ABL Kinase Domain Mutations Research Articles

New ABL1 Kinase Domain Mutations in BCR::ABL1-Positive Acute Lymphoblastic Leukemia.

Since the development of the first-generation Tyrosine Kinase Inhibitor (TKI), it has played a crucial role in the treatment of BCR::ABL1-positive acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML). However, ABL1 kinase domain (ABL1 KD) mutations confer resistance to several TKIs. These mutations have been extensively studied in chronic myeloid leukemia (CML) but less so in BCR::ABL1-positive acute lymphoblastic leukemia (ALL). Our study aimed to analyze the the ABL1 KD mutations in 97 consecutive newly-diagnosed adults with BCR::ABL1-positive ALL before therapy, in cytogenetic complete remission and at relapse with next generation sequencing (NGS). The relationship between ABL1 KD mutations and TKI selection was also analyzed. Previously unreported ABL1 KD mutations R239G, F401V/L, R516L and K262T were the most prevalent in pre-therapy and cytogenetic remission samples, whereas T315I/P and P-loop mutations were most prevalent in relapse samples. R239G, F401V/L, R516L and K262T are related to the BCR::ABL1 structure, whereas T315I/P and P-loop mutations directly alter kinase activity. BaF3 cells transfected with ABL1 KD F401V, K262T, R239G, or R516L mutations were resistant to imatinib but strongly inhibited by olverembatinib with IC50 values of 0.73 to 1.52nM. Meanwhile, olverembatinib had advantages in increasing complete molecular response (CMR) and good prognosis. Overall, our findings indicate the prevalence and impact of new ABL1 KD mutations in BCR::ABL1-positive ALL patients, highlighting the necessity for effective therapies targetingthese mutations.

Validation of a novel NGS based BCR::ABL1 kinase domain mutation detection assay in Indian cohort

The efficacy and treatment outcome of a CML patient are heavily dependent on BCR::ABL1 kinase domain (KD) mutation status. Next-generation sequencing technology is a bright alternative to the previously used sanger sequencing method due to its global presence in diagnostic setups, massive parallel sequencing ability, and far better sensitivity. In the present study, we have demonstrated a new protocol for kinase domain mutation analysis using the next-generation sequencing (NGS) method using the ion torrent sequencing platform. This protocol uses RNA as the starting material, followed by nested PCR to amplify the fusion transcript, which is subsequently used as a template for NGS. Initial validation and comparison of this assay with the sanger sequencing (SS) method yielded 95.23% agreement. CML samples (n = 121) with a failure to TKI response were subjected to this newly developed NGS-based assay to detect KD mutations, from which samples were found to have mutations with a sensitivity ranging from 2.32 to 93.41%. A total of 34.71% of samples (n = 42) were found to be positive for one or more KD mutations, whereas 65.29% of samples (n = 81) were found to be negative. Nine samples out of 42 positive samples, i.e., 21.42%, were found to have compound mutations. This is one of the first studies from India, which includes more than 160 samples and is analyzed by the NGS approach for KD mutation analysis.

Mutation analysis of BCR-ABL1 kinase domain in chronic myeloid leukemia patients with tyrosine kinase inhibitors resistance: a Malaysian cohort study

ObjectiveMutational analysis of BCR::ABL1 kinase domain (KD) is a crucial component of clinical decision algorithms for chronic myeloid leukemia (CML) patients with failure or warning responses to tyrosine kinase inhibitor (TKI) therapy. This study aimed to detect BCR::ABL1 KD mutations in CML patients with treatment resistance and assess the concordance between NGS (next generation sequencing) and Sanger sequencing (SS) in detecting these mutations.ResultsIn total, 12 different BCR::ABL1 KD mutations were identified by SS in 22.6% (19/84) of patients who were resistant to TKI treatment. Interestingly, NGS analysis of the same patient group revealed an additional four different BCR::ABL1 KD mutations in 27.4% (23/84) of patients. These mutations are M244V, A344V, E355A, and E459K with variant read frequency below 15%. No mutation was detected in 18 patients with optimal response to TKI therapy. Resistance to TKIs is associated with the acquisition of additional mutations in BCR::ABL1 KD after treatment with TKIs. Additionally, the use of NGS is advised for accurately determining the mutation status of BCR::ABL1 KD, particularly in cases where the allele frequency is low, and for identifying mutations across multiple exons simultaneously. Therefore, the utilization of NGS as a diagnostic platform for this test is very promising to guide therapeutic decision-making.

