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Background: Blockade of PD-1/PD-L1 pathways enhances anti-leukemic responses in pre-clinical studies. PD1 inhibition alone had limited clinical activity in AML. CLTA4 inhibition demonstrated encouraging single-agent CR's in postASCT patients (pts), especially for extra-medullary (EMD) relapses (Davis M et al, NEJM 2016). AZA+Nivo up-regulated CTLA4 on bone marrow (BM) CD8 cells in both responders (R) and non-responders (NR) on treatment (Daver N, et al, Cancer Discovery 2019), suggesting a triplet of AZA+Nivo+Ipi may abrogate PD-1 mediated resistance. Methods: Pts were eligible for the AZA+Nivo (cohort 1, n=59) if they had R/R AML, ECOG ≤ 2, and adequate organ function. This cohort has completed enrollment. A cohort of AZA+Nivo+Ipi was opened (cohort 2), with the same eligibility criteria. Ipi 1mg/kg Q6 weeks was added to the established AZA+Nivo schedule and found to be safe in a lead-in dose cohort. Results: Cohort 1 (59 R/R AML) pts were treated with Aza+Nivo (Daver et al., Cancer Discovery 2019). CR/CRi and OS were superior to contemporary historic HMA-based clinical trial controls at MDACC (Table 1). Pts with low pretherapy BM blasts ( Cohort 2 (36 R/R AML) pts were treated with Aza+Nivo+Ipi, with med age 67 years (25-83), secondary AML (50%), ELN unfavorable cytogenetics (67%), TP53 (36%), med salvage 2 (range, 1-3), and prior HMA based therapies in 67%. All 36 pts are evaluable. Per ELN 2017, CR/CRi was noted in 7 (19%) and PR in 1 (3%). Five (14%) pts had durable stable disease (SD) (defined as absence of CR, CRi, PR; with SD on treatment for ≥6 months), and 23 (64%) were NRs (Table 1). Interestingly, 4 pts with EMD were enrolled and 3 had CR/PR [med DOR 8 (5-13) mos]. The 4- and 8-week mortalities were 0 and 6%, respectively. Grade 3/4 immune toxicities noted in 8 pts (19%), including rash, pneumonitis, colitis, pyrexia. One pt required ICU stay, but no deaths attributed to immune toxicity. Other grade ≥2 toxicities were as expected for R/R AML population and were mostly infections/febrile neutropenia. Converse to AZA+Nivo, on CYTOF, R did not have a higher frequency of pre-therapy BM CD8+ infiltration, but did have progressive BM CD8+ infiltration on therapy, compared with NRs, demonstrating that Ipi (unlike Nivo) may be able to mobilize peripheral T-cells to the BM (Fig 1B). Expansion of a cluster of antigen experienced CD8+ T cells was associated with response (Fig 1B). In all salvage the med OS with Aza+Ipi+Nivo versus Aza+Nivo versus contemporary HMA-controls in R/R AML, were 7.6, 5.9, and 4.6 mos, respectively (P=0.01) (Fig 1C). The 1-year OS in R/R AML pts with AZA+Nivo+Ipi was 25%. The med OS with Aza+Ipi+Nivo was comparable to med OS 6-8 mos reported with HMA+VEN salvage in numerous studies. Conclusion: The OS with Aza+Nivo+Ipi was modestly improved over AZA+Nivo and HMA-controls in R/R AML. Single-cell cytokine profiling and single-cell RNA-seq from Aza+Nivo showed striking pre- and on-treatment differences among R and NR not only in T-cell fitness and clonality, but also in the tumor microenvironment. Ipi may be uniquely able to mobilize peripheral T-cell to BM and EMD. This underappreciated immune diversity suggests a critical need for biomarker-based trials (as we are doing with molecular therapies) for immunotherapies in AML. Disclosures Daver: Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Trovagene: Research Funding; Fate Therapeutics: Research Funding; ImmunoGen: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Trillium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Syndax: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Research Funding; Servier: Research Funding; Genentech: Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novimmune: Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Garcia-Manero:Merck: Research Funding; Onconova: Research Funding; Novartis: Research Funding; Jazz Pharmaceuticals: Consultancy; Helsinn Therapeutics: Consultancy, Honoraria, Research Funding; H3 Biomedicine: Research Funding; AbbVie: Honoraria, Research Funding; Acceleron Pharmaceuticals: Consultancy, Honoraria; Amphivena Therapeutics: Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. 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OffLabel Disclosure: Nivolumab and Ipilimumab based combinations for AML, will be discussed. These agents do not have an on label indication for AML at this time.

