A high proportion of patients with acute myeloid leukemia who achieve minimal residual disease (MRD) negative status ultimately relapse because a fraction of pathological clones remains undetected by standard methods. We designed and validated a high-throughput sequencing method for MRD assessment of cell clonotypes with mutations of NPM1, IDH1/2 and/or FLT3-SNVs. For clinical validation, 106 follow-up samples from 63 patients in complete remission were studied by NGS, evaluating the level of mutations detected at diagnosis. The predictive value of MRD status by NGS, multiparameter flow cytometry, or quantitative PCR was determined by survival analysis. The method achieved a sensitivity of 10-4 for SNV mutations and 10-5 for insertions/deletions and could be used in acute myeloid leukemia patients who carry any mutation (86% in our diagnosis data set). NGS-determined MRD positive status was associated with lower disease-free survival (hazard ratio [HR] 3.4, p=0.005) and lower overall survival (HR 4.2, p<0.001). Multivariate analysis showed that MRD positive status by NGS was an independent factor associated with risk of death (HR 4.54, p =0.005) and the only independent factor conferring risk of relapse (HR 3.76, p =0.012). This NGS based method simplifies and standardizes MRD evaluation, with high applicability in acute myeloid leukemia. It also improves upon flow cytometry and quantitative PCR to predict acute myeloid leukemia outcome and could be incorporated in clinical settings and clinical trials.

We evaluated the association of mutations in 34 candidate genes and response to azacitidine in 84 patients with myelodysplastic syndrome (MDS), with 217 somatic mutations identified by next-generation sequencing. Most patients (93%) had ≥1 mutation (mean=2.6/patient). The overall response rate to azacitidine was 42%. No clinical characteristic was associated with response to azacitidine. However, total number of mutations/patient was negatively associated with overall drug response (odds ratio [OR]: 0.56, 95% confidence interval [CI]: 0.33–0.94; p=0.028), and a positive association was found for having ≥1 mutation in a DNA methylation-related gene: TET2, DNMT3A, IDH1 and/or IDH2 (OR: 4.76, 95%CI: 1.31–17.27; p=0.017). Mutations in TP53 (hazard ratio [HR]: 3.88; 95%CI: 1.94–7.75) and EZH2 (HR: 2.50; 95%CI: 1.23–5.09) were associated with shorter overall survival. Meta-analysis of 6 studies plus present data (n=815 patients) allowed assessment of the association of drug response with mutations in 9 candidate genes: ASXL1, CBL, EZH2, SF3B1, SRSF2, TET2, DNMT3A, IDH1/2 and TP53. TET2 mutations predicted a more favorable drug response compared with ‘wild-type’ peers (pooled OR: 1.67, 95%CI: 1.14–2.44; p=0.01). In conclusion, mutations in the DNA methylation pathway, especially TET2 mutations, and low number of total mutations are associated with a better response to azacitidine.

BCR-ABL1-like B-cell precursor acute lymphoblastic leukemia (BCP-ALL) remains poorly characterized in adults. We sought to establish the frequency and outcome of adolescent and adult BCR-ABL1-like ALL using a novel RNA-Seq signature in a series of patients with BCP-ALL. To this end, we developed and tested an RNA-Seq custom panel of 42 genes related to a BCR-ABL1-like signature in a cohort of 100 patients with BCP-ALL and treated with risk-adapted ALL trials. Mutations related to BCR-ABL1-like ALL were studied in a panel of 33 genes by next-generation sequencing (NGS). Also, CRLF2 overexpression and IKZF1/CDKN2A/B deletions were analyzed. Twenty out of 79 patients (12-84 years) were classified as BCR-ABL1-like (25%) based on heatmap clustering, with significant overexpression of ENAM, IGJ, and CRLF2 (P ≤ 0.001). The BCR-ABL1-like subgroup accounted for 29% of 15-60-year-old patients, with the following molecular characteristics: CRLF2 overexpression (75% of cases), IKZF1 deletions (64%), CDKN2A/B deletions (57%), and JAK2 mutations (57%). Among patients with postinduction negative minimal residual disease, those with the BCR-ABL1like ALL signature had a higher rate of relapse and lower complete response duration than non-BCR-ABL1-like patients (P = 0.007). Thus, we have identified a new molecular signature of BCR-ABL1-like ALL that correlates with adverse prognosis in adult patients with ALL.