Tyrosine kinase inhibitor resistance in de novo BCR::ABL1–positive BCP-ALL beyond kinase domain mutations

AbstractA better understanding of ABL1 kinase domain mutation–independent causes of tyrosine kinase inhibitor (TKI) resistance is needed for BCR::ABL1–positive B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Although TKIs have dramatically improved outcomes, a subset of patients still experiences relapsed or refractory disease. We aimed to identify potential biomarkers of intrinsic TKI resistance at diagnosis in samples from 32 pediatric and 19 adult patients with BCR::ABL1–positive BCP-ALL. Reduced ex vivo imatinib sensitivity was observed in cells derived from newly diagnosed patients who relapsed after combined TKI and chemotherapy treatment compared with cells derived from patients who remained in continuous complete remission. We observed that ex vivo imatinib resistance was inversely correlated with the amount of (phosphorylated) BCR::ABL1/ABL1 protein present in samples that were taken at diagnosis without prior TKI exposure. This suggests an intrinsic cause of TKI resistance that is independent of functional BCR::ABL1 signaling. Simultaneous deletions of IKZF1 and CDKN2A/B and/or PAX5 (IKZF1plus), as well as deletions of PAX5 alone, were related to ex vivo imatinib resistance. In addition, somatic lesions involving ZEB2, SETD2, SH2B3, and CRLF2 were associated with reduced ex vivo imatinib sensitivity. Our data suggest that the poor prognostic value of IKZF1(plus) deletions is linked to intrinsic mechanisms of TKI resistance other than ABL1 kinase domain mutations in newly diagnosed pediatric and adult BCR::ABL1–positive BCP-ALL.

Clinical Value of Detecting ABL Kinase Domain Mutations in Patients with Chronic Myeloid Leukemia Based on High-Throughput Sequencing Technology

To compare the efficacy and clinical value of high-throughput sequencing (HTS) and Sanger sequencing in detecting ABL kinase domain mutations in patients with chronic myeloid leukemia (CML). A total of 198 samples of 147 CML patients from July 2017 to March 2021 in Henan Cancer Hospital were collected and underwent high-throughput sequencing and Sanger sequencing to detect the mutations in ABL kinase domain, and the relevant clinical data were collected for comparative analysis. The proportion of total mutations and ≥2 mutations detected by high-throughput sequencing were significantly higher than those detected by Sanger sequencing (P =0.01; P =0.046). ≥2 mutations were detected in 22 cases, of which 5 cases (22.7%) had compound mutations. High-throughput sequencing can detect low level mutations that cannot be detected by Sanger sequencing. In 198 samples, 25 (12.6%) were low level mutations, 33 (16.7%) were high level mutations and 10 (5.1%) were mixed high and low level mutations. In the analysis of related clinical factors, the total mutation rate and the low level mutation rate in the optimal period, failure period and warning period were gradually increased (total mutation rate, P =0.016; low level mutation rate, P =0.005). The mutation rate of the samples with additional chromosomal abnormalities was also significantly increased (P =0.009). The mutation rate of patients who received first- and second-line treatment was significantly lower than that of patients who received third- or higher-line treatment (P =0.006). Analysis based on variant allele frequency (VAF) of the mutation site was helpful to visually evaluate the clonal evolution status of TKI-resistance CML cells. High-throughput sequencing is more sensitive and accurate than Sanger sequencing in mutation detection, which is helpful to accurately and visually evaluate TKI treatment response and optimize treatment strategy for CML.

Erythroid variant evolving from chronic myeloid leukemia resistant to multiple tyrosine kinase inhibitors: a rare case report

BackgroundChronic myeloid leukemia (CML) is characterized by the presence of BCR::ABL1 fusion gene resulting from a reciprocal translocation, t(9;22)(q34;q11.2), leading to prominent granulocytic proliferation. The majority of patients initially present in chronic phase (CP), which may progress to advanced CML with predominantly granulocytic phenotypes in the absence of proper treatment or response to tyrosine kinase inhibitors (TKIs). We present an exceptionally rare case in which an erythroid variant emerged from a CML patient resistant to multiple TKIs. This variant is characterized by the detection of t(9;22) BCR::ABL1 fusion in erythroid precursors at various maturation stages and the absence of granulocytic progenitor hyperplasia typically seen in classical CML.Case presentationA 33-year-old female with CP-CML had received multiple TKI therapies since her initial diagnosis in 2015. Due to intolerable side effects and inconsistent adherence, she exhibited an inadequate response and developed new-onset pancytopenia. Bone marrow (BM) biopsy specimen revealed a hypercellular marrow with significant erythroid hyperplasia (90% of marrow cellularity) and a reversed myeloid-to-erythroid (M: E) ratio of 1:10. Both erythroid and myeloid cells displayed progressive maturation without dysplasia or excess blasts. Chromosomal analysis identified t(9;22) (q34;q11.2) in 19 out of 20 metaphase cells. BCR::ABL1 fusion transcript (p210 isoform) was confirmed by real-time quantitative polymerase chain reaction (RT-qPCR) and next-generation sequencing (NGS). Notably, no additional pathogenic cytogenetic abnormalities or ABL1 kinase domain mutations were detected. Here, we report the first published case of an erythroid variant emerging in a CML patient resistant to multiple TKIs—a distinct entity from the erythroid blast crisis evolving from CML.ConclusionThe erythroid variant of CML is distinguished by the presence of t(9;22) (q34;q11.2) BCR::ABL1 in predominant erythroid precursors at different stages of maturation. In a myeloid neoplasm showing predominant erythroid hyperplasia without typical CML features, it is vital to correlate morphology and t(9;22) BCR::ABL1 cytogenetic testing for accurate diagnosis, and to prevent confusion with PEL transformation in CML.

In vitro evidence of synergistic efficacy with asciminib combined with reduced dose of ATP-binding pocket tyrosine kinase inhibitors according to the ABL1 kinase domain mutation profile.

