ALBERT

Description: Plants lack the seemingly unlimited receptor diversity of a somatic adaptive immune system as found in vertebrates and rely on only a relatively small set of innate immune receptors to resist a myriad of pathogens. Here, we show that disease-resistant tomato plants use an efficient mechanism to leverage the limited nonself recognition capacity of their innate immune system. We found that the extracellular plant immune receptor protein Cf-2 of the red currant tomato (Solanum pimpinellifolium) has acquired dual resistance specificity by sensing perturbations in a common virulence target of two independently evolved effectors of a fungus and a nematode. The Cf-2 protein, originally identified as a monospecific immune receptor for the leaf mold fungus Cladosporium fulvum, also mediates disease resistance to the root parasitic nematode Globodera rostochiensis pathotype Ro1-Mierenbos. The Cf-2–mediated dual resistance is triggered by effector-induced perturbations of the apoplastic Rcr3pim protein of S. pimpinellifolium. Binding of the venom allergen-like effector protein Gr-VAP1 of G. rostochiensis to Rcr3pim perturbs the active site of this papain-like cysteine protease. In the absence of the Cf-2 receptor, Rcr3pim increases the susceptibility of tomato plants to G. rostochiensis, thus showing its role as a virulence target of these nematodes. Furthermore, both nematode infection and transient expression of Gr-VAP1 in tomato plants harboring Cf-2 and Rcr3pim trigger a defense-related programmed cell death in plant cells. Our data demonstrate that monitoring host proteins targeted by multiple pathogens broadens the spectrum of disease resistances mediated by single plant immune receptors.

Description: The micro-environment in multiple myeloma (MM) is highly immunosuppressive with increased numbers of regulatory T cells (Tregs) and myeloid derived suppressor cells (MDSCs) favoring tumorcell survival and hampering immunotherapeutic strategies such as dendritic cell vaccination. Immunomodulatory drugs (IMiDs) are known to enhance T- and NK-cell function. In this study we evaluated the effects of low dose (0.5 microM) lenalidomide (Len) and pomalidomide (Pom) on the functionality of CD8+ and CD4+ T cells, MDSCs, Tregs and ex-vivo generated mononuclear derived dendritic cells (moDCs) obtained from MM patients after first autologous stem cell transplantation (ASCT). Peripheral blood mononuclear cell fractions were obtained by leukapheresis from 9 MM patients (age 29-62 years), in very good partial response (4/9) or complete response (5/9) after ASCT. The magnitude of cytokine release (mean +/- standard error of the mean, in ng/ml) by purified CD8+ T cells after 144 hours stimulation with anti-CD3/anti-CD28 coated microbeads was significantly increased after addition of Len and Pom to the culture medium, respectively : IFN-gamma (217.5 +/- 62.1 and 437.1 +/- 137.1** vs 66.4 +/- 21.0) , TNF-alpha (21.4 +/- 5.4 and 44.9 +/- 9.4*** vs 4.9 +/- 1.7) and IL-2 (5.3 +/- 2.7 and 12.7 +/- 6.6 vs 1.9 +/- 1.7 ng/ml) (** p〈 0.01, *** p〈 0.001). We also evaluated the number of different types of cytokines/chemokines on a per cell basis by intracellular flow cytometry staining for IFN-gamma, TNF-alpha, IL-2 and MIP-1beta and observed increased polyfunctionality of CD8+ and CD4+ T cells. After 72 h of stimulation with anti-CD3/CD28 microbeads the number of single, double, triple or quadruple functional CD8+ T cells increased from 5.96 %, 2.82 %, 0.1 %, 0 % (culture medium alone) to 9.68 %, 7.57 %, 0.41%, 0.03 % (Len) and 12.57 %, 8.96 %, 0.81 %, 0.03 % (Pom), respectively. A similar observation was made for CD4+ T cells. A significant percentage, median 5.7 % (4.0-7.2 %) of CD4+ CD25high CD127low (Tregs) was found in the CD4+ T cell population in 8 out of 9 patients, demonstrating the highly suppressive immune environment in myeloma patients even with low disease burden. Effector T cells (Teffs) were