Ian Thomas

The World Health Organization fifth edition and International Consensus Classification for myeloid neoplasms both incorporate empirical numerical thresholds to morphologic and molecular features defining certain disease entities. However, the clinical implications of these thresholds remain unclear. We analyzed a large cohort (N = 6,976) of patients with myeloid neoplasms to evaluate the impact of proposed yet different numerical thresholds for variant allele frequency of genetic mutations or hematologic parameters set forth by the World Health Organization fifth edition and International Consensus Classification for classification of SF3B1-mutated myelodysplastic neoplasms, NPM1-mutated acute myeloid leukemia (AML), and oligomonocytic chronic myelomonocytic leukemia. Our analysis demonstrated that the clonal burden of SF3B1 mutation in myelodysplastic neoplasms informs classification and prognosis. Our findings support the notion that NPM1 mutation should be AML-defining regardless of blast percentage and highlight the adverse prognostic impact of the cumulative number of myelodysplasia-related mutations in NPM1-mutated AML. Finally, we provide evidence that integrating specific molecular signatures could improve the accuracy of oligomonocytic chronic myelomonocytic leukemia classification.Significance:Using comprehensive clinical and molecular profiling, this study provides a data-driven approach for evaluating numerical thresholds of variant allele frequency or hematologic parameters (i.e., blast percentage and absolute monocyte count) included in current classification schemas across a spectrum of myeloid malignancies, enabling refinement of disease classification and prognostication.

Comparison of clinical and molecular demographics and survival in patients with NPM1-mutated myeloid neoplasms with <20% blasts from the UK NCRI AML17/19 trials. (A) Density plots demonstrating no significant difference in baseline age or hematologic parameters between patients with <10% vs. 10% to 19% blasts. (B) Similarly, no significant difference was observed with respect to frequently identified mutations between patients with mutated NPM1 and <10% vs. 10% to 19% blasts. (C and D) Event-free and overall-survival was similar between patients with mutated NPM1 and <10% vs. 10% to 19% blasts. Reported p-values utilized the cox model Wald test.

Biplot of quantitative variables included in factor analysis of mixed data (FAMD) of myelodysplastic CMML cases (MD-CMML) including platelets, bone marrow blast (BM blasts) percentage, absolute neutrophil count (ANC), absolute monocyte count (AMC), relative monocytes, hemoglobin, and patient age at diagnosis. Arrow length and direction with respect to the circle indicates relative contribution to each quadrant. Closer proximity of an arrowhead to the circle indicates a stronger contribution of a variable to the quadrant.

WHO 2016 classification of included patients with NPM1-mutated myeloid neoplasms (MN)

Density plot depicting Bradley-Terry modeling of the estimated order of mutation acquisition in AML samples from the UKNCRI and AMLSG cohorts. Mutations in genes classified as MR mutations (SRSF2, ASXL1, RUNX1, U2AF1, ZRSR2, EZH2, STAG2, BCOR, SF3B1) tended to occur as earlier events relative to mutations in NPM1. VAF: Variant allele frequency

Molecular characteristics of SF3B1-mutated MDS. (A) Mixed violin/boxplot depicting the VAF of SF3B1 in patients with clonal vs. subclonal SF3B1 mutations. Box and whisker plot demonstrates median (solid bar), interquartile range, minimum, and maximum VAF values. (B) The number of cases based on the count of co-occurring mutations between SF3B1low vs. SF3B1high MDS. (C) Distribution of identified amino acid variants within the SF3B1 protein. Variant frequency is represented as Log2 values. Location of variants displayed within HEAT domains, conserved regions (1–20) of tandem repeats within the SF3B1 protein. Canonical MDS mutations are commonly associated with domains 4 to 7 (yellow highlight). (D) Barplot depicting clonality of SF3B1low vs. SF3B1high MDS cases. (E) Barplot depicting differences in canonical and non-canonical variants between SF3B1low vs. SF3B1high MDS. (F) Alternative SF3B1 variants identified in SF3B1low vs. SF3B1high MDS. VAF: variant allele frequency. Asterisk indicates Fisher’s exact p-value <0.05.

Density plot depicting Bradley-Terry modeling of the estimated order of mutation acquisition in AML samples from the UKNCRI and AMLSG cohorts. Mutations in genes classified as MR mutations (SRSF2, ASXL1, RUNX1, U2AF1, ZRSR2, EZH2, STAG2, BCOR, SF3B1) tended to occur as earlier events relative to mutations in NPM1. VAF: Variant allele frequency

Consort diagrams of included patient populations for each analysis representing the selection of patients based on filtering criteria from the IWG-PM cohort (N = 3,323 patients), the UK NCRI (N = 2,113), and AMSLG (N = 1,540) cohorts.

Biplot of quantitative variables included in factor analysis of mixed data (FAMD) of myelodysplastic CMML cases (MD-CMML) including platelets, bone marrow blast (BM blasts) percentage, absolute neutrophil count (ANC), absolute monocyte count (AMC), relative monocytes, hemoglobin, and patient age at diagnosis. Arrow length and direction with respect to the circle indicates relative contribution to each quadrant. Closer proximity of an arrowhead to the circle indicates a stronger contribution of a variable to the quadrant.

Comparison of clinical and molecular demographics and survival in patients with NPM1-mutated myeloid neoplasms with <20% blasts from the UK NCRI AML17/19 trials. (A) Density plots demonstrating no significant difference in baseline age or hematologic parameters between patients with <10% vs. 10% to 19% blasts. (B) Similarly, no significant difference was observed with respect to frequently identified mutations between patients with mutated NPM1 and <10% vs. 10% to 19% blasts. (C and D) Event-free and overall-survival was similar between patients with mutated NPM1 and <10% vs. 10% to 19% blasts. Reported p-values utilized the cox model Wald test.