In vitro evidence of synergistic efficacy with asciminib combined with reduced dose of ATP-binding pocket tyrosine kinase inhibitors according to the ABL1 kinase domain mutation profile.

BCR-ABL kinase domain mutations in CML patients, experience from a tertiary care center in North India

BackgroundChronic Myeloid Leukemia is characterized by the presence of the Philadelphia Chromosome (Ph) which contains the BCR::ABL1 fusion gene that occurs due to a reciprocal translocation between chromosomes 9 and 22. This accounts for up to 15 % of all adult leukemias [1]. Most patients treated with first line tyrosine kinase inhibitor (TKI) imatinib achieve durable response but may undergo relapse at some stage [2]. The most important mechanism that may confer imatinib resistance is point mutation within BCR::ABL kinase domain. Other generation ABL tyrosine kinase inhibitors such as dasatinib, nilotinib, bosutinib and ponatinib help to overcome imatinib resistance [3]. Sensitivity of the patient to each of the above TKIs depends upon the individual candidate mutation present. Thus, it is important to perform mutation analysis for effective therapeutic management of CML patients once they show imatinib resistance. We used direct sequencing to identify the different types of mutations responsible for resistance of imatinib treatment from north India. MethodsIn this study, the patient resistance for the imatinib were analyzed for BCR::ABL kinase domain mutation by direct sequencing and the detected mutations along with their percentage prevalence were reported. Results329 patients with CML-CP were analyzed for BCR::ABL kinase domain mutation. Total 66 (20.06 %) patients out of 329 had mutation in at least one of the domains of BCR::ABL conferring resistance to different generations of TKI. Mutations in BCR::ABL kinase domain was observed in different domain of BCR::ABL. ATP binding P-Loop (42.42 %), Direct binding site (36.36 %), C-Loop (10.60 %), A-Loop (6.06 %), SH2 contact (3.03 %), SH3 contact (1.51 %). ConclusionTotal 20.06 % patients (66/329) show mutation in at least one of the structural motifs of BCR-ABL kinase domain, which further confer the resistance to a particular generation of TKI.

Dynamic Changes in ABL1 Kinase Domain Mutations and Comparative Efficacy of Third-Generation Tyrosine Kinase Inhibitors across Different Stages in Acute Lymphoblastic Leukemia and Chronic Myeloid Leukemia Blast Crisis Patients

*Drs.Zixuan Li and Danyue Peng contributed equally. Drs.Mei Hong and Qiuling Wu are co-corresponding authors Background The development of first-generation and second-generation tyrosine kinase inhibitors (TKIs) has played a crucial role in the treatment of Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) and chronic myeloid leukemia (CML). However, the emergence of mutations in the kinase domain (KD) of BCR-ABL1 has increasingly compromised the efficacy of TKIs and become the primary cause of TKI resistance. Testing BCR-ABL1 KD mutations in early phase accurately is of great value in predicting response and guiding therapeutic decisions. Next-generation sequencing (NGS) provided a more comprehensive and elaborate understanding of mutation status. Third-generation (3G) TKI has been designed to overcome resistance problems caused by BCR-ABL1 KD mutations, and it exhibited unique pharmacological profiles and response patterns relative to different mutations and disease characteristics. Therefore, the potential clinical relevance of mutations detected by NGS and the efficacy of 3G TKI are worth exploring. Methods NGS was used to analyze the dynamic changes of both clinically unreported mutations and reported resistant mutations in the ABL1 KD during three stages of the disease course (pretreatment, remission, and relapsed periods) in Ph+ ALL (n = 97) and chronic myeloid leukemia blast crisis (CML-BP) (n = 21) patients. Meanwhile, the comparative efficacy of three generations of TKIs in Ph+ ALL (n = 92) and CML-BP (n = 23) and the relationship between ABL1 KD mutations and different TKI selection were analyzed. Results In Ph+ ALL patients, single ABL1 KD unreported mutations were detected in 30.49% of patients before therapy and 39.73% in remission, which accounted for the highest proportion among other mutation types. Conversely, single ABL1 KD resistant mutations were found to be the most prevalent (54.5%) in relapsed patients ( P < 0.0001). Logistic analysis revealed that the presence of F401V/L in untreated Ph+ ALL patients was associated with a higher risk of relapse ( P < 0.1). Furthermore, through analyzing patients who received TKI treatment, we observed that 3G TKIs like olverembatinib exhibited advantages in prognosis, including achieving deep molecular response, rapid remission reduction, and decreased relapse probability, for both newly diagnosed and relapsed Ph+ ALL patients. Notably, 3G TKIs not only demonstrated strong inhibition against the T315I mutation, as previously reported, but also displayed superior effectiveness against certain ABL1 KD unreported mutations, such as R516L, K262T, and F401V. Similar trends were observed in CML-BP, although statistical significance was not reached likely due to the limited number of patients. Conclusion Overall, our findings indicate the prevalence and impact of ABL1 KD mutations in Ph+ ALL and CML-BP patients, highlighting the necessity for effective therapies targeting these mutations. The promising outcomes observed with 3G TKIs suggest their potential as a treatment option for both newly diagnosed and relapsed Ph+ ALL patients, offering advantages in terms of molecular response depth, rapid remission, and reduced relapse risk. Further investigations are warranted to explore the clinical significance of unreported ABL1 KD mutations and validate the effectiveness of 3G TKIs against them.