stimulated with anti-CD3/CD28 microbeads and cocultured at varying ratios with purified Tregs. After 144 h of coculture, Len and Pom reduced the suppressive effects of Tregs on Teffs proliferation and IFN-gamma and TNF-alpha production (see figure). A similar effect was observed for MDSC but did not reach statistical significance (data not shown). TriMix DCs (moDCs matured by electroporation with mRNA encoding TLR4, CD40L and CD70) and cytokine matured moDCs were cocultured with autologous CD4+ and CD8+ T cells and anti-CD3 microbeads. Adding IMiDs resulted in more polyfunctional CD4+ and CD8+ T cells with both types of DCs but effects were most pronounced with the TriMix variant. Our study shows that Len and Pom restore effector cell functions in myeloma patients with low tumor burden after ASCT. These findings provide a immunomechanistic explanation for IMiD-based maintenance therapy. They also offer a rationale to combine IMiD-based maintenance with immunotherapeutic approaches such as dendritic cell vaccination in this particular setting. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1840 One of the greatest challenges in multiple myeloma (MM) treatment is to overcome drug resistance. More and more evidence showed that not only the MM tumor cells should be targeted but also the bone marrow (BM) micro-environment. Interactions of MM cells with the BM micro-environment have a pivotal role in MM cell proliferation, survival, migration, angiogenesis as well as drug resistance. Many pathways are involved including the conserved Notch signaling pathway. The interaction of Notch receptors and ligands between adjacent cells induces proteolytic cleavage and release of the intracellular domain of the Notch receptor, also called Notch intracellular domains (NICD). NICD will then enter the nucleus and modify the expression of downstream target genes. Notch receptors are expressed by MM cells and Notch ligand Dll1 is present on bone marrow (BM) stromal cells. We investigated whether Notch activation in myeloma cells by the interaction with Dll1 on stromal cells contributes to bortezomib resistance. We analyzed Notch1 and Notch2 surface expression by flow cytometry on MM cells after Dll1 interaction using a stromal cell line modified to overexpress Dll1. Notch1 surface expression was not disturbed on mouse 5T33MMvt and human MMS1 and LP-1 cells while Notch2 expression on MM cells was significantly decreased after Dll1 interaction for 2 days. Next, we investigated NICD1 and NICD2 expression by western blot after Dll1/Notch interaction. NICD1 did not change in murine 5T33MMvt and human LP-1 and MMS-1 cells, while NICD2 is increased after Dll1 interaction. These results suggest that Dll1 can activate Notch signaling likely through the Notch2 receptor. We investigated whether Dll1/Notch activation could contribute to MM bortezomib resistance. MM cells were cocultured on immobilized recombinant Dll1 ligand and treated with 5 nM bortezomib for 48h. Compared to control, MM cells cocultured with Dll1 ligand were less sensitive to bortezomib. Furthermore, blocking the Notch pathway by DAPT (a gamma secretase inhibitor, GSI) could reverse this effect and increased the sensitivity to bortezomib. To delineate the molecular mechanism of Dll1-induced bortezomib resistance, we performed a drug resistance and metabolism gene array and found that CYP1A1 was significantly upregulated by Dll1/Notch interaction. CYP1A1 is a member of the cytochrome P450 family and regulates drug metabolism. We further demonstrated that inhibiting CYP1A1 by either α-Naphthoflavone (inhibitor) or CYP1A1-siRNA increases the sensitivity of MM cells to bortezomib, suggesting that CYP1A1 is involved in bortezomib resistance. As also previously demonstrated, CD138- 5T33MM cells are less sensitive to bortezomib than CD138+ 5T33MM cells. We analyzed CYP1A1 expression and activity and observed a higher CYP1A1 amount in CD138- cells compared to CD138+ MM