Search

Dysregulation of immune-checkpoint receptors has been reported at diagnosis of chronic myeloid leukemia (CML), however, their role in the maintenance of remission after tyrosine kinase inhibitor (TKI) cessation is unclear. We assessed programmed cell death-1 (PD-1), T-cell immunoglobulin, and mucin-domain containing protein-3 (TIM-3), cytotoxic T-lymphocyte–associated protein-4 (CTLA-4), lymphocyte-activation gene-3 (LAG-3), and T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domains (TIGIT) expression on T-cell subsets, regulatory T cells (T-regs), and natural killer (NK) cells at the time of TKI cessation in 44 patients (22 patients sustained treatment-free remission [TFR] and 22 experienced molecular relapse [MolR]). We confirmed our previous finding that absolute numbers of T-regs are increased in patients who experienced MolR compared with those who sustained TFR. The immune-checkpoint receptors PD-1, CTLA-4, LAG-3, and TIGIT on T or NK cells were not differentially expressed between the MolR and TFR groups. However, TIM-3 was consistently upregulated on bulk T cells (CD3+) and T-cell subsets including, CD4+ T cells, CD8+ T cells, and T-regs, in patients who relapsed in comparison with those who maintained TFR after discontinuation. Furthermore, gene expression analysis from publicly available data sets showed increased TIM-3 expression on CML stem cells compared with normal hematopoietic stem cells. These findings suggest that among the targetable immune-checkpoint molecules, TIM-3 blockade may potentially improve effector immune response in patients with CML stopping TKI, while concomitantly targeting leukemic stem cells and could be a promising therapeutic strategy for preventing relapse after cessation of TKI in patients with CML.

Mutation detection is increasingly used for the management of hematological malignancies. Prior whole transcriptome and whole exome sequencing studies using total RNA and DNA identified diverse mu-tation types in cancer-related genes associated with treatment failure in patients with chronic myeloid leukemia. Variants included single-nucleotide variants and small insertions/deletions, plus fusion transcripts and partial or whole gene deletions. The hypothesis that all of these mutation types could be detected by a single cost-effective hybridization capture next-generation sequencing method using total RNA was assessed. A method was developed that targeted 130 genes relevant for myeloid and lymphoid leukemia. Retrospective samples with 121 precharacterized variants were tested using total RNA and/or DNA. Concordance of detection of precharacterized variants using RNA or DNA was 96%, whereas the enhanced sensitivity identified additional variants. Comparison between 24 matched DNA and RNA samples demonstrated 95.3% of 170 variants detectable using DNA were detected using RNA, including all but one variant predicted to activate nonsense-mediated decay. RNA identified an addi-tional 10 variants, including fusion transcripts. Furthermore, the true effect of splice variants on RNA splicing was only evident using RNA. In conclusion, capture sequencing using total RNA alone is suitable for detecting a range of variants relevant in chronic myeloid leukemia and may be more broadly applied to other hematological malignancies where diverse variant types define risk groups. Refereed/Peer-reviewed

Purpose: Imatinib induces a durable response in most patients with Philadelphia chromosome–positive chronic myeloid leukemia, but it is currently unclear whether imatinib reduces the leukemic stem cell (LSC) burden, which may be an important step toward enabling safe discontinuation of therapy. In this article, we use mathematical models of BCR–ABL levels to make inferences on the dynamics of LSCs.Experimental Design: Patients with at least 1 BCR–ABL transcript measurement on imatinib were included (N = 477). Maximum likelihood methods were used to test 3 potential hypotheses of the dynamics of BCR–ABL transcripts on imatinib therapy: (i) monoexponential, in which there is little, if any, decline in BCR–ABL transcripts; (ii) biexponential, in which patients have a rapid initial decrease in BCR–ABL transcripts followed by a more gradual response; and (iii) triexponential, in which patients first exhibit a biphasic decline but then have a third phase when BCR–ABL transcripts increase rapidly.Results: We found that most patients treated with imatinib exhibit a biphasic decrease in BCR–ABL transcript levels, with a rapid decrease during the first few months of treatment, followed by a more gradual decrease that often continues over many years.Conclusions: We show that the only hypothesis consistent with current data on progenitor cell turnover and with the long-term, gradual decrease in the BCR–ABL levels seen in most patients is that these patients exhibit a continual, gradual reduction of the LSCs. This observation may explain the ability to discontinue imatinib therapy without relapse in some cases. Clin Cancer Res; 17(21); 6812–21. ©2011 AACR.