Preclinical models have shown that blocking PD-1/PD-L1 pathways enhances antileukemic responses. Azacitidine upregulates PD-1 and IFNγ signaling. We therefore conducted this single-arm trial, in which patients with relapsed/refractory (R/R) acute myeloid leukemia (AML) were treated with azacitidine 75 mg/m2 days 1 to 7 intravenously or subcutaneously with nivolumab 3 mg/kg intravenously on days 1 and 14, every 4 to 6 weeks. For the seventy patients who were treated, the median age was 70 years (range, 22–90) and the median number of prior therapies received was 2 (range, 1–7). The overall response rate (ORR) was 33%, including 15 (22%) complete remission/complete remission with insufficient recovery of counts, 1 partial response, and 7 patients with hematologic improvement maintained >6 months. Six patients (9%) had stable disease >6 months. The ORR was 58% and 22%, in hypomethylating agent (HMA)–naïve (n = 25) and HMA-pretreated (n = 45) patients, respectively. Grade 3 to 4 immune-related adverse events occurred in 8 (11%) patients. Pretherapy bone marrow and peripheral blood CD3 and CD8 were significantly predictive for response on flow cytometry. CTLA4 was significantly upregulated on CD4+ Teff in nonresponders after 2 and 4 doses of nivolumab. Azacitidine and nivolumab therapy produced an encouraging response rate and overall survival in patients with R/R AML, particularly in HMA-naïve and salvage 1 patients. Pretherapy bone marrow aspirate and peripheral blood CD3 percentage may be biomarkers for patient selection. Significance: Azacitidine in combination with nivolumab appeared to be a safe and effective therapy in patients with AML who were salvage 1, prior hypomethylator-naïve, or had increased pretherapy CD3+ bone marrow infiltrate by flow cytometry or IHC. Bone marrow CD3 and CD8 are relatively simple assays that should be incorporated to select patients in future trials. This article is highlighted in the In This Issue feature, p. 305

Background Phenotypic characterization of immune cells in the bone marrow (BM) of patients with acute myeloid leukemia (AML) is lacking. Methods T‐cell infiltration was quantified on BM biopsies from 13 patients with AML, and flow cytometry was performed on BM aspirates (BMAs) from 107 patients with AML who received treatment at The University of Texas MD Anderson Cancer Center. The authors evaluated the expression of inhibitory receptors (programmed cell death protein 1 [PD1], cytotoxic T‐lymphocyte antigen 4 [CTLA4], lymphocyte‐activation gene 3 [LAG3], T‐cell immunoglobulin and mucin‐domain containing‐3 [TIM3]) and stimulatory receptors (glucocorticoid‐induced tumor necrosis factor receptor‐related protein [GITR], OX40, 41BB [a type 2 transmembrane glycoprotein receptor], inducible T‐cell costimulatory [ICOS]) on T‐cell subsets and the expression of their ligands (41BBL, B7‐1, B7‐2, ICOSL, PD‐L1, PD‐L2, and OX40L) on AML blasts. Expression of these markers was correlated with patient age, karyotype, baseline next‐generation sequencing for 28 myeloid‐associated genes (including P53), and DNA methylation proteins (DNA methyltransferase 3α, isocitrate dehydrogenase 1[IDH1], IDH2, Tet methylcytosine dioxygenase 2 [TET2], and Fms‐related tyrosine kinase 3 [FLT3]). Results On histochemistry evaluation, the T‐cell population in BM appeared to be preserved in patients who had AML compared with healthy donors. The proportion of T‐regulatory cells (Tregs) in BMAs was higher in patients with AML than in healthy donors. PD1‐positive/OX40‐positive T cells were more frequent in AML BMAs, and a higher frequency of PD1‐positive/cluster of differentiation 8 (CD8)‐positive T cells coexpressed TIM3 or LAG3. PD1‐positive/CD8‐positive T cells were more frequent in BMAs from patients who had multiply relapsed AML than in BMAs from those who had first relapsed or newly diagnosed AML. Blasts in BMAs from patients who had TP53‐mutated AML were more frequently positive for PD‐L1. Conclusions The preserved T‐cell population, the increased frequency of regulatory T cells, and the expression of targetable immune receptors in AML BMAs suggest a role for T‐cell–harnessing therapies in AML., T‐cell subsets are preserved in the bone marrow of patients with acute myeloid leukemia. The expression of targetable immune checkpoints by T cells suggests that therapies harnessing T cells may benefit these patients.