cells. The higher CYP1A1 expression in CD138- cells might be a possible mechanism for their decreased bortezomib sensitivity compared to CD138+ cells. In addition, an in vivo experiment was performed. Combination treatment of DAPT with bortezomib was able to increase bortezomib sensitivity and prolonged overall survival in the 5T33MM mouse model. In conclusion, our results suggest that Dll1/Notch activation contributes to bortezomib resistance by upregulating CYP1A1, a molecule involved in drug metabolism. Our data provide a potential strategy to overcome bortezomib resistance by combination with a Notch pathway inhibitor. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Approximately 20–40% of pts with NDMM present with RI, which is associated with a negative impact on survival (Rajkumar, 2005). In the pivotal phase 3 FIRST trial (median follow-up 37 months [mos]), continuous Rd improved progression-free survival (PFS) vs. melphalan-prednisone-thalidomide (MPT) in elderly NDMM pts by 28% (25.5 vs. 20.7 mos; HR = 0.72; P 〈 0.01) (Facon, Blood 2013). Although 121 pts receiving continuous Rd are still on Tx, the interim overall survival (OS) analysis showed a 22% reduction in the risk of death in favor of continuous Rd vs. MPT (HR = 0.78; P = 0.02). The present analysis was conducted to determine the impact of RI on PFS, OS, and time to 2nd antimyeloma Tx (AMT) as clinical study outcomes. Methods: Pts were randomized to 3 Tx arms: continuous Rd until progression (n = 535); Rd for 18 cycles (72 weeks) (Rd18; n = 541); or MPT for 12 cycles (72 weeks) (n = 547). Enrolled NDMM pts were categorized according to their renal function: 24% had normal renal function (creatinine clearance [CrCl] ≥ 80 mL/min), 44% presented with mild RI (≥ 50 and 〈 80 mL/min), 23% had moderate RI (≥ 30 and 〈 50 mL/min), and 9% had severe RI (〈 30 mL/min). Pts requiring dialysis were excluded. Lenalidomide starting dose was 25 mg QD for pts with normal renal function or mild RI, 10 mg QD for moderate RI, and 15 mg QOD for severe RI. Melphalan dose was reduced by 50% in pts with moderate or severe RI. The primary endpoint was PFS (continuous Rd vs. MPT); secondary endpoints were OS, overall response rate, time to response, duration of response, time to Tx failure, time to 2nd AMT, health-related quality of life, safety, and improvement in renal function from baseline. Improvement in RI was defined as shifts from baseline to most extreme post-baseline value of the calculated CrCl as a measure of renal function during the active Tx (N = 1484). Results: A PFS benefit favored continuous Rd vs. MPT irrespective of the degree of renal function (Table 1): there was a benefit in pts with normal renal function (HR = 0.72 (0.51–1.02); P = 0.06), and better in pts with mild RI (HR = 0.79 (0.62–1.00); P = 0.05) and moderate RI (HR = 0.62 (0.45–0.85); P 〈 0.01). A PFS benefit was also seen with continuous Rd vs. Rd18 (a secondary comparison) in pts with mild RI and moderate RI (P 〈 0.01 for both). An interim OS benefit with continuous Rd vs. MPT was observed in most renal subgroups. Similar results were observed between Rd18 and MPT in terms of PFS or interim OS in any of the renal subgroups. Continuous Rd, compared with Rd18 or MPT, extended time to 2nd AMT in most renal groups except severe RI (CrCl 〈 30mL/min) (Table 2). Improvement in RI was observed more frequently in pts treated with continuous Rd than those with Rd18 or MPT: improvement of mild RI, 48%, 43%, and 48%, respectively; of moderate RI, 67% 61%, and 62%; and of severe RI, 64%, 59%, and 56%. Overall, 〈 5% of pts in any Tx group experienced a worsening in renal function status during Tx (continuous Rd 2.2%; Rd18 2.8%; MPT 2.7%). The most common grade 3–4 adverse events (AEs) for these Txs were anemia, neutropenia, thrombocytopenia, deep-vein thrombosis/pulmonary embolism (DVT/PE), and peripheral sensory neuropathy (Table 3). Tx discontinuation due to AEs increased in pts with