Identification of Pre-existing Kinase Domain Mutations in Subclones of Ph-positive Leukemias

Background Single or multiple point mutations in the tyrosine kinase domain (TKD) of the BCR-ABL1 fusion gene in chronic myeloid leukemia (CML) and Ph-chromosome positive acute lymphoblastic leukemia represent the most important known mechanism of resistance to tyrosine kinase inhibitors (TKIs). It is conceivable that pre-existing point mutations in CML stem cells attributable to the genomic instability conferred by the BCR-ABL1 fusion protein give rise to the outgrowth of resistant subclones and onset of treatment-insensitive disease under the selection pressure of TKI therapy. Early detection of such subclones may therefore be of prognostic and therapeutic relevance. The recently published immunophenotype of CML stem cells (Hermann et al. Blood 2014), and our recent report on an NGS (next-generation sequencing)-based method facilitating sensitive detection and quantitative monitoring of BCR-ABL1 subclones carrying single or compound mutations (Kastner et al. European Journal of Cancer 2014) facilitate the screening for clinically relevant mutant subclones in stem cells or early progenitor cells.

Hypothesis Detection of mutant subclones within the stem cell or early progenitor compartments at diagnosis or early into therapy of Ph-positive leukemias, and monitoring of their proliferation kinetics, permit early prediction of resistant disease under the ongoing TKI treatment.

Experimental approach Bone marrow (BM) and peripheral blood (PB) samples will be collected upon informed consent from patients with Ph-positive leukemia at diagnosis (BM+PB), and subsequently at 3-month intervals (PB;BM upon availability) during the first year of therapy based on or including TKIs. Isolation of CD (cluster of differentiation) 34+ cells carrying additional phenotypic markers characterizing stem cells or early progenitor cells (CD38-/CD25+/CD26+ in CML, CD19 in Ph-ALL) will be performed by flow sorting. The entire BCR-ABL1 tyrosine kinase domain (TKD) will be amplified from cDNA (complementary DNA) or specific exons of interest from DNA by established protocols, and bidirectional sequencing will be performed by ultra-deep sequencing using NGS. Mutant subclones identified at diagnosis or after debulking of most of the treatment-sensitive leukemic burden early into treatment (3 month time point), will be monitored by NGS until month 12 of therapy. BCR-ABL1 transcripts will be monitored according to the International Scale (IS) in parallel. DNA isolated from fingernail clippings will be used as germline control for the ABL1 TKD. The testing will be performed in a blinded fashion to prevent treatment adjustments according to experimental data.