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Multicellular organisms remove damaged or superfluous cells through a highly regulated cellular process known as programmed cell death. There are two main forms of programmed cell death, apoptosis and necrosis. Necrosis (necroptosis) previously thought to be an unregulated death pathway was recently found to be highly regulated. The manner by which a cell dies has important implications. In apoptotic cell death, caspases digest the cell to cause implosion in an immunologically silent process. In necroptotic cell death, increased Rip kinase signaling effects rupture of the plasma membrane, cellular explosion, and the activation of an inflammatory response. Death receptors, such as the TNFα receptor, can activate either apoptotic or necroptotic death. The upstream activators and transducers including Caspase-8, Rip1, and Fadd, are common to both forms of cell death. Interestingly, Caspase-8 and c-FlipL, a caspase homolog, were recently shown to inhibit the necrotic pathway during embryonic development through the formation of a catalytically active complex. The BH3-only Bcl-2 family member, Bid is one of the strongest substrates of Caspase-8, placing it at the interface of the apoptotic and necroptotic pathways, and in position to mediate cell death fate. The role of apoptosis in hematopoietic homeostasis has been well characterized. We developed a mouse model of unrestrained necroptosis in order to determine how unrestrained necroptosis impacts hematopoietic homeostasis and bone marrow function. To do this we generated a mouse model in which apoptosis is prevented by the deletion of the pro-apoptotic effectors Bax and Bak. We further deleted the upstream activator Bid (VavBaxBakBid TKO mice). Surprisingly, these mice die of bone marrow failure due to unrestrained necroptotic cell death. TKO bone marrow displays necroptotic cells by electron microscopy, and markedly increased Rip1 expression by immunofluorescence. TKO mice die of bone marrow failure with marked myeloid dysplasia between the age of 3 and 12 months, and a small number develop leukemia, a phenotype that closely resembles MDS. Further analysis revealed expansion and increased BrdU incorporation of the SLAM-HSC population, consistent with increased HSC proliferation in response to death of more mature cells. To assess function of these HSCs, we performed competitive reconstitution assays. TKO bone marrow initially outcompetes WT bone marrow, but the mice eventually succumb to bone marrow failure beginning at 5 months post–transplantation, despite the presence of ~10-15% wild type bone marrow. These results demonstrate that increased necroptotic signaling results in a cell autonomous stem cell defect. In addition, the presence of necroptotic bone marrow also kills normal HSCs in a non cell-autonomous manner, due to a feed-forward inflammatory process. To further characterize how necroptotic cell death is regulated, we developed myeloid progenitor cell lines (MPCs) from the bone marrow of WT, Bid KO, BaxBak DKO, and BaxBakBid TKO mice to facilitate biochemical and mechanistic studies. Our studies demonstrated increased activation (phosphorylation) and markedly increased levels of Rip1 in the pronecrotic complex (Complex II) with Rip3, Caspase-8, and Fadd in our TKO MPCs following LPS treatment. This association of Rip1 with Complex II is abrogated by reintroduction of Bid by retrovirus into TKO MPCs, demonstrating that Bid inhibits Rip1 association with complex II, suggesting that Bid is a key factor that determines cell death fate. Increased bone marrow cell death is well documented in MDS. To determine if necroptosis plays a role in this bone marrow cell death, we evaluated RIP1 and Caspase 3 expression in 17 human MDS samples. Remarkably, we found increased RIP1 expression, but not activated caspase 3 in bone marrow samples from patients with the RCMD, RAEB-1, and RAEB-2 subtype of MDS, but not in 4 control bone marrow samples (normal lymphoma staging marrows). Our study thus demonstrates that increased necroptosis signaling can result in bone marrow failure with dysplasia, and that necroptotic cell death signaling is increased in bone marrow from MDS patients, highlighting the potential importance of this targetable signaling pathway in bone marrow failure disorders such as MDS. Disclosures No relevant conflicts of interest to declare.

