Single Molecule Real Time (SMRT™) Sequencing Sensitively Detects the Evolution of Polyclonal and Compound BCR-ABL Mutations in Patients Who Relapse On Kinase Inhibitor Therapy

Secondary kinase domain (KD) mutations represent the most well-documented mechanism of resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML). In CML, multiple TKIs with different mutation profiles are approved and the ability to detect KD mutations at the time of disease progression can impact therapy choice. To optimize clinical impact, second generation TKI selection must consider the majority TKI-resistant mutant population as well as smaller mutant sub-populations that may be selected with subsequent treatment. Sequential TKI therapy is associated with additional complexity: multiple mutations can coexist separately in an individual patient (“polyclonality”) or can occur in tandem on a single allele (“compound mutations”). Multiple mutations are associated with poor clinical outcome (Parker et al., Blood 2012). Compound mutations can cause in vitro resistance to ponatinib, the only TKI clinically active against the highly resistant T315I mutation (Eide, et. al, ASH 2012 abstract #1416). Currently, no clinically adaptable technology can distinguish polyclonal from compound mutations. Due to the size of the BCR-ABL KD, most next-generation sequencing platforms cannot generate reads of sufficient length to determine if mutations separated by ≥500 nt reside on the same allele. Pacific Biosciences RS Single Molecule Real Time (SMRT) sequencing technology is a third generation deep sequencing technology capable of achieving average read lengths of ∼1000bp and frequently >3000bp, enabling sensitive and accurate sequencing of the entire ABL KD on a single strand of DNA. Though allele-specific detection methods such as MassARRAY offer sensitivity as low as ∼0.5%, these assays are designed to detect a limited number (∼31) of mutations whereas SMRT sequencing offers an unbiased approach capable of detecting novel variants. We sought to (1) develop a potential clinically-applicable SMRT sequencing assay for the detection of BCR-ABLKD mutations capable of distinguishing polyclonal and compound mutations, and (2) compare the accuracy and sensitivity of this method to standard sequencing and MassARRAY.

We assessed 54 samples from 36 CML patients who had clinically relapsed on ABL kinase inhibitor therapy and were previously analyzed by standard sequencing, and in a subset, by MassARRAY. We amplified an 863bp area of the BCR-ABLKD from patient-derived cDNA with primers containing 5' barcodes, enabling sequencing of 6 to 8 patient samples on a single SMRT cell on a single run. On average, 2519 reads were obtained for each sample per run (range 330 to 10,240). All of 131 known mutations detected by MassARRAY were identified by SMRT sequencing using a p-value threshold of 1.03e–03. SMRT sequencing also identified all 107 known mutations detected by direct sequencing with a p-value threshold of 6.0e–08. In addition to these known mutations, SMRT sequencing detected an additional 1320 non-silent mutations across all patient samples using a strict p-value threshold cut-off of 6e–08, ranging in abundance from 0.2% to 17% (median 0.75%). Among 47 samples where >1 mutation was detectable by direct sequencing or MassARRAY, SMRT sequencing revealed that 40 (85%) had compound mutations detectable at a frequency of ≥1. In total, we detected 73 different compound mutations at a frequency of ≥1%. In all cases where compound mutations were detected and more than one treatment timepoint was available, at least one compound mutation clearly evolved from a mutation detectable at a prior timepoint. In the most complex case, 4 separate mutations yielded 8 different mutant alleles.

Pacific Biosciences RS SMRT sequencing detects KD mutations in patient samples with sensitivity comparable to or better than MassARRAY and can distinguish compound from polyclonal mutant clones. Among patient samples with multiple mutations, compound mutations were detectable in the vast majority of samples by SMRT sequencing, revealing a complex mutational landscape not demonstrable by other clinically viable sequencing methods and previously unappreciated. Given the growing numbers of patients exposed to multiple TKIs in a sequential manner, the ability to accurately and sensitively characterize drug-resistant alleles by SMRT sequencing promises to further facilitate a personalized approach to patient management and inform models of disease evolution.

Brown:Pacific Biosciences: Employment. Travers:Pacific Biosciences: Employment. Wang:Pacific Biosciences: Employment. Branford:Novartis : Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad : Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cepheid : Consultancy. Shah:ARIAD, Bristol Myers-Squibb: Consultancy, Research Funding; Novartis: Consultancy.