Unraveling the complexity of tyrosine kinase inhibitor-resistant populations by ultra-deep sequencing of the BCR-ABL kinase domain

In chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia, tyrosine kinase inhibitor (TKI) therapy may select for drug-resistant BCR-ABL mutants. We used an ultra-deep sequencing (UDS) approach to resolve qualitatively and quantitatively the complexity of mutated populations surviving TKIs and to investigate their clonal structure and evolution over time in relation to therapeutic intervention. To this purpose, we performed a longitudinal analysis of 106 samples from 33 patients who had received sequential treatment with multiple TKIs and had experienced sequential relapses accompanied by selection of 1 or more TKI-resistant mutations. We found that conventional Sanger sequencing had misclassified or underestimated BCR-ABL mutation status in 55% of the samples, where mutations with 1% to 15% abundance were detected. A complex clonal texture was uncovered by clonal analysis of samples harboring multiple mutations and up to 13 different mutated populations were identified. The landscape of these mutated populations was found to be highly dynamic. The high degree of complexity uncovered by UDS indicates that conventional Sanger sequencing might be an inadequate tool to assess BCR-ABL kinase domain mutation status, which currently represents an important component of the therapeutic decision algorithms. Further evaluation of the clinical usefulness of UDS-based approaches is warranted

Unraveling the complexity of tyrosine kinase inhibitor-resistant populations by ultra-deep sequencing of the BCR-ABL kinase domain

In chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia, tyrosine kinase inhibitor (TKI) therapy may select for drug-resistant BCR-ABL mutants. We used an ultra-deep sequencing (UDS) approach to resolve qualitatively and quantitatively the complexity of mutated populations surviving TKIs and to investigate their clonal structure and evolution over time in relation to therapeutic intervention. To this purpose, we performed a longitudinal analysis of 106 samples from 33 patients who had received sequential treatment with multiple TKIs and had experienced sequential relapses accompanied by selection of 1 or more TKI-resistant mutations. We found that conventional Sanger sequencing had misclassified or underestimated BCR-ABL mutation status in 55% of the samples, where mutations with 1% to 15% abundance were detected. A complex clonal texture was uncovered by clonal analysis of samples harboring multiple mutations and up to 13 different mutated populations were identified. The landscape of these mutated populations was found to be highly dynamic. The high degree of complexity uncovered by UDS indicates that conventional Sanger sequencing might be an inadequate tool to assess BCR-ABL kinase domain mutation status, which currently represents an important component of the therapeutic decision algorithms. Further evaluation of the clinical usefulness of UDS-based approaches is warranted

Deep sequencing of the BCR-ABL kinase domain reveals a frequency of 35INS insertion/truncation higher than expected

Background: The spectrum of Bcr-Abl kinase domain mechanisms that confer resistance to tyrosine kinase inhibitors (TKIs) in Philadelphia-positive (Ph+) Leukemia is quite heterogeneous. Not always molecular events underlying drug-resistance can be explained by presence of mutations; Bcr-Abl KD insertions/deletions can be an alternative mutational mechanisms. The recent development of \u201cdeep-amplicon sequencing\u201d (DS) technologies has opened the way to a more accurate characterization of molecular aberrations in Ph+ Leukemia with higher sensitivity of screening for know and unknown mutations. Aims: We took advantage of a DS approach in order to fully characterize the spectrum of insertions and deletions in CML and Ph+ ALL patients who had developed resistance to one or multiple lines of TKI therapy. Methods: We set up a Bcr-Abl KD mutation screening assay on the Roche GS Junior instrument that allows to reliably detect sequence variants and deletions or insertions with a lower detection limit of 1%. A total of 67 samples from 26 CML and 13 Ph+ ALL patients who had developed resistance to one or multiple TKIs (Imatinib, Dasatinib, Nilotinib) were selected for this analysis. In order to reconstruct the dynamics of growth of mutations we evaluated their presence in a serial follow-up samples collected during TKI therapy in 6 patients. Results: DS revealed a 35-base insertion (35INS) in 18/26 (69%) CML and 11/13 (84%) ALL Ph+ patients with an abundance from 1% up to 96% of all BcrAbl transcripts. Interestingly DS highlighted an increased expression of 35INS over time in 6 patients (growth ranged from 2% to 96% within a few months). This insertion is known to retain a stop codon which causes the loss of 653 Cterminal amino acids of Bcr-Abl resulting in early termination and a truncated Bcr-Abl1 protein missing a significant portion of the C-terminal regulatory regions. In addition DS detected 2 in-frame deletions in 3 samples, with an abundance from 2% to 19% . This not previously described variants include a 72-nt deletion (1233-1304) between the junction of Abl exon 6 and 7 that causes the loss of 24 amino acids (aa 359-383) and a 42-nt deletion in exon 7 (1258-1299) which leads to loss of 14 amino acids (aa 371-384). Summary / Conclusion: Our results show that DS technologies on the GS Junior instrument allow a more accurate characterization of mutational status of patients in comparison to conventional sequencing methods. The higher sensitivity of DS approach allowed to highlight, both in CML and in Ph ALL+ patients, a frequency of 35INS higher than previously reported (60%). The 35INS thus seems to be very frequent in CML and Ph+ ALL patients who develop resistance to one or multiple lines of TKI therapies but its abundance is dynamic in individual patients and seems not to be related to TKI therapy. In line with our results, recent 35INS in vitro studies have demonstrated that this insertion is kinase-inactive and should not contribute to TKI-resistance. Although this insertion does not predict for a specific TKIs-resistance its role in Ph+ Leukemia merit additional studies and further analysis of a larger number of samples will be needed to better understand its biological and clinical relevanc