Abstract 2687 Background: Patients with early stage (Stages I, II) diffuse large B cell (DLBCL) have been traditionally treated with either CHOP x6 alone or with CHOP followed by involved field external beam radiotherapy (IFRT). With the demonstration of the superiority of rituximab CHOP (RCHOP) in advanced stage DLBCL, RCHOP has been adopted as the chemoimmunotherapy standard of care in early stage disease with or without IFRT. Radioimmunotherapy (RIT) has the advantage of delivering high-energy, short path length radiation in a targeted approach using an anti-CD20 antibody as the carrier. Thus, radiation is potentially targeted to microscopic sites outside of known disease. Goals: To evaluate the complete response (CR) rate and functional CR rate (CR or CRu/PR and PET negative) to a treatment regimen of RCHOP followed by 90Y-ibritumomab tiuxetan (Zevalin, Spectrum Pharmaceuticals) radioimmunotherapy (RIT); only patients not achieving PET negative status after RIT were to be given IFRT. The study goal was to achieve a functional CR rate of ≥75%. Patients and Methods: Eligible patients were those with new, untreated Stages I or II DLBCL as determined by standard CT scanning, adequate blood counts, creatinine and bilirubin 45%, and a performance status 0–2. Stage I patients were required to have ≥1 adverse risk factor (≥60 years, bulky disease, elevated LDH or PS2). Patients received standard RCHOP21 × 2 cycles and then were restaged. Patients in CR received 2 additional cycles; those in PR or CRu received 4 additional cycles. At end of RCHOP a PET scan was repeated and centrally reviewed; those with a PR or functional CR proceeded to Zevalin RIT 0.4 mCi/kg (maximum 32 mCi) within 12 weeks of the last RCHOP. Patients were restaged 12 weeks post-RIT; PET negative patients were observed without maintenance; PET positive patients received 30Gy involved field radiation and then were restaged. Descriptive statistics were used to summarize the patient characteristics, treatment and response. The Kaplan-Meier method was used to estimate failure rates. Results: Between Dec 2004 and Nov 2008, 62 patients were enrolled; after pathology review 53 were considered eligible. 42% (22/53) patients were stage I/IE; 58% (31/53) were stage II/IIE. 57% (30/53) received 6 cycles of RCHOP; 40% (21/53) received 4 cycles; and 2 patients received 1 and 2 cycles, respectively. After immunochemotherapy, 79% (42/53) were in functional CR and 19% (10/53) PR; 1 was unevaluable. Of the 53 eligible patients who completed RCHOP therapy 91% (48/53) proceeded to RIT. After RIT, 87% (46/53; 95% CI: 75–95) were in CR/CRu and 89% (47/53; 95% CI:77–96%) were in functional CR. One patient proceeded to IFRT. The median follow-up is now 4.3 years. Seven (13%) of patients have had tumor progression but only 2 have died (1 of disease). At 4 years, 88% of patients remain progression free and 98% remain alive (Fig 1,2). The median has not been reached for either PFS or OS. There has been one case of myelodysplastic syndrome. Conclusions: Combination immunochemotherapy and RIT is an active regimen for patients with early stage DLBCL. Nearly all patients will achieve functional CR without the requirement of IFRT. The regimen of RCHOP and RIT for early stage DLBCL is worthy of further study. Disclosures: Witzig: Spectrum: Membership on an entity's Board of Directors or advisory committees, Research Funding, uncompensated Other. Off Label Use: Use of Zevalin in treating patients with Stage I-II DLBCL. Kahl:Roche: Consultancy; Genentech: Consultancy, Research Funding. Horning:Genentech: Employment; Roche: Equity Ownership.

