Molecular characterization of pediatric acute myeloid leukemia treated under the NOPHO-DBH-2012 protocol in the Netherlands Free

With chemotherapy intensification reaching its limits for pediatric acute myeloid leukemia (pAML), reflected by a ~5% mortality rate, there is a clear need for novel, more tailored treatment strategies. The advent of next generation sequencing (NGS) has significantly advanced the molecular characterization of pAML with the identification of new molecular subtypes such as NUP98::NDS1 and UBTF-ITD by large scale sequencing studies. In addition, NGS can uncover patient subgroups with actionable mutations and shared transcriptional profiles, potentially guiding targeted therapies such as Menin inhibitors.

In this unique retrospective study, we characterize 156 pAML patients diagnosed between 2014-2023 which were uniformly treated under the NOPHO-DBH 2012 protocol in the Netherlands by RNA sequencing (RNA-seq) and/or whole exome sequencing (WES). Both diagnostic (N=156) and relapse (N=27) material were sequenced when available. The aim of this study is to compare standard diagnostic methods with molecular characterization and assess patient eligibility for targeted therapies.

Patients with available material (N=156) had a median age of 8 years (0-18) and median leukocyte count of 16.8x10⁹/L. At diagnosis, patients were evaluated by flow cytometry, karyotyping and fluorescent in situ hybridization (FISH). Risk stratification was based on measurable residual disease (MRD) determined by flow cytometry; patients with MRD ≥15% after the first treatment course and ≥0.1% before consolidation chemotherapy were classified as high risk, as were patients with FLT3-ITD positivity and without NPM1 mutations (NPM1mt). High-risk patients underwent allogeneic stem cell transplantation.

NGS identified fusions or mutational drivers in 92% of patients (N=144) vs 60% (N=93) by standard diagnostics with the main drivers being KMT2Ar (28%), RUNX1r (15%), CBFB::MYH11 (10%) and NUP98r (8%). WES identified 101 mutations with 58% being recurrent. The most common mutations were NRAS, KRAS, WT1 and FLT3. IDH1/2 mutations were rare and detected in 4% of patients, namely in patients with NUP98::NSD1, NPM1mt or patients with unknown drivers (UnkDs). Notably, KIT mutations were absent in KMT2Ar, NUP98r, NPM1mt and UBTF-ITD patients. In the 11 UnkD patients, the most common mutations were in RUNX1 (36%) and PTPN11 (27%); 40% had moderate MEIS1 and HOXA9/10 expression and 60% experienced relapse.

Previous findings demonstrated the utility of MEIS1/HOXA9 expression to select patients for Menin inhibition (Rasouli et al., Hemasphere 2023; unpublished). This included 7 patients from our cohort selected based on high or low MEIS1/HOXA9 expression. Their bone marrow cells were treated with Revumenib in vitro for 2 weeks, which revealed that patients with high expression (2 UBTF-ITD, 1 DEK::NUP214 and 2 with UnkDs) were sensitive to Revumenib while those with low expression (1 RUNX1r, 1 CEBPAmt) were unresponsive. Therefore, we evaluated potential responsiveness to Menin inhibitors across the whole cohort by MEIS1/HOX gene expression analysis, normalized with DESeq2. Moderate MEIS1/HOXA9 expression was found in 55% of patients, namely those with NUP98r, UBTF-ITD, NPM1mt, nearly all KMT2Ar, 1 RUNX1r, and several with UnkDs. Roughly 30% of patients with MEIS1/HOXA9 expression experienced relapse. This indicates that a major portion of our cohort may have benefited from Menin inhibition.

Moreover, DESeq2 was used to compare patients who experienced relapse compared to patients in continuous complete remission (CCR). Genes LINC00461, SALL4 or PTPRD had elevated expression in a subset of relapsing patients while IGF2BP1 was highly expressed in a subset of CCR patients. Both LINC00461 and SALL4 have been associated with prognosis in AML. Genes elevated in relapse were not subtype specific. Patients with high IGF2BP1 expression included all GATA1mt patients and a subset of KMT2Ar, NPM1mt, patients with UnkDs and 1 RUNX1r patient.In conclusion, we identify genomic drivers in 92% of patients, including high risk groups such as NUP98r and UBTF-ITD. Among 11 patients with UnkDs, 36% had RUNX1 mutation and 27% PTPN11 with a relapse rate of 60%. Almost half of these unknown patients showed elevated MEIS1/HOXA9 expression indicating potential benefit to screen for elevated expression and a possible responsiveness to Menin inhibitors. NGS is now used as a standard method in the Princess Máxima Center for all newly diagnosed pAML patients with a turnaround time of 2 weeks.