From the laboratory perspective, effective management of patients with chronic myeloid leukemia (CML) requires accurate diagnosis, assessment of prognostic markers, sequential assessment of levels of residual disease and investigation of possible reasons for resistance, relapse or progression. Our scientific and clinical knowledge underpinning these requirements continues to evolve, as do laboratory methods and technologies. The European LeukemiaNet convened an expert panel to critically consider the current status of genetic laboratory approaches to help diagnose and manage CML patients. Our recommendations focus on current best practice and highlight the strengths and pitfalls of commonly used laboratory tests.

• MeSH
• Leukemia, Myelogenous, Chronic, BCR-ABL Positive * diagnosis   genetics   therapy   MeSH
• Protein Kinase Inhibitors * MeSH
• Humans MeSH
• Recurrence MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Protein Kinase Inhibitors * MeSH

One of the indications for BCR::ABL1 mutation testing in chronic myeloid leukemia (CML) is when tyrosine kinase inhibitor therapy (TKI) needs to be changed for unsatisfactory response. In this study, we evaluated a droplet digital PCR (ddPCR)-based multiplex strategy for the detection and quantitation of transcripts harbouring mutations conferring resistance to second-generation TKIs (2GTKIs). Parallel quantitation of e13a2, e14a2 and e1a2 BCR::ABL1 fusion transcripts enables to express results as percentage of mutation positive- over total BCR::ABL1 transcripts. We determined the limit of blank in 60 mutation-negative samples. Accuracy was demonstrated by further analysis of 48 samples already studied by next generation sequencing (NGS). Mutations could be called down to 0.5% and across 3-logs of BCR::ABL1 levels. Retrospective review of BCR::ABL1 NGS results in 513 consecutive CML patients with non-optimal response to first- or second-line TKI therapy suggested that a ddPCR-based approach targeted against 2GTKI-resistant mutations would score samples as mutation-negative in 22% of patients with warning response to imatinib but only in 6% of patients with warning response to 2GTKIs. We conclude ddPCR represents an attractive method for easy, accurate and rapid screening for 2GTKI-resistant mutations impacting on TKI selection, although ddPCR cannot identify compound mutations.

• MeSH
• Fusion Proteins, bcr-abl * MeSH
• Drug Resistance, Neoplasm MeSH
• Leukemia, Myelogenous, Chronic, BCR-ABL Positive * MeSH
• Protein Kinase Inhibitors MeSH
• Humans MeSH
• Mutation MeSH
• Polymerase Chain Reaction MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Fusion Proteins, bcr-abl * MeSH
• Protein Kinase Inhibitors MeSH

Somatic mutations are a common molecular mechanism through which chronic myeloid leukemia (CML) cells acquire resistance to tyrosine kinase inhibitors (TKIs) therapy. While most of the mutations in the kinase domain of BCR-ABL1 can be successfully managed, the recurrent somatic mutations in other genes may be therapeutically challenging. Despite the major clinical relevance of mutation-associated resistance in CML, the mechanisms underlying mutation acquisition in TKI-treated leukemic cells are not well understood. This work demonstrated de novo acquisition of mutations on isolated single-cell sorted CML clones growing in the presence of imatinib. The acquisition of mutations was associated with the significantly increased expression of the LIG1 and PARP1 genes involved in the error-prone alternative nonhomologous end-joining pathway, leading to genomic instability, and increased expression of the UNG, FEN and POLD3 genes involved in the base-excision repair (long patch) pathway, allowing point mutagenesis. This work showed in vitro and in vivo that de novo acquisition of resistance-associated mutations in oncogenes is the prevalent method of somatic mutation development in CML under TKIs treatment.