Background: Preclinically blocking PD-1/PD-L1 pathways enhanced anti-leukemic responses. PD-1 positive CD8 T-cells are increased in the bone marrow (BM) of patients (pts) with relapsed AML (Williams P et al., Cancer 2018). PD1 inhibition alone however had limited clinical activity in AML (Berger et al, Clin Cancer Res 2008). AZA up-regulates PD-1 and PD-L1 in AML and the up-regulation of these genes has been associated with emergence of resistance to AZA (Yang et al., Leukemia 2013). Methods: Pts were eligible for the AZA+Nivo (cohort 1) if they had relapsed/refractory AML (R/R AML), ECOG ≤ 2, and adequate organ function. 70 R/R AML pts were treated. This cohort is closed. A subsequent cohort of AZA+Nivo+Ipi was opened (cohort 2), with the same eligibility criteria. Ipi 1mg/kg Q6 weeks was added to the established AZA+Nivo schedule. This double CPI dosing was based on lung and melanoma dosing. Results: Cohort 1 of 70 R/R AML pts treated with Aza+Nivo (Daver et al., Cancer Discovery 2018). Response rates and OS were superior to contemporary historic HMA-based clinical trial controls at MDACC (Table 1), with most significant OS improvement in patients with low pretherapy BM blasts (10 with a statistically significant OS difference for PSI< vs > 10 (p= 0.0018) (Figure1B), suggesting this may be a more specific biomarker to prospectively select pts for T-cell therapy based trials. RNAseq and nanostring analysis on baseline R vs NR sample will be presented at the meeting. Cohort 2 of 31 R/R AML pts treated with Aza+Nivo+Ipi with median (med) age 71 years (26-86), secondary AML (49%), ELN unfavorable cytogenetics (65%), TP53 (38%), med salvage 2 (range, 1-4). 54% pts previously treated with HMA based therapies. 4 pts had prior alloSCT (med time from alloSCT 13 monts). 24 pts are evaluable, 7 too early. CR/CRi was noted in 9 (36%), additionally 2 (8%) had HI maintained >6 months, 4 (16%) stable disease (SD) (defined as absence of CR, CRi, PR, MLFS; with stable disease on treatment for at least 6 months), and 10 (40%) were NRs. The 4-week and 8-week mortality were 0 and 8%, respectively. In all salvage setting the med OS in Aza+Ipi+Nivo versus Aza+Nivo versus contemporary historical HMA-based clinical trial in R/R AML at MDACC, were 10.5, 6.4, and 4.6 months, respectively (P =0.0025) (Figure 1C). This median OS with Aza+Ipi+Nivo compares favorably even to DAC10+venetoclax in R/R AML (med OS: 7.1 months) reported from our center at ASH 2019. The 1-year OS in R/R AML pts with AZA+Nivo+Ipi was very encouraging at 45%. Grade 3/4 immune mediated toxicities were observed in 6 pts (25%), including rash, pneumonitis, and colitis. One pt required ICU stay but no deaths were attributed to immune toxicity. Other grade >2 toxicities were as expected for R/R AML population and were mostly infections/febrile neutropenia, and electrolyte disturbance. Conclusion: The CR/CRi rates and OS with Aza+Nivo+Ipi are encouraging, with median OS of >10 months in R/R high-risk AML, and the study is enrolling. PSI on pretherapy BM CD4 T-cells from pts treated with Aza+Nivo almost completely segregated Rs vs NRs (p=0.0317). This suggests a very significant and hitherto underappreciated immune diversity in AML and a critical need for biomarker based trials (as we are doing with molecular therapies) with immunotherapies in AML. Disclosures Garcia-Manero: Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding. Konopleva:Ablynx: Research Funding; Agios: Research Funding; Astra Zeneca: Research Funding; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Kisoji: Consultancy, Honoraria; Ascentage: Research Funding; Genentech: Honoraria, Research Funding; Calithera: