Interleukin-6 levels predict event-free survival in pediatric AML and suggest a mechanism of chemotherapy resistance by Alexandra M. Stevens, Jennifer M. Miller, Jaime O. Munoz, Amos S. Gaikwad, and Michele S. Redell BloodAdv Volume 1(18):1387-1397 August 8, 2017 © 2017 by The American Society of Hematology

Alexandra M. Stevens et al. Blood Adv 2017;1:1387-1397 © 2017 by The American Society of Hematology

Cytokine levels vary in pediatric AML at diagnosis. Cytokine levels vary in pediatric AML at diagnosis. Cytokine levels from BM plasma samples taken at the time of diagnosis for patients with untreated AML were compared with those of healthy children. After Bonferroni correction, 3 out of 41 tested cytokines and growth factors had levels at AML diagnosis that were significantly different when compared with normal controls. Levels of IL-6 (A), IL-10 (B), and IL-8 (C) were significantly elevated in a subset of AML patients at diagnosis compared with normal children. ***P < .001 by Mann-Whitney U test. The threshold of significance after Bonferroni correction was P = .0012. Alexandra M. Stevens et al. Blood Adv 2017;1:1387-1397 © 2017 by The American Society of Hematology

Elevated IL-6 levels are associated with inferior clinical outcome. Elevated IL-6 levels are associated with inferior clinical outcome. (A) Using a cut point of 12 pg/mL for the entire cohort (n = 45), 5-year EFS was 20.6% ± 10% for those with high IL-6 levels at diagnosis vs 43.4% ± 12% for those with low IL-6 levels (log-rank P = .036). (B) For LR patients (n = 27), a cut point of 12 pg/mL identified patients with inferior EFS (17.3% ± 11% vs 82.5% ± 11% for high vs low IL-6 levels, P = .0003). (C) Inferior OS was also noted for the LR patients (cut point of 12 pg/mL, 39% ± 14.8% vs 90.1% ± 8.7% for high vs low IL-6 levels, P = .007, n = 27). No other cytokines were associated with clinical outcome in our cohort. Notably, levels of IL-8 (D) and IL-10 (E) were not associated with EFS. Estimates of EFS are reported with corresponding Greenwood standard errors. Groups were compared for significant differences by the log-rank test. Alexandra M. Stevens et al. Blood Adv 2017;1:1387-1397 © 2017 by The American Society of Hematology

Alterations in cytokine levels return toward normal levels at remission in the majority of patients. Alterations in cytokine levels return toward normal levels at remission in the majority of patients. For 22 of the AML patients, matched remission BM plasma samples were available. BM plasma IL-6, IL-10, and IL-8 levels decreased in the majority of patients at remission compared with diagnosis. *P < .05, **P < .01, ***P < .001. #Unique patient number 14. Alexandra M. Stevens et al. Blood Adv 2017;1:1387-1397 © 2017 by The American Society of Hematology

OSM and sIL-6Rα do not predict survival in pediatric AML samples. OSM and sIL-6Rα do not predict survival in pediatric AML samples. For a subset of patients with available samples, levels of OSM and sIL-6Rα were evaluated. For OSM, levels between diagnostic AML BM plasma samples and normal controls differed significantly (A), though there was no difference between levels at diagnosis vs remission in a paired sample analysis (B). No cut point was identified that predicted outcome for OSM level (cut point of 50 pg/mL is shown in panel C). sIL-6Rα BM plasma levels were not significantly different between normal controls and AML samples at diagnosis (D) or between diagnostic and remission samples in a paired sample analysis (E). *P < .05; ns, not significant. Alexandra M. Stevens et al. Blood Adv 2017;1:1387-1397 © 2017 by The American Society of Hematology

Exposure to IL-6 reduces chemotherapy-induced apoptosis. Exposure to IL-6 reduces chemotherapy-induced apoptosis. The AML cell lines THP-1 (A) and NB4 (B) had less mitoxantrone-induced apoptosis when pretreated for 24 hours with IL-6 and sIL-6Rα at 50 ng/mL and 100 ng/mL, respectively (red), than vehicle-treated cells (blue) (THP-1, n = 5; NB4, n = 8; *P < .05, analysis of variance). (C) Representative primary sample demonstrating decreased mitoxantrone-induced apoptosis with IL-6 pretreatment, and (D) mean apoptosis rates of 7 primary AML samples with vs without IL-6 pretreatment. Duration of mitoxantrone exposure was 24 hours for cell lines and 18 hours for primary samples. Values shown are mean ± standard error of the mean; P < .05 by Wilcoxon signed-rank test. FITC, fluorescein isothiocyanate. Alexandra M. Stevens et al. Blood Adv 2017;1:1387-1397 © 2017 by The American Society of Hematology

IL-6–induced pY-STAT3 is increased in the leukemia stem cell enriched (LSCe) subpopulation. IL-6–induced pY-STAT3 is increased in the leukemia stem cell enriched (LSCe) subpopulation. (A) Histograms of sorted subpopulations of a representative diagnostic AML BM sample. Constitutive (yellow) and IL-6–induced (blue) pY-STAT3 are shown for the non–stem AML population (left) and the LSCe population (right). (B) A significant difference in the mean percentage of cells with IL-6–induced pY-STAT3 in the LSCe subpopulation compared with the non–stem AML subpopulation was found. No differences were seen in constitutive pY-STAT3 between sorted populations (n = 8). Bar graphs show mean ± standard error of the mean. *P < .05. PE, phycoerythrin. Alexandra M. Stevens et al. Blood Adv 2017;1:1387-1397 © 2017 by The American Society of Hematology