• Keywords
• TKI resistance, alt-nonhomologous end joining (alt-NHEJ), base excision repair (BER), chronic myelogenous leukemia (CML), oncogene,
• Publication type
• Journal Article MeSH

Chronic myeloid leukemia (CML) therapy has markedly improved patient prognosis after introduction of imatinib mesylate for clinical use. However, a subset of patients develops resistance to imatinib and other tyrosine kinase inhibitors (TKIs), mainly due to point mutations in the region encoding the kinase domain of the fused BCR-ABL oncogene. To identify potential therapeutic targets in imatinib‑resistant CML cells, we derived imatinib-resistant CML-T1 human cell line clone (CML-T1/IR) by prolonged exposure to imatinib in growth media. Mutational analysis revealed that the Y235H mutation in BCR-ABL is probably the main cause of CML-T1/IR resistance to imatinib. To identify alternative therapeutic targets for selective elimination of imatinib-resistant cells, we compared the proteome profiles of CML-T1 and CML-T1/IR cells using 2-DE-MS. We identified eight differentially expressed proteins, with strongly upregulated Na+/H+ exchanger regulatory factor 1 (NHERF1) in the resistant cells, suggesting that this protein may influence cytosolic pH, Ca2+ concentration or signaling pathways such as Wnt in CML-T1/IR cells. We tested several compounds including drugs in clinical use that interfere with the aforementioned processes and tested their relative toxicity to CML-T1 and CML-T1/IR cells. Calcium channel blockers, calcium signaling antagonists and modulators of calcium homeostasis, namely thapsigargin, ionomycin, verapamil, carboxyamidotriazole and immunosuppressive drugs cyclosporine A and tacrolimus (FK-506) were selectively toxic to CML-T1/IR cells. The putative cellular targets of these compounds in CML-T1/IR cells are postulated in this study. We propose that Ca2+ homeostasis can be a potential therapeutic target in CML cells resistant to TKIs. We demonstrate that a proteomic approach may be used to characterize a TKI-resistant population of CML cells enabling future individualized treatment options for patients.

• MeSH
• Fusion Proteins, bcr-abl metabolism   MeSH
• Drug Resistance, Neoplasm drug effects   MeSH
• Leukemia, Myelogenous, Chronic, BCR-ABL Positive drug therapy   metabolism   MeSH
• Homeostasis drug effects   MeSH
• Imatinib Mesylate pharmacology   MeSH
• Protein Kinase Inhibitors pharmacology   MeSH
• Humans MeSH
• Mutation drug effects   MeSH
• Cell Line, Tumor MeSH
• Proteome metabolism   MeSH
• Proteomics methods   MeSH
• Signal Transduction drug effects   MeSH
• Calcium metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Fusion Proteins, bcr-abl MeSH
• Imatinib Mesylate MeSH
• Protein Kinase Inhibitors MeSH
• Proteome MeSH
• Calcium MeSH

BACKGROUND: Imatinib-resistant chronic myeloid leukemia (CML) patients receiving second-line tyrosine kinase inhibitor (TKI) therapy with dasatinib or nilotinib have a higher risk of disease relapse and progression and not infrequently BCR-ABL1 kinase domain (KD) mutations are implicated in therapeutic failure. In this setting, earlier detection of emerging BCR-ABL1 KD mutations would offer greater chances of efficacy for subsequent salvage therapy and limit the biological consequences of full BCR-ABL1 kinase reactivation. Taking advantage of an already set up and validated next-generation deep amplicon sequencing (DS) assay, we aimed to assess whether DS may allow a larger window of detection of emerging BCR-ABL1 KD mutants predicting for an impending relapse. METHODS: a total of 125 longitudinal samples from 51 CML patients who had acquired dasatinib- or nilotinib-resistant mutations during second-line therapy were analyzed by DS from the time of failure and mutation detection by conventional sequencing backwards. BCR-ABL1/ABL1%(IS) transcript levels were used to define whether the patient had 'optimal response', 'warning' or 'failure' at the time of first mutation detection by DS. RESULTS: DS was able to backtrack dasatinib- or nilotinib-resistant mutations to the previous sample(s) in 23/51 (45 %) pts. Median mutation burden at the time of first detection by DS was 5.5 % (range, 1.5-17.5 %); median interval between detection by DS and detection by conventional sequencing was 3 months (range, 1-9 months). In 5 cases, the mutations were detectable at baseline. In the remaining cases, response level at the time mutations were first detected by DS could be defined as 'Warning' (according to the 2013 ELN definitions of response to 2nd-line therapy) in 13 cases, as 'Optimal response' in one case, as 'Failure' in 4 cases. No dasatinib- or nilotinib-resistant mutations were detected by DS in 15 randomly selected patients with 'warning' at various timepoints, that later turned into optimal responders with no treatment changes. CONCLUSIONS: DS enables a larger window of detection of emerging BCR-ABL1 KD mutations predicting for an impending relapse. A 'Warning' response may represent a rational trigger, besides 'Failure', for DS-based mutation screening in CML patients undergoing second-line TKI therapy.

