<i>BCR‐ABL1</i> kinase domain mutational analysis of CD34+ stem/progenitor cells in newly diagnosed CML patients by next‐generation sequencing
Authors: Milena Musilova, Filip Razga, Tomas Jurcek, Ivana Jeziskova, Marek Borsky, Veronika Nemethova, Daniela Zackova, Martin Culen, Dana Dvorakova, Jiri Mayer, Zdenek Racil
Journal: American Journal of Hematology (2014)

To the Editor: Mutations in the BCR-ABL1 kinase domain (KD) represent one of the most important causes of resistance to tyrosine kinase inhibitors (TKIs) treatment in patients with chronic myeloid leukemia (CML). The resistance-causing mutations are hypothesized to expand directly from the stem and/or progenitor cells and are present in CD34+ cells significantly earlier than they occur in bone marrow or peripheral blood. Thus, early detection of these mutations is of great importance, since it can affect the subsequent TKI therapy. We here report on a study performed to analyze the frequency and the spectrum of clinically relevant mutations in the stem cell/progenitor compartment represented by the CD34+ cell population at the time of CML diagnosis, using a next-generation sequencing (NGS) mutation screening platform. Sorted CD34+ cells obtained from bone marrow at the time of diagnosis of 50 de novo CML patients (median age 58 years; range: 26–92 years) were analyzed for the presence of BCR-ABL1 KD mutations. All samples were investigated using amplicon deep-sequencing during nine runs on a GS Junior Roche instrument. The sequencing steps and ABL1 reference alignment (GenBank accession number X16416.1) were performed with a variant frequency calculation as developed in the framework of the IRON-II (Interlaboratory Robustness of Next-generation sequencing) consortium. The lower detection limit of molecular variants was set at 2.0%. Overall, our analyses using the sensitive NGS technique revealed 10 missense mutations in the CD34+ samples from 4/50 (8.0%) de novo TKI-naive CML patients. Nine of ten mutations were detected with a low mutation burden (1.3–12.1%). Importantly, 5/10 mutations were associated with known in vitro TKI resistance. Three of these four mutation positive CML patients revealed at least one TKI-resistant mutation and had high risk scores (Table 1). In detail, the T315I mutation, known for its insensitivity to all approved TKIs, was detected in one case (No. 2). This patient also concomitantly harbored E255K and E255V mutations with remaining sensitivity to dasatinib. An additional two mutations, V339A and E255D, showing resistance to imatinib were observed in other two patients (No. 3 and 4). Moreover, two of three patients with a TKI-resistant mutation revealed warning signs of early therapy failure based on ELN 2013 criteria, that is, BCR-ABL1/ABL1 ratio > 10.0% at the third month (No. 2 and 3). These results indicate an infrequent occurrence of BCR-ABL1 KD mutations in CD34+ cells in TKI-naive CML patients. The presence of these mutations prior to TKI therapy is in most instances attributed to a clinical resistance to TKIs, which may result in subsequent overgrowth of the most resistant clones responsible for the ongoing therapy failure. The NGS technique enabling a robust limit of detection of ∼1–2% provides a detailed overview of the complexity of BCR-ABL1 KD mutations in the CD34+ cell compartment with a clear implication for clinical practice and further treatment management. The application of this method applied to CD34+ cells of CML patients promises to lead to novel clinical strategies preventing the expansion and further proliferation of clones harboring TKI-resistant BCR-ABL1 KD mutations. The authors would further like to thank the Interlaboratory Robustness of Next-generation sequencing (IRON) Phase II study group members for helpful discussions on the deep-sequencing assay. Milena Musilova,1 Filip Razga,1,2 Tomas Jurcek,1 Ivana Jeziskova,1 Marek Borsky,1 Veronika Nemethova,1,2 Daniela Zackova,1 Martin Culen,1,3 Dana Dvorakova,1,3 Jiri Mayer,1,3 and Zdenek Racil1,3 1Department of Internal Medicine, Hematology and Oncology, Faculty of Medicine, Masaryk University and University Hospital Brno, Brno, Czech Republic 2Department for Biomaterials Research, Polymer Institute of the Slovak Academy of Sciences, Bratislava, Slovakia 3CEITEC, Masaryk University, Brno, Czech Republic