Novel Insights in TKI Resistance in BCR:: ABL1-Positive B-Cell Acute Lymphoblastic Leukemia Beyond Kinase Domain Mutations

Background and Aim Despite the introduction of tyrosine kinase inhibitors (TKIs), approximately 30% of newly diagnosed pediatric and adult BCR:: ABL1-positive B-cell acute lymphoblastic leukemia (B-ALL) patients experience relapsed or refractory disease (Biondi et al, Lancet Haematology 2018; Bassan et al, Journal of Clinical Oncology 2010). ABL1 kinase domain mutations, hindering TKI binding, explain part of the relapses and are acquired during TKI therapy but are rarely detected in newly diagnosed BCR:: ABL1-positive B-ALL patients (Baer et al, Haematologica 2022). Intrinsic TKI resistance mechanisms in BCR:: ABL1-positive B-ALL present prior to TKI treatment are less well documented. Therefore, we aimed to identify potential biomarkers for TKI sensitivity at diagnosis using primary patient samples and newly generated cell lines made from patient-derived xenografts (PDX cell lines). Patients and Methods Leukemic cells from diagnosis of BCR:: ABL1-positive B-ALL patients without ABL1-kinase domain mutations were used to measure cell survival in an ex vivo co-culture assay after imatinib (1.95-125 μM), dasatinib (1.95-125 nM), or bosutinib (0.016-2 μM) exposure. TKI sensitivity was assessed using the area under the dose-response curve (AUC), with <80 arbitrary units (AU) classified as imatinib-sensitive, 80-120 AU as intermediate, and >120 AU as resistant. Multiplex ligation-dependent probe amplification, whole exome sequencing, and RNA sequencing were performed on diagnostic material if available. ABL1 phosphorylation and expression levels were measured using (phospho)flow cytometry in patient-derived xenografts from diagnostic samples and (PDX) cell lines. Results We determined ex vivo sensitivity to imatinib, dasatinib, and bosutinib in 32 pediatric and 19 adult BCR:: ABL1-positive B-ALL patient samples. We observed a substantial variability in ex vivo imatinib response in pediatric and adult patient samples (median AUC: 98 AU; range: 16 to 281 AU). The sensitivity towards imatinib strongly correlated with the sensitivity towards second-generation TKIs dasatinib and bosutinib (rho = 0.84; p < 0.001 and rho= 0.89; p <0.001). Moreover, imatinib's AUC of samples derived from newly diagnosed patients who relapsed after TKI treatment was higher compared with those derived from patients who did not suffer from a relapse (median AUC 123 vs 84 AU; p = 0.03). We observed that an increase in ex vivo imatinib resistance was correlated with a decreased amount of phosphorylated ABL1 protein (rho=-0.92; p=0.0013) and total ABL1 protein (rho=-0.82; p=0.01) in untreated, newly diagnosed samples. Ex vivo exposure to imatinib reduced the ABL1 phosphorylation levels in both sensitive and resistant samples but resistant cells exhibited lower levels of (p)ABL1 protein, indicating reduced dependence on BCR-ABL1 signaling. The ex vivo imatinib resistance in samples carrying deletions in B-cell development genes IKZF1 and/or PAX5 was higher than in samples without these deletions (median AUC: 122 versus 80 AU; p=0.01). In addition, one imatinib-resistant patient had a p.His1038Arg mutation in ZEB2, which is recurrent in B-ALL and this gene has also been described as B-cell development gene (Studd et al, Blood Cancer Journal 2021). Interestingly, another resistant patient had a mutation in SETD2,a gene previously associated with ex vivo TKI resistance in chronic myeloid leukemia cell lines (Sheng et al, Cell Proliferation 2019). Other secondary lesions included SH2B3 inactivation lesions and CRLF2 rearrangements. These lesions were present in intermediately sensitive and resistant BCR:: ABL1-positive samples and are expected to lead to increased JAK/STAT signaling, potentially serving as a resistance mechanism for TKI. Conclusion Our study revealed that the amount of total and phosphorylated ABL1 protein was correlated with ex vivo TKI sensitivity. Newly diagnosed TKI-resistant BCR:: ABL1 samples were further characterized by somatic lesions in B-cell development genes including IKZF1. These results link the known poor prognostic value of IKZF1 deletions in BCR:: ABL1-positive B-ALL patients (van der Veer et al. Blood 2014; Fedullo et al, Haematologica 2019) with intrinsic mechanisms of TKI resistance other than tyrosine kinase domain mutations.