Abstract 1603 Background: We have previously reported similar outcomes for patients with bulky stage I or II mediastinal HL treated with combined modality therapy either with ABVD + radiotherapy or the Stanford V regimen in the North American Intergroup trial E2496 (Advani et al, ASH 2010 abstract 416). In the current analysis, we compare the patterns of failure between the two groups. Methods: Patients with stage I/II bulky mediastinal HL (maximum mediastinal mass width > 1/3 of intrathoracic diameter) were randomized to receive chemotherapy (CT) on either Arm A (ABVD x 6–8 cycles administered q 28 days) or Arm B (12 weeks of Stanford V administered weekly). Two-3 weeks after completion of chemotherapy all patients received modified involved field radiotherapy (RT) (36 Gy) delivered to the mediastinum, hila, and supraclavicular regions. Patients on Stanford V arm also received involved field RT to any other sites ≥ 5 cm at diagnosis. Patients were assessed 3, 6 and 12 months after completing RT with computed tomography scanning and then every 6 months for 5 years. The primary end points were failure free survival (FFS) and overall survival (OS). Disease progression was defined as 'in-field', 'distant' or both relative to the radiation fields prescribed in the E2496 protocol. Distant sites of failure were further characterized as intra-thoracic, intra-abdominal or other (bone, bone marrow and axillae, if not previously irradiated). Results: Two hundred and sixty-seven patients were randomized: 136 on the ABVD arm and 131 on the Stanford V arm. Patient characteristics were well matched with no differences between two arms in overall response rates (ORR), FFS and OS. (Advani et al ASH 2010 abstract 416). At a median follow up of 5.5 years 40 patients have relapsed with no difference in ABVD (n=18, 13%) versus Stanford V (n=22, 17%) (p=0.49). Central review of RT fields available in 37/40 patients found major violations with under treatment of tumor noted in 7/37 (19%). Patterns of failure are shown in Table 1. There were no differences in patterns of relapse for the two study arms. In-field relapses occurred in Conclusion: For patients with stage I/II bulky mediastinal HL, combined modality therapy with either ABVD +RT or the Stanford V regimen results in excellent disease control. In-field relapse was uncommon. These results set a benchmark for assessing ongoing trials omitting RT in patients with stage I/II bulky mediastinal HL. Future research efforts should focus on risk stratification to identify the small subset of patients who are likely to fail standard upfront therapy. US cooperative group efforts in this subset of patients are ongoing that use interim PET-CT imaging based risk-adapted strategies. Disclosures: Horning: Genentech: Employment, Equity Ownership.

Abstract 416 Background: Standard therapy for locally extensive Hodgkin Lymphoma (HL) defined as stage I/II with bulky mediastinal disease [mediastinal mass ratio (MMR) > than 1/3 of the chest diameter on standing postero-anterior chest x-ray or 10 cm on computerized tomography] is combined modality therapy (CMT). E2496 was a North American intergroup, randomized phase III study comparing ABVD versus the Stanford V regimen for patients with locally extensive and advanced stage HL. In this subgroup analysis we compare two CMT approaches, ABVD + radiotherapy (RT) and the Stanford V regimen, in patients with stage I/II bulky mediastinal HL. Methods: Patients with stage I/II HL bulky mediastinal disease were randomized to receive chemotherapy (CT) on either Arm A (ABVD × 6–8 cycles administered q 28 days) or Arm B (12 weeks of Stanford V, administered weekly). Two-3 weeks after completion of chemotherapy, modified involved field RT was delivered at 36 Gy to the mediastinum to patients on ABVD as well as Stanford V. Patients on Stanford V also received involved field RT to any other sites >5 cm at diagnosis. The primary endpoint was failure free survival (FFS) defined as the time to either progression/relapse or death. The log-rank test was used to compare FFS and OS (overall survival) for all eligible patients. Results: Of the 812 eligible patients with advanced HL enrolled on the study 267 had locally advanced bulky mediastinal disease: 136 on ABVD and 131 on Stanford V. Patient characteristics between the two randomized groups were well matched with a median age of 30 years and 86% had stage II disease. On ABVD 61% of patients received 6 cycles, 29% 8 cycles of CT and 77% required some dose modification. In Stanford V 97% completed the assigned 12 weeks of CT and 76% required some dose modification. 82% received RT per protocol in ABVD versus 88% in Stanford V. The overall response (CR+PR) was 82% in ABVD and 86% in Stanford V. At a median follow-up of 5.47 years, there are 19 failures and 6 deaths in the ABVD group and 25 failures with 9 deaths in the Stanford V group. We failed to detect statistically significant differences between ABVD +RT and Stanford V for FFS (5y 85% versus 77% p=0.13, HR=1.56 95% CI (0.87, 2.88) and OS (5y 95% versus 92% p=0.31, HR=1.69 95% CI (0.60, 4.75). No difference in hematologic toxicity was observed between the two arms. Evaluation of pulmonary function and patterns of failure are pending. Conclusions: For stage I/II patients with bulky mediastinal disease, CMT with either ABVD +RT or the Stanford V regimen results in high cure rates and is highly effective. ABVD for 6–8 cycles plus 36 Gy RT remains the US standard of care. Longer follow up is required to assess RT related late effects. Future research efforts should focus on risk stratification to identify; a) the 15–20% of patients destined to relapse after standard therapy and evaluate treatment intensification strategies and b) the 80–85% of patients who are cured with standard therapy and determine whether a subset can achieve the same excellent outcomes with a reduction or elimination of radiation. Planned US cooperative group trials will address these questions, using ABVD as the chemotherapy backbone in conjunction with interim PET imaging for risk stratification. Disclosures: Blum: Seattle Genetics: Research Funding; Novartis: Research Funding; Celgene: Research Funding. Horning:Genentech: Employment.

