Clonal Evolution in NPM1 Mutated Acute Myeloid Leukemia (AML)

Background: Mutations in the nucleophosmin (NPM1) gene represent one of the most common gene mutations in AML, and are considered a founder event in the pathogenesis of AML. Recently, we gained novel insights into clonal architecture and clonal evolution in 53 NPM1 mutated (NPM1^mut) AML patients (pts). Here, we showed the highest stability for DNMT3A mutations (DNMT3A^mut) suggesting that these mutations represent an early event in leukemogenesis as well as an increase in genomic complexity.

Aims: To validate our previous results on clonal evolution in a larger cohort of NPM1^mutAML pts and to define relapse specific mutation patterns.

Methods: Paired samples at diagnosis and relapse from 129 NPM1^mut AML pts were assessed for additional mutations by comprehensive mutation analysis (FLT3-ITD, FLT3-TKD, DNMT3A, IDH1/2, NRAS, ASXL1, TP53, MLL-PTD). In addition, 11 AML cases with loss of NPM1^mut at the time of relapse and 12 cases with persisting NPM1^mut were subjected to whole exome sequencing (WES).

Results: At diagnosis, the incidence of concurrent gene mutations was as follows: FLT3-ITD^mut 32% (40/126), FLT3-TKD^mut 19% (22/118), DNMT3A^mut 69% (78/113), NRAS^mut 19% (22/117), IDH1^mut 21% (26/123) and IDH2^mut 19% (22/118). None of the pts analyzed exhibited a TP53 (n=53), MLL-PTD (n=63) or ASXL1 (n=59) mutation. At relapse, a significant shift in the genetic pattern was found in 74 pts (57%). FLT3-ITD^mut was lost in 10 pts and newly acquired in 24 pts,16 pts lost FLT3-TKD^mut.  DNMT3A^mut was lost in 2 pts and gained in 1 pt. NRAS^mut was lost in 12 pts and gained in 4 pts. IDH1^mut was lost in 3 pts and acquired in 4 pts, while IDH2^mut was lost in 2 pts and gained in only one pt. ASXL1^mut and TP53^mut were gained in 2 and 1 pts, respectively; gain of MLL-PTD^mut (n=4) was restricted to pts with loss of NPM1^mut at the time of relapse. Based on these findings we calculated the following stabilities: FLT3-ITD^mut 75%, FLT3-TKD^mut 27%, DNMT3A^mut 97%, NRAS^mut 45%, IDH1^mut 88% and IDH2^mut 91%, respectively. In total, 11 pts (9%) lost NPM1^mut at relapse while DNMT3A^mut was present in 9/11 pts at diagnosis and remained stable at relapse; 4/11 pts gained MLL-PTD^mut, one pt ASXL1^mut and 3 pts acquired RUNX1^mut.

By WES we observed a persistence of mutations known to be involved in clonal hematopoiesis, such as DNMT3A and TET2 mutations. These were usually seen during all analyzed time points (diagnosis, remission and relapse). In addition, we found distinct mutational patterns at the time of relapse compared to the time of diagnosis. For example, relapse- and diagnosis-specific mutations in NPM1^mut loss cases were significantly enriched for different signaling pathways.

Conclusions: 74 (57%) of 129 NPM1^mut AML pts showed clonal evolution at the time of relapse. DNMT3A^mut demonstrated the highest stability (97%) confirming our previous findings that DNMT3A^mut constitutes an early event which persists in preleukemic hematopoietic stem cells. High-resolution sequencing of selected cases is ongoing and will further unravel the clonal hierarchy in NPM1^mut AML.

Those authors equally contributed to work: SKS, SC, LB and KD.