Search

Deep sequence analysis for BCR-ABL revealed that native BCR-ABL decreased slowly after an exponential decrease within three months of tyrosine kinase inhibitor (TKI) treatment. BCR-ABL Ins35bp was present at diagnosis and increased to account for 15-30% of total BCR-ABL when IS BCR-ABL was reduced to 1%. Native BCR-ABL and BCR-ABL Ins35bp correspond to early relapse and fluctuating minimal residue disease, respectively, in the STOP-TKI trial. These findings highlight the clinical significance of quantifying BCR-ABL Ins35bp . We discovered pseudosplice sites at both ends of 35 bp within ABL intron 8, and TKI off-target effect inhibited RNAPII S2P and reduced native BCR-ABL expression but induced BCR-ABL Ins35bp mis-splicing.

To elucidate dynamic changes in native BCR-ABL and alternatively spliced tyrosine kinase inhibitor (TKI)-resistant but function-dead BCR-ABL Ins35bp variant, following commencement or discontinuation of TKI therapy, each transcript was serially quantified in patients with chronic myeloid leukemia (CML) by deep sequencing. Because both transcripts were amplified together using conventional PCR system for measuring International Scale (IS), deep sequencing method was used for quantifying such BCR-ABL variants. At the initial diagnosis, 7 of 9 patients presented a small fraction of cells possessing BCR-ABL Ins35bp , accounting for 0.8% of the total IS BCR-ABL, corresponding to actual BCR-ABL Ins35bp value of 1.1539% IS. TKI rapidly decreased native BCR-ABL but not BCR-ABL Ins35bp , leading to the initial increase in the proportion of BCR-ABL Ins35bp . Thereafter, both native BCR-ABL and BCR-ABL Ins35bp gradually decreased in the course of TKI treatment, whereas small populations positive for TKI-resistant BCR-ABL Ins35bp continued fluctuating at low levels, possibly underestimating the molecular response (MR). Following TKI discontinuation, sequencing analysis of 54 patients revealed a rapid relapse, apparently derived from native BCR-ABL + clones. However, IS fluctuating at low levels around MR4.0 marked a predominant persistence of cells expressing function-dead BCR-ABL Ins35bp , suggesting that TKI resumption was unnecessary. We clarified the possible mechanism underlying mis-splicing BCR-ABL Ins35bp , occurring at the particular pseudo-splice site within intron8, which can be augmented by TKI treatment through inhibition of RNA polymerase II phosphorylation. No mutations were found in spliceosomal genes. Therefore, monitoring IS functional BCR-ABL extracting BCR-ABL Ins35bp would lead us to a correct evaluation of MR status, thus determining the adequate therapeutic intervention., (© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Chronic myeloid leukemia (CML) is effectively treated with tyrosine kinase inhibitors (TKI) targeted against BCR-ABL. We previously reported the investigation of residual CML diseases during TKI treatment using FACS-sorting and quantitative RT-PCR of BCR-ABL among each population; total mononuclear cells, hematopoietic stem cells, and myeloid progenitors. The observations also implied that the second-generation of ABL-tyrosine kinase inhibitors (2nd TKIs), dasatinib or nilotinib therapy can be more promising approach for efficient reduction of the CML stem cells. Moreover, we need to develop the evaluation method of the residual CML diseases to establish rational therapy-cessation strategies in CML.

Treatment with tyrosine kinase inhibitors (TKI) may sequentially induce TKI-resistant BCR-ABL mutants in chronic myeloid leukemia (CML). Conventional PCR monitoring of BCR-ABL is an important indicator to determine therapeutic intervention for preventing disease progression. However, PCR cannot separately quantify amounts of BCR-ABL and its mutants, including alternatively spliced BCR-ABL with an insertion of 35 intronic nucleotides (BCR-ABL I ns35bp ) between ABL exons 8 and 9, which introduces the premature termination and loss of kinase activity. To assess the clinical impact of BCR-ABL mutants, we performed deep sequencing analysis of BCR-ABL transcripts of 409 samples from 37 patients with suboptimal response to frontline imatinib who were switched to nilotinib. At baseline, TKI-resistant mutations were documented in 3 patients, whereas BCR-ABL I ns35bp was detected in all patients. After switching to nilotinib, both BCR-ABL and BCR-ABL I ns35bp became undetectable in 3 patients who attained complete molecular response (CMR), whereas in the remaining all 34 patients, BCR-ABL I ns35bp was persistently detected, and minimal residual disease (MRD) fluctuated at low but detectable levels. PCR monitoring underestimated molecular response in 5 patients whose BCR-ABL I ns35bp was persisted, although BCR-ABL I ns35bp does not definitively mark TKI resistance. Therefore, quantification of BCR-ABL I ns35bp is useful for evaluating "functional" MRD and determining the effectiveness of TKI with accuracy., (© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)