Abstract 4632: Unraveling the complexity of tyrosine kinase inhibitor-resistant populations by ultra-deep sequencing of the BCR-ABL kinase domain in Philadelphia-positive leukemias.
Authors: Soverini Simona , De Benedittis Caterina , Machova Polakova Katerina , Brouckova Adela , Castagnetti Fausto , Papayannidis Cristina , Gugliotta Gabriele , Palandri Francesca , Klamova Hana , Iacobucci Ilaria , Venturi Claudia , Cattina Federica , Bresciani Paola , Coluccio Valeria , Salvucci Marzia , Tiribelli Mario , Binotto Gianni , Intermesoli Tamara , Luppi Mario , Bochicchio Maria Teresa , Ottaviani Emanuela , Russo Domenico , Gianantonio Rosti , Baccarani Michele , Martinelli Giovanni 
Published: August 2014

Abstract
Background & Aims - In chronic myeloid leukemia (CML) and Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL), tyrosine kinase inhibitor (TKI) therapy may select for drug-resistant BCR-ABL mutants, routinely assessed by Sanger sequencing (SS). We took advantage of ultra-deep sequencing (UDS) in order to: 1) resolve qualitatively and quantitatively the complexity of mutated populations surviving TKIs; 2) investigate their clonal structure and evolution in relation to time and treatment.
Methods - We retrospectively performed a longitudinal analysis of 111 samples from 32 CML or Ph+ ALL patients who had received sequential treatment with multiple TKIs and had experienced sequential relapses accompanied by selection of TKI-resistant mutations. All samples had already been scored by SS. UDS of the BCR-ABL KD was done using Roche 454 technology (lower detection limit, 0.1%). Seqnext software was used for alignment and variant identification; Jalview and Figtree softwares were used for haplotype and phylogenetic reconstruction.
Results - UDS showed that SS often misclassifies or underestimates BCR-ABL KD mutation status. In more than half of the cases, UDS revealed that up to five ‘minor’ mutations with 1-20% abundance were present, either in samples scored as wild-type by SS or in samples already bearing one or more dominant mutations. The high degree of complexity was even more evident when the clonal relationships of multiple mutations were reconstructed and the relative abundance of all mutant subclones coexisting at each timepoint was assessed. This revealed that identical mutations may be acquired in parallel by independent subclones (e.g., one wild-type and one already harboring a mutation), via the same or different nucleotide changes leading to the same amino acid substitution (convergent evolution). Longitudinal quantitative follow-up showed that the landscape of all competing mutant subclones is highly dynamic, and that dominant subclones may be replaced as quickly as within one month in case of selective pressure change. Earlier identification of emerging TKI-resistant mutants was made possible by UDS.
Conclusions - 1) sequential changes in the selective pressure exerted by TKIs may result in a heterogeneous mosaic of subclones harbouring different mutations or mutation combinations
2) The ‘ecosystem’ of mutant subclones is highly dynamic: acquisition of additional mutations dictates quick shrinkage or expansion of subclones over time
3) Inherent sensitivity to a specific TKI is necessary but not sufficient to determine the ‘fitness’ of a mutant subclone: competition with coexisting subclones also concurs to shape its fate
4) Reasoning on the basis of mutations detectable by SS may not always be sufficient to predict responsiveness to a TKI.
Supported by Fondazione CARISBO, PRIN, IGA MZCR NT11555.
Citation Format: Simona Soverini, Caterina De Benedittis, Katerina Machova Polakova, Adela Brouckova, Fausto Castagnetti, Cristina Papayannidis, Gabriele Gugliotta, Francesca Palandri, Hana Klamova, Ilaria Iacobucci, Claudia Venturi, Federica Cattina, Paola Bresciani, Valeria Coluccio, Marzia Salvucci, Mario Tiribelli, Gianni Binotto, Tamara Intermesoli, Mario Luppi, Maria Teresa Bochicchio, Emanuela Ottaviani, Domenico Russo, Rosti Gianantonio, Michele Baccarani, Giovanni Martinelli. Unraveling the complexity of tyrosine kinase inhibitor-resistant populations by ultra-deep sequencing of the BCR-ABL kinase domain in Philadelphia-positive leukemias. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4632. doi:10.1158/1538-7445.AM2013-4632