PDF file - 325K

CCR Translation for This Article from BCR–ABL Transcript Dynamics Support the Hypothesis That Leukemic Stem Cells Are Reduced during Imatinib Treatment

Refereed/Peer-reviewed Cancer treatment is constantly evolving from a one-size-fits-all towards bespoke approaches for each patient. In certain solid cancers, including breast and lung, tumor genome profiling has been incorporated into therapeutic decision-making. For chronic phase chronic myeloid leukemia (CML), while tyrosine kinase inhibitor therapy is the standard treatment, current clinical scoring systems cannot accurately predict the heterogeneous treatment outcomes observed in patients. Biomarkers capable of segregating patients according to outcome at diagnosis are needed to improve management, and facilitate enrollment in clinical trials seeking to prevent blast crisis transformation and improve the depth of molecular responses. To this end, gene expression (GE) profiling studies have evaluated whether GE signatures at diagnosis are clinically informative. Patient material from a variety of sources has been profiled using microarrays, RNA sequencing and, more recently, single-cell RNA sequencing. However, differences in the cell types profiled, the technologies used, and the inherent complexities associated with the interpretation of genomic data pose challenges in distilling GE datasets into biomarkers with clinical utility. The goal of this paper is to review previous studies evaluating GE profiling in CML, and explore their potential as risk assessment tools for individualized CML treatment. We also review the contribution that acquired mutations, including those seen in clonal hematopoiesis, make to GE profiles, and how a model integrating contributions of genetic and epigenetic factors in resistance to tyrosine kinase inhibitors and blast crisis transformation can define a route to GE-based biomarkers. Finally, we outline a four-stage approach for the development of GE-based biomarkers in CML.

The classification of myeloid neoplasms and acute leukemias was last updated in 2016 within a collaboration between the World Health Organization (WHO), the Society for Hematopathology, and the European Association for Haematopathology. This collaboration was primarily based on input from a clinical advisory committees (CACs) composed of pathologists, hematologists, oncologists, geneticists, and bioinformaticians from around the world. The recent advances in our understanding of the biology of hematologic malignancies, the experience with the use of the 2016 WHO classification in clinical practice, and the results of clinical trials have indicated the need for further revising and updating the classification. As a continuation of this CAC-based process, the authors, a group with expertise in the clinical, pathologic, and genetic aspects of these disorders, developed the International Consensus Classification (ICC) of myeloid neoplasms and acute leukemias. Using a multiparameter approach, the main objective of the consensus process was the definition of real disease entities, including the introduction of new entities and refined criteria for existing diagnostic categories, based on accumulated data. The ICC is aimed at facilitating diagnosis and prognostication of these neoplasms, improving treatment of affected patients, and allowing the design of innovative clinical trials.

Myeloid neoplasms and acute leukemias derive from the clonal expansion of hematopoietic cells driven by somatic gene mutations. Although assessment of morphology plays a crucial role in the diagnostic evaluation of patients with these malignancies, genomic characterization has become increasingly important for accurate diagnosis, risk assessment, and therapeutic decision making. Conventional cytogenetics, a comprehensive and unbiased method for assessing chromosomal abnormalities, has been the mainstay of genomic testing over the past several decades and remains relevant today. However, more recent advances in sequencing technology have increased our ability to detect somatic mutations through the use of targeted gene panels, whole-exome sequencing, whole-genome sequencing, and whole-transcriptome sequencing or RNA sequencing. In patients with myeloid neoplasms, whole-genome sequencing represents a potential replacement for both conventional cytogenetic and sequencing approaches, providing rapid and accurate comprehensive genomic profiling. DNA sequencing methods are used not only for detecting somatically acquired gene mutations but also for identifying germline gene mutations associated with inherited predisposition to hematologic neoplasms. The 2022 International Consensus Classification of myeloid neoplasms and acute leukemias makes extensive use of genomic data. The aim of this report is to help physicians and laboratorians implement genomic testing for diagnosis, risk stratification, and clinical decision making and illustrates the potential of genomic profiling for enabling personalized medicine in patients with hematologic neoplasms.

