The accurate detection of BCR-ABL1 kinase domain (KD) mutations is important for appropriate clinical management of CML patients on tyrosine kinase inhibitor therapy. Sanger sequencing (SS) is widely used for the clinical analysis of KD mutations. However, SS is relatively insensitive and mutant levels of approximately 15-20% are necessary for detection by this technique. Furthermore, it is not possible to determine whether or not two or more detected mutations are in the same subpopulation (in-cis; so-called composite mutations) or in separate subpopulations (in-trans). In order to validate the feasibility and potential advantages of next generation sequencing (NGS) for detecting BCR-ABL1KD mutations in a large volume clinical hematology reference laboratory, we developed an NGS assay on the Ion Torrent PGM platform (IT-NGS) and conducted a side-by-side comparison to SS in a series of clinical samples.


101 clinical samples that were evaluated for BCR-ABL1 KD mutations at our institution were collected from the archives. Of these, 74 were positive and 27 were negative for KD mutations by SS. 30 of 74 positive cases demonstrated 2 or more mutations by SS. These samples were subjected to a BCR-ABL1KD sequencing assay by IT-NGS then analyzed using Ion Torrent Server software, an off-the-shelf tool for the calling of sequence variants. All mutations that were detected by IT-NGS were quantitated. The sensitivity for detecting mutations by this method was validated to 4% and this was set as the threshold for variant calling.


100% of mutations that were detected by SS were also detected by IT-NGS. 11 of 74 positive cases (14.9%) demonstrated an additional 15 mutations by IT-NGS (range of mutation frequency = 4-21%) that were not previously detected by SS. This included 4 of 74 cases with 2 additional mutations. Two cases demonstrated mutations by IT-NGS that were not detected by SS because they were outside the range of coverage of the SS assay. In total, the 74 positive samples had 108 mutations detectable by SS and 125 mutations detectable by IT-NGS. 39 cases demonstrated 2 or more mutations by IT-NGS. In 32 of these 39 cases, it was possible to conclude the cis/trans configuration of specific mutation pairs, information that cannot be obtained from SS data. 16 total mutation pairs were in-cis and 17 mutation pairs were in-trans (1 case with 4 mutations had a pair which was in-cis and a pair which was in-trans). The ability to determine cis/transconfiguration was dependent upon whether or not multiple mutations were close enough in the sequence to be present in the same NGS reads. Of the 27 SS-negative cases, 1 demonstrated a low-level mutation (Y253F; 5.5%) missed by SS and three cases demonstrated low-level mutations outside the range of the SS assay. The total cost per sample for IT-NGS was comparable to SS when 10+ samples were multiplexed.


1. We describe an NGS-based BCR-ABL1 KD sequencing assay that is suitable, in terms of cost and performance, for routine use in a clinical hematology laboratory. 2. Data generated by this assay demonstrates that lower level mutations not detectable by SS (<20%) often accompany high-level mutations. 3. Information about the underlying complexity of BCR-ABL1 KD mutations is achievable in the context of routine clinical testing using these methods.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.