• Keywords
• BCR-ABL1, Chronic myeloid leukemia, Deep sequencing, Tyrosine kinase inhibitors, Warning,
• MeSH
• Fusion Proteins, bcr-abl genetics   MeSH
• Drug Resistance, Neoplasm MeSH
• Leukemia, Myelogenous, Chronic, BCR-ABL Positive drug therapy   genetics   MeSH
• Pharmacogenomic Variants MeSH
• Protein Kinase Inhibitors therapeutic use   MeSH
• Polymorphism, Single Nucleotide MeSH
• Humans MeSH
• Sequence Analysis, DNA methods   MeSH
• High-Throughput Nucleotide Sequencing methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Fusion Proteins, bcr-abl MeSH
• BCR-ABL1 fusion protein, human MeSH Browser
• Protein Kinase Inhibitors MeSH

Bone marrow transplantation or ponatinib treatment are currently recommended strategies for management of patients with chronic myeloid leukemia (CML) harboring the T315I mutation and compound or polyclonal mutations. However, in some individual cases, these treatment scenarios cannot be applied. We used an alternative treatment strategy with interferon-α (IFN-α) given solo, sequentially or together with TKI in a group of 6 cases of high risk CML patients, assuming that the TKI-independent mechanism of action may lead to mutant clone repression. IFN-α based individualized therapy decreases of T315I or compound mutations to undetectable levels as assessed by next-generation deep sequencing, which was associated with a molecular response in 4/6 patients. Based on the observed results from immune profiling, we assumed that the principal mechanism leading to the success of the treatment was the immune activation induced with dasatinib pre-treatment followed by restoration of immunological surveillance after application of IFN-α therapy. Moreover, we showed that sensitive measurement of mutated BCR-ABL1 transcript levels augments the safety of this individualized treatment strategy.

• MeSH
• Fusion Proteins, bcr-abl drug effects   genetics   MeSH
• Leukemia, Myelogenous, Chronic, BCR-ABL Positive drug therapy   genetics   MeSH
• Adult MeSH
• Precision Medicine MeSH
• Protein Kinase Inhibitors administration & dosage   pharmacology   MeSH
• Interferon-alpha administration & dosage   pharmacology   MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Antineoplastic Combined Chemotherapy Protocols administration & dosage   pharmacology   MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Treatment Outcome MeSH
• High-Throughput Nucleotide Sequencing methods   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Case Reports MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Fusion Proteins, bcr-abl MeSH
• BCR-ABL1 fusion protein, human MeSH Browser
• Protein Kinase Inhibitors MeSH
• Interferon-alpha MeSH

Identification and quantitative monitoring of mutant BCR-ABL1 subclones displaying resistance to tyrosine kinase inhibitors (TKIs) have become important tasks in patients with Ph-positive leukemias. Different technologies have been established for patient screening. Various next-generation sequencing (NGS) platforms facilitating sensitive detection and quantitative monitoring of mutations in the ABL1-kinase domain (KD) have been introduced recently, and are expected to become the preferred technology in the future. However, broad clinical implementation of NGS methods has been hampered by the limited accessibility at different centers and the current costs of analysis which may not be regarded as readily affordable for routine diagnostic monitoring. It is therefore of interest to determine whether NGS platforms can be adequately substituted by other methodological approaches. We have tested three different techniques including pyrosequencing, LD (ligation-dependent)-PCR and NGS in a series of peripheral blood specimens from chronic myeloid leukemia (CML) patients carrying single or multiple mutations in the BCR-ABL1 KD. The proliferation kinetics of mutant subclones in serial specimens obtained during the course of TKI-treatment revealed similar profiles via all technical approaches, but individual specimens showed statistically significant differences between NGS and the other methods tested. The observations indicate that different approaches to detection and quantification of mutant subclones may be applicable for the monitoring of clonal kinetics, but careful calibration of each method is required for accurate size assessment of mutant subclones at individual time points.