usc Refereed/Peer-reviewed Purpose: Genetic variants causing aberrant premessenger RNA splicing are increasingly being recognized as causal variants in genetic disorders. In this study, we devise standardized practices for polymerase chain reaction (PCR)-based RNA diagnostics using clinically accessible specimens (blood, fibroblasts, urothelia, biopsy). Methods: A total of 74 families with diverse monogenic conditions (31% prenatal-congenital onset, 47% early childhood, and 22% teenage-adult onset) were triaged into PCR-based RNA testing, with comparative RNA sequencing for 19 cases. Results: Informative RNA assay data were obtained for 96% of cases, enabling variant reclassification for 75% variants that can be used for genetic counseling (71%), to inform clinical care (32%) and prenatal counseling (41%). Variant-associated mis-splicing was highly reproducible for 28 cases with samples from ≥2 affected individuals or heterozygotes and 10 cases with ≥2 biospecimens. PCR amplicons encompassing another segregated heterozygous variant was vital for clinical interpretation of 22 of 79 variants to phase RNA splicing events and discern complete from partial mis-splicing. Conclusion: RNA diagnostics enabled provision of a genetic diagnosis for 64% of recruited cases. PCR-based RNA diagnostics has capacity to analyze 81.3% of clinically significant genes, with long amplicons providing an advantage over RNA sequencing to phase RNA splicing events. The Australasian Consortium for RNA Diagnostics (SpliceACORD) provide clinically-endorsed, standardized protocols and recommendations for interpreting RNA assay data.

Purpose of Review The chronic myeloid leukemia (CML) treatment success story is incomplete as some patients still fail therapy, leading to end-stage disease and death. Here we discuss recent research into CML incidence, the role of comorbidities on survival and detecting patients at risk of failing therapy. Recent Findings The incidence of CML has fallen markedly in high social-demographic index (SDI) regions of the world but there is disturbing evidence that this is not the case in low and low-middle SDI countries. Now that CML patients more frequently die from their co-morbid conditions than from CML the Adult Comorbidity Evaluation-27 score can assist in risk assessment at diagnosis. Non-adherence to therapy contributes greatly to treatment failure. A good doctor-patient relationship and social support promote good adherence, but patient age, gender, and financial burden have negative effects, suggesting avenues for intervention. Mutations in cancer-associated genes adversely affect outcome and their detection at diagnosis may guide therapeutic choice and offer non-BCR::ABL1 targeted therapies. A differential gene expression signature to assist risk detection is a highly sought-after diagnostic tool being actively researched on several fronts. Summary Detecting patients at risk of failing therapy is being assisted by recent technological advances enabling highly sensitive genomic and expression analysis of insensitive cells. However, patient lifestyle, adherence to therapy, and comorbidities are critical risk factors that need to be addressed by interventions such as social and financial support.

Atypical BCR-ABL1 transcripts occur in around 3% of patients with chronic phase chronic myeloid leukemia (CML) [1]. Due to the rarity of each individual transcript type there are limited data on th...

Approximately half of patients with chronic myeloid leukemia (CML) in sustained deep molecular response who discontinue tyrosine kinase inhibitors (TKIs) remain in treatment-free remission (TFR). Some of these patients have measurable residual disease (MRD) by BCR-ABL1 mRNA testing, and most have detectable BCR-ABL1 DNA by highly sensitive methods. We used fluorescence-activated cell sorting and BCR-ABL1 DNA PCR to investigate the lineage of residual CML cells in TFR. Twenty patients in TFR for >1 year provided blood for sorting into granulocytes, monocytes, B cells, T cells, and NK cells. MRD was identified predominantly in the lymphoid compartment and never in granulocytes. B cells were more often BCR-ABL1 positive than T cells (18 vs 11/20 patients) and at higher levels (median 10−4.9 vs 10−5.7; P = 0.014). In 13 CML patients studied at diagnosis lymphocytes expressing BCR-ABL1 mRNA comprised a small proportion of total leukocytes. These data improve our understanding of TFR biology, since it is now clear that MRD in the blood of TFR patients need not imply the persistence of multipotent CML cells. Lineage-specific assessment of MRD could be explored as a means to improve the prediction of TFR Refereed/Peer-reviewed

