Katerina Machova Polakova

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.

A certified plasmid reference material for the standardisation of BCR–ABL1 mRNA quantification by real-time quantitative PCR

Serial quantification of BCR–ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR–ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 106, 1.08±0.11 × 105, 1.03±0.10 × 104, 1.02±0.09 × 103, 1.04±0.10 × 102 and 10.0±1.5 copies/μl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR–ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR–ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f).

Role of Epigenetics in Chronic Myeloid Leukemia

The efficacy of therapeutic modalities in chronic myeloid leukemia (CML) depends on both genetic and epigenetic mechanisms. This review focuses on epigenetic mechanisms involved in the pathogenesis of CML and in resistance of tumor cells to tyrosine kinase inhibitors leading to the leukemic clone escape and propagation. Regulatory events at the levels of gene regulation by transcription factors and microRNAs are discussed in the context of CML pathogenesis and therapeutic modalities.

Imatinib as the first-line treatment of patients with chronic myeloid leukemia diagnosed in the chronic phase: Can we compare real life data to the results from clinical trials?†

Imatinib (IM) dramatically improved the prognosis of chronic myeloid leukemia (CML), particularly with newly diagnosed patients in a chronic phase (CP) [1]. The most robust source of data about IM efficacy in this setting is the IRIS trial. However, every day clinical practice data are still scarce. We analyzed IM efficacy and safety in the first-line therapy of 152 consecutive adult CP-CML patients from a defined region. The estimated 4-year cumulative incidences of complete hematologic, complete cytogenetic, major, and complete molecular responses were 95.3%, 80.6%, 65.4%, and 39.2%, respectively. The 4-year probability of overall and progression-free survival (PFS) defined as with the IRIS [2] was 91.5% and 78.1%, respectively. We thus confirmed very good IM efficacy also in patients not participating in clinical trials. However, the estimated 4-year event-free survival (EFS), which also counted failure events according to valid recommendations [3] or IM discontinuation due to intolerance, was only 60.7%. The 4-year probability of an alternative treatment-free survival, our newly defined parameter, which better reflects the proportion of patients remaining on IM despite an event, was 67.6%. Therefore, more appropriate selection and unification of survival analyses end-points is desirable to describe and compare IM real efficacy.

Next-generation sequencing for sensitive detection of BCR-ABL1 mutations relevant to tyrosine kinase inhibitor choice in imatinib-resistant patients.

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.

The predictive value of human organic cation transporter 1 and ABCB1 expression levels in different cell populations of patients with de novo chronic myelogenous leukemia

In this study, we investigated the predictive value of pretreatment mRNA expression levels of hOCT-1 and ABCB1 in different cell populations with regard to the response to therapy at 6 and 12 months of IMA therapy. Expression levels were assessed in peripheral blood (PB) leukocytes (LEU, n = 30), polymorphonuclear cells (PMNC, n = 23), and mononuclear cells (MNC, n = 21) of PB LEU obtained from 30 patients with de novo chronic myelogenous leukemia (CML). Moreover, the available bone marrow cells (BM) were also included and analyzed (BM, n = 11). The PB and BM samples were obtained, processed, and analyzed as previously described. Responses to therapy were classified according the European LeukemiaNet 2009 (ELN) criteria: responders show an optimal response at 6 months and 12 months, while non-responders reflect a suboptimal response or therapy failure. The assessed pretreatment expression levels were stratified into two groups according to the median-a low-mRNA expression group below the median and a high-mRNA expression group equal to or above the median. The statistical evaluation of the data obtained was performed using the Fisher’s exact tests and summarized in Table 2.

Discontinuation of tyrosine kinase inhibitor therapy in chronic myeloid leukaemia (EURO-SKI): a prespecified interim analysis of a prospective, multicentre, non-randomised, trial

