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Letters to the Editor
Figure 2. Droplet digital polymerase chain reaction can detect subclonal T681I mutation in clinical samples at diagnosis. Droplet digital polymerase chain reaction (ddPCR) experiments including positive T681I, wild-type and no template controls in the left panel. In the right panel, three EBF1-PDGFRB patients were found to have subclonal T681I mutationd at diagnosis by ddPCR. Patient #1 had four droplets containing the mutant T681I out of 20,879 total generated droplets (0.019%); Patient #2 had three positive droplets out of 17,987 generated (0.017%) and patient #3 had five positive droplets out of 22,799 generated (0.022%).
patients without a subclonal T681I mutation, although there was a trend towards a higher likelihood of relapse in the T681I-positive group versus the T681I-negative group (100% vs. 35%; P=0.0678) (Online Supplementary Table S2).
To the best of our knowledge, our study is the first to report that KD mutations represent a potential mecha- nism of acquired resistance in children with EBF1- PDGFRB Ph-like ALL. The gatekeeper T6811 mutation was the predominant KD mutation in our in vitro screens which was resistant to both imatinib and dasatinib, but could be rescued by ponatinib as predicted. The paucity of KD mutations in EBF1-PDGFRB recovered in the dasa- tinib mutational screen was similar to that in other BCR- ABL1 mutational screens, since dasatinib is active against most imatinib-resistant KD mutations.10 However, to our surprise, the gatekeeper mutation was the only KD muta- tion in EBF1-PDGFRB retrieved in the imatinib mutation- al screen, while over 90 imatinib-resistant KD mutations have been reported with BCR-ABL1.11 This finding could be explained by the higher dose that was used in our screen compared to previous reports, but it is also known that imatinib is much more potent in PDGFR family fusions than in the BCR-ABL1 fusion. The IC50 of ima- tinib for EBF1-PDGFRB in our hands was 15.74 nM, while Cools et al. reported that the IC50 of imatinib for cells expressing FIP1L1-PDGFRA was 3.2 nM, whereas the IC50 for BCR-ABL1 was 582 nM.12 Thus, mutations that impart a modest degree of imatinib resistance may not have been detected by our screens.
The analogous N666S mutation has not been previous- ly reported in BCR-ABL1 in vitro screens with either ima- tinib or dasatinib. However, the residue N666 in EBF1- PDGFRB is adjacent to its analogous residue V299 in BCR-ABL1, which represents the third most common
contact residue where KD mutations to dasatinib arise, after T315 and F317 amino acid residues.10 Smith et al. identified the N676S mutation in FLT3-ITD in their in vitro mutagenesis screen with the FLT3 inhibitor PLX3997, but only N676K/T mutations rather than N676S were isolated from adult acute myeloid leukemia patients with acquired clinical resistance to PLX3997.7 Moreover, FLT3 N676K mutations have been identified in core-binding factor leukemia at diagnosis and may rep- resent a cooperating mutation in leukemogenesis. The FLT3 N676K mutant alone can induce cytokine-indepen- dent growth in Ba/F3 cells and confer resistance to FLT3 inhibitors.13
In contrast to the report by Zhang et al.,14 our EBF1- PDGFRB in vitro saturation mutagenesis screen did not identify the C843G KD mutation that was seen in AGGF1-PDGFRB Ph-like ALL. In their experiments, the reported IC50 of AGGF1-PDGFRB C843G and EBF1- PDGFRB C843G to dasatinib was 0.78 nM and 0.121 nM, respectively. Thus, we may not recover this mutant in our screens even at 25 nM of dasatinib, the lowest dasatinib concentration used in our screen, which is more than 200-fold above the measured IC50 of EBF1-PDGFRB C843G.
The detection of drug-resistant KD mutations at diag- nosis has been reported in 21% to 40% of cases of TKI- naïve chronic myelogenous leukemia with advanced dis- ease and in Ph+ ALL samples.8,15 The frequency of T315I mutation at diagnosis ranges from 12.5% to 17%,15 which is in keeping with the frequency of the analogous gatekeeper T681I mutation in our cohort of EBF1-PDGFRB patients. Nevertheless, the clinical and prognostic significance of pre-existing KD mutation detected by sensitive technologies prior to TKI remains unclear. Willis et al. showed that mutation detection at
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