The presence of BCR-ABL kinase domain mutations below the detection limit of Sanger sequencing (low level mutations, LLM) predicts the outcome of subsequent therapy in patients (pts) with imatinib resistance (Parker et al. JCO 2011, Blood 2012). The incidence of LLM in the ENEST1st trial, in which newly diagnosed chronic phase CML pts were treated with nilotinib, was 51%. Presence of LLM could not be correlated with clinical endpoints (Franke et al.; ASH 2014), but only 41 pts samples were available for analysis. We therefore investigated the importance of LLM in a much larger cohort of pts within the German CML V (TIGER) trial, in which newly diagnosed CP CML pts were treated with nilotinib or nilotinib plus interferon. Study endpoints are rates of MMR at 18 months of treatment and after TKI discontinuation. We report here on the incidence and type of LLM in this cohort of pts.


467 TIGER study pts provided 10ml of peripheral blood or 2ml bone marrow after written informed consent. CD34+ selection was carried out by MACS® (Miltenyi Biotec) and the CD34+purity was determined by fluorescent activated cell sorting (FACS). Aliquots of 105 CD34+ were used for RNA extraction, cDNA synthesis and BCR-ABL amplification followed by Ligation PCR (L-PCR) for mutations T315I, Y253H, E255K/V, and F359V. This method has previously been shown to achieve a dynamic detection range of 100% to <0.1% mutant allele (3-3.5 log). For 343 of 467 pts (75%) >= 105 CD34+ cells with a purity of >50% were available for BCR-ABL amplification.


Mean CD34+ purity was 86% and the rate of successfully amplified samples was 70% (328/467 samples). A total of 1640 L-PCR assays of CD34+ cells identified 75 (4.6%) mutations in 60/328 pts (18.3%). The most frequently detected LLM was E255K (n=45), followed by Y253H (n=22). The T315I mutation was detected in 6 pts and E225V in 2 pts. No pt was positive for F359V. Forty-seven pts had only one LLM, 11 had two LLM and 2 pts had 3 LLM. The median quantitative level of mutant alleles in CD34+ cells was 0.085 (range 0.02-4.96) BCR-ABL mutant/ BCR-ABL unmutated.


LLM potentially causing resistance to nilotinib and other tyrosine kinase inhibitors can be detected in newly diagnosed CML pts. Nearly twenty percent harbor one or more LLM at diagnosis, with 1.7% displaying the T315I mutation. The significance of this observation will be evaluated in regards to the study endpoints of the TIGER trial.


Franke: Takeda: Consultancy; Pfizer: Honoraria; Novartis: Honoraria, Research Funding. Hochhaus: BMS: Research Funding; ARIAD: Research Funding; Novartis: Research Funding; Incyte: Research Funding; Pfizer: Research Funding; MSD: Research Funding. Lange: Novartis: Honoraria, Research Funding.

Author notes


Asterisk with author names denotes non-ASH members.

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