In Vitro Evidence of Synergistically Enhanced Efficacy of Axitinib When Combined with Asciminib in T315I Mutated Chronic Myeloid Leukemia

Background: Development of ABL1 kinase domain (KD) mutation is one of resistance mechanism to ATP-binding pocket inhibitor (ABPI) therapy. T315I mutation in ABL KD confers broad-spectrum resistance to all 1st- and 2nd-generation ABPIs including Imatinib (IMA), Dasatinib (DAS), Nilotinib (NIL) and Bosutinib (BOS). Ponatinib (PON), a 3 rd generation inhibitor, can overcome T315I-mediated resistance in chronic myeloid leukemia (CML) treatment. However, its increased risk of cardiovascular toxicity limits the broader use of PON in the clinic. Asciminib (ASC) is a Specifically Targeting ABL1 Myristoyl Pocket (STAMP) inhibitor and has been approved for CML treatment in the patients who failed at least two previous lines of TKI therapy. ASC, which blocks the myristoylate pocket in ABL1 KD protein, can potentially overcome ABL1 KD mutation (KDM)-mediated tyrosine kinase inhibitor (TKI) resistance by combining with ABPIs which blocks ATP binding pockets in ABL1 KD protein. However, in vitro evidence to support double blockade approach is still to be further investigated including combination with ABPIs as well with other drug compounds. Axitinib (AXI), which is approved for the treatment of advanced renal cell carcinoma (RCC), is an ABPI that can specifically bind to the ATP-binding pocket in ABL1 KD protein structurally harboring the T315I mutation. In the Cancer Therapeutics Response Portal (CTRP) database, which integrates drug compound and cell line experiment databases, AXI showed very low IC 50 in CML cell lines compared to other cancer cell lines, including RCC. The present study attempted to provide in vitro evidence of the synergistic enhancement of therapeutic efficacy of AXI combined with ASC to overcome T315I mutated CML. Methods and Materials: We analyzed IC 50 values for 649 cell lines tested with AXI using the CTRPv2 database. The drug response of AXI in K562/Wild-Type (WT), K562/T315I mut, BaF3/WT, and BaF3/T315I mut cell lines was evaluated using the WST-8 assay. From the measured IC 50 value in those cell lines as the baseline concentration (conc), AXI and ASC conc were serially diluted. The dose-response matrix and ZIP synergy score were analyzed using SynergyFinder. Results We compared the sensitivity of 649 different cell lines to AXI in the CTRPv2 database and confirmed that the IC 50 of AXI in CML (median IC 50 = 0.31 uM) is 96 times lower than that of various cell lines derived from RCC (median IC 50 = 29.85 uM) (Fig. 1A). This result implies that AXI is more effectively inhibit CML cells at a lower conc than RCC cell lines or other cell lines. When measuring the inhibitory activityof AXI (Fig. 1B), AXI treatment showed 2.22 times lower IC 50 in the K562/T315I mut (IC 50 = 111.6nM) than in the K562/WT (IC 50 = 248nM). Also AXI treatment showed 4.73 times lower IC 50 in the BaF3/T315I mut (IC 50 = 84nM) than in the BaF3/WT (IC 50 = 397.4nM). Overall, the T315I carrying CML cell lines were much more sensitive to lower dose AXI than wild type CML cell lines. In the dose-response matrix analysis (Fig. 1C), AXI treatment alone at 100 nM inhibited the growth of the K562/T315 mut cell line by 38.42%, and ASC treatment alone at 100 nM, by 36.52%. Of note, the combination of AXI at 25nM and ASC at 12.5nM inhibited cell growth by 38.1%, and the combination of AXI at 12.5nM and ASC at 25nM showed 38.42% inhibition, which is similar to the inhibition achieved by 100nM of each drug alone. The result suggests that the combination of reduced dose of each drug, by 25% or 12.5% from the baseline IC 50 dose, showed equivalent inhibitor activity to the baseline IC 50 concentration of each drug monotherapy in the K562/T315 mut cell line. Similarly, when AXI and ASC were combined at a reduced concentration by a half or a quarter and tested in the BaF3/T315I mut cell line, a higher inhibitory activity was observed compared to the baseline IC 50 concentration of each drug monotherapy. The ZIP score was calculated as 31.7 and 20.64 in K562/T315 mut and BaF3/T315I mut cell line. Given that ZIP score was above 10 each, synergistic enhancement of inhibitory activity was demonstrated against T315I mutant CML cells between AXI and ASC. Conclusion: This result suggests synergistically enhanced inhibitory activity of dual blockade using AXI combined with ASC specifically for T315I mutant CML. This approach will be promising against CML cells carrying compound mutation with T315I mutation, which is highly resistant even to Ponatinib or Asciminib.

Impact of Mutations in Blood Cancer-Related Genes on Clinical Outcomes in Chronic Myeloid Leukemia in Chronic Phase (CML-CP) after ≥2 Tyrosine Kinase Inhibitors (TKIs) in the Ascembl Trial

