Rapid identification of compound mutations in patients with Philadelphia-positive leukaemias by long-range next generation sequencing

An emerging problem in patients with Philadelphia (Ph)-positive leukaemias is the occurrence of cells with multiple mutations in the BCR-ABL1 tyrosine kinase domain (TKD) associated with high resistance to different tyrosine kinase inhibitors. Rapid and sensitive detection of leukaemic subclones carrying such changes, referred to as compound mutations, is therefore of increasing clinical relevance. However, current diagnostic methods including next generation sequencing (NGS) of short fragments do not optimally meet these requirements. We have therefore established a long-range (LR) NGS approach permitting massively parallel sequencing of the entire TKD length of 933 bp in a single read using 454 sequencing with the GS FLX+ instrument (454 Life Sciences). By testing a series of individual and consecutive specimens derived from six patients with chronic myeloid leukaemia, we demonstrate that long-range NGS analysis permits sensitive identification of mutations and their assignment to the same or to separate subclones. This approach also facilitates readily interpretable documentation of insertions and deletions in the entire BCR-ABL1 TKD. The long-range NGS findings were reevaluated by an independent technical approach in select cases. Polymerase chain reaction (PCR) amplicons of the BCR-ABL1 TKD derived from individual specimens were subcloned into pGEM®-T plasmids, and >100 individual clones were subjected to analysis by Sanger sequencing. The NGS results were confirmed, thus documenting the reliability of the new technology. Long-range NGS analysis therefore provides an economic approach to the identification of compound mutations and other genetic alterations in the entire BCR-ABL1 TKD, and represents an important advancement of the diagnostic armamentarium for rapid assessment of impending resistant disease.