Research Funding; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Cellectis: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Eli Lilly: Research Funding; Forty-Seven: Consultancy, Honoraria. Pemmaraju:incyte: Consultancy, Research Funding; affymetrix: Research Funding; sagerstrong: Research Funding; Daiichi-Sankyo: Research Funding; plexxikon: Research Funding; novartis: Consultancy, Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; cellectis: Research Funding; mustangbio: Consultancy, Research Funding; abbvie: Consultancy, Honoraria, Research Funding; celgene: Consultancy, Honoraria; samus: Research Funding. Kadia:Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Research Funding; Jazz: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bioline RX: Research Funding; BMS: Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding. DiNardo:celgene: Consultancy, Honoraria; syros: Honoraria; notable labs: Membership on an entity's Board of Directors or advisory committees; abbvie: Consultancy, Honoraria; agios: Consultancy, Honoraria; jazz: Honoraria; daiichi sankyo: Honoraria; medimmune: Honoraria. 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7041 Background: Combining PD-1/PD-L1 blockade with hypomethylating agents (HMA) shows encouraging preliminary efficacy in AML, but immune features predictive of response are lacking. Methods: We treated 11 relapsed/refractory (R/R) AML patients with azacitidine (AZA) and nivolumab (Nivo) in a phase 2 clinical trial ( Daver N et al Cancer Discovery 2018). Patient characteristics: median (med) age 65 years (47-73), 63% adverse cytogenetics, 27% TP53 mutated. Pretreatment bone marrow aspirates had immune-phenotypic 17-color flow analysis and NanoString RNA quantification of 1469 immune-relevant genes. Results were correlated with clinical, pathological and molecular data. Results: The median courses of AZA+Nivo administered was 3 (range 1-17). The CR/CRi rate was 45% (including 2 CR, 1 CRi, 1 CRN and 1 CRP), with a median time to response of 1.8 months (range 0.8-4.9 months). The median overall survival was 13 months with 27% patients alive at 1 year. We found significant positive correlations between proportions of T-effector cells at baseline, and CD3+, CD8+, and T-regulatory cells at end of cycle (EOC) 1 (r > 0.75, p < 0.01 for all). At EOC2, these correlations were no longer significant. However, there was a significant positive correlation between T-effector cells at baseline and T-regulatory cells (r = 1, p < 0.001) at EOC4. Using NanoString analysis, we found 105 differentially expressed genes (fold change = 1.5, p < 0.05) between responders (5/11) and non-responders (6/11) at pretreatment. IL2-STAT5, TP53 and TNF Hallmark pathways and immune response from GO gene sets were highly enriched (q < 5x10-4) in responders. We then utilized z-score distribution analysis to quantify the degree of activation of known immunologic pathways. We found that signatures highly specific to neutrophils, NK cells, T-cells and eosinophils were significantly (p < 0.05) upregulated in patients with CR compared to non-responders at pretreatment. Conclusions: Our data demonstrates that a signature suggestive of lymphocyte activation in the pretreatment BM may be associated with augmented clinical response to PD-1 based therapies. Similar underlying pathways that have consistently predicted for responses to PD-1 inhibition in solid cancers, primarily IL2-STAT5 genes, may have predictive relevance in AML. Such pretreatment flow and NanoString signatures may help select AML patients most likely to benefit from PD-1 blockade plus HMA, further enhancing the benefit-risk ratio with such therapies.