moderate and severe RI, regardless of the type of Tx (Table 3). Conclusions: PFS, OS (at interim analysis), and time to 2nd AMT outcomes generally improved continuous Rd vs. Rd18 or MPT in transplant-ineligible NDMM pts with normal renal function, and in those with mild or moderate RI. The small number of pts in the severe RI group precluded a meaningful conclusion. Continuous Rd was generally well tolerated and renal function improved in the majority of pts during Tx with continuous Rd vs. Rd18 or MPT. Disclosures Dimopoulos: Celgene Corporation: Consultancy, Honoraria. Off Label Use: Lenalidomide used in newly diagnosed multiple myeloma patients. Roussel:Celgene: Consultancy, Lecture fees Other, Research Funding. van der Jagt:Celgene Corporation: Research Funding. Jaccard:Celgene Corporation: Honoraria, Research Funding. Tosikyan:Celgene: Consultancy. Karlin:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sandoz: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bensinger:Celgene Corporation: Consultancy, Research Funding. Schots:Celgene: Research Funding. Chen:Celgene Corporation: Employment. Marek:Celgene Corporation: Employment, Equity Ownership. Ervin-Haynes:Celgene Corporation: Employment. Facon:Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Description: Abstract 3567 Autologous transplantation significantly improves the progression free survival (PFS) and overall survival (OS) of patients with relapsed or resistant follicular (rFL) lymphoma compared with chemotherapy alone (Schouten H, et al. J Clin Oncol 2003;21:3918–27). Small phase II trials suggest, that rituximab (R) given peritransplant further improves survival outcome. Whilst the role of maintenance R post chemotherapy in FL is established, the benefit and safety of maintenance R following autologous transplant is unknown. In this randomised prospective study the efficacy and safety of R as in vivo purging pretransplant and as maintenance treatment immediately post transplant was assessed. From Oct 1999 to Apr 2006, 280 of a planned 420 R naïve patients with rFL in first (n=16), second (n= 222) or third remission (n=41) who achieved either a complete remission (n=83) or a very good partial remission (n=196) to induction chemotherapy, with limited bone marrow infiltration ( 0.5 × 109 /L was prompt, median 14.3 days (range 10–115) and platelets 〉 50 × 109/L,median 25.1 days (range 9–190). Time to engraftment and early or late toxicities did not differ significantly between the groups apart from a lower neutrophil count at 3 months in patients on maintenance. No graft failures or late neutropenia was reported. Transplant related mortality was 0.5%. Only 3 infection related deaths have been reported post 100 days. Two hundred and seventeen patients are alive on continued follow-up. Median follow-up is 6.4 years. PFS at 5 years was 62.9% for patients receiving RP + RM versus 37.6 % for patients receiving no R (logrank PFS; p=0.004; HR 0.76, 95%CI: 0.66 – 0.93). OS at 5 years was 79.5% % versus 78.4 % for patient receiving RP + RM versus no R (logrank PFS; p〉0.1). Multivariate analysis was not able to define a high or low risk patient group. R in vivo purging and maintenance results in superior PFS compared to no R. R does not adversely affect peripheral blood stem cell harvesting or engraftment and maintenance R post transplant is safe. The impressive OS suggests that relapsed FL patients can be effectively salvaged post R purging and maintenance. R Purging + R Maintenance R Maintenance R Purging No R Pt number 69 69 72 70 Median PFS NR@ 6.4 y 7.23 y 4.03 y 3.34 y 5y PFS 62.9 % 56 % 46 % 37.6 % 5y OS 79.5 % 80.5 % 84.8 % 78.4 % Disclosures: Pettengell: Roche: Honoraria. Schmitz:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Gisselbrecht:Roche: Research Funding. Walewski:Roche: Honoraria, Research Funding. Geisler:Roche: Research Funding. Kimby:Roche: Honoraria, Research Funding. Goldstone:Roche: Honoraria, Speakers Bureau.

