Mutational Phasing: Clinical Relevance in Tyrosine Kinase Domain Mutations Using Next Generation Sequencing in Chronic Myeloid Leukemia

Background: Tyrosine kinase mutation analysis in BCR/ABL1 gene is important for management of patients with chronic myeloid leukemia. Sanger Sequencing has been the mainstay for testing with Next Generation Sequencing (NGS) now becoming the primary technology. In this study we show a comparison between NGS versus Sanger Seqencing based ABL kinase domain mutation analysis with a likely trend of clinical relevance based on a compound versus polyclonal state of mutational distribution which may also need to be considered for patient management and therapy.

Methodology:

A total of 213 Imatinib-resistant patients with CML for BCR-ABL1 mutation analysis were processed on both technologies.Initial blood counts were assessed and RNA was extractedfollowed by cDNA conversion. NGS libraries were prepared with 400bp multiplexed amplicons to allow optimal phasing.

Results:

179 samples were negative by both technologies. A total of only 20 samples were positive and concordant by both technologies(58.2%). Mutations in 14 other samples however were only detected in NGS(41.17%). In these 14 samples (41.17%), NGS was able to detect 23 mutations with mutation frequencies of 3-28%, which were missed by Sanger.

Conclusions:

Moreover 11/34 patients had 2 or >2 mutations. An inhouse script delineated mutations as compound or polyclonal from NGS data. 2/11 cases demonstrated compound mutations (Mutations in the same clone) while 7/11 cases were polyclonal per NGS. Sanger sequencing cannot differentiate between polyclonal and compound mutations. 2/11 cases appeared to have polyclonal and compound mutations. 4/11 patients presented in a blast crisis or accelerated phase CML. Interestingly, most of these patients hadat leasttwo mutations and were polyclonal(3/4).

Significantly previously archived samples patients with polyclonal mutations showed polyclonality at extremely low frequency percentages in initial samples.

None of the single mutation patients had presented in a blast crisis or an accelerated phase.