Ultra-Deep Amplicon Sequencing Using Roche 454 Technology Allows High Sensitivity Bcr-Abl Kinase Domain Mutation Screening and Anticipates Emerging Mutations Leading to Resistance to Tyrosine Kinase Inhibitors in Philadelphia-Positive Leukemia Patients

Definite spectra of point mutations in the Bcr-Abl kinase domain (KD) are known to confer resistance to the tyrosine kinase inhibitors (TKIs) imatinib (IM), dasatinib (DAS) and nilotinib (NIL) in Philadelphia-positive (Ph+) leukemias. Until the recent advent of next-generation sequencing (NGS) technologies, no method was available that could conjugate high sensitivity, possibility to screen for any known or unknown sequence variation and accurate quantitation of mutated subclones. Because of these key technical limitations, the clinical relevance of early detection of small mutated subclones could never be fully elucidated. We have set up and optimized a Bcr-Abl KD mutation screening assay taking advantage of the Roche 454 NGS technology on a GS Junior instrument, that allows parallel pyrosequencing of a hundred thousand clonally amplified DNA molecules of 400 bp average length. We have thus designed 4 partially overlapping amplicons covering the whole KD of the Bcr-Abl transcript (a.a. 240–520) to be generated by nested RT-PCR using sequence-specific primers conjugated with multiplex identifiers – allowing to pool and sequence different samples from one or multiple patients (pts) in a single run. The assay proved capable to identify, characterize and quantitate sequence variations in samples from pts already known to harbour mutations as assessed by conventional Sanger sequencing with 100% concordance. Given that the number of sequence reads generated in each run is relatively constant, the lower detection limit is inversely correlated with the number of samples that can simultaneously be analyzed. Sensitivity could thus be easily modulated – analyzing a single sample per run allowed to routinely achieve lower detection limits ranging between 0.02 and 0.05% (that is, to detect as little as 2 to 5 mutated Bcr-Abl transcripts in a total of 10,000 Bcr-Abl transcripts). With this target sensitivity, we first retrospectively analyzed samples collected at the time of diagnosis from selected chronic myeloid leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL) cases known to have developed a T315I mutation 3 to 9 months after TKI start. We found that the T315I could not always be traced back to the time of diagnosis. We also found that, both in CML and in Ph+ ALL pts, several low-level mutations (in the range of 0.05 to 1%) with apparently no clinical relevance (e.g., never reported in association with TKI resistance) were routinely detectable – and appeared not to be predictors of a higher level of genetic instability. More interestingly, when used for mutation monitoring of de novo Ph+ ALL pts enrolled in clinical trials with DAS or NIL, NGS could highlight emerging resistant clones far earlier than D-HPLC or conventional sequencing. Also, some IM-resistant CML and Ph+ ALL pts who had been scored as unmutated by conventional sequencing could be shown by NGS to harbour DAS- or NIL-resistant mutations at 1–10% level – a phenomenon that is known to happen because of the clonal deselection resulting from the temporary lack of any TKI selective pressure in the interval between IM discontinuation and DAS or NIL start. Analyses will be presented in detail. We can conclude that: 1) the 454 NGS technology on the GS Junior instrument is a reliable and cost-effective method to perform Bcr-Abl KD mutation screening of Ph+ leukemia pts – with a target sensitivity of 0,1%, 4 samples can simultaneously be analyzed with costs comparable to those of conventional Sanger sequencing and the advantage to quantitatively follow the dynamics of mutated subclones over time; 2) in line with a recent report, small mutated subclones with apparently no clinical relevance can be detected both in newly diagnosed CML and Ph+ ALL pts before TKI start – which further underlines that mutation screening of pts at diagnosis is uninformative, if not misleading; 3) during TKI therapy, the high sensitivity of NGS allows to detect emerging Bcr-Abl mutant subclones earlier than D-HPLC or conventional sequencing; this is particularly relevant: i) for mutation monitoring of Ph+ ALL pts, that are highly prone to develop resistance and mutations while on TKI therapy, and ii) in IM-resistant CML and Ph+ ALL pts, where NGS may uncover the presence of DAS- or NIL-insensitive mutations, and may thus help choosing the second-line therapeutic strategy most likely to be successful.