Background: Preclinically blocking PD-1/PD-L1 pathways enhanced anti-leukemic responses. PD-1 positive CD8 T-cells were increased in the bone marrow (BM) of pts with AML (Daver et al, ASH 2016). Azacitidine up-regulates PD-1 and interferon-gamma signaling (Yang et al., Leukemia 2013). Methods: Pts were eligible for the AZA+Nivo (cohort 1) if they had relapsed/refractory AML (R/R AML), ECOG ≤ 2, and adequate organ function. The recommended phase 2 dose was established as AZA 75mg/m2 Days 1-7 with Nivo 3mg/kg Day 1 and 14. Courses were repeated approximately every 4-5 weeks indefinitely. 70 R/R AML pts have been treated; cohort 1 is closed. A subsequent cohort of AZA+Nivo+Ipi in R/R AML was opened (cohort 2), with the same eligibility criteria as cohort 1 except that enrollment was restricted to pts with salvage 1 and 2 AML. Ipi 1mg/kg Q6 weeks was added to the established AZA+Nivo schedule. This dosing was based on lung and melanoma dosing for Ipi + Nivo. Responses in both cohorts included best response within 3 months of therapy initiation. Results: Cohort 1: 70 R/R AML pts with median (med) age 70 years (range, 22-90), secondary AML (44%), poor risk cytogenetics (34%), med salvage 2 (range, 1-7) were enrolled. The ORR was 33% including 15 CR/CRi (22%) (4 CR, 11 CRi), 1 PR, and 7 HI maintained on study >6 months (10%) without allogeneic stem cell transplant. Additionally, 6 pts (9%) remained on study with stable disease (SD) >6 months. The remaining 41 pts (58%) had no response. 8-week (wk) mortality was 10%. Patients who achieved a CR/CRi/PR/HI/SD (n=29; 42%) had significantly improved overall survival (OS) compared with NRs (n=41; 58%) (16.1 versus 4.1 months; p Flow-cytometry (MFC) was performed on pre- and post-therapy BMAs, after 2 doses (end of cycle 1 or EOC1) and 4 doses (EOC2) of nivol in 19 of 23 responders (R) (CR/CRi/PR/HI) (83%) and 28 of 41 NRs (68%). 36 parameter CyTOF was performed at same time-points in 5R and 5NRs. On pretherapy BMAs, R had a higher frequency of CD3+ (p=0.04) and CD8+ cells (p=0.09) on MFC (Fig 2A). CD3+ in the pretherapy BMA with a cut-off of 13.2%, had a sensitivity of 74% and a specificity of 65% for predicting response. The CD3+ was >13.2% in 26 of 47 pts (55%) who had an evaluable pretherapy BMA. Immunohistochemistry confirmed that CD3+ cells were increased in pretherapy BMs in R. PhenoGraph clustering of all CD3-gated cells revealed 24 meta-clusters (Fig 2B). The frequency of an effector CD8+ T cell cluster (C2) expressing CD45RA+PD1loTbethiEomeslo was significantly higher in the pretherapy BMAs of R versus NRs (11.2% versus 2.5%; p=0.002), and was further expanded in R not in NRs in the EOC1, EOC2, and EOC4 BMAs (Fig 2C). Cohort 2: 20 pts with R/R AML, med salvage 2 (range 1-2), med age 68 (range, 25-83), secondary AML (36%), adverse cytogenetics (25%), and common molecular mutations TP53 in 5, RAS in 4, DNMT3A/ASXL1 in 2 pts each, were enrolled. 14 pts enrolled before May 1, 2018 were evaluable for response: 6 CR/CRp/CRi (43%) (including 2 CR, 2CRp, 2CRi), 2 stable disease > 6 mths, and 6 no response. The 8-wk mortality was 0. Grade 3/4 irAEs noted in 26% of the pts, most common pneumonitis. Med cycles to response of 2 (1-3). The med OS for all pts was not reached and the projected 1-yr OS in R/R AML was encouraging at 58% (Fig 1). Conclusion: AZA+Nivo was effective in pts with AML in Salvage 1 or those with increased pretherapy CD3+ BM infiltrate by MFC or IHC. BM CD3 and CD8 are simple assays to select pts for future trials of AZA+PD1 inhibitors in AML. AZA+Nivo+Ipi had a encouraging CR/CRp/CRi rate (43%) and med OS (NR) in pts with salvage 1/2 AML and is enrolling. Disclosures Daver: BMS: Research Funding; ImmunoGen: Consultancy; Sunesis: Consultancy; Incyte: Research Funding; Novartis: Consultancy; Karyopharm: Consultancy; Daiichi-Sankyo: Research Funding; Novartis: Research Funding; Pfizer: Consultancy; Karyopharm: Research Funding; Pfizer: Research Funding; Kiromic: Research Funding; ARIAD: Research Funding; Otsuka: Consultancy; Sunesis: Research Funding; Incyte: Consultancy; Alexion: Consultancy. Cortes:novartis: Research Funding. Ravandi:Bristol-Myers Squibb: Research Funding; Astellas Pharmaceuticals: Consultancy, Honoraria; Sunesis: Honoraria; Jazz: Honoraria; Xencor: Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Honoraria, Research Funding, Speakers Bureau; Astellas Pharmaceuticals: Consultancy, Honoraria; Xencor: Research Funding; Sunesis: Honoraria; Seattle Genetics: Research Funding; Seattle Genetics: Research Funding; Macrogenix: Honoraria, Research Funding; Macrogenix: Honoraria, Research Funding; Abbvie: Research Funding; Abbvie: Research Funding; Amgen: Honoraria, Research Funding, Speakers Bureau. Kadia:Takeda: Consultancy; Abbvie: Consultancy; BMS: Research Funding; BMS: Research Funding; Pfizer: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Celgene: Research Funding; Pfizer: Consultancy, Research Funding; Celgene: Research Funding; Abbvie: Consultancy; Jazz: Consultancy, Research Funding; Novartis: Consultancy; Takeda: Consultancy; Amgen: Consultancy, Research Funding; Novartis: Consultancy; Jazz: Consultancy, Research Funding. Konopleva:Stemline Therapeutics: Research Funding; Immunogen: Research Funding; abbvie: Research Funding; cellectis: Research Funding. DiNardo:Celgene: Honoraria; Medimmune: Honoraria; Abbvie: Honoraria; Karyopharm: Honoraria; Bayer: Honoraria; Agios: Consultancy. Pemmaraju:daiichi sankyo: Research Funding; SagerStrong Foundation: Research Funding; Affymetrix: Research Funding; novartis: Research Funding; abbvie: Research Funding; stemline: Consultancy, Honoraria, Research Funding; plexxikon: Research Funding; samus: Research Funding; celgene: Consultancy, Honoraria; cellectis: Research Funding.

Background: Blocking PD-1/PD-L1 pathways enhances anti-leukemia responses by enabling T-cells in murine models of AML (Zhang et al, Blood 2009). PD-1 positive CD8 T-cells are increased in bone marrow (BM) of pts with AML (Daver et al, AACR 2016). PD1 inhibition has shown activity in AML (Berger et al, Clin Cancer Res 2008). We hypothesized that addition of nivolumab to an induction regimen of ara-C and idarubicin may prolong relapse-free survival (RFS) and overall survival (OS); this study was designed to determine the feasibility of this combination. Methods: Pts with newly diagnosed acute myeloid leukemia (by WHO criteria; ≥20% blasts) and high risk MDS (≥10% blasts) were eligible to participate if they were 18-65 yrs of age and had adequate performance status (ECOG ≤3) and organ function (LVEF ≥ 50%; creatinine ≤ 1.5 g mg/dL, bilirubin ≤ 1.5 mg/dL and transaminases ≤ 2.5 times upper limit of normal). Treatment included 1 or 2 induction cycles of ara-C 1.5 g/m2 over 24 hours (days 1-4) and Idarubicin 12 mg/m2 (days 1-3). Nivolumab 3 mg/kg was started on day 24 ± 2 days and was continued every 2 weeks for up to a year. For pts achieving complete response (CR) or CR with incomplete count recovery (CRi) up to 5 consolidation cycles of attenuated dose ara-C and idarubicin was administered at approximately monthly intervals. Eligible pts received an allogeneic stem cell transplant (alloSCT) at any time during the consolidation or thereafter. Results: 3 pts with relapsed AML were treated at a run-in phase with a dose of nivolumab 1 mg/kg without specific drug-related toxicity. Subsequently, 32 pts (median age 53 yrs; range, 26-65) were treated as above including 30 with AML (24 de novo AML, 2 therapy-related AML, 3 secondary AML and 1 therapy-related secondary AML) and 2 high risk MDS. Pre-treatment genetic risk by ELN criteria was 11 adverse, 16 intermediate, and 5 favorable, including 2 FLT3 -ITD mutated, 5 NPM1 mutated, and 7 TP53 mutated. All 32 pts were evaluable for response and 23 (72%) achieved CR/CRi (19 CR, 4 CRi). The 4-week and 8 week mortality was 6% and 6%. The median number of doses of nivolumab received was 6 (range, 0-13); one pt did not receive nivolumab due to insurance issues. 