CONCLUSIONS Somatic mutations in blood cancer-related genes are commonly detected in CML-AP. The most frequently mutated genes are RUNX1, ASXL1IKZF1 (submicroscopic deletions), and BCR:: ABL1 (kinase domain mutations). ASXL1 is the most frequently detected mutation at CML diagnosis (≈9%). However, the incidence and impact of mutations in blood cancer-related genes in patients (pts) with CML-CP previously treated with ≥2 TKIs have not been described since genomic studies have focused on pts at diagnosis or at progression. With >2 years of follow-up in the phase 3 ASCEMBL study, asciminib (ASC) has continued to demonstrate superior efficacy vs bosutinib (BOS) in pts with CML-CP after ≥2 prior TKIs, and analysis of cancer gene somatic mutations in this population has the potential to reveal additional insights into pts' response to study treatments or characteristics of the disease in later lines of therapy. Here we present a descriptive analysis of variants in the ASCEMBL pt population, including frequencies, co-occurrences, and association of mutations with clinical outcomes, specifically major molecular response (MMR) and treatment failure (TF; defined as discontinuation due to lack of efficacy or intolerance). Adults with CML-CP previously treated with ≥2 TKIs with intolerance of their last TKI or lack of efficacy per 2013 European LeukemiaNet recommendations and without BCR:: ABL1 T315I or V299L mutations were randomized 2:1 to ASC 40 mg twice daily or BOS 500 mg once daily. DNA was isolated from blood collected at baseline (BL) and end of treatment (EOT; defined as treatment discontinuation for any reason or completion of the study). A next-generation sequencing panel of 89 commonly mutated genes in blood cancers, including ABL1 and IKZF1 deletion, was used to detect somatic mutations that met criteria for pathogenicity. Only samples with BCR::ABL1IS ≥2%, which was the level demonstrated to detect mutations in leukemic cells, were tested. A multivariable Cox proportional hazards regression model adjusted for age, sex, treatment, and reason for prior TKI discontinuation (intolerance vs lack of efficacy) was used to determine the prognostic value of BL mutations. A total of 233 pts were randomized to ASC (n=157) or BOS (n=76). Of 162 pts (ASC, n=110; BOS, n=52) with available sequencing data at BL, 102 had ≥1 mutation: 69 (63%) on ASC and 33 (63%) on BOS. Overall, 159 mutations were detected in 23 genes at BL, ranging from 0 to 5 per pt. The median variant allele frequency (VAF) was 19.6% (range, 0.9%-58%). ASXL1 (in 31% of pts) and ABL1 kinase domain (in 17% of pts) mutations were the most frequently detected. Of 27 pts with ≥1 ABL1 mutation at BL, 85% had ≥1 other mutated cancer gene. Co-occurrence of ASXL1 and ABL1 mutations at BL was reported in 12 of 162 pts (7.4%), which was higher than the expected 2.9% based on these genes' individual frequencies in this data set. RUNX1 and IKZF1 mutations were rare in CML-CP at BL. Of 162 pts tested at BL, 73 had TF and available samples for analysis at EOT with only 3 pts discontinuing due to disease progression. The presence of any mutation at BL was significantly prognostic of TF in the total cohort of 162 pts (Figure 1). In an unadjusted analysis, the presence of an ASXL1 mutation with VAF >5% at BL was a prognostic factor of TF. In 73 pts with TF, most BL mutations were also detected at EOT (110/116 mutations in 48/54 carriers) (Figure 2). Of 15 pts who acquired ≥1 mutation at EOT, 9 acquired ABL1 mutations. No single mutations at an individual or gene level were prognostic of MMR. Mutations were not predictive of MMR or TF when pts were examined by their assigned treatment of ASC or BOS. ASXL1 mutations are most frequently detected at CML diagnosis and were enriched at BL in this study of pts with CML-CP previously treated with ≥2 TKIs, which is consistent with a role in TKI resistance. RUNX1 and IKZF1 mutations, which are associated with progression to accelerated or blast phase in CML, were very rare in this pt population, supporting their role in CML disease progression. The high frequency of co-occurrence of BCR:: ABL1 mutations and blood cancer gene mutations in CML-CP is an important finding since cancer gene mutations could modify response to TKIs in individual pts. The presence of any somatic blood cancer mutation at BL was prognostic of TF. These mutations may serve as potential biomarkers that can identify pts who may be at higher risk of TF in later lines of therapy.

Olverembatinib (HQP1351) Combined with Chemotherapy Is an Effective and Safe Treatment in Patients with Philadelphia Chromosome-Positive (Ph +) Acute Lymphoblastic Leukemia (ALL) and Chronic Myeloid Leukemia in Lymphoid Blast Phase (CML-LBP) That Failed TKI-Based Regimens

