Clonal evolution in relapsed acute myeloid leukaemia revealed by whole-genome sequencing

Abstract

Most patients with acute myeloid leukaemia (AML) die from progressive disease after relapse, which is associated with clonal evolution at the cytogenetic level. To determine the mutational spectrum associated with relapse, we sequenced the primary tumour and relapse genomes from eight AML patients, and validated hundreds of somatic mutations using deep sequencing; this allowed us to define clonality and clonal evolution patterns precisely at relapse. In addition to discovering novel, recurrently mutated genes (for example, WAC, SMC3, DIS3, DDX41 and DAXX) in AML, we also found two major clonal evolution patterns during AML relapse: (1) the founding clone in the primary tumour gained mutations and evolved into the relapse clone, or (2) a subclone of the founding clone survived initial therapy, gained additional mutations and expanded at relapse. In all cases, chemotherapy failed to eradicate the founding clone. The comparison of relapse-specific versus primary tumour mutations in all eight cases revealed an increase in transversions, probably due to DNA damage caused by cytotoxic chemotherapy. These data demonstrate that AML relapse is associated with the addition of new mutations and clonal evolution, which is shaped, in part, by the chemotherapy that the patients receive to establish and maintain remissions.

Figure 1. Somatic mutations quantified by deep sequencing of capture validation targets in 8 acute myeloid leukemia primary tumor and relapse pairs

a) Summary of tier 1–3 mutations detected in 8 cases. All mutations shown were validated using capture followed by deep sequencing. Shared mutations are in gray, primary tumor specific mutations in purple, and relapse-specific mutations in red. The total number of tier 1–3 mutations for each case is shown above the light gray rectangle. b) Mutant allele frequency distribution of validated mutations from tier 1–3 in the primary tumor and relapse of case UPN 933124 (left). Mutant allele frequencies for 5 primary tumor specific mutations were obtained from a 454 deep readcount experiment. Four mutation clusters were identified in the primary tumor, and one was found at relapse. Five low-level mutations from known copy number variable regions were excluded from clustering analysis. Non-synonymous mutations from genes that are recurrently mutated in AML are shown. The change of mutant allele frequencies for mutations from the 5 clusters is shown (right) between the primary tumor and relapse. c) The mutation clusters detected in the primary tumor and relapse samples from 7 additional AML patients. The relationship between clusters in the primary tumor and relapse samples are indicated by lines linking them. Cluster: a group of mutations with similar allele frequencies, likely derived from the same clone. Clone: a group of cells with identical mutation content.

Figure 2. Graphical representation of clonal evolution from the primary tumor to relapse in UPN 933124, and patterns of tumor evolution observed in 8 primary tumor and relapse pairs

a) The founding clone in the primary tumor in UPN 933124 contained somatic mutations in DNMT3A, NPM1, PTPRT, SMC3, and FLT3 that are all recurrent in AML and probably relevant for pathogenesis; one subclone within the founding clone evolved to become the dominant clone at relapse by acquiring additional mutations, including recurrent mutations in ETV6 and MYO18B, and a WNK1-WAC fusion gene. HSC: hematopoietic stem cell. b) Examples of the two major patterns of tumor evolution in AML: Model 1 shows the dominant clone in the primary tumor evolving into the relapse clone by gaining relapse-specific mutations; this pattern was identified in 3 primary tumor and relapse pairs (UPNs 804168, 573988, and 400220). Model 2 shows a minor clone carrying the vast majority of the primary tumor mutations survived and expanded at relapse. This pattern was observed in 5 primary tumor and relapse pairs (UPNs 933124, 452198, 758168, 426980, and 869586).

Figure 3. Comparison of mutational classes between primary tumors and relapse samples

a) Fraction of the primary tumor and relapse-specific mutations in each of the transition and transversion categories. b) Transversion frequencies of the primary tumor and relapse-specific mutations from 8 AML tumor and relapse pairs. 456 relapse-specific mutations and 3590 primary tumor mutations from 8 cases were used for assessing statistical significance using proportion tests.