9 pts underwent an alloSCT. After a median follow-up of 8.3 mths (range, 1.5-17.0) the median RFS among the responding pts has not been reached (range, 0.1 - 15.8 mths) and the median OS has not been reached (range 0.5-17.0 mths). Grade 3/4 immune mediated toxicities have been observed in 5 pts and include rash, pancreatitis, and colitis. Other grade 3/4 toxicities thought to be potentially related to nivolumab include cholecystitis in one pt. 9 pts proceeded to an alloSCT. Donor source was matched related in 2, matched unrelated in 6 and haplo-identical in 1 pt. Conditioning regimen was Fludarabine plus busulfan-based in 8, and fludarabine plus melphalan in 1 pt. 4 pts developed graft versus host disease (GVHD)(grade I/II in 3, grade III/IV in 1), which responded to treatment in 3. Multicolor flow-cytometry studies are conducted by the Immunotherapy Platform on baseline (prior to first dose of nivolumab) and on-treatment BM aspirate and peripheral blood to assess the T-cell repertoire and expression of co-stimulatory receptors and ligands on T-cell subsets and leukemic blasts, respectively. The baseline BM was evaluated on 23 of the 32 evaluable pts, including 18 responders and 5 non-responders. Pts who achieved a CR/CRi had a trend of higher frequency of live CD3+ total T cell infiltrate as compared to non-responders in the baseline BM aspirates (Fig 1A). We evaluated expression of immune markers on T cell subsets: CD4 T effector cells [Teff]: CD3+CD4+CD127lo/+Foxp3-, CD4 T regulatory cells [Treg]: CD3+CD4+CD127-Foxp3+, and CD8 T cells. At baseline, BM of non-responders had significantly higher percentage of CD4 T effector cells co-expressing the inhibitory markers PD1 and TIM3 (p Conclusion: Addition of nivolumab to ara-C and anthracycline induction chemotherapy is feasible and safe in younger pts with AML. Among the pts proceeding to alloSCT the risk of GVHD is not significantly increased. Figure 1 Figure 1. Disclosures Daver: Pfizer Inc.: Consultancy, Research Funding; Otsuka America Pharmaceutical, Inc.: Consultancy; Sunesis Pharmaceuticals, Inc.: Consultancy, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Bristol-Myers Squibb Company: Consultancy, Research Funding; Kiromic: Research Funding; Karyopharm: Consultancy, Research Funding; Jazz: Consultancy; Immunogen: Research Funding; Daiichi-Sankyo: Research Funding; Incyte Corporation: Honoraria, Research Funding. Thompson: Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees. Jabbour: Bristol-Myers Squibb: Consultancy. Takahashi: Symbio Pharmaceuticals: Consultancy. DiNardo: Novartis: Honoraria, Research Funding; Daiichi-Sankyo: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Sharma: Jounce: Consultancy, Other: stock, Patents & Royalties: Patent licensed to Jounce; Astellas: Consultancy; EMD Serono: Consultancy; Amgen: Consultancy; Astra Zeneca: Consultancy; GSK: Consultancy; Consetellation: Other: stock; Evelo: Consultancy, Other: stock; Neon: Consultancy, Other: stock; Kite Pharma: Consultancy, Other: stock; BMS: Consultancy. Cortes: BMS: Consultancy, Research Funding; Sun Pharma: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding; ImmunoGen: Consultancy, Research Funding; ARIAD: Consultancy, Research Funding. Kantarjian: Delta-Fly Pharma: Research Funding; Amgen: Research Funding; ARIAD: Research Funding; Novartis: Research Funding; Bristol-Meyers Squibb: Research Funding; Pfizer: Research Funding.