Background The prognosis of relapsed or refractory (R/R) Philadelphia Chromosome-Positive (Ph+) acute lymphoblastic leukemia (ALL) and TKI-resistant chronic myeloid leukemia in lymphoid blast phase (CML-LBP) remains poor, with lower rates of complete remission (CR) and shorter durations of response. Olverembatinib is a novel third-generation tyrosine kinase inhibitor (TKI) that has been demonstrated to effectively target a wide range of BCR::ABL1 kinase mutations, especially T315I, in patients with CML in the chronic or accelerated phase resistant to either imatinib or second-generation TKIs. However, data on olverembatinib-based regimens in R/R Ph+ ALL and CML-LBP are limited. Here, we report the efficacy and safety of olverembatinib, combined with chemotherapy, in patients with Ph+ ALL or CML-LBP that failed prior TKI-based therapies. Methods A retrospective study was conducted in adults with Ph + ALL or CML-LBP who failed imatinib- or second-generation TKI-based chemotherapy and subsequently received olverembatinib-based chemotherapy. Per NCCN 2023 guidelines: refractory disease was defined as CR not achieved at the end of induction; relapsed disease, reappearance of blasts in the blood or bone marrow (> 5%) or any extramedullary site after CR; molecular resistance, persistent or rising BCR::ABL1 transcript (> 1-log) by real-time quantitative polymerase chain reaction (RT-qPCR); and complete molecular response (CMR), BCR::ABL1 transcript < 0.01% by RT-qPCR. The primary endpoints of this study were CR rates in R/R patients and CMR rates in molecular-resistant patients. Results From February 2022 to May 2023, 31 patients were included in this study, including 24 (77%) with TKI-based chemotherapy-failed Ph + ALL; 7 (23%) with TKI-resistant CML-LBP; 15 (48%) with R/R disease; and 16 (52%) with molecular-resistant disease. A total of 17 (57%) patients were male, and the median (range; IQR) age was 46 (20-72; 30-64) years. Among 30 patients screened for BCR::ABL1 kinase domain mutations before administration of olverembatinib, 6 (19%) had no mutations; 15 (48%) had T315I; and 9 (29%) had other mutations, including E255K (n = 2), G250E(n = 2), D241E (n = 1), Y253H (n = 1), F317L (n = 1), F359C(n = 1), and Y253Fplus M351T (n = 1). The 15 R/R patients (11, Ph + ALL; 4, CML-BP) received olverembatinib 30 or 40 mg on alternate days combined with VP (vindesine 4 mg once per week for 4 weeks and prednisone 1 mg/kg for 3 weeks and tapered at the fourth). A total of 13 (86%) patients achieved CR after 4-week induction therapy. At a median (IQR) follow-up of 8 (4-13) months, 5 patients received allogeneic transplantation in CR, and were alive in CMR. A total of 8 patients received olverembatinib-based consolidation chemotherapy of either VP (n = 4) or hyper-CVAD (n = 4) after achieving CR. Six patients relapsed, of whom 4 died, and 1 remained in CMR. The 1-year relapse-free survival (RFS) and 1-year survival rates were 52% (95% CI, 21-83) and 68% (95% CI, 37-98), respectively. Among the 16 patients with molecular resistance to TKI-based chemotherapy (imatinib [n = 4]; dasatinib [n = 9]; flumatinib [n = 3]), 2 received olverembatinib monotherapy, 7 olverembatinib plus VP, and 7 hyper-CVAD, of whom 8 received subsequent allogeneic transplantation. At a median (IQR) follow-up of 9 (5-11) months, 1 of the patients receiving olverembatinib monotherapy progressed to hematologic relapse and was switched to CAR T-cell therapy; the other patient did not achieve CMR and underwent transplantation. Of the 14 patients receiving olverembatinib plus chemotherapy, 7 (50%) achieved CMR and 3 (21.4%) progressed to relapse, of whom 1 had hematological relapse and 2 developed central nervous system leukemia (CNSL) with bone marrow remission. A total of 7 patients received allogeneic transplantation, of whom 3 achieved and 4 did not achieve CMR before transplantation. After transplantation, all the 7 patients were in CMR except one who developed CNSL. The 1-year RFS and survival rates were 67% (95% CI, 42-91) and 93% (95% CI, 79-100), respectively. Among all patients, hematologic adverse events were readily manageable. Treatment-related nonhematologic severe adverse events were observed in 3 patients, including (each) stable angina pectoris, severe pneumonia, and fatal Klebsiella sepsis. Conclusions Olverembatinib-based chemotherapy is effective and safe in patients with R/R and molecular resistant Ph + ALL or CML-LBP.

Asciminib Enhances Its Treatment Efficacy Synergistically in the Treatment of Chronic Myeloid Leukemia Harboring ABL1 Kinase Domain Mutation When Combined with a Reduced Dose of Ponatinib, Dasatinib, or Bosutinib, but Not with Nilotinib or Imatinib