DEEP SEQUENCING OF THE BCR-ABL KINASE DOMAIN REVEALS A FREQUENCY OF 35INS INSERTION/TRUNCATION HIGHER THAN EXPECTED

Background: The spectrum of Bcr-Abl kinase domain mechanisms that con- fer resistance to tyrosine kinase inhibitors (TKIs) in Philadelphia-positive (Ph+) Leukemia is quite heterogeneous. Not always molecular events underlying drug-resistance can be explained by presence of mutations; Bcr-Abl KD inser- tions/deletions can be an alternative mutational mechanisms. The recent devel- opment of “deep-amplicon sequencing” (DS) technologies has opened the way to a more accurate characterization of molecular aberrations in Ph+ Leukemia with higher sensitivity of screening for know and unknown mutations. Aims: We took advantage of a DS approach in order to fully characterize the spectrum of insertions and deletions in CML and Ph+ ALL patients who had developed resistance to one or multiple lines of TKI therapy. Methods: We set up a Bcr-Abl KD mutation screening assay on the Roche GS Junior instrument that allows to reliably detect sequence variants and deletions or insertions with a lower detection limit of 1%. A total of 67 samples from 26 CML and 13 Ph+ ALL patients who had developed resistance to one or multi- ple TKIs (Imatinib, Dasatinib, Nilotinib) were selected for this analysis. In order to reconstruct the dynamics of growth of mutations we evaluated their presence in a serial follow-up samples collected during TKI therapy in 6 patients. Results: DS revealed a 35-base insertion (35INS) in 18/26 (69%) CML and 11/13 (84%) ALL Ph+ patients with an abundance from 1% up to 96% of all Bcr- Abl transcripts. Interestingly DS highlighted an increased expression of 35INS over time in 6 patients (growth ranged from 2% to 96% within a few months). This insertion is known to retain a stop codon which causes the loss of 653 C- terminal amino acids of Bcr-Abl resulting in early termination and a truncated Bcr-Abl1 protein missing a significant portion of the C-terminal regulatory regions. In addition DS detected 2 in-frame deletions in 3 samples, with an abundance from 2% to 19% . This not previously described variants include a 72-nt deletion (1233-1304) between the junction of Abl exon 6 and 7 that caus- es the loss of 24 amino acids (aa 359-383) and a 42-nt deletion in exon 7 (1258-1299) which leads to loss of 14 amino acids (aa 371-384). Summary / Conclusion: Our results show that DS technologies on the GS Junior instrument allow a more accurate characterization of mutational status of patients in comparison to conventional sequencing methods. The higher sensitivity of DS approach allowed to highlight, both in CML and in Ph ALL+ patients, a frequency of 35INS higher than previously reported (60%). The 35INS thus seems to be very frequent in CML and Ph+ ALL patients who devel- op resistance to one or multiple lines of TKI therapies but its abundance is dynamic in individual patients and seems not to be related to TKI therapy. In line with our results, recent 35INS in vitro studies have demonstrated that this insertion is kinase-inactive and should not contribute to TKI-resistance. Although this insertion does not predict for a specific TKIs-resistance its role in Ph+ Leukemia merit additional studies and further analysis of a larger number of samples will be needed to better understand its biological and clinical rele- vance