ALBERT

Description: Article The synthesis of molecular nanostructures often requires the variation of several parameters, such as stoichiometry, pH, counter-ion etc. Here, the authors report a flow reaction array with algorithmic control which is used as a ‘search engine’ to isolate six nanoscale clusters from a massive parameter space. Nature Communications doi: 10.1038/ncomms4715 Authors: Hong-Ying Zang, Andreu Ruiz de la Oliva, Haralampos N. Miras, De-Liang Long, Roy T. McBurney, Leroy Cronin

Description: Abstract 3123 Background: Multiple myeloma (MM) is a plasma cell malignancy with variable prognosis depending on disease features such as β2m level and cytogenetics. High dose therapy and stem cell transplantation (SCT) remains the current standard of care for MM, however the role of tandem SCT is controversial, particularly in the era of novel induction therapy. Methods: The Virginia Commonwealth University BMT program has a practice of risk-stratified transplant allocation in MM patients referred for SCT; those with high-risk (HR) disease (β2m 〉5.5, adverse cytogenetics, 〉1st remission) are preferentially assigned tandem SCT, and those with standard-risk (SR), a single SCT. Between 2008 and 2011, 146 MM patients underwent SCT, 64 (44%) SR patients received a single SCT (SRS), 32 (22%) HR patients received tandem SCT (HRT) & 50 (34%) a single SCT (HRS). Median age at SCT was 58 years. Maintenance therapy was administered in 48% SRS, 53% HRT & 63% HRS patients. Results: Disease status at day 100 post-transplant was complete response (CR) or very good partial response (VGPR) in 84%, 75% and 72% patients in the SRS, HRT, and HRS groups respectively. Patients in the HRT group (41%) were more likely to achieve CR than in the HRS group (18%) (P=0.04). At a median follow up of 23 months, there was no difference in the relapse incidence (Figure 1) between the HRT and SRS groups (Log Rank P=0.22) or between the HRT and HRS groups (Log Rank P=0.64), however the relapse incidence for SRS was lower than for HRS group (Log Rank P=0.01). Correspondingly, there is no difference in the estimated survival between the HRT and SRS (Log Rank P=0.12), or between the HRT and HRS groups (Log Rank P=0.57), though the survival for the SRS group is superior to HRS group (Log Rank P=0.025) (Figure 2). Estimated 2-year survival rates are 0.96 (95% CI: 0.88, 1.00), 0.87 (0.73, 1.0), and 0.79 (0.65, 0.94) in the SRS, HRT and HRS groups respectively, where the SRS survival is significantly higher than the HRS group (Log Rank P=0.047). This was attributable to a lower 1-year relapse rate of 0.10 (0.02, 0.19), 0.07 (0.00, 0.16) in the SRS, HRT groups compared to 0.29 (0.15, 0.43) in the HRS group (Log Rank P

Description: Severe graft versus host disease (GVHD) continues to compromise transplant outcomes following reduced intensity (RI) allogeneic stem cell transplantation (SCT). ATG reduces the risk of GVHD in unrelated donor (URD) stem cell transplant (SCT) recipients. Here we report the results of a randomized phase II clinical trial evaluating two doses of rabbit ATG (Thymoglobulin, Sanofi Aventis) 5.1 (ATG5) and 7.5 mg/kg (ATG7.5) administered along with 450 cGy total body irradiation (TBI). This study followed an earlier trial which examined the optimal schedule of ATG administration in this regimen (Toor et al, BMT 2008). ATG was administered from day -9 to day -7, and fractionated TBI on day -1 and 0. Tacrolimus and mycophenolate mofetil were administered for GVHD prophylaxis. Forty-one patients with advanced hematologic