was low in S2&S3 both before and after in vitro OFA exposure (median dMFI 3 and 4 respectively) with significantly higher levels in S1 (median dMFI 94, p=0.002). C3b binding was higher in all CLL cells treated with ALM (median dMFI>358). OFA treatment resulted in marked decreases in serum complement levels in S2 (median 86%, range 65–98%) and S3 (median 78% range 61–88%) compared to S1. The median serum OFA concentration was 18.5 μg/ml (range 7.3–50.4) in S2 and 19.8 μg/ml (range 0–32.3) in S3. Conclusions: Circulating CLL cells from patients treated with OFA are resistant to in vitro OFA CDC primarily because of low levels of expression of CD20. The reduction in complement titers could limit in vivo CDC but we found no evidence that low levels of OFA were important. Cells surviving OFA in vivo do retain sensitivity to ALM CDC. Our data support the previous description of trogocytosis in CLL patients treated with RTX and suggest that lower doses of these mAb, that promote far lower levels of trogocytosis, could be more effective in sustaining CDC. In addition, our study provides pre-clinical data to support a clinical trial of combination therapy with OFA and ALM for CLL. Acknowledgment: This study was supported by funding from GlaxoSmithKline and the University of Iowa/Mayo Clinic Lymphoma SPORE (CA097274). Disclosures: Taylor: Genmab: Consultancy; Glaxo Smith Kline: Research Funding. Zent:GlaxoSmithKline: Research Funding; Genentech: Research Funding; Genzyme: Research Funding.

Abstract It is believed that malignant cells “condition” the microenvironment to facilitate tumor cell survival. We hypothesized that crosstalk between CLL B-cells and marrow stromal cells impacts both cell types bi-directionally and ultimately contributes to leukemic cell apoptotic resistance. To test this hypotheses, bone marrow stromal cells from core bone biopsies from CLL patients were isolated and cultured using methods we have previously described (Leuk Res 2007 31(7):899). Subsequently, we determined the impact of co-culture on CLL B-cell features including apoptosis and CD38 expression. In addition, we evaluated the release of angiogenic cytokines on co-culture and signal events in the stromal cells. Immunophenotyping demonstrated that cultured bone biopsy derived stromal cells were CD73+, CD105+, CD146+, CD14−, CD45−, CD34−, HLA-DR-, suggesting they were mesenchymal stem cells (MSC). Co-culture of these MSC with CLL B-cells protected CLL B-cells from both spontaneous apoptosis (SA) and drug-induced (fludarabine and chlorambucil) apoptosis (DA). For SA, the mean survival of CLL B-cells with or without co-culture of MSC for 5 days were 56.9 ± 10.0 and 7.7 ±3.7 (p<0.05), respectively. When CLL B cells were treated with fludarabine or chlorambucil, the fraction of CLL cells tightly adherent to MSC (TA-CLL) showed higher survival than a less adherent but viable fraction of CLL B-cells. The mean survival of TA-CLL cells treated with 10 μM of fludarabine for 48 hours in the presence of MSC were 67.5 ± 3.6 vs 29.8 ± 11.1 without MSC (P<0.05), respectively. When CLL cells with evidence for CD38 expression were co-cultured with MSC, both the percentage of CD38 positive cells and level of expression of CD38 per cell were up-regulated (mean fold change: CD38 percentage, 2.7, p<0.05; CD38 MFI, 1.9, p<0.05) after 2 weeks. In contrast, the CD38 percentage and expression were not changed in cells with minimal CD38 expression when these CLL B-cells were co-cultured with MSC. In addition, co-culture of MSC with CLL cells induced rapid ERK and AKT phosphorylation (within 30 min) in the MSC on immunoblot analysis. When CLL B cells and MSCs were cultured in transwells, the activation of ERK and AKT in MSC occurred at similar levels, indicating that activation of MSC was mediated by soluble factors. In addition, co-culture led to increased secretion of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) as well as a decrease of thrombospondin-1 (TSP-1) in the culture medium. These findings confirm that co-culture of CLL B-cells and MSC culminates in “angiogenic switch.” Taken together, these results strongly suggest interactions between MSC and CLL B cells are a bi-directional process. In leukemic cells, the interaction not only protects against spontaneous and drug induced apoptosis but also leads to an increase in CD38 expression consistent with an activated status. In MSC, the interaction leads to activation of ERK and AKT. Co-culture also facilitates angiogenic switching. These results underscore the dynamic and complex nature of the interactions between bone marrow stromal cells and CLL B-cells. Further studies are needed to dissect how crosstalk between CLL B-cells and MSC relates to disease progression, and determines whether these interactions can be targeted with therapeutic intent.

Abstract The interactions between leukemic cells and the bone marrow microenvironment play a critical role in angiogenesis, disease progression and cell adhesion mediated drug resistance (CAM-DR). VLA-4 (CD49d) is one of the most important adhesion receptors involved in these interactions. The binding of VLA-4 to fibronectin was shown to protect CLL B-cell from apoptosis (de la Fuente, 1999). The introduction of monoclonal antibodies targeting VLA-4 provides the opportunity for the development of novel treatment strategies. We examined the ability of VLA-4 blocking antibodies to induce apoptosis and overcome CAM-DR of primary B cells from CLL patients. Methods: Blood samples were obtained from consenting CLL patients. Mononuclear cells were isolated by density gradient centrifugation. Only samples with over 80% CLL B cells as assessed by flow cytometry (minimum 80% CD19+/CD5+ cells) were used. HS-5 human stromal cell line was cultured in 24 well plates (10% FCS DMEM media) and grown until they reached confluence. The confluent cells were then washed with PBS and incubated with AIM-V media 24 hours before CLL cells were co-cultured. CLL B cells were incubated in AIM-V media with and without fludarabine (1 mmol/L) for 24 hours, spun and resuspended in AIM-V. CLL B cells were then incubated with or without anti VLA-4 antibodies (BD Biosciences) at 10 μg/106 CLL cells for 2 hours and directly added into a co-culture with HS-5 cells. The CLL B cells remained in the co-culture for 24 hours, and were then collected and analyzed by flow cytometry for apoptosis/cell death by annexin/propidium iodide assay (i.e., total culture time 48 hours). Paired T-test was used to compare B cell viability results. Results: After 48 hour culture, the viability cells cultured in media alone or fludarabine for the first 24 hours were 40.6% (18.2%–73.7%) and 8.5% (0.2%–17.9%) respectively. Significant rescue from fludarabine induced cell death was seen if CLL B cells were co-cultured with HS-5 stromal cells for the second 24 hours rather than media alone (86.5% vs. 8.5%; p<0.001). The addition of anti VLA-4 antibodies, reduced stromal cell rescue from fludarabine induced apoptosis by 30% (61.5% vs. 86.5%; p= 0.0053). The addition of VLA-4 antibodies to untreated CLL B cells cultured with stromal cells had no effect on cell viability (90.2% vs. 91.2 p=0.59). Conclusion: Stromal cell adhesion rescues CLL B-cells from fludarabine induced apoptosis. Stromal cell rescue of CLL B-cells from drug induced apoptosis can be significantly reduced by blocking VLA-4 mediated cell adhesion of CLL B-cells to stromal cells. Blocking VLA-4 has no effect on the survival of untreated CLL B cells cultured with stromal cells suggesting the interaction mediated by VLA-4 protects against drug induced cell death rather than directly inducing apoptosis. These findings suggest anti VLA-4 antibodies are unlikely to be useful as a single agent therapy for treatment of CLL. However, blocking VLA-4 may enhance the efficacy of other agents used to treat CLL by overcoming adhesion mediated protection against drug induced cell death.