With the focus of leukaemia management shifting to the implications of low-level disease burden, increasing attention is being paid on the development of highly sensitive methodologies required for detection. There are various techniques capable of identification of measurable residual disease (MRD) either evidencing as relevant mutation detection [e.g. nucleophosmin 1 (NPM1) mutation] or trace levels of leukaemic clonal populations. The vast majority of these methods only permit detection of a single clone or mutation. However, mass spectrometry and next-generation sequencing enable the interrogation of multiple genes simultaneously, facilitating a more complete genomic profile. In the present review, we explore the methodologies of both techniques in conjunction with the important advantages and limitations associated with each assay. We also highlight the evidence and the various instances where either technique has been used and propose future strategies for MRD detection. Refereed/Peer-reviewed

With treatment-free remission (TFR) rapidly becoming the ultimate goal of therapy in chronic myeloid leukemia (CML), there is a need to develop strategies to maximize sustained TFR by improving our understanding of its key determinants. Chronic-phase CML patients attempting TFR were evaluated to identify the impact of multiple variables on the probability of sustained TFR. Early molecular response dynamics were included as a predictive variable, assessed by calculating the patient-specific halving time of BCR-ABL1 after commencing tyrosine kinase inhibitor (TKI) therapy. Overall, 115 patients attempted TFR and had ≥12 months of follow-up. The probability of sustained TFR, defined as remaining in major molecular response off TKI therapy for 12 months, was 55%. The time taken for the BCR-ABL1 value to halve was the strongest independent predictor of sustained TFR: 80% in patients with a halving time of 21.85 days (last quartile) (P < .001). The e14a2 BCR-ABL1 transcript type and duration of TKI exposure before attempting TFR were also independent predictors of sustained TFR. However, the BCR-ABL1 value measured at 3 months of TKI was not an independent predictor of sustained TFR. A more rapid initial BCR-ABL1 decline after commencing TKI also correlated with an increased likelihood of achieving TFR eligibility. The association between sustained TFR and the time taken for BCR-ABL1 to halve after commencing TKI was validated using an independent dataset. These data support the critical importance of the initial kinetics of BCR-ABL1 decline for long-term outcomes.

Refereed/Peer-reviewed Response to tyrosine kinase inhibitor (TKI) therapy in CML can be measured by haematological, cytogenetic and molecular criteria. Achieving haematological and cytogenetic response is still a desirable early measure of response. However measurement of the level of BCR-ABL1 transcripts in the blood by qRT-PCR, adjusted to the international scale (IS), is now the primary means of monitoring response. Achieving time-dependent molecular targets is a strong predictor of survival as well as indicating the longer-term prospects of achieving eligibility for treatment-free remission (TFR). Optimal response can be determined solely by the achievement and maintenance of time-dependent molecular response levels. Likewise, treatment failure is largely defined by a failure to achieve molecular milestones or loss of that molecular response, except in cases where treatment failure is evident by progression to advanced-phase disease. Treatment failure mandates a switch in therapy, usually to a more potent TKI, if possible. In between optimal response and treatment failure, there is a “warning category” where outcomes are generally inferior and therapeutic decisions are more complex. Recently, the importance of determining the dynamics of response, not just the BCR-ABL1 level at one specified time point, has been recognized, particularly when determining the probability of achieving sustained TFR in eligible patients.

Blast crisis (BC) predicts dismal outcomes in patients with chronic myeloid leukaemia (CML). Although additional genetic alterations play a central role in BC, the landscape and prognostic impact of these alterations remain elusive. Here, we comprehensively investigate genetic abnormalities in 136 BC and 148 chronic phase (CP) samples obtained from 216 CML patients using exome and targeted sequencing. One or more genetic abnormalities are found in 126 (92.6%) out of the 136 BC patients, including the RUNX1-ETS2 fusion and NBEAL2 mutations. The number of genetic alterations increase during the transition from CP to BC, which is markedly suppressed by tyrosine kinase inhibitors (TKIs). The lineage of the BC and prior use of TKIs correlate with distinct molecular profiles. Notably, genetic alterations, rather than clinical variables, contribute to a better prediction of BC prognosis. In conclusion, genetic abnormalities can help predict clinical outcomes and can guide clinical decisions in CML., In chronic myeloid leukaemia (CML), the drivers of blast crisis and resistance to tyrosine kinase inhibitors are not fully characterised. Here, the authors analyse a cohort of CML samples with genomic technologies and find that at least one driver alteration is associated with progression and worse prognosis.