malignancies were randomized and conditioned with ATG-TBI, stratified according to donor type, HLA-matched related (MRD) or unrelated (URD) donor. ATG5 had 10/19 URD recipients compared to 12/22 ATG7.5. Median age of the patients was 57; 9 patients had MM, 15 NHL, 12 CLL/PLL and 6 MDS/AML. Twenty-six were in CR at SCT (11 ATG5, 15 ATG7.5), 13 were in PR/SD (7 and 6 in the two arms) and 2 (1 in each arm) were in untreated relapse. Median number of prior therapies was 4 in each arm; 6 (ATG5) and 9 (ATG7.5) patients had a prior autologous SCT. Myeloid engraftment occurred at a median of 12 days after stem cell infusion in both arms; median neutrophil chimerism at 4, 8, 12 and 24 weeks was 0% recipient in both arms. Persistent mixed T cell chimerism was observed more frequently in the ATG7.5 arm with a trend toward full donor chimerism over time in the ATG5 patients (Figure 1); mean T cell chimerism was 14% in ATG5 and 25% in ATG7.5 at 4 weeks; 8 and 26 (repeated measures ANOVA p=0.058) at 8 weeks; 9 and 31 (RMANOVA p=0.016) at 12 weeks; and 6 and 27 (p=0.029) at 24 weeks respectively. Mean donor derived T cell counts (ddCD3; product of absolute CD3+ cell count and donor T cell chimerism) were 182/microliter, 1080, 872, 876 in ATG5 and 182, 290, 296, 445 in ATG7.5 patients at the corresponding time points, being significantly larger in the ATG5 arm compared to ATG7.5 at 8 (p = 0.008) and 12 weeks (p = 0.052). The changes in ddCD3 between successive months were significantly larger in the ATG5 cohort between 1 and 2 months (p = 0.004). Further, 2 patients in the ATG7.5 arm developed secondary graft loss indicating more robust engraftment in the ATG5 cohort, likely due to superior immune reconstitution. There was no significant difference in the survival between arms (Log-Rank P = 0.8). The 2-year survival rates were 64% (95%CI: 61%, 67%) ATG5 and 54% (95%CI: 50%, 58%) ATG7.5. Day 100 TRM was 0% in both arms; classical onset grade III-IV acute GVHD was not observed in either arm, 2 patients in the ATG7.5 arm developed grade II acute GVHD. Delayed onset acute GVHD occurred in 5 patients in the ATG5 cohort, (3 Grade III-IV) and 4 ATG7.5 (2 Grade III-IV) following early tapering of immunosuppression. Chronic GVHD occurred in 2 patients in ATG5 (1 mild, 1 severe) compared to 7 in ATG7.5 cohort (2 mild, 1 moderate, 4 severe). Overlap syndrome occurred in 2 in ATG5 patients and none in ATG7.5 cohort. GVHD was observed more frequently in patients with higher ddCD3 counts at 8 weeks. Relapse occurred in 26% vs 27%, ATG5 and ATG7.5 (Grey's test P=0.9). Two patients in the ATG5 cohort needed donor lymphocyte infusions (DLI) compared to 8 in ATG7.5 (Grey's test P = 0.07); 6 of these patients received prophylactic DLI for declining donor chimerism (5 in the ATG 7.5 cohort). A composite index (relapse, DLI, non-relapse mortality) was not significantly different between the cohorts (Log Rank P = 0.9). High-dose valacyclovir was used for CMV prophylaxis; CMV reactivation (〉1000 copies/microl) developed in 2 ATG5 patients and 3 in ATG7.5 arm; EBV reactivation (〉10 copies/microl) developed in 1 and 2 patients respectively in the two cohorts. In summary our clinical trial demonstrates that ATG5 in combination with low dose TBI yields optimal T cell engraftment, with an excellent safety profile and good clinical outcomes, and may serve as a platform for adoptive immunotherapy. Phase III clinical trial comparing this regimen with more established regimens for RI-SCT is being planned. Disclosures: Toor: Sanofi Avnetis: Research Funding.

