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TP53 subclones in AML
TP53 aberrations are somatically acquired, constituting an early leukemogenic event, germline mutations are increas- ingly being recognized, predominantly in patients with therapy-related AML.8,9 Aberrations of TP53 are associated with an exceedingly adverse prognosis as demonstrated by several independent reports.5,10,11 Recently, it was shown that TP53 mutations and deletions encompassing the TP53 locus have a different prognostic impact in AML, with only mutations but not deletions significantly influencing sur- vival of these patients.12 As a consequence, testing for TP53 mutations has been introduced into the 2017 recommen- dations of the European LeukemiaNet.13
However, in the studies performed so far, TP53 muta- tions were assessed as a dichotomous variable only. With the advent of next-generation sequencing technologies, mutational subclones can now be detected with high sen- sitivity. Here, we aimed to investigate the clinical charac- teristics associated with subclonal TP53 mutations and their prognostic impact in a large cohort of AML patients prospectively treated within studies of the “German- Austrian AML Study Group” (AMLSG).
Methods
The study was approved by the ethics committees of the University of Ulm, Germany, and the Medical University of Graz, Austria and conducted in accordance with REMARK guidelines (“REporting recommendations for tumor MARKer prognostic studies”).14
Study participants
Data from a total of 1537 intensively treated AML patients enrolled in three prospective, multicenter, clinical trials of the AMLSG were analyzed.15-17 Details of these studies as well as a list of AMLSG investigators and centers are provided in the Online Supplementary Appendix.
Sequence analysis
Genetic profiling of a total of 1,537 diagnostic AML specimens using a targeted sequencing approach with 111 genes associated with myeloid neoplasms was reported previously for this com- bined cohort.2 Sequencing data were deposited in the European Genome-Phenome Archive (www.ebi.ac.uk/ega, accession number EGAS00001000275) and retrieved for the present study. Methodological details are provided in the Online Supplementary Methods together with information on ultradeep sequencing used for the analysis of selected, sequential patients’ samples.
Statistical analysis
The study was designed to assess differences in overall survival between AML patients exhibiting a TP53 wild-type status and those with subclonal TP53 mutations. Based on data from Papaemmanuil et al.2 an absolute reduction of 35% in 3-year over- all survival compared to that of patients with a TP53 wild-type sta- tus should be detectable using a sample size of 574 patients assum- ing an overall survival rate of 55% for TP53 wild-type patients and a frequency of 5% for TP53 muted subclones with a variant allele frequency (VAF) as low as 5% (power=90%; α=0.05).
The main outcome parameters assessed were overall survival and event-free survival, as defined by the European LeukemiaNet.13 We determined median survival times and esti- mated 3-year survival rates along with their 95% confidence inter- vals (95% CI). Survival rates of patients with a TP53 wild-type sta- tus and patients with TP53 mutations were plotted using the
Kaplan-Meier method and compared by the log-rank test. In addi- tion, TP53-mutated patients were further categorized according to their VAF (>40%, 20%-40%, <20%). This categorization into three different groups according to the TP53 VAF was based on different biological features regarding concomitant chromosomal aberrations as outlined in the “Results” section. It also allowed a comparison with previous reports on the impact of TP53 VAF in patients with myelodysplastic syndromes.18,19 Univariable and multivariable Cox regression analyses were performed to identify relevant prognostic factors. We assessed age, white blood cell count, cytogenetic risk group and type of AML (de novo, secondary or therapy-related) in addition to the TP53 status. Hazard ratios (HR) are presented along with their 95% CI. To compare patients’ characteristics among the groups defined by their TP53 status and VAF, we performed a Fisher exact test for categorical parameters and the Kruskal-Wallis or Mann-Whitney-U tests, respectively, for continuous parameters. If the overall test between the three TP53 VAF groups showed statistically significant differences (α=0.05), post-hoc tests were performed. Due to the multiple groups tested, we employed a Bonferroni correction and considered a P-value of <0.017 as statistically significant. All statistical analyses were con- ducted using R version 3.4.4 (https://www.r-project.org).
Results
Characteristics of TP53-mutated subclones in acute myeloid leukemia
A total of 1,537 patients, enrolled in the AMLSG trials HD98A, HD98B and 07-04, were evaluated in this study. Their clinical characteristics are shown in Table 1. Of those patients, 1,408 (91.6%) had de novo AML, 61 (4.0%) had secondary AML following myelodysplastic syn- dromes and 68 (4.4%) had therapy-related AML. The median follow-up of all patients was 25 months (range, 0 - 219.6).
When analyzing diagnostic AML specimens, 108 patho- genic TP53 mutations were found in 98 (6.4%) patients. Seven patients exhibited two TP53 mutations each and one patient had four. A detailed presentation of the muta- tions detected in this cohort is given in Online Supplementary Table S1. When categorizing the 98 patients with TP53 mutations according to their maximum VAF, we found 61 (62.2%) with a VAF >40% representing the major AML clone, 19 (19.4%) with a VAF between 20% and 40% and 18 (18.4%) with a VAF <20%, this last group representing subclones. The vast majority of TP53 muta- tions in all groups were missense mutations located in the DNA binding domain of the gene. Neither type of the mutation nor its location showed a statistically significant difference among the three TP53-mutated subgroups (P=0.279 for mutation type and P=0.687 for mutation location) (Figure 1). As compared to AML patients with TP53 wild-type, those with clonal and subclonal TP53 mutations showed significantly lower white blood cell counts as well as peripheral blood and bone marrow blasts (Table 1).
It is well documented that clonal TP53 mutations are highly associated with complex karyotypes as well as marker chromosomes arising from chromothripsis.20,21 As shown in Table 2, a significant association with complex karyotypes was observed for TP53 mutations with a VAF of >40% and between 20% and 40%, but not for subclon- al ones with a VAF of <20%. There was also a statistically significant difference between TP53 wild-type and TP53
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