Introduction: For over two decades, asparaginase-based regimen have contributed to the improved outcome of young adults with Ph-negative (Ph-) acute lymphoblastic leukemia (ALL). The use of E. coli-derived asparaginase (ASPA) can be responsible for numerous adverse effects, including silent hypersensitivity, in up to 50% of patients (pts), which has been associated with enzyme inactivation. In the GRAALL-2014 trial, patients (pts) received ASPA in induction and late intensification (LI) phases, with activity monitoring and assessments for anti-ASPA antibodies. Those exhibiting low activity, any clinical allergy to ASPA, or the presence of anti-ASPA antibodies were considered for switching to Erwinase (ERW) during LI. Here we report on the results of this strategy.Patients and methods: Between 2014 and 2020, the multicenter French-Belgian-Swiss GRAALL-2014 trial enrolled 743 adult pts with newly diagnosed Ph- ALL, including 489 with BCP-ALL and 254 with T-ALL. During induction and LI, ASPA was administered as follows: for pts <45y, ASPA was given at 6000 IU/m²/day for 8 infusions every two days (D8,10,12 then 20,22,24,26,28), and for pts 45y+, it was given for 6 infusions. Pts with initial central nervous system (CNS) involvement received only 5 infusions regardless of age during induction.Therapeutic drug monitoring (TDM) included measurement of asparaginase activity 48 hours after the third (D14) and sixth infusions (D26), with low ASPA activity defined as <100 IU/L. Anti-ASPA antibodies (Abs) were detected using an ELISA test, and were measured at baseline, at the end of induction (week 6), and twice during consolidation (week 10, week 12). A switch for ERW 25.000 IU/m²/infusion on the same schedule as ASPA was recommended in the event of clinical allergy (any grade), ASPA activity < 100 UI/L, or Ab detection (any of the 3 post-induction time points).Results: Median age was 35y (range: 18-59) with 239 pts 45y+. Initial median white blood cell count was 11x109/L (range: 0-712) and 87 pts had CNS involvement (12%). Among the 574 pts who underwent TDM assessment on D14, the median asparaginase activity was 660 IU/L (range: 2-2545), with 97.0% achieving levels of ≥100 IU/L. On D26, among 573 assessed pts, the median asparaginase activity was 551 IU/L (range: 2-4741), with 93.4% achieving levels of ≥100 IU/L. The planned schedule of ASPA infusions was administered to 554/737 pts (75.2%). Multivariable analysis identified increased body mass index (OR 0.94, 95%CI[0.91-0.98]), CNS involvement (OR 0.70, 95%CI[0.53-0.93]), and B- versus T-cell phenotype (OR 0.58, 95%CI[0.39-0.67-]) as associated with incomplete ASPA schedule administration.Anti-ASPA Abs (Ab+) were detected in 293/565 pts (51.9%) at the end of induction. This rate was stable at week 10 (49.8%) and week 12 (49.5%). In pts with clinical allergy during induction (n=19), anti-ASPA Abs were more frequently observed, in 79.0%, 89.5%, and 94.1% at week 6, 10, and 12, respectively (p<0.001 at week 12).Among the 447 pts who started LI, anti-ASPA Abs were detected in 240/438 pts (54.8%). Among the 198 Ab- patients,168 (84.8%) started IR with ASPA. Among the 240 Ab+ pts, 163 (67.9%) started IR with ERW, including all pts with clinical allergy during induction, while 57 (23.8%) started IR with ASPA despite switch recommendations. At D14 of LI, median asparaginase activity was 428.5 UI/L (range: 0-2171) and 294/322 assessed pts (91.3%) achieved an activity ≥100 IU/L. Unexpectedly, no difference in asparaginase activity was observed in pts receiving ASPA between Ab+ (669 IU/L, range 42-1418) and Ab- (753.5 IU/L, range 0-2171, P=0.13). On the other hand, Ab+ pts had inferior asparaginase activity if they received ERW (227.5 IU/L, range 2-2037 IU/L, p<0.001). The rate of pts with asparaginase activity ≥100 IU/L was 98.3% for ASPA/Ab-, 95.4% for ASPA/Ab+, and 83.6% for ERW/Ab+. Finally, Ab+ pts who received ERW had a higher cumulative incidence of relapse than those exposed to ASPA (Fine-Gray subdistribution hazard ratio 2.0, 95%CI[1.03;3.97]) with no significant impact on disease-free or overall survival.Conclusion: In the GRAALL-2014 study, about half of the pts exposed to ASPA developed anti-ASPA Abs rapidly after induction phase. In contrast to prior observation, the presence of Abs did not predict ASPA inactivation. Pts who switched to ERW during LI had a higher risk of relapse, possibly due the different pharmacology profile between both asparaginases.