Clonal Hematopoiesis Landscape in Patients with De Novo Acute Myeloid Leukemia By Deep Sequencing

Purpose: Acute myeloid leukemia (AML) is associated with progressive accumulation of genetic alterations in hematopoietic progenitors. Massive sequencing allows inference of the clonal architecture of hematologic malignancies, by determining the presence of subclones, their genetic composition and evolution. The objective of the present study was to determine the spectrum of mutations present at relapse and define the proportion of cellular clones and the genetic architecture of the evolution of patients with de novo AML.

Methods: Paired samples (diagnostic/relapse) of 44 patients of the University Hospital La Fe with de novo AML and treated with consecutive PETHEMA schemes were studied. The samples were provided by the Biobanco La Fe. The median age was 59 years (range 17 - 89); 21M/13F; 17 patients with normal karyotype; 14 patients with FLT3-ITD positive and 9 with mutations in NPM1. Using an amplicon panel (Ampliseq, Life Technologies) for deep sequencing (10.000x) with an Ion Torrent Proton, the complete coding regions of the following genes were sequenced, BCOR, BRAF, CDKN2A, CEBPA, DNMT3A, ETV6, EZH2, GNAS, LUC7L2, NF1, PHF6, PTPN11, RAD21, RPS14, SF1, SF3A1, SMC3, SPARC, SRSF2, STAG2 and ZRSR2, as well as, the hotspot regions of ASXL1, MPL, NPM1, JAK2, KRAS, NRAS, TET2, U2AF1, KIT, IDH1, RUNX1, IDH2, SETBP1, TP53, WT1, CBL, SF3B1 and FLT3. Primary bioinformatic analysis was performed using an in-house protocol and variants were selected based on VAF ≥ 1%, its absence in the healthy population (UCSC Common SNPs; MAF < 0.01) and its putative effect on the protein.

Results: At least one alteration was detected in 98% of patients, (n = 43). At a mean sequencing depth of 8967x, in total, 249 mutations were detected with an average of 3.3 mutations per patient and sample (range 0 - 8). Comparing the two time points, we noted that 45% of the mutations were present at both moments, with rather similar VAF values. However, 24% were acquired during progression while 31% went missing at the time of relapse. Regarding the mutated genes analyzed at diagnosis, in 8 of 44 patients one single gene clone was detected, in 26 two subclones and in 10 three or more subclones. In addition, two different patterns of clonal evolution were detected. In model 1 the dominant founder clone persisted at relapse (n = 32, 71%), occasionally acquiring new changes, either in the same clone (n = 5) or in a new subclone (n = 17). In model 2 the founder clone was displaced at relapse by new subclones (n = 12, 29%), probably due to selective pressure through competition between subclones or as a consequence of the chemotherapy. Furthermore, the clonal hematopoiesis models did not show an association with clinical variables or prognostic impact on OS or EFS (P = 0.317; P = 0.12, respectively).

Conclusions: AML cells can acquire additional mutations at relapse. Some of those may contribute to the clonal selection responsible for disease progression. Two models of clonal evolution were observed: model 1, where the dominant founder clone persists during relapse, and, model 2, where the founder clone is displaced by new cell subclones, displaying, both models, a similar impact on outcome.

Financed by the Spanish Foundation of Hematology (FEHH), PI12/01047, RD12/0036/0014, PIE13/00046, PI13/01640, PI13/02837, PT13/0010/0026, PI14/01649, ACOMP2015/0335 and PROMETEOII/2015/study/025.