Abstract 415 Background: The ability to cure patients (pts) with advanced Hodgkin's Lymphoma (HL) with combination chemotherapy (CC) (MOPP and variants, ABVD and variants) represented a major milestone in oncology research, and CC became a paradigm for other malignancies. Further, the rationale for combined modality therapy (CMT) (radiation (RT) and CC) in HL evolved based on the high frequency of relapse in initially involved sites. As response rates and survival improved, newer treatments such as the combined modality Stanford V regimen were developed to shorten the duration of chemotherapy, add RT to sites of disease and reduce toxicity while maintaining or improving the cure rate. Indeed, the Stanford V regimen was tested and validated in a Phase II co-operative group trial (E1492) (J Clin Oncol 2000; 18:972). In order to investigate this approach against “standard” therapy, we conducted a randomized Phase III Intergroup trial of ABVD vs. the Stanford V regimen for patients with locally extensive or advanced HL. Objectives: The trial was designed to detect a 33% reduction in the failure free survival (FFS) hazard rate with Stanford V compared with ABVD, which corresponds to a difference in five-year FFS of 64% vs. 74%. Method: Patients with locally extensive (defined as clinical Ann Arbor Stage I-IIA/B and bulky mediastinal disease (BMD) (mass > 1/3 maximum intrathoracic diameter on standing postero-anterior chest x-ray or >/−10 cm on computerized tomography) or advanced (Ann Arbor Stage III or IV) HL were randomized to receive either ABVD × 6–8 cycles (C) (51% had 6 C, 35% had 8 C, 14% had 5cm or for macroscopic splenic disease). The log-rank test was used to compare FFS for all eligible patients stratified on extent of disease (locally extensive vs. advanced), and number of International Prognostic Factor Project (IPFP) risk factors (0–2 vs. 3–7). An extended Cox model was also used to address non-proportional hazard between the two arms. Results: 854 pts enrolled from April, 1999 to June, 2006 and 812 were eligible for analysis. 404 pts were randomized to ABVD and 408 to Stanford V. Median age was 33 yrs in both arms (range 16–83). 53% were men and 47% women; 4% had Stage I, 31% had Stage II, 39% had Stage III and 25% had Stage IV disease by Ann Arbor criteria. 35% of pts on ABVD and 35% on Stanford V had BMD. Three % of pts had nodular lymphocyte predominant HL, 77% of pts had nodular sclerosis HL, 14% had mixed cell HL. Age, stage, pathology and risk factors (0–2 vs. 3–7) were similar in both arms. In total, 65% were IPFP score 0–2 and 33% were 3–6. Response rate. There was no difference in response rates (RR) between the two arms (ABVD=72% CR+ CCR, 7.7% PR, 7.9% SD; Stanford V= 69 % CR +CCR, 7% PR and 10 % SD. 8% were not evaluable for response on ABVD and 9% on Stanford V. Toxicity was similar in both groups. The most frequent Grade 3 + 4 toxicity was neutropenia, and was similar between the 2 groups (76% Grade 3 + 4 in ABVD and 70% Grade 3+ 4 in Stanford V). Grade 5 toxicity was Conclusion: In the largest Phase III intergroup trial of HL in North America, there was no significant difference in RR, FFS, OS, and 5-year toxicity when ABVD (+ RT for BMD) is compared with Stanford V (+RT for nodal sites >5 cm and macroscopic splenic disease). There was more Grade 3 lymphopenia (p< 0.0001) and more Grade 3 + 4 sensory neuropathy (p< 0.0001) on Stanford V. Thus ABVD (plus RT for BMD) remains the standard of care because Stanford V did not meet the objective of 33% improvement in FFS. For some patients, Stanford V, when given as described with RT, remains an acceptable alternative. Disclosures: Friedberg: Genentech: Honoraria. Blum:Seattle Genetics: Research Funding; Novartis: Research Funding; Celgene: Research Funding. Horning:Genentech: Employment.