Simple Summary This study provides new insights into the changing transcriptomic and epigenomic landscapes in chronic myeloid leukaemia (CML) patients who are receiving tyrosine kinase inhibitor (TKI) therapy (often life-long). Alternative splicing, vital for cellular homeostasis, is dysregulated in human cancers. Remarkably, we found abnormal splicing patterns despite molecular remission in peripheral blood cells of chronic-phase CML patients. This phenomenon is independent of the TKI drug used and in striking contrast to the normalisation of gene expression and DNA methylation patterns. Abstract Vast transcriptomics and epigenomics changes are characteristic of human cancers, including leukaemia. At remission, we assume that these changes normalise so that omics-profiles resemble those of healthy individuals. However, an in-depth transcriptomic and epigenomic analysis of cancer remission has not been undertaken. A striking exemplar of targeted remission induction occurs in chronic myeloid leukaemia (CML) following tyrosine kinase inhibitor (TKI) therapy. Using RNA sequencing and whole-genome bisulfite sequencing, we profiled samples from chronic-phase CML patients at diagnosis and remission and compared these to healthy donors. Remarkably, our analyses revealed that abnormal splicing distinguishes remission samples from normal controls. This phenomenon is independent of the TKI drug used and in striking contrast to the normalisation of gene expression and DNA methylation patterns. Most remarkable are the high intron retention (IR) levels that even exceed those observed in the diagnosis samples. Increased IR affects cell cycle regulators at diagnosis and splicing regulators at remission. We show that aberrant splicing in CML is associated with reduced expression of specific splicing factors, histone modifications and reduced DNA methylation. Our results provide novel insights into the changing transcriptomic and epigenomic landscapes of CML patients during remission. The conceptually unanticipated observation of widespread aberrant alternative splicing after remission induction warrants further exploration. These results have broad implications for studying CML relapse and treating minimal residual disease.

Refereed/Peer-reviewed Chronic myeloid leukemia (CML) represents the disease prototype of genetically based diagnosis and management. Tyrosine kinase inhibitors (TKIs), that target the causal BCR::ABL1 fusion protein, exemplify the success of molecularly based therapy. Most patients now have long-term survival; however, TKI resistance is a persistent clinical problem. TKIs are effective in the BCR::ABL1-driven chronic phase of CML but are relatively ineffective for clinically defined advanced phases. Genomic investigation of drug resistance using next-generation sequencing for CML has lagged behind other hematological malignancies. However, emerging data show that genomic abnormalities are likely associated with suboptimal response and drug resistance. This has already been supported by the presence of BCR::ABL1 kinase domain mutations in drug resistance, which led to the development of more potent TKIs. Next-generation sequencing studies are revealing additional mutations associated with resistance. In this review, we discuss the initiating chromosomal translocation that may not always be a straightforward reciprocal event between chromosomes 9 and 22 but can sometimes be accompanied by sequence deletion, inversion, and rearrangement. These events may biologically reflect a more genomically unstable disease prone to acquire mutations. We also discuss the future role of cancer-related gene mutation analysis for risk stratification in CML.

Accurate quantification of minimal residual disease during treatment of chronic myeloid leukaemia guides clinical decisions. The conventional minimal residual disease method, RQ-PCR for BCR-ABL1 mRNA, reflects a composite of the number of circulating leukemic cells and the BCR-ABL1 transcripts per cell. BCR-ABL1 genomic DNA only reflects leukemic cell number. We used both methods in parallel to determine the relative contribution of the leukemic cell number to molecular response. BCR-ABL1 DNA PCR and RQ-PCR were monitored up to 24 months in 516 paired samples from 59 newly-diagnosed patients treated with first-line imatinib in the TIDEL-II study. In the first 3 months of treatment BCR-ABL1 mRNA values declined more rapidly than DNA. By 6 months the two measures aligned closely. The expression of BCR-ABL1 mRNA was normalized to cell number to generate an expression ratio. The expression of e13a2 BCR-ABL1 was lower than that of e14a2 transcripts at multiple time points during treatment. BCR-ABL1 DNA was quantifiable in 48% of samples with undetectable BCR-ABL1 mRNA, resulting in minimal residual disease being quantifiable for an additional 5-18 months (median 12 months). These parallel studies show for the first time that the rapid decline in BCR-ABL1 mRNA over the first 3 months of treatment is due to a reduction in both cell number and transcript level per cell, whereas beyond 3 months falling levels of BCR-ABL1 mRNA are predominantly due to depletion of leukaemic cells. Refereed/Peer-reviewed

