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O.K. Weinberg et al.
A
B
C
D
Figure 1. Examples of typical morphological dysplastic features found in de novo acute myeloid leukemia (AML). Megakaryocytes often show separated lobes (A) and small size micromegakaryocytes (B). Dysplastic changes in myeloid cells, including hypogranular cytoplasm and abnormal nuclear lobation (C). Dysplastic ery- throid cells are shown with irregular nuclear contors (D).
ary-type mutation was present at >5% variant allele frequency (VAF)] implied de novo AML, and all other cases were designated as having a pan-AML mutation pattern.14
Subclonal analysis
The presence of a leukemia subclone was defined as having at least one mutation at a frequency of at least 10% less than the mutation with highest VAF, and with the sum of the highest muta- tion and subclonal mutation VAF over 55%.19
Statistical analysis
Fisher exact test and the Wilcoxon rank sum test were used to compare categorical and continuous variables, respectively. In the event that a continuous variable was compared across 3 or more groups, the Kruskall-Wallis test was used. The Kendall coefficient of concordance, W, with a correction for ties, was used to compare the 3 observers’ score of dysplasia percentages and individual dys- plastic feature scores; Kendall W ranges from 0 (no agreement) to 1 (complete agreement).
The association between each degree of dysplasia in all three lineages was assessed by both continuous and categorical analy- ses. Individual dysplastic findings assessed by categorical analysis explored different cut offs (>0, ≥2, and ≥3) for each evaluable dys- plastic feature. We explored the associations between age, WBC, peripheral blasts (%), BM cellularity (%), BM blasts (%), platelet count, and hemoglobin level, considering individual mutations present more than 5 patients (>3%), mutation pathways, molecu-
lar ontogeny, and subclone status. There were 29 planned analyses based on mutation data for each of the morphological measures and patients' clinical characteristics; therefore, a false discovery rate adjustment (q-value) was made for the planned analyses with- in the respective outcome. All tests were two-sided and a q-value (FDR-adjusted P-value) <0.05 was considered significant
Event-free survival (EFS), defined as the time in months from the date of diagnosis to the date of disease progression or death, was explored in the subset of patients that received induction chemotherapy. Nominal P-values are presented for univariate analyses. Cox proportional hazards models with transplant as a time varying covariate were considered to evaluate the association of mutation and morphological findings with EFS. Mutation data and morphological findings underwent univariate analysis with transplant-adjusted Cox models. Multivariable Cox models were considered with mutations and morphological findings with a uni- variate P-value <0.2 and the backwards elimination method was used to select a final model retaining transplant and variables with a P-value <0.1.
Results
Patients
A total of 168 cases of de novo AML without WHO- defined cytogenetic abnormalities were identified with a median age of 60.6 years (range 19.9-86.7). A total of 137
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