Description: Background This study aims to describe transplant outcomes and disease-specific clinical outcomes of allogeneic cord blood transplantation (CBT) in leukodystrophies. Leukodystrophies are rare inborn errors of metabolism (IEM) in which the development and maintenance of brain myelin is primarily affected. They are characterized by rapid neurological deterioration. Hematopoietic stem cell transplantation (HSCT) has been shown to halt disease progression for selected leukodystrophies, including adrenoleukodystrophy (ALD), Krabbe disease (globoid cell leukodystrophy) and metachromatic leukodystrophy (MLD). The fast availability of unrelated cord blood (UCB) makes HSCT feasible in leukodystrophy patients who lack an HLA-matched sibling. Patients and methods All patients transplanted for leukodystrophies with related and unrelated umbilical cord blood in EBMT centers between 1996 and 2012 were included. HSCT data were collected from the EUROCORD database. An additional questionnaire on disease-specific clinical outcomes was sent to participating centers. Included in the questionnaire were enzyme- and (for ALD) fatty acid levels, general disease status, performance score, MRI/MRS status, peripheral neuropathy, school attendance and performance, mental development testing, adrenal insufficiency (for ALD only), vision and hearing. Kaplan-Meier estimates were used to calculate overall survival (OS) and cumulative incidence methods for secondary outcomes, including neutrophil and platelet engraftment, incidence of graft-versus-host disease (GvHD), donor chimerism and disease-specific characteristics at most recent follow-up. The two-sided log-rank test was used for univariate comparisons. Results Seventy patients (31 ALD, 5 Krabbe, 34 MLD) were available for analysis, with a median age at transplant of 6.5 years (range 0-43 years). Median follow-up for survivors was 46 months. Overall survival at 4 years was 60% (±6%). Cumulative incidences of neutrophil and platelet engraftment were 88% (±4%) at day 60, and 73% (±6%) at day 180, respectively. Ten patients experienced graft failure and 5 had autologous recovery; 4 received a 2nd HSCT (2 CBT, 1 Haplo, 1 BM) and of those 3 were alive in a median of 60 months after HSCT. Acute-GvHD (grade II-IV) occurred in 16 patients (22% ±5%) at day 100 and chronic GvHD in 9 patients (13% ±5%). Out of the 24 patients who died, 18 (67%) died of transplant-related causes and 6 (33%) died of disease progression. Higher survival was seen in patients who had no or 1 HLA mismatch (OS 81%), compared to patients who received UCBT with 2 HLA mismatches (OS 47%, p 0.02). On 37 patients (53%), information on disease-specific characteristics was available. OS among these patients was 68% (±8), which was not significantly different from patients without disease-specific information (p=0,307). Of the 37 patients, 19 (51%) were asymptomatic at transplant, 14 (38%) had mild disease and 4 (11%) were severely affected. Overall survival was dramatically worse in patients with severe disease at transplant; all 4 severely affected patients died, while 79% of patients with mild disease and 76% of asymptomatic patients survived. At most recent follow-up, disease status was stable in 15 (57.7%), had improved in 2 (7.7%) and worsened in 9 (34.6%) surviving patients. The majority of patients showed abnormalities on MRI-scanning pre-HSCT (n=25, 68%). In patients with normal MRI-scans pre-transplant OS was 69%; in patients with abnormal MRI-scans 75%. At most recent follow-up, MRI-scans were stable in 12 (43.9%), improved in 5 (17.8%) and worsened in 11 (39.3%) patients. Eighteen patients (50%) showed peripheral neuropathy pre-HSCT, of which 13 were mildly affected and 5 severely affected. Of patients who had no signs of peripheral neuropathy pre-HSCT, 70% survived, versus 77% of patients who were mildly affected and 40% of those severely affected. At most recent follow-up, peripheral neuropathy was stable in 14 (56%), had improved in 3 (12%) and worsened in 8 (32%) patients. Conclusion We found an overall survival of 60% at 4 years after CBT for leukodystrophies, with relatively low rates of GvHD. Use of a mismatched donor (〉1 HLA mismatch) negatively impacts survival. Data on clinical characteristics suggest that overall survival is strongly influenced by disease status at transplant. Disclosures: No relevant conflicts of interest to declare.

