Using next-generation sequencing, we have found that the proportion of neoplastic bone marrow cells is indistinguishable in myelodysplastic-syndrome and secondary-AML samples, suggesting that the myelodysplastic syndromes are as clonal as secondary AML, even with a myeloblast count of zero. Although clonality is not sufficient to define malignant transformation, it is a cardinal manifestation of most human cancers, and our findings suggest that the myelodysplastic syndromes and secondary AML are both highly clonal hematologic cancers.31
Analysis of the proportion of mutant cells in samples obtained from the same subject before and after progression to secondary AML allowed us to compare the clonal architecture and genes that were mutated in order to gain insight into the genetics of these diseases. Robust detection of mutation clusters was possible because hundreds of mutations per genome were identified by whole-genome sequencing, and the allele burdens were quantified by deep resequencing at two time points. If we had analyzed only the secondary-AML samples or only the tier 1 (i.e., exomic) variants, this complexity could not have been elucidated. In the samples from all seven subjects, the secondary-AML genomes were oligoclonal. The preexisting myelodysplastic-syndrome founding clone always persisted in secondary AML, although it was outcompeted by daughter subclones in some cases. With the acquisition of each new set of mutations, all the preexisting mutations were carried forward, resulting in subclones that contained increasing numbers of mutations during evolution. On the basis of our experimental design, we cannot exclude the possibility that there were additional subclones in the myelodysplastic-syndrome samples that were not present in the secondary-AML samples, and one genome (UPN298273) suggests that this could occur.
A unique aspect of the biology of leukemia is that hematopoietic cells freely mix and recirculate between the peripheral blood and the bone marrow. Clones that persist and grow over time must retain the capacity for self-renewal. Mutations in new clones must confer a growth advantage for them to successfully compete with ancestral clones. The result is that these secondary-AML samples are not monoclonal but are instead a mosaic of several genomes with unique sets of mutations; this mosaic is shaped by the acquisition of serial mutations and clonal diversification. Similarly, recent analysis of de novo AML samples with the use of whole-genome sequencing showed that relapse after chemotherapy is associated with clonal evolution and acquisition of new mutations.32
Analysis of individual cancer cells may reveal additional layers of genetic complexity. Recent studies of B-cell acute lymphoblastic leukemia have shown that serial acquisition of cytogenetic abnormalities in that disease most often occurs through a branching hierarchy and only rarely follows a simple linear path.33,34
Extending this work to include the full complement of mutations discovered by whole-genome sequencing will be a major goal for future studies of cancer genetics.
Our study has several clinical implications. First, the distinction between the myelodysplastic syndromes and secondary AML currently relies on manual enumeration of bone marrow myeloblasts, a standard that is subject to interobserver bias but nonetheless drives major decisions about treatment for patients with small differences in myeloblast counts. Ultimately, identifying the patterns of pathogenic mutations and their clonality in bone marrow samples from patients with myelodysplastic syndromes should lead to greater diagnostic certainty and improved prognostic algorithms. On this note, two of the subjects in our study had progression from myelodysplastic syndromes to secondary AML in 1 month. This progression was based on an increase in the myeloblast count from 7% to 66% in one subject and from 13% to 43% in the other, despite an absence of change in the number of clones (two in each case) and only minor increases in point mutations (<2% were gained during progression to secondary AML in these cases) when the same specimens were analyzed by means of next-generation sequencing. Second, our finding that the dominant secondary-AML clone was derived from a myelodysplastic-syndrome founding clone in all cases suggests that therapies targeted to these early mutations might be the most effective strategy for eliminating disease-propagating cells and improving the rate of response to traditional chemotherapy for patients with secondary AML.35,36
Finally, it is possible that disease progression in patients with myelodysplastic syndromes is driven not only by the presence of recurrent mutations, which have recently been shown to have prognostic value,37
but also by the clone (i.e., founding vs. daughter) in which they arise. Coupling genotyping of myelodysplastic-syndrome samples for prognostically important mutations with analysis of the clonal architecture may yield more informative biomarkers and a better understanding of the pathogenesis of the myelodysplastic syndromes.