Description: Anti-thymocyte globulin (ATG) is widely used for in vivo T cell depletion and immunomodulation in unrelated donor (URD) stem cell transplantation (SCT) to reduce the risk of graft vs. host disease (GVHD). However, despite the reduction in GVHD risk, outcomes are generally not superior to matched related donor (MRD) SCT conditioned without ATG. This is primarily because of defective immune reconstitution and high rates of opportunistic infections in ATG recipients. We have previously reported equivalent outcomes in URD SCT recipients conditioned with ATG when compared to MRD recipients. We now report immune reconstitution in an expanded cohort of these patients. Patients with AML, ALL, MDS, MPD (n=142) transplanted between 2004 and 2011 were included in this retrospective review. Seventy eight received either bone marrow or peripheral blood stem cell (PBSC) grafts from URD and received either 10 or 7.5 mg/kg rabbit ATG (Thymoglobulin, Sanofi-Aventis) during conditioning, those with MRD did not. Conditioning was myeloablative in all patients. Lymphoid recovery was equivalent in the two cohorts during the first year following SCT except in the first month (Figure), when URD recipients had lower absolute lymphocyte count (μ-URD=0.6x103/ μ L, μ-MRD=1.1; repeated measures mixed model p=0.022). Age, CD3/34 cell dose infused, stem cell source and conditioning intensity were not associated with ALC recovery post transplant. In a subset of patients lymphocyte subset enumeration was performed following withdrawal of immunosuppression, at an average 237 days post-SCT. ATG recipients had significantly lower mean CD4+ counts (μ-URD=267 (n=30), μ-MRD =434/ μ L (n=27); ANCOVA p = 0.003), however no significant differences were observed in CD3+, CD8+, CD19+ or CD56+ cell recovery. ATG recipients were significantly more likely to have complete donor T cell chimerism at 1 (OR = 12.5, CI= 2.4, 66.0, p = 0.001) and 2 months post-SCT (OR = 6.5 , CI=1.5, 27.4, p = 0.013), however by 9 months following SCT this trend had reversed with a greater likelihood of mixed T cell chimerism (OR 〉 100; p = 0.017), suggesting re-emergence of recipient derived T cell clones. Consequently, donor lymphocyte infusions were given significantly more often to ATG recipients (12/78) than to non-recipients (2/64) (OR = 5.64, CI = 1.21, 26.20, p=0.027). High grade CMV viremia (1000 copies/ μL) was significantly more likely in CMV sero-positive ATG recipients (n=18/55) than in non-recipients (n=7/48) (OR = 2.8, CI 1.1, 7.6, p = 0.032). Reduced intensity conditioning and PBSC were associated with higher CMV reactivation in ATG recipients and there was a lower likelihood of survival in these individuals than in those who did not receive ATG (HR: 0.53, CI: 0.31, 0.92; p = 0.024). EBV reactivation was observed more often in susceptible ATG recipients (n=22/58) than in non-recipients (n=5/43), (OR=4.6, CI=1.6, 13.6, p= 0.003). The median peak EBV viral load in ATG recipients (u=1545 copies/ μL, IQR: 288, 2,302) was significantly higher than in non-recipients (u=120 IQR: 57, 169, p = 0.005). PBSC stem cell source (p = 0.049) and HLA mismatch (p =〈 0.001) were associated with EBV reactivation in ATG recipients but not in non-recipients. ATG recipients were also more likely to experience a fungal infection (OR=2.8, CI=1.1, 6.7, p=0.023). 1-month ALC was predictive of disease free survival whereby it had a significant negative effect on relapse (HR = 0.33; 95% CI: 0.16, 0.66; p = 0.002). As 1-month ALC increased by one-tenth, the odds of relapse decreased by over 3% and survival increased by 3%. In conclusion, high doses of ATG used during conditioning are associated with an early retardation of lymphoid recovery post-SCT, and with late mixed T cell chimerism accompanied by a delay in CD4+ T cell recovery. This is associated with a higher rate of viral reactivation in PBSC recipients and of fungal infections in general. Lower doses of ATG should be used in SCT and in ATG conditioned SCT, early intervention with DLI, particularly CD8+ cell depleted DLI as reported by others, may help restore T cell repertoire and improve SCT outcomes and survival. Disclosures: Toor: Sanofi Avnetis: Research Funding.

Description: Abstract 2926 Patients with multiple myeloma (MM) undergoing high dose therapy and autologous stem cell transplantation (SCT) remain at risk for disease progression. Maintenance therapy may delay recurrence but is associated with toxicities, highlighting the need for alternative strategies for long-term disease control. Malignant plasma cells in MM patients occasionally express highly immunogenic cancer testis antigens (CTA). CTA expression is regulated by DNA methylation, and may be increased by 5-azacitidine (Celgene corp., Summit, NJ) (Aza), a DNA hypomethylating agent. The addition of lenalidomide (Celgene corp.) (L) may augment any ensuing adoptive CTA-specific immune response. These immune effector cells may then be collected and adoptively transferred in a setting of lympho-depletion and minimal residual disease following SCT, serving a maintenance function. To demonstrate the feasibility