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E. Onecha et al.
toriness and relapse.
The genes most frequently mutated at diagnosis in
patients showing subsequent primary refractoriness were CBL, KIT, NRAS, RUNX1 and TP53, and those more fre- quently mutated in relapsed patients were DNMT3A, IDH1/2, KMT2A and SF3B1. Thus signaling, transcription and tumor suppression pathways were the more affected biological categories at diagnosis in the treatment refractory group, whereas methylation and splicing were the path- ways most affected at diagnosis in the relapsed group. This perhaps indicates that methylation and splicing are rescue pathways used by leukemia cells to develop resistance to treatment. Our findings may lead to the development of a new focused therapeutic approach for patients belonging to the high-risk cytogenetics group at relapse but who have nevertheless achieved CR, because the oncologist could anticipate maintenance treatments based on drugs targeting methylation and splicing pathways, for example, hypomethylants or splicing inhibitors. Rescue pathways were not identified for cases of primary refractoriness, as clones remained stable and alterations were found in signal- ing and tumor suppressor genes, which are the most clinical- ly relevant.
We identified new potential therapeutic targets at the leukemia relapse stage affecting IDH2, SF3B1, KRAS, KIT and JAK2 genes, all of which are targets for approved drugs or available within clinical trials. However, some identified variants in SF3B1 and FLT3 are categorized as variant of uncertain (or unknown) significance and for clinical decision making only pathogenic or probably pathogenic variants can be used. Supporting our findings, a recent study described de novo mutations in transcription factors, signal- ing, cohesin and splicing pathways at the time of leukemia relapse in the t(8;21) AML patient subgroup;23 however, mutations detected at diagnosis in epigenetic regulators and genes involved in cell cycle control were stable or disap- peared.23
We also detected an equal percentage of cases (32.3%) where the dominant clone changed within the refractory group versus the leukemia relapsed group with evolution of the following genes: ETV6, VHL, EPOR, JAK2, TP53, and PHF6. Consequently, therapeutic approaches must be tar- geted specifically to these clones if they are detected at diag- nosis.
The impact of subclonal mutations detected at diagnosis and their usefulness as MRD markers is not yet defined. In our AML series, 55% of subclones detected at diagnosis were lost in failure samples and the other 45% remained as subclones. Only the PHF6 mutation became the predomi- nant clone in a failure sample, supporting the concept that treatment can result in subclonal eradication, but whether a resistance-mediating mutation determines the presence of a corresponding subclone from diagnosis could found a future relapse.
We observed an increased mutational load as a strong molecular feature of relapse, as previously established in other hematological malignancies.24 In addition, the authors of the aforementioned study suggest that the knowledge of the tumor burden is important in the identification of sub- clones, with the aim of targeted and specific therapies to eradicate them and to follow their evolution.
In an analysis of over 4,000 patients with newly diag- nosed AML, several biological-clinical variables (age, per- formance status, white blood cell count, secondary disease, cytogenetic risk and NPM1/FLT3-ITD mutational status)
were each strongly and independently associated with resistance (P<0.001); however, their ability to predict resist- ance was only fair.25 Our study includes other molecular markers (AMA) that improve the prediction of failure to response to leukemia relapse treatment (P=0.066), although it does not predict it for refractoriness (P=not signifcant). This is likely justified by the fact that the refractory group was enriched with high-risk cytogenetics features at diagno- sis, whereas the relapsed group was associated with a greater number of pathogenetic or likely pathogenetic vari- ants at diagnosis. In contrast to other reported findings,26 we did not detect differences in the age of the patients between the group with persistence of these mutations and the group without these mutations.
We found persisting DNMT3A, TET2 and ASXL1 variants in CR, which have been previously reported,26 and are relat- ed to clonal hematopoiesis of indeterminate potential (with oncogenic potential).27 Other mutations, such as those involving KMT2A, CBL and NRAS, could be associated with AML transformation from a prior clonal disease, as described by Bejar.28 These differ from reported mutations that were involved in leukemic hematopoiesis in NPM1, PHF6, SRSF2, RUNX1 and TP53,27,29-36 although de novo cases predominated in our series.
To the best of our knowledge, we provide the first clini- cal evidence that clonal evolution defined as AMA is a fea- ture associated with cases that achieve CR and that have a better prognosis for disease-free survival. A previous study in AML provided a contrary prediction, which in an analo- gous manner defined the clonal evolution as additional cyto- genetic abnormalities, and observed a worse prognosis.37 Our prediction agrees with reported findings in NPM1- mutated AML,14 whereby AML patients with clonal evolu- tion (NPM1-) at relapse have a significantly longer remission duration than patients without clonal evolution (NPM1+). The better prognosis associated with patients with new clones (AMA) might be due to the fact that the treatment has been effective in the basal clone.
Previous studies have shown an association between per- sisting clonal cytogenetic markers in first remission and an increased risk of relapse.38,39 Somatic mutations that activate signaling pathways (FLT3, KRAS, or NRAS) were usually cleared on day 30, suggesting that subclones containing these mutations may be more sensitive to induction chemotherapy.2 Although new advances in induction treat- ment for AML have improved the rates of CR and overall survival, most patients ultimately relapse without effective post-remission therapy.40
The utility of clonal dynamics studies can be tested with new treatments such as FLT3, IDH1 and IDH2 inhibitors. The recommended study time to perform the monitoring of clonal evolution would be at diagnosis, at the end of the induction in CR, and at the refractory or relapsed stages, with the main utility in those patients with AML who achieve CR or blast clearance. Although some conclusions obtained need to be validated in another wide series, the results of the relevance of clonal kinetics and its implications are robust.
Our results suggest that the monitoring of clonal evolution by genomic approaches can help to select post-remission strategies to target AML, and may improve prediction of clonal evolution and response of treatment.
Disclosures
No conflicts of interest to disclose.
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haematologica | 2021; 106(9)