usc Background: Some patients receiving a tyrosine kinase inhibitor (TKI) for the first-line treatment of chronic phase chronic myeloid leukemia (CML-CP) experience intolerable adverse events. Management strategies in-clude dose adjustments, interrupting or discontinuing therapy, or switching to an alternative TKI. Methods: This multicenter, single-arm, Phase IIIb study included CML-CP patients intolerant of, but responsive to, first-line treatment with imatinib or dasatinib. All patients were switched to nilotinib 300 mg bid for up to 24 months. The primary endpoint was achievement of MR4.5 (BCR-ABL transcript level of ≤ 0.0032% on the International Scale) by 24 months. Results: Twenty patients were enrolled in the study (16 imatinib-intolerant, 4 dasatinib-intolerant); which was halted early because of low recruitment. After the switch to nilotinib 300 mg bid, MR4.5 at any time point up to month 24 was achieved in 10 of 20 patients (50%) in the full analysis set. Of the non-hematological adverse events associated with intolerance to prior imatinib or dasatinib, 74% resolved within 12 weeks of switching tonilotinib 300 mg bid. Conclusion: Nilotinib 300 mg bid shows minimal cross intolerance in patients with CML-CP who have prior toxicities to other TKIs and can lead to deep molecular responses. Refereed/Peer-reviewed

Asciminib (previously ABL001), which binds the myristate-binding pocket of the Bcr-Abl kinase domain, is in phase I clinical trials as monotherapy and in combination with imatinib, nilotinib and dasatinib for the treatment of patients with refractory CML or Ph+ ALL. Asciminib sensitivity was evaluated in asciminib naïve BCR-ABL1+ cell lines K562 (negligible ABCB1/ABCG2 expression), K562-Dox (ABCB1-overexpressing through doxorubicin exposure) and K562-ABCG2 (ABCG2 overexpression via transduction) with results demonstrating asciminib efflux by both ABCB1 and ABCG2 transporters. K562-Dox and K562-ABCG2 cells demonstrated increased LD50 asciminib vs K562 control cells: 256 and 299 nM respectively vs 24 nM, p < 0.001. Sensitivity was completely restored with specific inhibitors cyclosporine (ABCB1) and Ko143 (ABCG2): K562-Dox LD50 asciminib+cyclosporine = 13 nM, K562-ABCG2 LD50 asciminib+Ko143 = 15 nM (p < 0.001). When asciminib resistance was modelled in vitro, ABCB1 and ABCG2 overexpression was integral in the development of asciminib resistance. In K562 asciminib-resistant cells, ABCG2 expression increased prior to BCR-ABL1 overexpression and remained high (up to 7.6-fold greater levels in resistant vs control cells, p < 0.001). K562-Dox asciminib-resistant cells had increased ABCB1 expression (2.1-fold vs control cells p = 0.0033). KU812 asciminib-resistant cells overexpressed ABCB1 (5.4-fold increase, p < 0.001) and ABCG2 (6-fold increase, p < 0.001) before emergence of a novel myristate-binding pocket mutation (F497L). In all three cell lines, asciminib resistance was reversible upon chemical inhibition of ABCB1, ABCG2 or both (p < 0.001). When K562 asciminib-resistant cells were treated with asciminib in combination with clinically achievable doses of either imatinib or nilotinib, reversal of the resistance phenotype was also observed (p < 0.01). Overexpression of efflux transporters will likely be an important pathway for asciminib resistance in the clinical setting. Given the lack of evidence for ABCG2-mediated transport of nilotinib or imatinib at clinically relevant concentrations, our data provide an additional rationale for using asciminib in combination with either TKI. Refereed/Peer-reviewed

