Torghabeh, Mehrdad Hefazi

Abstract BACKGROUND Efficacy of chimeric antigen receptor T cell (CART) therapy remains limited in solid tumors. Given the heterogeneity of surface receptor expression and immunosuppressive stromal microenvironment, strategies to target tumor neovasculature and tumor stromal cells are needed to help overcome CART inhibition by GBM. Eph receptors are the largest family of receptor tyrosine kinases and are integral to cell adhesion, migration, and axon guidance, during development and homeostasis. EphA3 is a receptor tyrosine kinase which is lowly expressed in adult tissues but is highly expressed in tumor neovasculature and tumor stromal cells in GBM and other solid tumors. EphA3 is over-expressed in up to 40% of GBM samples. We aimed to develop CART cells directed against EphA3 to use in targeting tumor neovasculature and tumor stromal cells in GBM. METHODS we developed a second generation CD28 co-stimulated CAR construct in a third generation lentivirus backbone to generate EphA3 CART cells using the single chain variable fragment of ifabotuzumab, a monoclonal antibody directed against EphA3. Patient derived GBM xenograft cell lines were used in these experiments. RESULTS EphA3 directed CART cells exhibited specific and potent antitumor activity against EphA3+ GBM cell lines with variable transcriptome EphA3 expression indicating its broader applicability in patients with GBM. Killing over 24-hour incubation was significant at low effector: target ratio: 52.5% killing at 1.25:1 against cell lines with 25.05% EphA3 expression and 37.1% killing at 1.25:1and 90% killing at 5:1 ratio against a cell line with 19.28% expression. Conversely, when co-cultured with UTD controls there was significantly lower killing, or growth of tumor cells. CONCLUSION We demonstrate for the first time that targeting EphA3 with CART cells is feasible, efficacious and represents a novel therapeutics strategy to target GBM. Data from in vivo and combinatorial CART experiments will be reported at the meeting.

Abstract Background The impact of cytomegalovirus (CMV) infection on the risk of relapse after Hematopoietic Cell Transplantation (HCT) remains controversial, with some studies demonstrating lower risk (Green et al, Blood 2013), while others demonstrating no impact (Schmidt-Hieber et al, Blood 2013), or lower relapse risk only in myeloablative (MA) (Manjappa et al, BBMT, 2014) or in reduced intensity conditioning (RIC) (Cichocki et al, ASH 2014) HCT. Furthermore, the majority of studies have used antigenemia for CMV monitoring, while there is limited data with the use of Quantitative Nucleic Acid Testing (QNAT). Aim To determine the impact of CMV infection, as detected by QNAT, on transplant-related outcomes for patients with acute myeloid leukemia (AML), stratified by MA or RIC HCT. Methods After IRB approval, consecutive adults with AML who underwent allogenic HCT from HLA-matched peripheral blood (PB) grafts at our institution between 2006 and 2014 were retrospectively reviewed. All grafts were T-cell replete. Early CMV infection was defined as any positive CMV result by PCR assay during the first 100 days post HCT. Relapse incidence (RI), non-relapse mortality (NRM), disease-free survival (DFS), and overall survival (OS) were compared between patients with and without CMV infection, and stratified according to MA or RIC. DFS and OS were estimated using the Kaplan-Meier method, and compared with log-rank test. Cumulative incidence of relapse and NRM were analyzed as competing events, using Gray's test. Multivariable analyses adjusting for patient's age, disease status at transplant, cytogenetic risk, and ABO incompatibility were performed using Cox's proportional hazards regression, and the Fine and Gray approach. Results A total of 190 consecutive patients, including 94 (49%) MA and 96 (51%) RIC HCT were analyzed. Patients, disease, and transplant characteristics are shown in table 1. Of the 94 MA HCT (median age 45 y, 50% male), 66 (70%) received total body irradiation. After a median follow up of 18 m, 36 (38%) CMV infection, 16 (17%) relapses, 22 (23%) NRM and 35 (37%) deaths occurred in the MA group. Median time to CMV infection and to relapse in this group was 38 (range 8-241) and 140 (29-1690) days, respectively. Among the recipients of MA HCT, patients with early CMV infection showed no difference in the RI (figure 1), but had a significant trend towards higher NRM and significantly lower DFS and OS in both univariate and multivariable analysis (figure 2) (P values shown in table 2). Among 96 RIC HCT (median age 59 y, 56% male), fludarabine plus melphalan was used in 62 (65%) and fludarabine plus busulfan in 34 (35%) patients. In this group, early CMV infection occurred in 33 (34%), relapse in 25 (26%), NRM in 25 (26%), and deaths in 46 (48%) patients after a median follow up of 21 m (1-91 m). Among the recipients of RIC HCT, median time to CMV infection and to relapse was 40 (17-175) and 291 (19-1971) days, respectively. In the RIC group, early CMV infection was associated with a significant trend towards lower RI (figure 1) and higher NRM in both univariable and multivariable analysis, but no difference in DFS and OS (figure 2). Conclusion Our study of a large homogenous group of AML patients who underwent a uniform HLA-matched PB HCT demonstrates a significantly lower risk of relapse associated with early CMV infection detected by QNAT following RIC, but not MA HCT. Early CMV infection in MA HCT is associated with higher NRM and therefore inferior survival, whereas in the RIC HCT, there is no impact on survival as the benefit of lower relapse is offset by the higher NRM. Table 1. Patients, disease and transplant characteristics according to CMV infection All Patients Patients with CMV Viremia Patients without CMV Viremia P value Patients, no (%) 190 (100) 69 (36) 121 (64) - Median age, y (range) 54 (18-72) 54 (26-70) 54 (18-72) 0.75 Male Patients, no (%) 104 (55) 30 (43) 74 (61) 0.02 D/R CMV Serostatus, no (%) <0.0001 -/- 29 (15) 1 (1) 28 (23) +/+ 64 (34) 35 (51) 29 (24) +/- 13 (7) 1 (1) 12 (10) -/+ 84 (44) 32 (47) 52 (43) Conditioning 0.65 MA 96 (51) 33 (48) 63 (52) RIC 94 (49) 36 (52) 58 (48) Disease status at Transplant 0.96 CR1 129 (68) 46 (67) 83 (69) CR2 34 (18) 13 (19) 21 (17) Other 27 (14) 10 (14) 17 (14) Cytogenetic 0.65 Favorable 8 (4) 2 (3) 6 (5) Intermediate 130 (68) 46 (67) 84 (69) Unfavorable 52 (28) 21 (30) 31 (26) Disclosures Al-Kali: Novartis: Research Funding.

