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Radiosensitivity differs in humans and likely among closely-related primates. Reasons for variation in radiosensitivity are not well known. We examined preirradiation gene expression in peripheral blood among male and female rhesus macaques which did or did not survive (up to 60 days) after whole-body irradiation with 700 cGy (LD66/60). RNA samples originated from a blinded randomized Good Laboratory Practice study in 142 irradiated rhesus macaques. Animals were untreated (placebo), or treated using recombinant human IL-12, G-CSF or combination of the two. We evaluated gene expression in a two-phase study design where phase I was a whole genome screen [next generation sequencing (NGS)] for mRNAs (RNA-seq) using five RNA samples from untreated male and female animals per group of survivor and non-survivor (total n = 20). Differential gene expression (DGE) was defined as a statistically significant and ≥2-fold up- or downregulation of mRNA species and was calculated between groups of survivors and non-survivors (reference) and by gender. Altogether 659 genes were identified, but the overlapping number of differentially expressed genes (DGE) observed in both genders was small (n = 36). Fifty-eight candidate mRNAs were chosen for independent validation in phase II using the remaining samples (n = 122) evaluated with qRT-PCR. Among the 58 candidates, 16 were of significance or borderline significance (t test) by DGE. Univariate and multivariate logistic regression analysis and receiver operating characteristic (ROC) curve analysis further refined and identified the most outstanding validated genes and gene combinations. For untreated male macaques, we identified EPX (P = 0.005, ROC=1.0), IGF2BP1 (P = 0.05, ROC=0.74) and the combination of EPX with SLC22A4 (P = 0.03, ROC=0.85) which appeared most predictive for the clinical outcome for treated and combined (untreated and treated) male macaque groups, respectively. For untreated, treated and both combined female macaque groups the same gene (MBOAT4, P = 0.0004, ROC = 0.81) was most predictive. Based on the probability function of the ROC curves, up to 74% of preirradiation RNA measurements predicted survival with a positive and negative predictive value ranging between 85-100% and associated odds ratios reflecting a 2-3-fold elevated risk for surviving per unit change (cycle threshold value) in gene expression. In conclusion, we identified gender-dependent genes and gene combinations in preirradiation blood samples for survival prediction after irradiation in rhesus macaques.

Recombinant human interleukin-12 (rHuIL-12, HemaMaxTM) is being developed for mitigation of HSARS under the FDA Animal Rule using a NHP model of HSARS for proof of efficacy and clinical studies in healthy human subjects to demonstrate safety. We have shown previously that a single injection of rHuIL-12 administered 24-25 hours after lethal total body irradiaton (TBI), in the absence of antibiotics, fluids or blood products, resulted in improved survival while filgrastim (G-CSF) did not provide any survival benefit in our NHP HSARS model (Basile et al 2012, Gluzman-Poltorak et al 2014, Gluzman-Poltorak et al 2014). The mechanism by which IL-12 mitigates HSARS following TBI appears to involve multiple effects of IL-12 on hematopoieses. In our previous studies, animals treated with rHuIL-12 showed statistically significant reductions in the occurrence of severe neutropenia and severe thrombocytopenia, as well as attenuated nadirs for lymphocytes, neutrophils, platelets, and reticulocytes. To characterize further relationship between survival and hematological nadirs (lymphocytes, neutrophils, platelets, RBCs and reticulocytes) in the rhesus model of HSARS and to better understand the effects of rHuIL-12 versus G-CSF on blood cell nadirs, we undertook a meta-analysis analysis across three studies in irradiated rhesus monkeys. Animals used in this analysis were irradiated (700 cGy) and treated with a single subcutaneous injection of vehicle (n=64) or rHuIL-12 50-500ng/kg (n=108) 24-25 hours after irradiation, or daily subcutaneous injections of G-CSF at 10μg/kg/d for 18 days starting 24-25 hours after irradiation (n=26). Males and females were equal in each group. Lymphocytes, neutrophils and platelets were significantly lower in decedents versus monkeys that survived to day 60 overall and this was true in each treatment group (p Lymphocytes nadir appears to be the strongest and most consistent predictor of death followed by neutrophils and platelets (Spearman’s rank correlation). RBCs and reticulocytes are less informative in terms of predicting survival status. In addition, the receiver operating characteristic (ROC) curve area under the curve (AUC) by nadir value was assessed. The operating characteristics for lymphocytes, neutrophils and platelets allow for relatively large positive predictive values (PPV) of death with relatively high sensitivity. A cutoff value for lymphocytes nadir of 0.08x109/L (values less than or equal predicting death and higher values predicting life) allows for largest PPVs (97.2% and 92.5%) with 76.1% and 62.7% sensitivities for control and rHuIL-12 treatments, respectively. A cutoff value for neutrophils nadir of 0.03x109/L permits for 84% and 71.1% PPVs with sensitivities of 91.3% and 91.5 % for control and rHuIL-12 treatments, respectively. A cutoff value for platelets nadir of 9x109/L permits for 84.1% and 76.8% PPVs with sensitivities of 80.4% and 72.9 % for control and rHuIL-12 treatments, respectively. RBCs and reticulocytes were found less informative. To conclude, in the rhesus model of HSARS we have observed that an augmented hematological nadirs generally predicts an increased potential for survival as this effect reflects early bone marrow regeneration. The nadir for lymphocytes appears to be the strongest and most consistent predictor of death. Decrease of lymphocyte counts has been established as a best marker of bone marrow damage in a large database of human victims of acute radiation (METREPOL, Fliedner et al 2001). Thus, the correlation of our results with the human data supports the validity of our animal model as an accurate representation of human HSARS and its ability to predict effectiveness in humans exposed to lethal radiation. These data also suggest that the significant increase in early bone marrow regeneration seen in our studies, resulting in increases in nadir values for all major blood cell types, may be the main mechanism of action by which rHuIL-12 mitigates the lethality of HSARS. This project has been entirely funded with Federal funds from BARDA/ASPR/DHHS under Contract No. HHSO100201100037C. Disclosures Gluzman Poltorak: Neumedicines Inc.: Employment, Equity Ownership. Vladimir:Neumedicines Inc.: Consultancy, Employment, Equity Ownership. Basile:Neumedicines Inc.: Employment, Equity Ownership, Neumedicines Inc. Patents & Royalties.