ALBERT

Description: Abstract 1004 Data from single institutional clinical studies have indicated that the content of CD34+ cells, T-cells, and dendritic cells in a bone marrow allograft are associated with clinical outcomes. The relationship between the cellular constituents of hematopoietic allografts and clinical outcomes was prospectively studied in BMT CTN 0201 in which patients with hematological malignancies (and their donors) were randomized to transplantation with bone marrow or G-CSF-mobilized peripheral blood stem cell (PB) allografts. A secondary objective of this study was to correlate graft characteristics with overall survival (OS). 551 patients were enrolled at 50 North American centers between January 2004 and September 2009 in a BMT CTN-sponsored randomized clinical trial supported by the NHLBI and NCI. Of the 278 subjects randomized to BM, aliquots from 173 BM allografts collected in North America were analyzed at a central laboratory for the content of CD34+ progenitors and immune cell subsets by flow cytometry, and of 151 of these BM transplants had complete clinical data and are the subject of this analysis. The subset of 151 patients were similar to the total group of patients transplanted in BMT CTN 0201, with a median age of 38, and 42% of patients with AML, 26% with ALL, 10% CML, and 21% MDS. 74% of patients had low risk disease, and 47% received 12 Gy TBI and cyclophosphamide with the remainder receiving myeloablative busulfan-based (48%) or fludarabine/melphalan conditioning (5%). GvHD prophylaxis was predominately a calcineurin inhibitor plus methotrexate (91%). 26.5% of patients received grafts from HLA mismatched donors. Clinical outcomes, including OS, acute GVHD III–IV and chronic GVHD, were similar between the subset of patients with analysis of BM cells and the 127 BM recipients without product analysis. 46 progenitor and immune cell subsets were selected for study based upon the absence of a strong correlation with another graft subset (Pearson or Spearman correlation 〉0.8) and a priori interest or the number of evaluable patients. Graft characteristics were then described separately for survivors and those who died and compared using a nonparametric Mann-Whitney Wilcoxon test. P-values were not adjusted for multiple comparisons, but only covariates for which the q value is or ≤ 1.3 × 10E6/kg) or Right: donor pDC precursors (〉 or ≤ 0.3 × 10E6/kg) in the BM graft. Among patients receiving unrelated donor bone marrow grafts, higher numbers of donor cell that have important activity in regulating post-transplant immunity, including an unexpected role for CD8N in reducing severe acute GvHD, are associated with improved clinical outcomes. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2960 Background: Donor cell engraftment following allogeneic bone marrow transplantation (BMT) is affected by several factors, including immunological major histocompatibility complex (MHC) barriers, the intensity of the conditioning regimen, and the content of T-cells in the graft. The current model for engraftment in allogeneic BMT is that host dendritic cells (DCs) activate donor T-cells which promote engraftment by eliminating radio-resistant cytotoxic host immune cells, especially natural killer (NK) cells and T-cells. To explore the interaction between donor T-cell and host antigen-presenting cells (APC) in engraftment in allogeneic BMT, we focused on the role of interleukin-12 (IL-12), a key cytokine produced mainly by DCs that drives the development of donor type 1 helper T cells (Th1) and type 1 cytotoxic T lymphocytes (Tc1). Methods: Radiation chimeras with 〉95% donor chimerism were created by transplanting 5 × 106 bone marrow (BM) cells from IL-12 knock out (IL-12 KO) or wild type (WT) B6 (H-2Kb, CD45.2) donors into congenic BL6 Pepboy (B6.SJL-PtprcaPep3b/BoyJ, H-2Kb, CD45.1) mice following lethal 11 Gy irradiation. A second allogeneic BMT was conducted 2 months later using MHC mismatched FVB (H-2q, CD45.1), BA.B10 (H-2Kk, CD45.2, CD90.1) or B10.BR (H-2Kk, CD45.2, CD90.2) donor cells. In vivo bioluminescent imaging (BLI) was performed to analyze the number and in vivo distribution of luciferase+ donor T-cells. The whole-body bioluminescent signal was used as a marker of the donor T cell expansion. Engraftment of donor myeloid cells was determined by flow cytometry using mAbs for specific leukocyte markers expressed on donors and recipients (CD45.1, CD45.2, H-2Kb). Intracellular cytokine expression (IL-4, IL-10, IFN-g) by donor CD4+ and CD8+ T cells was analyzed by flow cytometry. Results: WT BL6→BL6 radiation chimeras recipients showed greater expansion of luciferase+ donor T-cells compared with IL-12 KO BL6→BL6 radiation chimeras recipients and FVB→FVB syngeneic recipients at early time point (2 wks) following 9 Gy re-irradiation and transplantation of 3 × 105 luciferase+ FVB-L2G85 T-cells in combination with 5 × 106 T cell depleted (TCD) BM cells from FVB mice following (Fig 1). At 4 weeks post