Detection of Twelve Nucleotides Instertion in the BCR-ABL Kinase Domain in an Imatinib-Resistant but Dasatinib-Sensitive Patient with Bi-Phenotypic Acute Leukemia.
Authors: Hayette Sandrine , Chabane Kaddour , Tchirkov Andrei , Berger Marc G , Magaud jean-Pierre , Nicolini Franck E , Tournilhac Olivier 
Published: April 2020

Abstract
Although targeted inhibition of BCR-ABL with imatinib (IM) is an effective therapy for patients with Philadelphia chromosome-positive leukemia, a minority of patients (most of them in advanced phase) acquire mutations in the BCR-ABL kinase domain (KD) and relapse. The spectrum of the mutations described to date consists in-almost exclusively-single nucleotide substitutions affecting IM binding or impairing the conformational changes of the BCR-ABL KD. Recently, rare cases of intron/truncation mutations of 35 nucleotides, presumably inserted by alternative splicing events but inducing no conservation of the open reading frame (ORF), have been described in CML patients undergoing IM therapy (Laudadio et al. J Mol Diagn.2008; 10:177–180). Here, we describe a novel mutation involving the BCR-ABL KD, acquired at the moment of the IM resistance, consisting in an insertion of 12 nucleotides leading to the conservation of the ORF. The 57-year old female patient was diagnosed in July 2006 with bi-phenotypic acute leukemia based on peripheral blood and bone marrow findings (hyperleukocytosis at 48 G/l with 47% of blasts exhibiting myeloid and lymphoid features (CD13+, CD33+, CD19+, CD10+ and CD22+), on karyotypic analysis found a t(9;22)(q34;q11) in all mitosis and on molecular analysis detected M BCR-ABL transcript. There was no sibling donor identified. Induction chemotherapy (Hyper C-VAD) and IM at 800mg/day were given. She achieved a complete remission (CR), a complete cytogenetic response and the BCR-ABL/ABL ratio was 2.3% (RT-RQ PCR performed according to the ELN recommendations with ICF 0.51). Consolidation therapy with alternating high dose methotrexate plus cytarabine and Hyper C-VAD plus IM was given and the BCR-ABL/ABL ratio dropped to 0.06%. After a successful PBSC collection with G-CSF alone, she was autografted in April 2007. IM was re-introduced at 600mg/day leading to a sustained drop in transcript level to 0.035% 6 months later. Then the transcript ratio rose rapidly within 3 months (0.7%;2.4%;8.1%) and a mutation screen performed in April 2008 revealed the insertion of 12 nucleotides in 100% of the BCR-ABL transcripts inducing the insertion of 4 amino acid residues (A, F, G and P) between A293 and K294 in the BCR-ABL KD. This insertion into exon 5 of ABL in the BCR-ABL transcript sequences did not seem related to alternative splicing events or to a constitutional polymorphism because this insertion was present at the genomic level in DNA extracted from leukemic cells but was missing in genomic DNA extracted from the patient’s epithelial mouth cells. Moreover, retrospective analysis with a fluorescent probe and allele specific oligonucleotide performed by RQ-PCR on the cDNA from patient at diagnosis did not show the insertion at diagnosis, suggesting that the mutational mechanism was induced or selected by IM therapy. The increase of IM up to 800mg/day during six weeks led to the loss of hematologic response. Dasatinib was introduced at 70mg twice daily in may 2008 and molecular monitoring performed monthly showed a strong reduction of the BCRABL/ABL ratio to 0.7%; 0.2% and 0.07% respectively. The mutant ratios performed on the latest follow-up did not show any change in the proportion (100%) of the mutated BCR-ABL transcripts among total BCR-ABL transcripts suggesting no resurgence of the wild-type BCR-ABL clone. Therefore dasatinib seems to efficiently target the mutated clone as well as the wild-type leukemic clone and appears to be highly effective on this kind of mutation.