The Role Of Early TP53 Mutations On The Evolution Of Therapy-Related AML

Therapy-related acute myeloid leukemia (t-AML) and therapy-related myelodysplastic syndrome (t-MDS) are well-recognized complications of cytotoxic chemotherapy and/or radiotherapy. Compared to de novo AML, there is a higher incidence of TP53 mutations, abnormalities of chromosomes 5 or 7, and complex cytogenetics in t-AML. The response to chemotherapy is also reduced compared to de novo AML, and long-term remissions are rare. From a pathogenesis perspective, the key difference between de novo AML and t-AML is the prior exposure of t-AML patients to cytotoxic therapy during treatment of their primary cancer. We previously reported the initial findings of our study to sequence the genome of 22 cases of t-AML (Ramsingh et al, Abstract #784, 2012). These data were compared to the genomic sequence of 49 cases of de novo AML (TCGA, NEJM, 2013). Surprisingly, the data showed that the total number of somatic single nucleotide variants (SNVs) and the percentage of transversions (which are associated with chemotherapy-induced DNA damage) were similar in t-AML and de novo AML. Thus, there is no evidence that chemotherapy induces genome-wide DNA damage in t-AML.

We extended this data set by sequencing 149 genes of interest in an additional 89 patients with t-AML/t-MDS. Mutations of TP53 were the most frequent (31.5%), and TP53 was the only gene mutated at a higher frequency in t-AML or t-MDS compared with de novo AML or MDS respectively. The mechanism by which TP53 mutations are selectively enriched in t-AML/t-MDS is unclear. Since t-AML/t-MDS has a similar mutation burden compared to de novo AML, it is not likely that chemotherapy directly induces TP53 mutations. We recently reported that individual HSCs accumulate somatic mutations as a function of age. By age 50, there are on average 5 coding gene mutations per HSC clone (Welch et al, Cell, 2012). As it is estimated that there are 10,000 HSCs in humans, it is possible that HSC clones harboring aging-related TP53 mutations are present in a subset of healthy individuals. Based on these observations, we propose a model in which rare TP53 mutant-bearing HSC clones have a selective growth advantage in patients undergoing chemotherapy. This results in their clonal expansion, with additional mutations in the HSC clone undoubtedly required for full leukemic transformation. This model predicts that TP53 mutations are present in many patients years prior to the development of t-AML/t-MDS. To test this prediction, we developed a next generation sequencing technique able to detect rare subclones harboring TP53 mutations to as low as 1 in 1,000 cells. We identified 7 cases of t-AML/t-MDS with specific TP53 mutations for whom we had blood or bone marrow banked 3-10 years prior to the development of t-AML/t-MDS. The specific TP53 mutations were detected in two cases. In patient UPN 530447, bi-allelic mutations of TP53 were identified in mobilized peripheral blood leukocytes 6 years prior to the development of t-AML at frequencies of 0.5 and 0.7%. In patient UPN 341666, a heterozygous TP53 mutation was identified in mobilized peripheral blood leukocytes 3 years prior to t-MDS diagnosis at a frequency of 0.1%. In at least some cases, we have clear evidence that the TP53 mutations occurred prior to the acquisition of other driver mutations or the development of cytogenetic abnormalities. To directly test the hypothesis that TP53 mutations confer a clonal advantage after chemotherapy, we generated mixed bone marrow chimeras containing wild type and Tp53^+/- cells. No clonal advantage of Tp53^+/- cells was identified in untreated chimeras. However, upon treatment with N-ethyl-N-nitrosourea, Tp53^+/- HSCs had a significant growth advantage. Collectively, these data suggest that HSCs that acquire heterozygous TP53 mutations as a function of normal aging may be selected for in the presence of cytotoxic therapy. This provides a potential mechanism for the high incidence of TP53 mutations in t-AML/t-MDS. The early acquisition of TP53 mutations in the founding clone likely contributes to the frequent cytogenetic abnormalities and poor response to chemotherapy that are typical of t-AML/t-MDS.