Abstract Background: Molecular profiling has revolutionized the field of hematology, and in particular myeloid neoplasms. NPM1+/FLT3- mutational status has been shown to have a favorable prognostic impact in patients with acute myeloid leukemia (AML). However, little is known about its interaction with age. Methods: We retrospectively analyzed the overall survival (OS) in newly diagnosed AML patients, whose FLT3 and NPM1 data were available, and were treated with cytotoxic chemotherapy at our institution between 2003 and 2013. Patients 18 to 60 years of age were categorized as adult, and those above 60 years were classified as elderly. Each of the elderly or adult groups were further divided according to the presence or absence of combined NPM1+/FLT3- mutational status. Estimated probabilities of overall survival were calculated using the Kaplan-Meier method, with the log-rank test to compare groups. Demographics and continuous variables were compared using the Wilcoxon rank sum test. Appropriate IRB approval was obtained. Results: Out of 835 patients with AML, 86 patients with both known FLT3/NPM1 mutation status and receiving induction chemotherapy were found. NPM1+/FLT3- mutational status was present in 11 elderly (median age 68 yr, hemoglobin (Hb) 8.7 g/dL, white blood cells (WBC) 9.6 x109/L, platelets (PLT) 100 x109/L, peripheral blood blasts percentage (PB%) 31), and in 11 adult patients (median age 49 years, Hb 9.1 g/dL, WBC 14 x109/L, PLT 67 x109/L, and PB% 21). Twenty-eight elderly patients (median age 66 yr, Hb 9.4 g/dl, WBC 15.7 x109/L, PLT 66 x109/L, and PB% 33), and 36 adult patients (median age 49 years, Hb 9.1 g/dL, WBC 14 x109/L, PLT 67 x109/L, and PB% 21) were carrying genotypes other than NPM1+/FlT3-. There was no statistical significant difference in the median age, Hb, WBC, PLT, and PB% at the time of diagnosis. Out of 86 patients, 79 had intermediate, 6 had adverse, and 1 had favorable cytogenetic risk. All elderly and adult patients with NPM1+/FLT3- genotype had intermediate risk cytogenetics. When treated with cytotoxic chemotherapy, elderly patients with NPM1+/FLT3- genotype demonstrated a significantly better overall survival compared to elderly patients without this genotypes (p=0.015), and interestingly also to adult AML carrying other genotypes (p=0.028). Surprisingly, there was not an improved survival in adults with NPM1+/FLT3- genotype compared to adults carrying other genotypes (p=0.14), but this may have been related to the small numbers in each group. No significant difference in the overall survival was observed between elderly and adult patients who were carrying NPM1+/FLT3- mutational status (p=0.4). The estimated 5-year survival rates for elderly with and without NPM1+/FLT3- status were 71% vs. 14%, and for adults with and without this genotype were 49% vs. 19%, respectively. Conclusion: Age does not have an impact on the OS in AML patients with NPM1+/FLT3- mutational status, arguing strongly for intense chemotherapy in this group. Elderly AML patients with NPM1+/FLT3- genotype have a superior OS compared to both adult and elderly patients carrying other genotypes, when treated with cytotoxic chemotherapy. Further validation in large prospective studies is warranted. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.