of this approach, we initiated a phase II clinical trial of Aza administered sequentially with L in patients with residual disease following initial therapy (NCT01050790). Three cycles of Aza (75 mg/m2 day 1–5 SQ) and L (10 mg PO daily, day 6–21) were administered every 4 weeks; autologous lymphocytes (AL) were collected around day 21 of the 2nd and 3rd cycles of Aza-L and cryopreserved. Subsequent stem cell mobilization was followed by melphalan 200 mg/m2 and SCT. GM-CSF was used post-transplant for hematopoietic engraftment. Autologous lymphocyte infusion (ALI) was performed between day +30 to +60. Twelve patients have been enrolled; median age is 60 years (range 40–69). Eight are African American, 10 had disease in first partial remission (PR) and 2 in very good PR (VGPR) at the time of initiation of therapy. Median of 2 prior regimens had been administered (range 1–2) and 6 had prior therapy with L. Stage at diagnosis was II (n=6) and III (n=6) and 4 had abnormal cytogenetics. Eight patients have completed all three cycles of Aza-L; 2 developed grade 3 neutropenia, no other grade 3 to 4 toxicity has been observed. Eight patients have gone through both AL collections, 21 days following cycles 2 and 3 of Aza-L, yielding 0.90±0.41 and 0.81±0.36 ×108 CD3+ cells/kg (mean ± SD) with the first and second procedures. Three patients had further disease response, (1 near complete remission, 2 VGPR) and 5 had stable disease following three months of Aza-L, representing a median 18% decline in para-protein levels before and after therapy. So far 8 patients have undergone stem cell mobilization with either GCSF alone (n=6) or GCSF + plerixafor (n=2), and have collected 11.2±3.3 ×106 CD34+ cells/kg body weight. To date 6 patients have undergone SCT (tandem SCT in 1). Neutrophil engraftment was at median of 14 days (13–14), and no unexpected post transplant toxicities were observed. Four patients received ALI at a median 42 days following SCT with no immediate or remote infusional toxicities. With a median follow up of 9 months post-transplant, all four ALI recipients are progression free with either complete remission (n=1) or VGPR (n=3). Quantitative RT-PCR evaluating a panel of 10 CTA in unfractionated bone marrow specimens collected before and after Aza-L from five patients demonstrated 6–8 discrete CTA induced in each patient (Figure 1). This expression was seen in CD138+ plasma cells when tested. In one patient with an induced increase in NY-ESO 1 expression following Aza-L, an antigen specific immune response was recorded (IFN-g release assay) when blood mononuclear cells were co-cultured with recombinant NY-ESO 1 pulsed monocyte derived dendritic cells. This response was maintained for several months post-transplant (Figure 2). Consistent with this observation, CD3+ T cell counts before and after ALI demonstrated a marked increase in T cell counts at two (mean 959/μl; n=4) and eight (1277/μl) weeks, compared with baseline (414/μl; P=0.05); no difference was seen in the NK cell counts at these times. Further studies to confirm these observations in the remaining patients are ongoing. In conclusion, we demonstrate the safety and feasibility of epigenetic modification resulting in over-expression of antigenic targets in MM. This may then be exploited in formulating adaptive immunotherapy protocols in these patients. Adoptively transferred cells may maintain long-term surveillance against malignant plasma cells in patients with MM. Disclosures: Toor: Celgene corporation: Research Funding. Off Label Use: azacitidine in multiple myeloma. Manjili:Celgene corporation: Research Funding.

Description: Abstract 3043 In patients undergoing T cell depleted stem cell transplantation (SCT) a high rate of mixed chimerism is seen. Further, chimerism analysis does not consistently predict risks of GVHD or relapse upon withdrawal of immunosuppression (IS) in these patients. Thus, a reliable predictor for the expected evolution of mixed T cell chimerism is needed to help in clinical decision-making for withdrawal of IS and donor lymphocyte infusion (DLI). An alternative immune parameter is T cell recovery post-transplant. We combined this measure with T cell chimerism and examined the predictive value of a calculated donor-derived T cell count for clinical outcomes following SCT. Patients were enrolled in a randomized clinical trial examining two different doses of rabbit anti-thymocyte globulin (ATG 2.5 or 1.7 mg/kg/day; Thymoglobulin®, Genzyme, Cambridge, MA) given IV on day –9 through –7, followed by total body irradiation (4.5 Gray), administered in 3 doses on day –1 and 0 (NCT00709592). Donor engraftment was measured by PCR of short tandem repeat alleles in each donor and patient at 4, 8, 12, and 24 weeks following SCT on whole blood, granulocytes, and total T cells. Between 2008 and 2011, 25 patients were enrolled in this trial, and 22 patients were eligible for this analysis. Nine of the 22 patients had mixed T cell chimerism (≥5% recipient DNA) at 8 weeks post-transplant, and of these 33% (n=3) became fully donor T cell chimeric after withdrawal of IS over the ensuing