BACKGROUND Tyrosine kinase inhibitors (TKIs) have improved the survival of patients with chronic myeloid leukaemia. Many patients have deep molecular responses, a prerequisite for TKI therapy discontinuation. We aimed to define precise conditions for stopping treatment. METHODS In this prospective, non-randomised trial, we enrolled patients with chronic myeloid leukaemia at 61 European centres in 11 countries. Eligible patients had chronic-phase chronic myeloid leukaemia, had received any TKI for at least 3 years (without treatment failure according to European LeukemiaNet [ELN] recommendations), and had a confirmed deep molecular response for at least 1 year. The primary endpoint was molecular relapse-free survival, defined by loss of major molecular response (MMR; >0·1% BCR-ABL1 on the International Scale) and assessed in all patients with at least one molecular result. Secondary endpoints were a prognostic analysis of factors affecting maintenance of MMR at 6 months in learning and validation samples and the cost impact of stopping TKI therapy. We considered loss of haematological response, progress to accelerated-phase chronic myeloid leukaemia, or blast crisis as serious adverse events. This study presents the results of the prespecified interim analysis, which was done after the 6-month molecular relapse-free survival status was known for 200 patients. The study is ongoing and is registered with ClinicalTrials.gov, number NCT01596114. FINDINGS Between May 30, 2012, and Dec 3, 2014, we assessed 868 patients with chronic myeloid leukaemia for eligibility, of whom 758 were enrolled. Median follow-up of the 755 patients evaluable for molecular response was 27 months (IQR 21-34). Molecular relapse-free survival for these patients was 61% (95% CI 57-64) at 6 months and 50% (46-54) at 24 months. Of these 755 patients, 371 (49%) lost MMR after TKI discontinuation, four (1%) died while in MMR for reasons unrelated to chronic myeloid leukaemia (myocardial infarction, lung cancer, renal cancer, and heart failure), and 13 (2%) restarted TKI therapy while in MMR. A further six (1%) patients died in chronic-phase chronic myeloid leukaemia after loss of MMR and re-initiation of TKI therapy for reasons unrelated to chronic myeloid leukaemia, and two (<1%) patients lost MMR despite restarting TKI therapy. In the prognostic analysis in 405 patients who received imatinib as first-line treatment (learning sample), longer treatment duration (odds ratio [OR] per year 1·14 [95% CI 1·05-1·23]; p=0·0010) and longer deep molecular response durations (1·13 [1·04-1·23]; p=0·0032) were associated with increasing probability of MMR maintenance at 6 months. The OR for deep molecular response duration was replicated in the validation sample consisting of 171 patients treated with any TKI as first-line treatment, although the association was not significant (1·13 [0·98-1·29]; p=0·08). TKI discontinuation was associated with substantial cost savings (an estimated €22 million). No serious adverse events were reported. INTERPRETATION Patients with chronic myeloid leukaemia who have achieved deep molecular responses have good molecular relapse-free survival. Such patients should be considered for TKI discontinuation, particularly those who have been in deep molecular response for a long time. Stopping treatment could spare patients from treatment-induced side-effects and reduce health expenditure. FUNDING ELN Foundation and France National Cancer Institute.

Current survival measures reliably reflect modern sequential treatment in CML: Correlation with prognostic stratifications

Using the data of 723 chronic myeloid leukemia (CML) patients in the chronic phase, we analyzed the prognostic value of the Sokal, Euro, and EUTOS scores as well as the level of BCR‐ABL1 and the achievement of complete cytogenetic response (CCgR) at 3 months of imatinib therapy in relation to the so‐called current survival measures: the current cumulative incidence (CCI) reflecting the probability of being alive and in CCgR after starting imatinib therapy; the current leukemia‐free survival (CLFS) reflecting the probability of being alive and in CCgR after achieving the first CCgR; and the overall survival. The greatest difference between the CCI curves at 5 years after initiating imatinib therapy was observed for the BCR‐ABL1 transcripts at 3 months. The 5‐year CCI was 94.3% in patients with BCR‐ABL1 transcripts ≤ 10% and 57.1% in patients with BCR‐ABL1 transcripts > 10% (P = 0.005). Therefore, the examination of BCR‐ABL1 transcripts at 3 months may help in early identification of patients who are likely to perform poorly with imatinib. On the other hand, CLFS was not significantly affected by the considered stratifications. In conclusion, our results indicate that once the CCgR is achieved, the prognosis is good irrespective of the starting prognostic risks. Am. J. Hematol. 88:790–797, 2013.

Interferon-α Revisited: Individualized Treatment Management Eased the Selective Pressure of Tyrosine Kinase Inhibitors on BCR-ABL1 Mutations Resulting in a Molecular Response in High-Risk CML Patients.

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.

No clinical evidence for performing trough plasma and intracellular imatinib concentrations monitoring in patients with chronic myelogenous leukaemia

This multicentre study focused on monitoring imatinib mesylate (IMA) trough plasma (Ctrough) and intracellular (IMA Cintrac) concentrations in 228 chronic myelogenous leukaemia patients. The median of measured IMA Ctrough in our patient group was 905.8 ng ml (range: 27.7–4628.1 ng/ml). We found a correlation between IMA Ctrough and alpha 1‐acid glycoprotein plasma concentrations (rS = 0.42; p < 0.001). All other analysed parameters revealed only weak (gender, dose of IMA per kg) or not significant (age, albumin, creatinine plasma concentration or body mass index) impact on measured IMA Ctrough. The IMA Ctrough decreased during the first 6 months and significantly increased later during treatment. The IMA Ctrough at the first month of therapy did not differ between patients with and without an optimal response at the 12th (p = 0.724) and 18th month (p = 0.135) of therapy. There were no significant differences in medians of IMA Ctrough between both groups measured during the first year of treatment. The IMA Cintrac during the first month were not different between patients with and without an optimal response at the 6th (p = 0.273) and the 12th month (p = 0.193) of therapy. Our data obtained from real life clinical practice did not find a benefit of routine and regular IMA Ctrough nor IMA Cintrac therapeutic drug monitoring in chronic myelogenous leukaemia patients or for subsequent adjustments of the IMA dose based on these results. Moreover, actual alpha 1‐acid glycoprotein plasma concentration should be used for proper interpretation of IMA Ctrough results. Copyright