European LeukemiaNet laboratory recommendations for the diagnosis and management of chronic myeloid leukemia

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

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

Standardization of molecular monitoring of CML: results and recommendations from the European treatment and outcome study

Standardized monitoring of BCR::ABL1 mRNA levels is essential for the management of chronic myeloid leukemia (CML) patients. From 2016 to 2021 the European Treatment and Outcome Study for CML (EUTOS) explored the use of secondary, lyophilized cell-based BCR::ABL1 reference panels traceable to the World Health Organization primary reference material to standardize and validate local laboratory tests. Panels were used to assign and validate conversion factors (CFs) to the International Scale and assess the ability of laboratories to assess deep molecular response (DMR). The study also explored aspects of internal quality control. The percentage of EUTOS reference laboratories (n = 50) with CFs validated as optimal or satisfactory increased from 67.5% to 97.6% and 36.4% to 91.7% for ABL1 and GUSB, respectively, during the study period and 98% of laboratories were able to detect MR4.5 in most samples. Laboratories with unvalidated CFs had a higher coefficient of variation for BCR::ABL1(IS) and some laboratories had a limit of blank greater than zero which could affect the accurate reporting of DMR. Our study indicates that secondary reference panels can be used effectively to obtain and validate CFs in a manner equivalent to sample exchange and can also be used to monitor additional aspects of quality assurance.</p

Clinical impact of low-burden BCR-ABL1 mutations detectable by amplicon deep sequencing in Philadelphia-positive acute lymphoblastic leukemia patients

Although the prognosis of Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) has significantly improved upon the advent of tyrosine kinase inhibitors (TKIs) targeting the deregulated BCR-ABL1 tyrosine kinase activity, disease recurrence remains a major problem.1 The long-term stability of responses is frequently undermined by the selection of BCR-ABL1 mutants that can survive TKI treatment and trigger relapse.2 Imatinib-resistant Ph+ ALL patients frequently harbor the T315I mutation,2 against whom second-generation TKIs (2GTKIs; dasatinib and nilotinib) are ineffective. Even in imatinib-resistant patients who have 2GTKI-sensitive mutants, responses may be short-lived because of the high genetic instability fostering the acquisition of additional mutations, often in a ‘compound’ form (that is, two mutations gained by the same clone).2 Recently, however, effective salvage options for T315I mutation-positive or multi-TKI-resistant cases have become available—including the third-generation TKI ponatinib,3 as well as monoclonal antibodies such as blinatumomab.4 To prevent hematologic relapse with timely and rational therapeutic intervention, minimal residual disease (MRD) monitoring by real time quantitative polymerase chain reaction (RQ-PCR) and BCR-ABL1 mutation screening of patients who do not achieve MRD negativity can thus be expected to have an important role