BackgroundAdministration of recombinant G-CSF following cytoreductive therapy enhances the recovery of myeloid cells, minimizing the risk of opportunistic infection. Free G-CSF, however, is expensive, exhibits a short half-life, and has poor biological activity in vivo.MethodsWe evaluated whether the biological activity of G-CSF could be improved by pre-association with anti-G-CSF mAb prior to injection into mice.ResultsWe find that the efficacy of G-CSF therapy can be enhanced more than 100-fold by pre-association of G-CSF with an anti-G-CSF monoclonal antibody (mAb). Compared with G-CSF alone, administration of G-CSF/anti-G-CSF mAb complexes induced the potent expansion of CD11b+Gr-1+ myeloid cells in mice with or without concomitant cytoreductive treatment including radiation or chemotherapy. Despite driving the dramatic expansion of myeloid cells, in vivo antigen-specific CD8+ T cell immune responses were not compromised. Furthermore, injection of G-CSF/anti-G-CSF mAb complexes heightened protective immunity to bacterial infection. As a measure of clinical value, we also found that antibody complexes improved G-CSF biological activity much more significantly than pegylation.ConclusionsOur findings provide the first evidence that antibody cytokine complexes can effectively expand myeloid cells, and furthermore, that G-CSF/anti-G-CSF mAb complexes may provide an improved method for the administration of recombinant G-CSF.

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Although PD-1 blockade has revolutionized cancer immunotherapy, immune-related adverse events (irAEs) present life-threatening complications. Recent reports of aplastic anemia (AA) as irAEs implicate PD-1/PD-L1 as important in preventing immune-mediated destruction of the hematopoietic niche. Infusion of PD-1-deficient (PD-1 knockout [KO]) lymph node (LN) cells into minor-antigen mismatched mice resulted in early mortality, as well as more severe bone marrow (BM) hypoplasia, anemia, and BM microarchitecture disruption in PD-1 KO LN-infused mice relative to mice that received B6 LN cell infusion. Mice that received PD-1 KO LN cells had more CD8 T-cell infiltration of the BM and greater expansion of H60-specific CD8 T cells than did their B6 LN-infused counterparts. In the spleen, CD8 T cells were skewed to an effector memory phenotype, suggesting accelerated differentiation of PD-1 KO T cells. Our data suggest that PD-1 dysregulation has a role in murine BM failure and vigilance in irAE monitoring may be desirable to treat early AA and related cytopenias.

4638 Thrombotic thrombocytopenic purpura (TTP), a syndrome characterized by thrombocytopenia and microangiopathic hemolytic anemia due to deficiency of the von Willebrand factor cleaving protease ADAMTS13, was once a highly fatal condition, but therapeutic plasma exchange (PEX) has dramatically improved outcomes. We report the results of a retrospective chart review of TTP patients treated with PEX at our institution from September 2006 through December 2011. Complete demographic, clinical, laboratory, treatment, and response data were collected to examine the influence of race on clinical outcomes. A total of 49 patients were treated for TTP. This cohort was predominantly female (71%) and African American (61%). Three patients were of Hispanic ethnicity. The median age was 50 years (14–81), and the median number of comorbidities at presentation was 4 (range 0–14). Assays for ADAMTS13 activity with reflex to ADAMTS13 inhibitor were performed by the Blood Center of Wisconsin. All patients were treated with PEX (1–1.5 plasma volume using fresh frozen plasma as replacement fluid) once daily until plateau in the platelet count, and then PEX frequency was tapered over 2 weeks in remitting patients. The median number of PEX procedures for the first episode was 8 (1–22). The median total PEX for all episodes was 9 (1–56). The majority of patients, 37 (75.5%), received corticosteroids, and rituximab was administered to 19 (38.8%) of patients at some point during their treatment, usually for slow response or relapse. An initial response to PEX, defined by normalization of platelet count (above 140×109/L) and resolution of signs of hemolysis, was achieved in 36/49 (73.5%) patients. The response rate was not significantly different between African Americans (73.3%) and Caucasians (68.8%, p=0.742), and all 3 Hispanic patients responded to initial treatment. Pre-treatment ADAMTS13 activity was severely deficient (<10% of normal) in 20/39 (51.3%) patients. An inhibitor of ADAMTS 13 was detected (>0.4 Inhibitor Units) in 19/23 (82.6%) patients tested. African Americans were more likely to have severely deficient ADAMTS13 activity (16/25, 64%) than Caucasians (2/13, 15.4%; p=0.004). The initial response rate was higher in patients with severely deficient ADAMTS 13 activity (90.0%) in comparison to those with >10% activity (68.4%), however the difference did not reach statistical significance (p=0.067). Of the patients with severely deficient ADAMTS 13 activity and detectable inhibitor levels, 89.5% had an initial response to PEX. All four patients with severely deficient ADAMTS 13 activity but undetectable inhibitor levels also responded to PEX. The total number of relapse events was 16 (32.7%), and the median time to relapse was 19.4 months. All three Hispanic patients, 4 (25%) Caucasians, and 9 (30%) African Americans relapsed. One-year relapse free survival was similar among Caucasians and African Americans at 72% and 75% respectively (p=0.852). All three Hispanic patients relapsed within the first year. One-year overall survival was similar for Caucasians and African Americans at 81.3% and 83.3% respectively (p=0.968). A total of 10 deaths were documented. The causes of death were relapse of TTP (3/10), sepsis (4/10), GI bleeding (2/10), and unknown etiology (1/10). In this unusual TTP cohort with 61% African Americans, we found that African Americans were more likely to have severely deficient ADAMTS13 activity than Caucasians, but response to PEX, relapse, and survival for Caucasian and African Americans were very similar. This study also supports pre-treatment assays for ADAMTS13 activity and its inhibitor, not only for the diagnosis of TTP, but also for predicting response to PEX therapy. Disclosures: No relevant conflicts of interest to declare.

