RAS pathway mutations have been linked to relapse and chemotherapy resistance in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, comprehensive data on the frequency and prognostic value of subclonal mutations in well-defined subgroups using highly sensitive and quantitative methods are lacking. Targeted deep sequencing of 13 RAS pathway genes was performed in 461 pediatric BCP-ALL cases at initial diagnosis and in 19 diagnosis-relapse pairs. Mutations were present in 44.2% of patients, with 24.1% carrying a clonal mutation. Mutation frequencies were highest in high hyperdiploid, infant t(4;11)-rearranged, BCR-ABL1-like and B-other cases (50–70%), whereas mutations were less frequent in ETV6-RUNX1-rearranged, and rare in TCF3-PBX1- and BCR-ABL1-rearranged cases (27–4%). RAS pathway-mutated cells were more resistant to prednisolone and vincristine ex vivo. Clonal, but not subclonal, mutations were linked to unfavorable outcome in standard- and high-risk-treated patients. At relapse, most RAS pathway mutations were clonal (9 of 10). RAS mutant cells were sensitive to the MEK inhibitor trametinib ex vivo, and trametinib sensitized resistant cells to prednisolone. We conclude that RAS pathway mutations are frequent, and that clonal, but not subclonal, mutations are associated with unfavorable risk parameters in newly diagnosed pediatric BCP-ALL. These mutations may designate patients eligible for MEK inhibitor treatment.

NUTM1 is a recurrent fusion gene partner in B-cell precursor acute lymphoblastic leukemia associated with increased expression of genes on chromosome band 10p12.31-12.2 For 20-25% of patients with pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), the driving cytogenetic aberration is unknown. Identification of the primary lesion could provide better risk stratification and even identify possible treatment options. We therefore aimed to find novel recurrent genetic aberrations in BCP-ALL cases. We identified an in-frame SLC12A6-NUTM1 fusion, resulting in expression of 3' exons of NUTM1, and six additional NUTM1-rearranged fusion cases. These NUTM1-rearranged cases were associated with high expression of a cluster of genes on chromosome band 10p12.31-12.2, including the BMI1 gene. Our data point to NUTM1 fusions as a new entity of BCP-ALL negative for known genetic abnormalities. Pediatric BCP-ALL consists of many cytogenetic subtypes, each with a different prognosis. 1 ETV6-RUNX1 fusions, high hyperdiploid, and TCF3-PBX1 fusions have a favorable prognosis, while BCR-ABL1 fusions and KMT2A/MLL-rearrangements have a poor prognosis.

Pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is associated with a high frequency of copy number alterations (CNAs) in IKZF1, EBF1, PAX5, CDKN2A/B, RB1, BTG1, ETV6, and/or the PAR1 region (henceforth: B-cell development genes). We aimed to gain insight in the association between CNAs in these genes, clinical outcome parameters, and cellular drug resistance. 71% of newly diagnosed pediatric BCP-ALL cases harbored one or more CNAs in these B-cell development genes. The distribution and clinical relevance of these CNAs was highly subtype-dependent. In the DCOG-ALL10 cohort, only loss of IKZF1 associated as single marker with unfavorable outcome parameters and cellular drug resistance. Prednisolone resistance was observed in IKZF1-deleted primary high hyperdiploid cells (~1500-fold), while thiopurine resistance was detected in IKZF1-deleted primary BCR-ABL1-like and non-BCR-ABL1-like B-other cells (~2.7-fold). The previously described risk stratification classifiers, i.e. IKZF1plus and integrated cytogenetic and CNA classification, both predicted unfavorable outcome in the DCOG-ALL10 cohort, and associated with ex vivo drug cellular resistance to thiopurines, or L-asparaginase and thiopurines, respectively. This resistance could be attributed to overrepresentation of BCR-ABL1-like cases in these risk groups. Taken together, our data indicate that the prognostic value of CNAs in B-cell development genes is linked to subtype-related drug responses.