Introduction: Treatment Free Remission (TFR) is the ultimate goal of therapy for most CML patients. Despite adopting consensus eligibility criteria of a sustained deep molecular response and more than 4 years of TKI therapy, the relapse rate after TKI cessation is still around 50%. More sensitive detection of residual leukaemia has the potential to improve our capacity to predict TFR outcomes for individual patients. Aim: To correlate droplet digital PCR (ddPCR) assay results with TFR outcome, especially in the setting of undetectable levels measured by qRT-PCR. Method: ddPCR was performed on blood samples from 51 TFR-eligible CML patients at the time of TKI cessation. 5 µg RNA per sample was used in 8 wells/sample using the BioRad QXDx BCR-ABL %IS kit on QX200 ddPCR system which yielded BCR-ABL1% (IS) directly. All these patients achieved MR4.5 that was sustained for ≥ 2 years. Results: 100% of patient were in MR4.5 via qRT-PCR at the time of stopping. 61% of the 51 patients evaluated relapsed within 12 months. Median duration of TKI therapy for the whole group was 5.8 years (range 2.2- 14 years). 20 patients achieved TFR success with a median follow up of 24 months (TFR group; sustained BCR-ABL1 0.1% (IS) after stopping; median time of relapse 3 months, range 1-10 months). A ROC curve analysis correlated TFR outcome with ddPCR results, with BCR-ABL1 level ≥0.003% via ddPCR at the time of stopping identified as an optimal cut-off. Kaplan-Meier analysis showed that 89% of the patients with ddPCR ≥0.003 relapsed after TKI cessation, whereas the ddPCR We next assessed other known predictors of TFR success relative to ddPCR results in a Cox proportional hazard model. We have previously demonstrated that the BCR-ABL1 halving time after commencing therapy is highly predictive of TFR. At a univariate level, transcript type (e13a2 versus e14a2, p=0.01), BCR-ABL1 halving time (p>0.0001), and mRNA quantitation by ddPCR ≥ 0.003% (p=0.02) were all significantly associated with clinical outcome. Other variables including gender, age, ELTS score, Sokal score, MR4.5 duration and TKI duration were not associated with clinical outcome in this cohort (Figure 1B). In the multivariate analyses (Figure 1C), ddPCR remained an independent predictor after adjusting for ELTS, TKI duration and MR4.5 duration. Interestingly, ddPCR was not an independent predictor after adjusting for BCR-ABL1 transcript type or halving time. Conclusion: QXDx ddPCR assay is a promising tool for molecular residual disease monitoring in CML, especially when the BCR-ABL1 is undetectable by conventional method. The CML patients with levels of detectable BCR-ABL1 ≥0.003% measured by ddPCR have a significantly higher probability of relapse compared to patients with lower levels of the transcript. The ≥0.003% BCR-ABL1 level cut-off value could be a potential tool to aid decision-making when attempting TKI discontinuation in CML. However, even though a measurable level of BCR-ABL1 above 0.003% via ddPCR identified patients at high risk of relapse after a TFR attempt, it does not rule out the possibility of TFR; and a negative ddPCR result does not exclude the risk of molecular relapse. ddPCR may be most useful where other TFR predictive factors including BCR-ABL1 transcript type and halving time are not available. In-kind support was received from Bio-Rad for this study. Figure 1 Figure 1. Disclosures Branford: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Cepheid: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Qiagen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hughes: Novartis: Honoraria, Research Funding; Incyte: Honoraria; BMS: Honoraria, Research Funding. Yeung: BMS: Honoraria, Research Funding; Amgen: Honoraria; Novartis: Honoraria, Research Funding; Pfizer: Honoraria.

Chronic myeloid leukaemia (CML) is driven by the activity of the BCR-ABL1 fusion oncoprotein. ABL1 kinase inhibitors have improved the clinical outcomes for patients with CML, with over 80% of patients treated with imatinib surviving for more than 10 years. Second-generation ABL1 kinase inhibitors induce more potent molecular responses in both previously untreated and imatinib-resistant patients with CML. Studies in patients with chronic-phase CML have shown that around 50% of patients who achieve and maintain undetectable BCR-ABL1 transcript levels for at least 2 years remain disease-free after the withdrawal of treatment. Here we characterize ABL001 (asciminib), a potent and selective allosteric ABL1 inhibitor that is undergoing clinical development testing in patients with CML and Philadelphia chromosome-positive (Ph +) acute lymphoblastic leukaemia. In contrast to catalytic-site ABL1 kinase inhibitors, ABL001 binds to the myristoyl pocket of ABL1 and induces the formation of an inactive kinase conformation. ABL001 and second-generation catalytic inhibitors have similar cellular potencies but distinct patterns of resistance mutations, with genetic barcoding studies revealing pre-existing clonal populations with no shared resistance between ABL001 and the catalytic inhibitor nilotinib. Consistent with this profile, acquired resistance was observed with single-agent therapy in mice; however, the combination of ABL001 and nilotinib led to complete disease control and eradicated CML xenograft tumours without recurrence after the cessation of treatment. Refereed/Peer-reviewed