Dynamics and Characteristics of BCR-ABL Multiple Mutations In Tyrosine Kinase Inhibitor Resistant Chronic Myeloid Leukemia.

Abstract 3443

BCR-ABL kinase domain (KD) point mutation causes resistance to tyrosine kinase inhibitors (TKI) in CML patients through impaired binding of TKI to the target site. One of the characteristics of patients with BCR-ABL kinase domain point mutations is the fact that some patients have multiple mutations. However there have not been many studies showing that data about clinical relevance or dynamics of multiple mutation during CML treatment.

From January 2002 to June 2010 at Seoul St Mary's Hospital, 277 CML patients were screened for mutation analysis due to sign of resistance to tyrosine kinase inhibitors including imatinib, nilotinib, dasatinib or bosutinib. We found that 95 patients have point mutation in BCR-ABL kinase domain through direct sequencing or ASO-PCR. Among them, 17 patients showed multiple mutation containing more than one type of point mutations in BCR-ABL KD.

We investigated the patients with multiple mutations to characterize its clinical relevance and dynamics. Once mutation found, follow-up samples from the corresponding patients were collected and analyzed prospectively, or mutation status was analyzed retrospectively with cryopreserved samples if they were available.

Status of the patients with multiple mutation is shown in Table 1. In order to investigate whether the multiple mutations are on same clone or on separated clone, we cloned serial samples from the 17 patients. Cloning of cDNA region corresponding to BCR-ABL KD into plasmid was performed and followed by transformation into competent cells, colony formation, plasmid preparation of 20 colonies from each sample, and then direct sequencing.

Multiple mutations of 88% patients (15 out of 17) existed compound mutation which means the individual mutant types are located on the same BCR-ABL molecule. In addition of major mutation types which were detectable in direct sequencing analysis, all the patients showed to have minor types of mutations which were found only through BCR-ABL KD cloning and subsequent colony sequencing. To make sure that this minor mutation types were not caused by sequencing error, we also analyzed of 3 patients who showed TKI resistance, but had no BCR-ABL mutation. In addition, samples from 3 normal persons were analyzed with the same method. The frequency of appearance of the minor types of point mutation was reduced in the patient group who showed TKI resistance, but had no BCR-ABL mutation, and then dramatically decreased in the normal person group, indicating that BCR-ABL gene in patients with point mutation are relatively unstable.

Analysis of serial samples from a same patient provided evidence of dynamic change of portion of compound mutation. In most case, portion of the clone containing compound mutation was increased as treatment went on, indicating the clone harboring compound mutation can take survival advantage over TKI treatment in comparison of the clone containing individual type of mutation. In addition, some patients showed change in individual mutation type comprising multiple mutation as treatment went on. Currently investigation of clinical relevance of compound mutation and other analyses are being carried on and more results will be provided in detail at the conference.