Background Despite significant improvements in the outcome of children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL), therapeutic strategies for high risk and relapsed patients are limited and cause severe side effects. Reliable risk assessment and new therapeutic targets with high specificity are therefore warranted. The RAS pathway is the most frequently mutated pathway in cancer, and the RAF-MEK-ERK kinase axis is crucial for mediating the oncogenic effects of RAS. We and others have previously shown that in pediatric BCP-ALL, RAS pathway mutations can be retrospectively linked to relapse and chemotherapy resistance. However, data on the frequency of (sub-)clonal mutations at diagnosis and hence information about the prognostic relevance at initial diagnosis is lacking. Aim Guide therapy adaptation in pediatric BCP-ALL by evaluating the prognostic relevance of RAS pathway mutations and investigating the sensitivity to MEK inhibition. Methods We performed targeted next-generation sequencing of mutational hotspots in 13 RAS pathway genes to determine the frequency and clonality of RAS pathway mutations in a large, clinically and biologically characterized cohort of BCP-ALL patients. Initial diagnosis samples of 461 patients and 19 matched diagnosis-relapse sets were included. Mutations were considered clonal at ≥25% variant allele frequency, and high coverage allowed detection of subclones with down to 1% variant allele frequency. Clinical outcome was evaluated in 244 patients treated according to a contemporary, minimal residual disease (MRD)-based protocol (DCOG ALL10). The evolution of RAS pathway mutations was studied in 19 matched sets from diagnosis and relapse. Ex vivo sensitivity of RAS pathway mutated cells towards chemotherapeutic agents and trametinib was evaluated in an MTT-based cytotoxicity assay. Results Variants in RAS pathway genes were observed in 44% of initial diagnosis pediatric BCP-ALL cases, mostly affecting NRAS, KRAS, PTPN11, and FLT3. Clonal and subclonal mutations were found in 24% and 20% of patients, respectively. The mutation frequency was highest in high hyperdiploid, infant t(4;11)-positive, BCR-ABL1-like, and B-other cases (50-70%), whereas mutations were rare in ETV6-RUNX1-positive (27%), TCF3-PBX1-positive (8%) and BCR-ABL1-positive cases (4%). In matched diagnosis-relapse sets, clonal mutations at diagnosis were preserved at relapse, whereas the kinetics of subclones was variable. Interestingly, most RAS pathway mutations at relapse were clonal and exclusive. Cells carrying RAS pathway mutations, especially KRAS G13 mutations, were more often ex vivo resistant to prednisolone and vincristine. No association was found with ex vivo response to daunorubicine, L-asparaginase, 6-mercaptopurine, and 6-thioguanine. Mutant primary leukemic cells were ex vivo sensitive to the MEK-inhibitor trametinib. In addition, trametinib could enhance the cytotoxic effect of prednisolone ex vivo. In DCOG-ALL10 and COALL-97/-03 patients with clonal but not subclonal mutations, MRD levels tended to be more often high compared to wildtype cases (31% vs. 19%, p=0.057), while other risk factors (age, gender, white blood cell count, CNS, prednisone response) where not different. Event-free survival was lower in the standard risk and high risk arms of the DCOG ALL10 protocol (69% vs. 96%, p=0.027 and 56% vs. 100%, p=0.015, respectively). Conclusions Collectively, analysis of 461 diagnostic BCP-ALL patient samples identified RAS pathway mutations in 44% of patients, and one out of four carried a clonal mutation. MRD was the only risk factor associated with clonal RAS pathway mutations. MRD is essential to treatment stratification in many contemporary protocols, such as the DCOG ALL10 protocol, where only patients with negative MRD after induction courses are treated with a reduced regimen (standard risk arm). Given their unfavorable event-free survival, therapy should be adapted for mutated patients in future protocols. Since treatment intensification is not feasible for high risk or relapsed cases, addition of MEK inhibitors may be of benefit especially because they enhance the cytotoxicity of prednisolone. RAS pathway mutation status may therefore serve as biomarker to select patients for MEK-inhibitor treatment in new treatment protocols for children with BCP-ALL. Disclosures No relevant conflicts of interest to declare.