Background:
Philadelphia chromosome-positive(Ph+) acutelymphoblastic leukemia (ALL) is characterized by thereciprocal
translocation t(9;22)(q34;q11).Theadvent of tyrosinekinaseinhibitors (TKIs) has markedly improved the outcome
of Ph+ ALL patients and indeed changed the natural history of the disease. At present,TKIs represent the gold
standard treatment for patients with Ph+ ALL, with or without chemotherapy
1
. Despitetheefficacy of targeted
therapy with TKIs, some patients show drug resistance dueto the onset of BCR::ABL1 kinase domain (KD) point
mutations. In particular, the most deleterious oneis represented by theT315I, which causetreatment failure with
firstand second generation TKI-based therapies.Sanger sequencing (SS) is the gold standard tool for BCR::ABL1
KD mutational screening.The digital droplet PCR (ddPCR) technology,a third generation PCR, may representa
valid alternativeto SS for the detection of BCR::ABL1 KD mutations.
Aims:
Theaim of this study was to evaluateif ddPCR is at leastas sensitiveas SS for the detection of theT315I mutation,
and if the ddPCR can detect the mutation also atvery low levels of minimal residual disease(MRD), ultimately
anticipating molecular relapse.
Methods:
Thestudy comprised samples from patients enrolled in the phaseII GIMEMA LAL2116 chemotherapy-free
protocol for newly diagnosed adult Ph+ ALL and based on theadministration of thesecond generation TKI
dasatinib followed by the bispecific monoclonal antibody blinatumomab
2
. A BCR::ABL1 KD mutational screening
was carried out in all patients with a MRD increase by both SS and ddPCR. In mutation-positive patients, thetimepoint (TP) preceding the MRD increase was evaluated by ddPCR to assess theearlier presence of the mutation.
DdPCR was performed as described
3
.
Results:
Wecarried outa BCR::ABL1 KD mutational screening by SS in 16 patients enrolled in the GIMEMA LAL2116
protocol. Overall, of the 16 patients with a MRD increase(10 during theinduction phase, 6 in theconsolidation
phase), 8 resulted wild type(WT) while mutations were detected in 8 patients (7 harboring theT315I mutation and
1 theE255K). In theT315I positive patients, theevaluation by ddPCR of thesameTP samples,confirmed in all
cases the presence of the mutation. Moreimportantly, theanalysis ata previous TP, when MRD levels werelower,
showed that ddPCR could detect theT315I BCR::ABL1 KD mutation in 6/7 cases, while 1 positive non-quantifiable
(PNQ) case proved WT(Table 1). In order to determineif ddPCR could anticipatethe detection of mutations
compared to SS, the previous TPs werealso evaluated by SS in 4 cases with available material. In 2 cases the
mutation was detected also by SS, whilein 2 it could not befound (Table 1).Likewise, ddPCR for theT315I
mutation was also performed in samples that proved negative by SS,and theabsence of mutations was
confirmed; this is particularly relevant from a clinical standpointand was corroborated by thefact that only 1/8
experienced a central nervous system relapsein this last cohort,and is currently alivein second complete
remission (CR).
Summary/Conclusion:The ddPCR proved as reliableand accurateas SS to detect theT315I BCR::ABL1 KD
mutation.Furthermore, the ddPCR proved to be moresensitivefor predict molecular relapse beforetheincrease
in MRD where, in somecases, theSS failed to detect theT315I mutation.Further efforts are ongoing to expand
this screening also to other ABL1 mutations,considering thealways morefrequent use of ponatinib in thefirst-line
setting.
1.Foà & Chiaretti, NEJM 2022
2.Foà et al, NEJM 2020
3.Soverini et al,Leukemia 202