Description: We recently reported the efficacy of darbepoetin alpha (DA)(Aranesp®) and intravenous (IV) iron (Venofer®) on erythroid reconstitution following autologous hematopoietic cell transplantation (auto-HCT). Although short-term disease progression, infections or other safety events were not influenced by DA or IV iron, a longer follow-up was required as some meta-analyses suggest increased mortality in patients with cancer receiving erythropoiesis-stimulating agents (ESA), particularly when no concomitant chemotherapy is administered. Besides, some in vitro and animal studies raised the possible role of iron in tumor growth. Therefore, we analyzed long-term outcome to verify the safety of DA and IV iron. We included 127 patients with myeloma (n=76), Hodgkin’s lymphoma (n=8), aggressive (n=26) or indolent (n=12) non-Hodgkin’s lymphoma and other diagnoses (n=5). Groups were well balanced for age, disease, disease stage and disease status at time of transplantation. They were randomized between no erythropoietic therapy (n=25), DA from day 28 post-auto-HCT (n=52) or DA + IV iron on days 28, 42 and 56 (n=50). We collected data about infections and other complications, disease status and overall survival (OS) from auto-HCT (between March 2004 and January 2008) up to January 2014. Patients given a second transplant were censored at that time. During long-term follow-up (mean: 3.4 years), the incidence of infection remained comparable in the 3 groups. Specifically, 3 patients in the control group (12%), 8 in the DA group (15%) and 6 in the DA+iron group (12%) experienced at least one infection. The number of infections per patient between day 126 (end of the study) and the last follow-up was 0.2 ± 0.7, 0.2 ± 0.4 and 0.1 ± 0.3 in control, DA and DA+iron groups, respectively (NS). During follow-up, 4/25 (16%) patients in control group, 13/52 (25%) in DA group and 6/50 (12%) in the DA+iron group experienced at least one complications (NS). While 3 patients (2 in the control group and 1 in the DA+iron group) presented a thrombosis on study, no other thrombo-embolic event occurred during follow-up. Two patients (1 in control group and 1 in DA group) developed a benign tumor, whereas 4 patients (3 in DA group and 1 in DA+iron group) presented a secondary malignancy (NS). Other complications were: hypertension (n=2, 1 in control and 1 in DA groups), ischemic cardiomyopathy (3 in DA group) with 1 myocardial infarction, 4 arrhythmias (1 in control and 3 in DA groups) with pacemaker requirement in 2 (1 in control and 1 in DA groups), peripheral arteriopathy (1 in DA group), diabetes (n=3, 2 in DA and 1 in DA+iron groups), radiopneumonitis (1 in control group), vasculitis (1 in DA group), nephritis (1 in DA and 1 in DA+iron groups), thyroiditis (1 in DA group), spondylarthritis (1 in DA+iron group), jaw osteonecrosis in myeloma patients treated with zoledronic acid (1 in DA and 1 in DA+iron groups), hip osteonecrosis (1 in DA+iron group), peripheral neuropathy (1 in DA and 1 in DA+iron groups), sudden transient deafness (1 in DA group) and sarcoidosis (1 in DA+iron group). We did not observe any difference in survival. Indeed, 1-year OS were 100%, 88% and 100% and 5-year OS were 86%, 78% and 91% in the control, DA and DA+iron groups, respectively (p=0.43). Progression-free survival (PFS) were also similar in the 3 groups: 1-year and 5-year PFS were 85% and 71% in the control group, compared to 86% and 57% in the DA group and 94% and 78% in the DA+iron group (p=0.30). In conclusion, DA and IV iron therapy following auto-HCT did not affect safety, disease outcome or survival in long-term analyses. Disclosures Beguin: Amgen: Consultancy, Speakers Bureau; Vifor: Consultancy, Speakers Bureau.