Absolute Quantification of Mutant T315I BCR-ABL Transcripts At 6 Months Identifies Imatinib Resistant CML Patients with a Low Probability to Achieve MMR After 12 Months on Second Line Tyrosine Kinase Therapy
Authors: Lange Thoralf , Ernst Thomas , Pfirrmann Markus , Maier Jacqueline , Müller Martin C. , Gruber Franz , Niederwieser Dietger , Hochhaus Andreas 
Published: October 2019

Abstract
Abstract 1679
The BCR-ABL T315I mutation is associated with resistance against imatinib (IM), nilotinib (NI) and dasatinib (DA) in patients with chronic myeloid leukemia (CML). Although this mutation can be detected by different techniques and at different levels of the mutated clone, the prognostic significance of the absolute amount of the mutated allele is widely unknown. We therefore sought to determine absolute T315I allele levels after 6 months on second line NI or DA therapy to identify a cut off value for predicting the achievement of major molecular response after 12 months (MMR12; ≤0.1% BCR-ABL transcripts at international scale).
Forty randomly selected BCR-ABL positive CML patients (n=24 male, median age 64, range 39–78 years) in chronic (n=31), accelerated (n=7), or blastic phase (n=2) with failure to IM were treated with DA 140 mg/day (n=20) or NI 800 mg/day (n=20) and analyzed by denaturing high-performance liquid chromatography (D-HPLC) and high-sensitive allele-specific ligation PCR (L-PCR) for quantification of mutated T315I BCR-ABL allele normalized by beta-glucuronidase (GUS) expression. This method was previously shown to obtain a dynamical detection range of 100% to <0.1% mutant T315I allele (3–3.5 log). The levels of the BCR-ABL T315I alleles at 6 months were correlated with the achievement of a MMR12 on second line treatment. The L-PCR investigator was blinded for the MMR12 results.
On second line therapy, MMR12 was achieved by 12 (20%) patients. After 6 months, 37 (10 with MMR12) patients were available for D-HPLC assessment and showed a T315I mutation in 3 (8%) patients. None of these patients achieved MMR12. In comparison, 35 evaluable patients (8 with MMR12) showed >10−5 BCR-ABLT315I/GUS (n=10, 20%), <10−5 (n=8, 22%) or no (n=17, 48%) T315I mutation by L-PCR. All patients with >10−5 BCR-ABLT315I/GUS did not achieve MMR12, all patients with <10−5 developed MMR12 (p<0.0001). This pre-defined value of >10−5 BCR-ABLT315I/GUS as a negative predictor for MMR12 was applied to a new second independent group of 40 IM resistant patients (n=17 male, median age 60, range 32–79 years) all in first chronic phase, MMR12 n=9, 22.5%) equally distributed to NI and DA. In this group 12 (30%) patients showed >10−5 BCR-ABLT315I/GUS after 6 months and all but one did not achieve MMR12. In contrast, 5 patients with <10−5 showed MMR12, 3 patients did not (p=0.0181).
In conclusion, absolute quantification of the mutant T315I allele is helpful to identify patients with a low chance of MMR12 on second line nilotinib or dasatinib treatment. Therefore, relevant mutations should be quantified into clinical trial protocols to identify certain amounts with prognostic significance of the mutated clones for a specific mutations on a given TKI treatment in CML.

Disclosures:
Lange: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hochhaus:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.