Role of F-araA PK and PG on HCT outcome:
None of F-araA PK parameters was associated with OS, EFS, TRM, and RRTs in 169 patients for whom PK data was available. Interestingly, patients carrying the NT5E promoter variant (rs2295890) showed a trend to no rejection (0% vs 7.7%, p=0.07), better EFS (87.5% vs 75.7%, p=0.1), lower late TRM D+100 (0.3% vs 12.5%, p=0.08), better OS (89.7% vs 78%, p=0.25) and lower early TRM D+30 (0.3% vs 8.8%, p=0.2) (Figure 2) . Additionally, none of the patients carrying the NT5E promoter variant developed SOS compared to those with wild-type genotype for this variant (0% Vs 19%, p=0.0007). Logistic regression using the penalized maximum likelihood estimation method highlighted that NT5Epromoter variant (rs2295890) has a protective effect on HCT outcome(Table-3) . No association was observed between DCK polymorphism and outcomes.
Discussion :
Although the toxicity-reduced conditioning regimen containing F-araA/Treo/Thiotepa has a favorable toxicity profile and has shown to improve HCT outcome in high-risk TM patients 1, graft rejection and RRTs still present a roadblock in a subset of patients3,4. In this first single centre study, we have evaluated the PK and PG as well as the dose-exposure-response relationship to F-araA in a large uniform cohort of patients with high-risk TM undergoing HCT.
F-araA PK has been reported previously in patients undergoing HCT for both malignant, benign conditions and in various combination7–11,29–34. Despite wide IIV in F-araA PK in the present study, none of the biochemical or demographic parameters explained this variability. Previous F-araA PopPK studies have identified Glomerular filtration rate (GFR) 11,34 and creatinine clearance 8,10 as significant predictors of F-araA CL. In the present study, we did not include GFR as a covariate in the PopPK model as the patients enrolled in the study had a normal renal function and all the patients received a fixed initial dose of F-araAMP. The dose of F-araA used and the PK parameters in the present study are comparable to the existing reports (Table 4) .
Although the dose-exposure response relationship has been explored previously for Bu 35–40, Cy 41,42, and Treo 43, no such attempt had been made for F-araA in patients with TM. Several studies have described the influence of F-araA PK in HCT outcomes, albeit majority of the studies were conducted in patients undergoing HCT for malignant conditions6,7,10. A recent study carried out in a mixed cohort of patients with malignant and non-malignant 34conditions did not identify any relationship between F-araA PK and HCT outcomes. Despite significant IIV in F-araA PK observed in the present study, none of the F-araA PK parameters was associated with HCT outcomes. This could probably be because of the decreased incidence of events such as rejection or TRM in this non-malignant condition. A recent study in F-araA PK also predicted optimal cumulative exposure of 20 mg*h/L for better EFS, lower TRM, and lower rejection44. However, the study cohort was heterogeneous, and the optimal exposure range was not confirmed in an independent cohort44.
Genetic variants in drug-metabolizing enzymes and transporters may also contribute to PK variability, which in turn could influence HCT outcome. Similar to our previous report on F-araA PK in patients with AA/FA undergoing HCT 9, the patients carrying rs2295890 variant genotype exhibited significantly lower plasma F-araA CL compared to those with wild-type genotype in the present study (Figure 1) . This variant also explained 4.5% of the IIV in F-araA clearance in the POPPK model. Apart from its role in the biotransformation of F-araA45, NT5E/CD73 is a multifunctional ectoenzyme involved in immunosuppression 46, cancer progression47, and tumor microenvironment48,49. When we compared the role of this polymorphism on HCT outcomes, we observed that patients carrying the rs2295890 variant genotype showed better OS, EFS, lower rejection and lower TRM, consistent with our previous finding in AML cohort 50. Low NT5E activity has been reported to be associated with a good prognosis in many malignancies 46,51,52 probably due to the production of less adenosine that suppresses antitumor immunity and by not contributing to metastasis. The role of NT5E activity in HCT setting has not been explored except for few mice model studies, where it was suggested that low NT5E activity could lead to GvL/GvT (Graft Versus Leukemia/Tumor) phenomenon favoring HCT outcome, again reinstating the probable role of Adenosine 5’-triphosphate (ATP)-Adenosine axis in transplant immunology 53–56. We could thus hypothesize that due to the reduced NT5E activity in patients carrying the variant genotype for this polymorphism, there is a lower production of adenosine and higher extracellular ATP activity, which in turn could prevent graft rejection and help in immunosuppression, eventually favoring better HCT outcome. In addition, we observed that none of the patients carrying the rs2295890 variant genotype had SOS. This could also be due to decreased NT5Eactivity in patients carrying the variant genotype for this polymorphism resulting in decreased production of adenosine, thus protecting the liver from fibrosis 57,58. However, the exact mechanism between decreased NT5E activity and SOS needs to be explored further for its implication in pharmacogenetics testing as a plausible biomarker for HCT outcome.