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LETTERS TO THE EDITOR
The EBF1-PDGFRB T681I mutation is highly resistant to imatinib and dasatinib in vitro and detectable in clinical samples prior to treatment
EBF1-PDGFRB accounts for 3% of cases of childhood Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL),1 represents the most common fusion gene in the Ph-like ABL-class subtype,2 and is notoriously associated with high rates of induction fail- ure.1-3 EBF1-PDGFRB fusions exhibited exquisite sensitiv- ity to ABL tyrosine kinase inhibitors (TKI) in preclinical models,3 and durable remissions have been reported in patients harboring EBF1-PDGFRB when treated with either imatinib or dasatinib.4 Collectively, these observa- tions provide a compelling rationale for investigating the incorporation of ABL TKI in combination with conven- tional chemotherapy for Ph-like ABL-class ALL patients in clinical trials. However, the emergence of kinase domain (KD) mutations as the primary mechanism of acquired resistance to TKI has been well described and occurs in many adults with relapsed/refractory Philadelphia chromosome-driven leukemias.5 The mech- anisms of TKI resistance in Ph-like ABL-class ALL have not been extensively studied, although we hypothesize that similar resistance mechanisms may occur between the two subsets. Hence, we sought to characterize the spectrum of TKI-resistant KD mutations in EBF1- PDGFRB Ph-like ALL as a mechanism of acquired resist-
ance by using a validated in vitro saturation mutagenesis screen, as previously described.6
Among 245 imatinib-resistant and 416 dasatinib-resis- tant colonies isolated from our in vitro screens, 233 (95%) and 363 (87%) colonies, respectively, harbored a single KD mutation. The predominant recurrent single KD mutation was the gatekeeper T681I point mutation for both imatinib (n=233/245, 95%) and dasatinib (n=338/416, 81%). The next most common recurrent KD mutation was N666S (n=18/416, 4%), which conferred resistance to dasatinib only. The T681I mutation in EBF1-PDGFRB is analogous to the gatekeeper mutation T315I in BCR-ABL1, while the N666S mutation is analo- gous to the N676S mutation in FLT3-ITD.7 The full spec- trum of KD mutations in EBF1-PDGFRB identified from the in vitro saturation mutagenesis screens with imatinib and dasatinib is reported in Online Supplementary Table S1.
We then focused on the two most common KD muta- tions to assess their proliferative properties and charac- terize their biochemical resistance to the relevant TKI. Introduction of EBF1-PDGFRB T681I and N666S mutant isoforms into Ba/F3 cells rendered them independent of interleukin-3, illustrating that the transforming capacity of the EBF1-PDGFRB fusion gene is preserved in the pres- ence of these mutations. In viability assays, the T681I mutation was highly resistant to imatinib and dasatinib, while the N666S mutation showed intermediate resist- ance to dasatinib. The half maximal inhibitory concentra-
Table 1. Clinical characteristics and outcomes of the 23 EBF1-PDGFRB patients with or without a subclonal T681I mutation at diagnosis, as determined by droplet digital polymerase chain reaction.
ID#
1
2
3
4
5 6* 7
8
9 10 11 12 13 14 15 16* 17 18 19 20* 21 22
23
Age at diagnosis (years)
3
12
14
7
17
17
12
19
14
11
6
14
9
6
12
12
4
19
18
14
8
16
16
WBC at diagnosis x109/L
18.4
114.3 419.8 79.9 396 13.4 32.5 54.8 41.7 28.2 80.7 3.3 39 212 17 5 49
BM blasts (%)
95
98
92
85
69
96
89
97
90
85
91
90
74
98
68 Unknown 95
CR EOI Relapse MRD (%)
Yes >1 No
IF >1 BM IF >1 No IF >1 No Yes 0.1-0.99 CNS Yes >1 BM Yes >1 BM IF >1 No
Unknown >1 No IF >1 No IF >1 No
Unknown Unknown No IF >1 No
Yes Unknown BM/CNS Yes Unknown No Yes Unknown BM Yes >0.1 No Yes >10 CNS Yes >10 No Yes >10 BM Yes >10 BM
Months to relapse
12
27 28 32
31
39
40
18 50
HSCT Status
Yes Alive (11.1 years)
Yes Died of disease (1.2 years) Yes Died in remission (1.2 years) Yes Died in remission (1.5 years)
No Alive (7.4 years) Yes Alive (6.8 years) Yes Alive (6.8 years) Yes Alive (6.2 years) Yes Alive (6.0 years) Yes Alive (5.2 years) Yes Alive (5.6 years)
No Induction death (19 days) Yes Alive (5.0 years)
No Died of disease (5.0 years) No Alive (7.6 years)
No Alive (7.4 years)
No Alive (7.5 years)
No Alive (6.8 years) Yes Alive (5.2 years)
No Died of disease (1.8 years)
No Alive (3.8 years)
No Died in remission (4 months)
No Died of disease (3 months)
8 99
10272IF>10No
3
26
34
68 95 Unknown >0.01 No
Unknown 94
99
2242
ID#: identification number; WBC: white blood cell; BM: bone marrow; CR: complete remission defined as M1 marrow or <5% blasts on microscopic assessment; EOI MRD: end of induction minimal residual disease; HSCT: hematopoietic stem cell transplantation; IF: induction failure; CNS: central nervous system, *patients with subclonal T681I mutation.
haematologica | 2021; 106(8)