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5041 Background: Novel biological agents, particularly bortezomib (B) and the immunomodulatory agents thalidomide (T) and lenalidomide (L), have improved the response rates and the survival in multiple myeloma (MM). However, patients refractory to, or progressing after, treatment with one or more new agents have a very poor prognosis. Alkylating agents are often avoided in the initial management of MM patients who are eligible for autologous hematopoietic stem cell (HSC) transplantation due to concerns for impairment of HSC mobilization. We hypothesized that high doses of cyclophosphamide (HiCy) administered without HSC support may be an effective and safe treatment to rescue patients with relapsed or refractory MM previously treated with novel biological agents. Methods: We performed a retrospective single institution review of all recent patients receiving high dose cyclophosphamide (3000mg/m2) after failure of one or more new biological agents. Data extracted included patient demographics, disease characteristics, treatment history, toxicity, response, and survival data. This study received institutional review board approval. Results: Between 03/2009 and 06/2010, 11 patients were treated with cyclophosphamide 3,000mg/m2 for relapsed or refractory MM. Supportive care included administration of mesna to prevent hemorrhagic cystitis and PEG-filgrastim to minimize the duration and depth of neutropenia. All patients received antibiotic prophylaxis with ciprofloxacin, acyclovir, and fluconazole. The median age of patients was 52 years (range 26–73). Among these, 7 patients (64%) had stage 2 (international staging system), and 4 patients (36%) had stage 3 disease. Metaphase cytogenetics and/or fluorescence in situ hybridization (FISH) was available for 10 patients. In all but one patient, MM cells had FISH abnormalities. Cytogenetic subgroups included five cases with complex karyotype, 6 cases with a deletion of chromosome 13, 3 cases with abnormalities involving 17p, 2 cases with t(11;14), 2 hyperdiploid cases, and 1 case with t(14;16). The median number of prior treatments was 3 (range 1–6) with two patients previously receiving autologous HSC transplantation. All patients were refractory to the last therapeutic regimen and had failed to respond or were refractory to a regimen containing B. Seven patients had also previously received an immunomodulatory agent (2 patients received T and 6 patients received L). At the time of treatment 5 patients had plasma cell leukemia and 5 patients had renal failure (including 2 patients on dialysis). All patients developed grade 4 neutropenia, and 9 patients developed thrombocytopenia requiring transfusion support. Six patients (55%) developed fever and neutropenia requiring intravenous antibiotics, and one patient (9%) had documented bacteremia. One patient died from sepsis 7 weeks after cyclophosphamide treatment. Median follow up was 13.4 weeks (range 4.4–69.7) from HiCy administration. Overall, 7 patients had a partial response or better (64%), including 3 patients with a very good partial response (27%). Four of the 7 responding patients subsequently underwent autologous (3) or allogeneic (1) HSC transplantation. Median overall survival was not reached, and the estimated one year survival was 52.5% (+/− 20.4%). Conclusion: High-dose cyclophosphamide can be an effective salvage therapy for young, high-risk MM patients refractory to new biological agents. HiCy is associated with significant toxicity, and careful patient selection is necessary. Disclosures: No relevant conflicts of interest to declare.