transplant, more WT BL6→BL6 radiation chimeras achieved myeloid engraftment than IL-12 KO BL6→BL6 radiation chimera recipients(75.0% versus 33.3% respectively, p = 0.086), and the former group had better erythroid engraftment than the latter group (RBC 8.65 ± 1.88 × 1012/L versus 5.67 ± 2.22 × 1012/L respectively, p = 0.011). However, when FVB, WT BL6→BL6 or IL-12 KO BL6→BL6 radiation chimeras recipients were conditioned with a larger dose of irradiation prior to the second transplantation (10 Gy) and received a larger dose of donor T-cells (5 × 105), both the WT BL6→BL6 and IL-12 KO BL6→BL6 radiation chimeras recipients achieved full donor engraftments (85.7% versus 87.5% respectively, p = NS). Donor T cells in allogeneic BMT recipients were Th1/Tc1 polarized, there were no differences in frequencies and total numbers of Th1/Tc1 donor CD4+ and CD8+ T cells comparing recipients of WT BL6→BL6 and IL-12 KO BL6→BL6 radiation chimeras. In spite of an increased irradiation dose and larger number of donor T-cells in the second transplant regimen, no increase in graft versus host disease (GVHD) clinical scores and GVHD-mortality were observed in the recipients of WT BL6→BL6 radiation chimeras compared with recipients of IL-12 KO BL6→BL6 radiation chimeras. Conclusion: These data support a role for host BM-derived IL-12 in facilitating engraftment in allogeneic BMT following a reduced dose (9 Gy) radiation. The lack of host BM-derived IL-12 expression led to allograft rejection. Rejection could be overcome by increasing the dose of pre-transplant irradiation and the content of donor T-cells without causing lethal GVHD. As the main source of host BM-derived IL-12, recipient APC thus play an important role in donor T-cell activation. As has been previously demonstrated in a murine BMT model, the addition of IL-12 in the peri-transplant period helped to separate graft versus leukemia effects from the GVHD-promoting activity of donor T-cells (Yang, 1997). Patients predicted to be high risk of graft failure may benefit from treatment strategies that contribute to production of IL-12 during the early phases of hematopoietic engraftment. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4837 Background: The tumor microenvironment includes tumor cells, and host-derived endothelial cells, fibroblasts, innate and adaptive immune cells. Tumors may induce neo-vascularization that supports local tumor growth or immune suppression and tolerance that facilitates tumor metastasis. We hypothesized that the patients with higher numbers of circulating CD34+ endothelial progenitor cells (CD34+/CD146+/CD45-, CEC), a cellular bio-marker for vasculogenesis, would have worse post-treatment outcomes and patients with more hematopoietic progenitor cells (CD34+/CD45+/CD45dim/CD133+, HPC) and Immune cells including T-cells would have better outcomes. Methods: We analyzed blood samples from sixty-two patients with advanced NSCLC at 3 time points: before chemotherapy, after cycle one, and at completion of treatment or progression of disease, in an IRB-approved protocol. CEC, HPC, and immune subsets were measured by high throughput multi-parameter flow cytometry, 2.5,000,000 events were acquired using a lyse, no-wash method optimized for rare event detection. Primary outcomes were progression free survival(PFS) and Overall Survival(OS) from the time of study entry. The patient population was stratified into groups based on optimum cut-off point for each cell subset of interest. Statistical analysis was done with log-rank test and Cox regression. Results: Mean age at diagnosis was 64 (37–87 years), 30 events (death) occurred with median follow-up of 9.3 months. Forty-six patients (74%) had disease progression with a median follow-up of 4.7 months. At baseline lower numbers of WBC, Neutrophil lymphocyte ratio(NLR), CEC, HPC were associated with better PFS, while only WBC and Neutrophil lymphocyte ratio (NLR) were associated with a favorable OS. While lower numbers of Immune cells were associated with worse PFS and OS (increased HR death or relapse) in univariate analysis as noted in the Table. Only covariates that were significant and non collinear were entered in the Multivariable model adjusted for age, gender, smoking, race, TNM stage, pathology, and performance status at diagnosis. This showed that baseline numbers of CD4+ T-cell (HR 0.46; 95% CI 0.33–0.98; p= 0.045), Myeloid DC (HR 0.38; 95% CI 0.39–0.81; p=0.012), HPC (continuous variable) (HR 0.78; 95% CI 0.64–0.93; p= 0.008) were significant for disease progression, while NLR was significant for death after study entry (Figure; HR 3; 95% CI 1.45–6.25; p=0 0.003). Conclusions: In patients with advanced NSCLC, lower numbers of HPC and NLR were associated with improved PFS and OS respectively. Lower numbers of immune subsets at diagnosis were associated with inferior outcomes to treatment, supporting the role for immune-mediated disease control. Disclosures: No relevant conflicts of interest to declare.