Abstract 1884: Resistance to second-line tyrosine kinase inhibitor treatment in imatinib-resistant Philadelphia-positive leukemia patients can be anticipated by ultra-deep sequencing of the BCR-ABL kinase domain using Roche 454 technology
Authors: Soverini Simona , Benedittis Caterina De , Gnani Alessandra , Iacobucci Ilaria , Castagnetti Fausto , Gugliotta Gabriele , Papayannidis Cristina , Cattina Federica , Russo Domenico , Pantaleo Maria Abbondanza , Rosti Gianantonio , Baccarani Michele , Martinelli Giovanni 
Published: August 2012

Abstract
Definite spectra of point mutations in the Bcr-Abl kinase domain (KD) are known to confer resistance to the 1st-generation tyrosine kinase inhibitor (TKI) imatinib (IM) and to the 2nd-generation TKIs dasatinib (DAS) and nilotinib (NIL) in Philadelphia-positive (Ph+) leukemias. Currently, European LeukemiaNet guidelines recommend mutation analysis to be performed by denaturing-high performance liquid chromatography (D-HPLC) and/or direct sequencing. Using Roche 454 next-generation sequencing (NGS) technology on a GS Junior instrument, we investigated whether an ultra-deep amplicon sequencing strategy for mutation screening of the Bcr-Abl KD could highlight emerging clones harbouring critical mutations, ultimately anticipating the development of drug-resistance. A lower detection limit of 0.05% - 5 mutated Bcr-Abl transcripts in a total of 10,000 Bcr-Abl transcripts - could routinely be achieved. Serially collected samples from IM-resistant CML and Ph+ ALL pts who received DAS and/or NIL as 2nd- or 3rd-line therapy and developed DAS- or NIL-resistant mutations were retrospectively analyzed from the time of switchover to the time of relapse. NGS was able to confirm all mutations known to be present in &gt;20% of cells, allowed to follow quantitatively the dynamics of mutated clones over time and showed that i) 9/12 IM-resistant pts who failed to achieve a satisfactory response to 2nd-line therapy (primary resistance) were already harbouring mutations known to be insensitive to DAS (T315I, F317L/V) or NIL (E255K/V, Y253H, F359V/C) at 1-10% level before the TKI start - despite they had been scored as wild-type by D-HPLC; ii) in 9/13 IM-resistant pts who relapsed after an initial response (acquired resistance), the emerging resistant clone(s) carrying DAS- or NIL-insensitive mutation(s) could be identified up to six month in advance than when D-HPLC and conventional sequencing were used. In addition, when used for mutation monitoring of newly diagnosed Ph+ ALL pts enrolled in clinical trials with DAS or NIL, NGS again highlighted emerging resistant clones in 7/8 pts who later relapsed. We conclude that the higher sensitivity of NGS allows to detect emerging mutant clones earlier than the currently used methods; this is particularly relevant: i) in IM-resistant CML and Ph+ ALL pts before switching to and during treatment with a 2nd-line TKI, where NGS may anticipate the presence of DAS- or NIL-insensitive mutations, ensuring the most effective and timely therapeutic decisions; ii) more in general, for mutation monitoring of Ph+ ALL pts, that are known to be highly genetically unstable, hence markedly prone to develop resistance and mutations while on TKI therapy. Based on our data, further evaluation of the routine applicability of NGS is warranted. Supported by AIL, AIRC, PRIN, ELN.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1884. doi:1538-7445.AM2012-1884