weeks. One of the 9 patients had improved donor chimerism after DLI. Of the 13 patients who were full donor chimeric in T cells, one patient reverted from full donor to mixed chimerism. Donor-derived CD3+ T cell count (dd CD3) was calculated from T cell chimerism and absolute blood CD3+ T cell count. Median dd CD3+ cell count at 8 weeks following SCT was 433 ƒýL (range: 3–2464). After calculating the sum of receiver operating characteristic area under the curves (AUC), a dd CD3 of 110ƒýL was found to be the optimal cut-off value with both the highest AUC sum and the highest AUC for each measure (cumulative acute and chronic GVHD: 0.79, remission: 0.74, whole blood chimerism: 0.88), and was subsequently used to distinguish between low (110; n=14) values of dd CD3. A significant correlation was found between this single dd CD3 level discriminator and higher rates of cumulative GVHD (Fishers Exact Test: p = 0.024), remission (p = 0.0524) and full donor whole blood chimerism at ≥12 weeks following SCT (p 〈 0.0001) in patients with high-dd CD3 cell counts. Furthermore, Bayesian analysis showed higher whole blood mixed chimerism rates (Posterior Probability = 0.99), lower GVHD rates (PP = 0.99), and lower remission rates (PP = 0.99) in the low-dd CD3 group. Both overall survival and time to relapse favored the high-dd CD3 group (vs. low-dd CD3 group). However, while the relationship was significant for relapse (p = 0.028), it was only marginally significant for overall survival (p = 0.07). When measured after withdrawal of IS, declining donor or persistent mixed T cell chimerism was observed in patients who had mixed T cell chimerism and low- dd CD3 at eight weeks post SCT (Fig 1A), whereas those with high- dd CD3 had increasing donor T cell chimerism (Fig 1B). Granulocyte chimerism too was modulated by dd CD3 in patients with mixed chimerism and early DLI arrested the decline of donor hematopoiesis in a patient with low-dd CD3. NK cell counts were significantly higher in the high-dd CD3 group than in the low-dd CD3 group at 8 weeks (p = 0.044; PP = 0.99). No significant relationships were found between dd CD3 levels and donor age, donor type, total ATG dose, and infused graft CD34+ or CD3+ cell dose.Figure 1AFigure 1A. In conclusion, we report the effect of an easily calculable measure of donor T cell reconstitution on clinical outcomes following SCT (particularly GVHD), stability of engraftment and disease relapse. Donor-derived CD3+ cell count may help in the decision-making regarding IS withdrawal and DLI timing in allogeneic SCT recipients. We will be confirming the optimal dd CD3 cell count parameter in larger patient cohorts with comparable disease biology for validation in regimens of varying myeloablative intensities. Disclosures: Toor: Genzyme: Research Funding. Off Label Use: ATG in stem cell transplantation.

Description: Abstract 4330 Objective: Acute myeloid leukemia (AML) that arises from an antecedent hematologic disorder (most often myelodysplastic syndrome, MDS) or that is related to prior chemotherapy (therapy-related AML, t-AML) carries a poor prognosis. Secondary AML occurs in 16–50% of patients with MDS and in

Description: Planktonic foraminifera are a major contributor to the deep carbonate-flux and the planktonic biomass of the global ocean. Their microfossil deposits form one of the richest databases for reconstructing paleoenvironments, particularly through changes in their taxonomic and shell composition. Using an empirically-based foraminifer model that incorporates three known major physiological drivers of foraminifer biogeography – temperature, food and light – we investigate (i) the global redistribution of planktonic foraminifera under anthropogenic climate change, and (ii) the alteration of the carbonate chemistry of foraminifer habitat with ocean acidification. The present-day and future (2090–2100) 3-D distributions of foraminifera are simulated using temperature, plankton biomass, and light from an Earth system model forced with historical and a future (IPCC A2) high CO2 emission scenario. The broadscale patterns of present day foraminifer biogeography are well reproduced. Foraminifer abundance and diversity are projected to decrease in the tropics and subpolar regions and increase in the subtropics and around the poles. In the tropics, the geographical shifts are driven by temperature, while the vertical shifts are driven by both temperature and food availability. In the high-latitudes, vertical shifts are driven by food availability, while geographical shifts are driven by both food availability and temperature. Changes in the marine carbon cycle would be expected in response to (i) the large-scale rearrangements in foraminifer abundance, and (ii) the reduction of the carbonate concentration in the habitat range of planktonic foraminifers: from 10–30 μmol kg−1 in the polar/subpolar regions to 30–70 μmol kg−1 in the subtropical/tropical regions. High-latitude species are most vulnerable to anthropogenic change: their abundance and available habitat decrease and up to 10% of their habitat drops below the calcite saturation horizon.