Single Molecule Real Time (SMRT™) Sequencing Sensitively Detects Polyclonal and Compound BCR-ABL in Patients Who Relapse on Kinase Inhibitor Therapy,

Secondary kinase domain (KD) mutations are the most well-recognized mechanism of resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) and other cancers. In some cases, multiple drug resistant KD mutations can coexist in an individual patient (“polyclonality”). Alternatively, more than one mutation can occur in tandem on a single allele (“compound mutations”) following response and relapse to sequentially administered TKI therapy. Distinguishing between these two scenarios can inform the clinical choice of subsequent TKI treatment. There is currently no clinically adaptable methodology that offers the ability to distinguish polyclonal from compound mutations. Due to the size of the BCR-ABL KD where TKI-resistant mutations are detected, next-generation platforms are unable to generate reads of sufficient length to determine if two mutations separated by 500 nt reside on the same allele. Pacific Biosciences RS Single Molecule Real Time (SMRT) circular consensus sequencing technology is a novel third generation deep sequencing technology capable of rapidly and reliably achieving average read lengths of ∼1000bp (Travers et al, 2010) and frequently beyond 3000bp, allowing sequencing of the entire ABL KD on single strand of DNA. We sought to address the ability of SMRT sequencing technology to distinguish polyclonal from compound mutations using clinical samples obtained from patients who have relapsed on BCR-ABL TKI treatment.

We analyzed an 863bp area of the BCR-ABL KD in 6 patients who had clinically relapsed on ABL kinase inhibitor therapy. SMRT sequencing detected mutations at a sensitivity of ∼1–2% of the total sequenced population, and successfully distinguished polyclonal from compound BCR-ABL KD mutations in several patient samples. Results were largely consistent with those obtained by PCR subcloning and sequencing, although SMRT sequencing detected additional mutations and/or mutation combinations. In the most complex case, 7 distinct mutation-bearing alleles were detected in an individual patient after sequential relapse on imatinib and dasatinib. Mutant clones contained single and compound mutations combining distinct mutations (Y253H, T315F, T315A, T315I, T319A, E355G). Three distinct substitutions at residue T315 were detected: T315A, T315I and T315F. Notably, these findings are clinically important as the T315A mutation confers resistance to dasatinib but not imatinib, while the T315F and T315I mutations are resistant to all three clinically approved BCR/ABL inhibitors (imatinib, dasatinib, and nilotinib). Phospho-flow analysis for p-Crkl, a direct substrate of BCR-ABL, was conducted following ex vivo exposure of patient cells from the same time point to all three BCR-ABL inhibitors, and demonstrated the existence of distinct populations of cells with varying sensitivity to each drug (i.e. polyclonal drug sensitivity), underscoring the potential clinical importance of distinguishing polyclonal from compound mutations. Additionally, SMRT sequencing routinely detected alleles harboring compound mutations not detectable by conventional direct sequencing. Data analysis of samples from additional patients is ongoing and will be presented.

Pacific Biosciences RS SMRT sequencing sensitively detects KD mutations in patient samples and can distinguish TKI-resistant clones containing compound mutations to reveal a complex mutational landscape in an individual patient not detectable by conventional sequencing. SMRT sequencing of the BCR-ABL KD can feasibly be developed into a rapid and economical clinical test with the additional advantages of increased sensitivity and reliability over current methods. Given the growing numbers of patients exposed to multiple TKIs in a sequential manner, the ability to accurately and sensitively characterize drug-resistant alleles promises to further facilitate a personalized approach to patient management.

Brown:Pacific Biosciences: Employment. Chin:Pacific Biosciences: Employment. Travers:Pacific Biosciences: Employment. Wang:Pacific Biosciences: Employment. Kasarskis:Pacific Biosciences: Employment, Equity Ownership. Schadt:Pacific Biosciences: Employment, Equity Ownership.