Molecular characterization of AML with RUNX1-RUNX1T1 at diagnosis and relapse reveals net loss of co-mutations

Abstract

AML with RUNX1-RUNX1T1 fusion is a WHO entity with a favorable outcome following intensive chemotherapy. The absence of RUNX1-RUNX1T1 transcripts in remission defines complete molecular response and correlates with a superior survival. However, a significant proportion of patients still relapses and defining molecular risk factors that identify patients at diagnosis or at molecular remission that are at risk of relapse could help tailor treatment strategies for those high risk patients. Here, we analyze a cohort of 94 patients that reach a molecular remission (MR) following intensive treatment and identify 21 patients that relapse despite achieving MR. Using targeted sequencing of 63 genes implicated in hematologic malignancies we show that at diagnosis patients who relapse following MR have a higher burden of co-mutated genes than patients that do not relapse (median = 2 vs median = 0; P = 0.0156). This resulted in a relapse free survival rate of 65% vs 86% at 2 years, respectively (≥1 co-mutation vs no co-mutation, P = 0.02) with a trend for inferior overall survival (n.s.). Applying sensitive sequencing to reassess mutations at relapse in paired samples of 17/21 patients we demonstrate a net loss of co-mutations at relapse: median 2 (range 0-5) vs 1 (0-4) at diagnosis and relapse (P = 0.048). At relapse more patients had no detected co-mutation compared to diagnosis (47% vs 17%, P = 0.034). Co-mutations at diagnosis, therefore, might represent a general susceptibility of the AML clone to acquire mutations and the true nature of 2nd hit mutations that drive leukemia has to be defined for AML with RUNX1-RUNX1T1 fusion.

Figure 1

The number of co-mutations with RUNX1-RUNX1T1 at diagnosis is associated with relapse. A) Samples of 42 patients that never relapsed and 21 patients that relapsed were available for sequencing. The absolute number of mutated genes in patients with relapse and without relapse (red dot, mean) is significantly different (P = 0.0156). B) The VAF of mutations of patients that relapse and patients without relapse is shown (n.s., red dot, mean).

Figure 2

The number of co-mutations is associated with relapse free survival (RFS). A) RFS of patients with no co-mutated gene vs patients with ≥1 co-mutated gene. B) Overall survival (OS) of patients with no co-mutated gene vs patients with ≥1 co-mutated gene. C) Survial of patients post relapse according to number of co-mutated genes. Table: Relatively more patients were transplanted following relapse in the group of patients with more co-mutated genes (n.s.).

Figure 3

Clonal evolution of paired samples at diagnosis and relapse. For 17/21 patients who relapsed following molecular remission paired samples at diagnosis and relapse were available for sequencing analysis. A) Heatmap of mutated genes at diagnosis and relapse: shown are genes with ≥1 mutation at diagnosis or relapse. B) Patient based mutation tracking: shown is the variant allele frequency (VAF) at diagnosis (D) and at relapse (R) of the indicated gene. Mutations that are lost at relapse are shown in red, mutations that are gained at relapse in green, stable mutations in black. 2 patients had no mutation at diagnosis or relapse and are not shown. For FLT3-ITD the mutant/wildtype ratio is given. 4 patients with evidence of relapse in centrally reviewed morphology are indicated with m.

Figure 4

Net loss of co-mutations at relapse. For 17/21 patients who relapsed following MR paired diagnostic and relapse samples were available for sequencing. A) Mutations in known leukemia associated genes are lost at relapse. Shown is the number of mutations in the indicated genes that are lost at relapse. B) The number of mutations at relapse is significantly reduced. Shown is the absolute number of mutations at diagnosis and at relapse (P = 0.0485), red dot, mean. C) The change in VAF at diagnosis and at relapse of persisting mutations is given (n.s., P = 0.389). D) The VAF at diagnosis of mutations that persist at relapse and the VAF at diagnosis of mutations that are lost at relapse is compared (P = 0.0148), red dot, mean. E) The RUNX1-RUNXT1/ABL qPCR ratios were comparable at diagnosis and relapse: shown is the change of RUNX1-RUNXT1/ABL ratios for each patient at diagnosis and at relapse (n.s., P = 0.117).

Figure 5

A gain of co-mutations at relapse is associated with reduced RFS. A) RFS of patients with no gained co-mutations (n = 13) vs patients with gained co-mutations at relapse (n = 4, P = 0.009). B) Overall survival (OS) of patients with no gained co-mutations vs patients with gained co-mutations at relapse (n.s.).