• Keywords
• BCR-ABL1, CML, LD-PCR, NGS, pyrosequencing, quantitative analysis of mutant subclones,
• MeSH
• Fusion Proteins, bcr-abl chemistry   genetics   MeSH
• Leukemia, Myelogenous, Chronic, BCR-ABL Positive genetics   pathology   MeSH
• DNA analysis   genetics   metabolism   MeSH
• Humans MeSH
• Polymerase Chain Reaction * MeSH
• Sequence Analysis, DNA * MeSH
• Comparative Genomic Hybridization MeSH
• High-Throughput Nucleotide Sequencing * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• Fusion Proteins, bcr-abl MeSH
• DNA MeSH

In chronic myeloid leukemia (CML) and Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) patients who fail imatinib treatment, BCR-ABL1 mutation profiling by Sanger sequencing (SS) is recommended before changing therapy since detection of specific mutations influences second-generation tyrosine kinase inhibitor (2GTKI) choice. We aimed to assess i) in how many patients who relapse on second-line 2GTKI therapy next generation sequencing (NGS) may track resistant mutations back to the sample collected at the time of imatinib resistance, before 2GTKI start (switchover sample) and ii) whether low level mutations identified by NGS always undergo clonal expansion. To this purpose, we used NGS to retrospectively analyze 60 imatinib-resistant patients (CML, n = 45; Ph+ ALL,n = 15) who had failed second-line 2GTKI therapy and had acquired BCR-ABL1 mutations (Group 1) and 25 imatinib-resistant patients (CML, n = 21; Ph+ ALL, n = 4) who had responded to second-line 2GTKI therapy, for comparison (Group 2). NGS uncovered that in 26 (43%) patients in Group 1, the 2GTKI-resistant mutations that triggered relapse were already detectable at low levels in the switchover sample (median mutation burden, 5%; range 1.1%-18.4%). Importantly, none of the low level mutations detected by NGS in switchover samples failed to expand whenever the patient received the 2GTKI to whom they were insensitive. In contrast, no low level mutation that was resistant to the 2GTKI the patients subsequently received was detected in the switchover samples from Group 2. NGS at the time of imatinib failure reliably identifies clinically relevant mutations, thus enabling a more effective therapeutic tailoring.

• Keywords
• BCR-ABL1, Next-generation sequencing, acute lymphoblastic leukemia, chronic myeloid leukemia, tyrosine kinase inhibitors,
• MeSH
• Precursor Cell Lymphoblastic Leukemia-Lymphoma drug therapy   genetics   MeSH
• Fusion Proteins, bcr-abl genetics   MeSH
• Drug Resistance, Neoplasm genetics   MeSH
• Leukemia, Myelogenous, Chronic, BCR-ABL Positive drug therapy   genetics   MeSH
• Dasatinib therapeutic use   MeSH
• Adult MeSH
• Imatinib Mesylate therapeutic use   MeSH
• Protein Kinase Inhibitors therapeutic use   MeSH
• Middle Aged MeSH
• Humans MeSH
• DNA Mutational Analysis methods   MeSH
• Retrospective Studies MeSH
• Aged MeSH
• High-Throughput Nucleotide Sequencing methods   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Fusion Proteins, bcr-abl MeSH
• BCR-ABL1 fusion protein, human MeSH Browser
• Dasatinib MeSH
• Imatinib Mesylate MeSH
• Protein Kinase Inhibitors MeSH