Introduction: The expression of co-stimulatory (costim) receptors/ligands in the bone marrow (BM) and peripheral blood (PB) in patients (pts) with AML has not been defined. Identification of immune-checkpoint pathways that dominate in AML will guide the rational selection of specific antibodies for clinical trials. Methods: Between March, 2015 and October 2015 we performed 17-color multi-parameter flow-cytometry (MFC) on 15 untreated AML and 25 relapsed AML to assess expression of costim ligands (4-1BBL, B7-1, B7-2, ICOSL, PDL-1, PDL-2, OX40L) on leukemic blasts and costim receptors (4-1BB, CTLA-4, ICOS, PD-1, OX40, GITR, LAG-3, TIM-3) on T cell subsets: CD4 T effector cells [Teff]: CD3+CD4+CD127lo/+Foxp3−, CD4 T regulatory cells [Treg]: CD3+CD4+CD127−Foxp3+, and CD8 T cells: CD3+CD8+. Four healthy human BMs were used as control. Expression is denoted by percentage of specific T-cell subset or gated AML blasts positive for the marker indicated. PB mononuclear cells (PBMCs) and blasts were evaluated at the same time-point. Results: OX40+ Teffs and OX40+ CD8 cells were higher in untreated AML BM as compared to healthy donor BM (median [med]: 7.2% versus [vs] 0.26%; P = 0.0005 and med: 2.03% vs 0.06%; P = 0.07, respectively). PD1+ Tregs and OX40+ Tregs were also higher in untreated AML BM as compared to healthy donor BM (med: 19.7% vs 7.5%; P = 0.03 and med: 10.3% vs 0.5%; P = 0.02, respectively). PD1+ Teffs, OX40+ Teffs, and ICOS+ Teffs were higher in relapsed AML BM as compared to healthy donor BM (med: 17.7% vs 6.7%; P = 0.047, med: 12% vs 0.27%, P = 0.002, and med: 13.3% vs 1.1%; P = 0.07, respectively). OX40+ CD8 cells, ICOS+ CD8 cells, TIM3+ CD8 cells (med: 5.0% vs 0.07%; P = 0.04, med: 18.5% vs 2.6%; P = 0.09, and med: 2.7% vs 0.7%; P = 0.01, respectively) and OX40+ Tregs (med: 15.5% vs 0.5%; P Conclusions: Clinically targetable checkpoint receptors including PD1, OX40, and ICOS appear to be overexpressed in the BM of pts with AML as compared to healthy donor BM. Relapsed AML had higher expression of costim receptors than untreated AML. Three anti-PD1 based clinical trials for pts with AML are enrolling at our institution (NCT02397720, NCT02464657, NCT02532231). Citation Format: Naval Daver, Sreyashi Basu, Guillermo Garcia-Manero, Jorge Cortes, Farhad Ravandi, Steven Kornblau, Marina Konopleva, Michael Andreeff, Gautam Borthakur, Nitin Jain, William Wierda, Srdan Verstovsek, Peter Ruvolo, Tapan Kadia, Jairo Matthews, Wilmer Flores, Hui Yang, Carlos Bueso-Ramos, Narmeen Somani, Jorge Blando, James Allison, Hagop Kantarjian, Padmanee Sharma. Defining the immune checkpoint landscape of acute myeloid leukemia (AML). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3205.