Title: Cladribine, idarubicin and cytarabine (CLIA1) in patients with acute myeloid leukemia (AML): a phase 2 single-arm trial.

Introduction: Several studies have demonstrated that the addition of cladribine (CdA) to standard cytarabine containing induction regimens improve outcomes for patients with newly diagnosed and relapsed/refractory (RR) acute myeloid leukemia (AML) patients. Based on this experience, we conducted a phase-2 clinical trial to study the efficacy of CdA combined with a higher dose of cytarabine (ARA-C) and idarubicin (IDA) as induction and consolidation treatment in younger patients with AML. Aim and Methods: In this single-arm, open-label, single-centre phase 2 study, we enrolled 153 patients aged ≤ 65 years with AML who had adequate organ function and an Eastern Cooperative Oncology Group (ECOG) performance status of 2 or less. Patients were enrolled under three cohorts: frontline, secondary AML (s-AML), and relapsed/refractory (R/R). All patients). All patients underwent cytogenetic analysis and molecular testing at baseline using a customized next generation sequencing (NGS) panel including known AML related mutations. All patients received induction treatment. Induction regimen consisted of CdA 5 mg/m2 intravenously (IV) over 30 minutes on days 1-5, followed by ARA-C 1 g/m2 IV on days 1-5, and IDA 10 mg/m2 IV days 1-3 (CLIA). Patients who had remission during this induction regimen moved on to consolidation therapy (up to 5 more cycles of CdA 5 mg/m2 IV over 30 minutes on days 1-3 with ARA-C 750 mg/m2 IV on days 1-3 and IDA 8 mg/m2 IV on days 1-2). Sorafenib was added in FLT3-ITD mutated patients during induction and consolidation treatments. The primary outcome measure was overall response rate. Secondary outcomes were overall survival, relapse free survival, toxicity, and induction mortality. All patients were included in the analyses. This trial is ongoing and is registered with ClinicalTrials.gov, number NCT02115295. Results: Between May 31, 2014, and June 2, 2017, 153 patients were enrolled and treated, among whom 65 (42%) belonged to frontline, 15 (10%) to s-AML, and 73 (48%) to R/R cohorts, respectively. In the frontline cohort, the molecular subgroup presenting the higher CR rate was the NPM1 (94%), followed by CEPBA (90%), IDH2 (83%), RAS (81%) and FLT3-ITD (78%). The lower CR rate was observed in patients harboring DNMT3A (69%), TP53 (67%) and ASXL1 (33%) mutation. According to the cytogenetic risk stratification, patients with an adverse risk at baseline showed an ORR of 71%, while patients with in favorable and intermediate risk the ORR was 89% and 93%, respectively (p=0.01). Among R/R patients cohort the ORR rates in NPM1, IDH1, IDH2 and FLT3-ITD cohorts were 58%, 40%, 58% and 70%, respectively. None of patients with TP53 mutation responded to treatment. Also in this cohort, cytogenetic adverse risk (p=0.0028) and complex karyotype (p=0.0092) at baseline significantly affected the ORR rate. At a median study follow up of 24.8 months among survivors, median overall survival estimates were NR, 4.5, and 7.8 months for the frontline, s-AML, and R/R cohorts, respectively. In the frontline group, FLT3-ITD patients, showed a 1- and 2- year OS of 86% and 75% and a CR rate of 95% at 1- and 2-year with a median duration of CR duration not reached. Among R/R setting, patients with complex karyotype had a significantly shorter median OS compared to others (3.3 vs 12.1 months; p=0.0001); according to molecular mutations, no difference was shown in patients harboring FLT3-ITD (8.8 vs 6.7 months; p=0.843), NPM1 (4.7 vs 8.1 months; p=0.213) IDH1 (5.5 vs 7.8 months; p=0.567), and DNMT3A (6.7 vs 10.9 months; p=0.739) compared to their negative molecular counterpart in terms of survival. According to Papaemmanuil genomic classification, the groups of AML with NPM1 mutation and AML with no driver mutations were associated with improved OS when compared to other groups (TP53/aneuploidy, driver mutations but no detected class-defining lesions, mutated chromatin, RNA splicing genes) either in the whole population (p=0.002) or in the R/R setting (p=0.017). Conclusions:: The combination of CLIA constitutes an effective and safe induction regimen in younger AML patients either in frontline or in relapse/refractory setting. Genomic subgrouping identifies high-risk AML groups, particularly TP53/aneuploidy, driver mutations, and chromatin/RNA-splicing genes, in which there exists a critical need for risk-adapted therapeutic approaches.