Background: Asciminib (ASC) monotherapy has been approved for chronic myeloid leukemia (CML) treatment in the patients who failed at least 2 lines of tyrosine kinase inhibitor (TKI) therapy. Theoretically, dual inhibition of the ABL1 kinase protein is plausible by combining ASC with ATP-binding pocket inhibitors (ABPIs) including Imatinib (IMA), Dasatinib (DAS), Nilotinib (NIL), Bosutinib (BOS) or Ponatinib (PON). A phase 1 study determined the appropriate dose of ASC with fixed doses of ABPIs (IMA, DAS, NIL). However, this approach of using full dose ABPIs with variable dose of ASC would raise a concern of tolerability given that all the toxicity from the doses of ABPI will be augmented by adding even half dose of ASC, evidenced by the FASCINATION trial (Ernst et al. EHA 2023). Questions still remained such as what is the best dose for combination? Is the combination of full dose ASC with reduced dose ABPIs effective? Is this combination effective to other ABL1 kinase domain mutant CML? For the ABL1 kinase domain (KD) mutant CML, which ABPI and what dose is more effective and more synergistic than other ABPIs? Methods and Materials: To investigate the combined effect of ASC with ABPIs, we determined the IC 50 values for ASC and ABPIs in the wild-type (WT) BaF3 BCR-ABL+ cell line, and utilized the IC 50 as baselines throughout the study. From the baseline IC 50s of ASC (3nM), IMA (150nM), NIL (10nM), BOS (10nM), DAS (0.8nM) and PON (0.6nM), serially diluted ASC alone, ABPIs alone or in combination were administered to BaF3 cell lines. We conducted IC 50 determining experiment in different combination of ASCs/ABPIs/combination in different concentration (conc) on eleven different KD mutant (KDM) BaF3 cell lines, including WT, G250E, E255K, T315A, M351T, F317L, F317V, H396P, Y253F, M244V, and T315I mutant cell lines. ZIP synergy scores for these cell lines were analyzed using SynergyFinder. Results We observed that the addition of 3 nM ASC to ABPI at baseline IC 50 conc led to a higher response (% cell growth inhibition) compared to treatment with ABPI alone at baseline conc, irrespective of the mutation. Moreover, the combined treatment of ABPI at half baseline conc with 3nM ASC demonstrated similar or even higher effects than ABPI alone at IC 50 baseline conc. These results suggest that double blockade allows for the reduction of ABPI dose by half while maintaining cell inhibition activity. According to its sensitivity to ASC monotherapy and combination, we have stratified the cell lines into 3 categories. High sensitive cell lines to ASC monotherapy (i.e. WT, G250E, and E255K) achieving 100% of response rate when combined with ABPI. Also, T315A mutation showed a significant synergistic effect (ZIP = 12.91~21.75) with the combination of ASC 3nM with a half conc of ABPI, resulting in 1.90-2.43 times higher inhibitory effect than ABPI monotherapy.Intermediate sensitive cell lines (i.e. M351T and F317L) displaying 69-85% inhibition efficiency with ASC 6nM combined with ABPI, without significant increases in all ABPI conc.Low sensitive cell lines (i.e. F317V, H396P, Y253F, M244V, and T315I) exhibiting unique resistance profiles that varied with different combinations. Notably, F317V and F317L mutations showed resistance to DAS. When combined with ASC 3nM, F317V showed restored sensitivity with IMA or NIL than with DAS, while F317L better with DAS. Moreover, the H396P, Y253F, and M244V mutations demonstrated higher sensitivities with BOS or DAS. Interestingly, in the combination treatment with ASC at 3 or 6 nM, with exception of the F317V and T315I mutations, both DAS and PON exhibited a potent efficacy. High resistance of T315I to ABPIs can be overcome with combination of ASC at baseline conc with reduced dose PON. In the case of T315I, the combination of 3nM ASC with a half conc PON resulted in a higher inhibitory effect than baseline conc PON alone. Conclusion: The present study demonstrated that a half of baseline conc ABPIs in combination with ASC is as effective as other ABPIs not just in WT but also in other ABL1 KDM CML cell lines. Different CML cell lines harboring different ABL KDM has different treatment spectrum on optimal ABPI drug as well as optimal drug conc. This result will be useful to design future clinical trial of dual blockade of ASC with other ABPIs considering the ABL KDM profiles. It also emphasizes the importance of ABL KDM testing prior to ASC monotherapy or combination with other ABPIs.

ASC2ESCALATE: A US Phase 2, Open-Label, Single-Arm, Dose-Escalation Study of Asciminib (ASC) Monotherapy in Patients (Pts) with Chronic Myeloid Leukemia in Chronic Phase (CML-CP) As Second-Line (2L) and First-Line (1L) Treatment

CONCLUSIONS Adenosine triphosphate (ATP)-competitive tyrosine kinase inhibitors (TKIs) have increased survival in CML, although resistance and intolerance remain challenges, which often necessitate switch to another TKI. Many pts discontinue 1L treatment due to resistance/intolerance and switch to 2L therapy, to which resistance is typically higher. A need exists for effective, safe treatment options for pts in 1L and 2L. ASC is thefirst approved BCR::ABL1 inhibitor that Specifically Targets the ABL Myristoyl Pocket (STAMP), with activity against most BCR::ABL1 kinase domain mutations conferring resistance to ATP-competitive TKIs, potentially providing a new early-line treatment option. ASC was first approved in the United States for pts with CML-CP after ≥2 TKIs at 80 mg once daily (QD) or 40 mg twice daily (BID) and for pts with CML-CP with the T315I mutation at 200 mg BID. A large, phase 1, dose-escalation trial (NCT02081378) demonstrated efficacy and tolerability of ASC across a broad range of dosages. Also, with >2 years of follow-up, ASC demonstrated superior efficacy and better safety and tolerability compared with bosutinib in pts with CML-CP without the T315I mutation after ≥2 prior TKIs in the phase 3 ASCEMBL study (NCT03106779). We describe the phase 2 ASC2ESCALATE trial investigating efficacy and safety of ASC with dose escalation in pts with CML-CP receiving ASC as their 2L or 1L therapy. This is a phase 2, open-label, multicenter, single-arm, dose-escalation study with sites in the United States (NCT05384587) (Figure). Pts receiving ASC as their 2L TKI must be adults with non-T315I CML-CP who discontinued their first ATP-competitive TKI for warning response ( BCR::ABL1IS >1%-10% after 6 months or >0.1%-1% after 12 months of 1L treatment), resistance ( BCR::ABL1IS >10% after 6-12 months or >1% or loss of major molecular response [MMR] after >12 months of 1L treatment), or intolerance (with BCR::ABL1IS >0.1% at screening). In addition to pts receiving ASC as their 2L TKI, a separate cohort of newly diagnosed pts will be included, and these pts may receive up to 4 weeks of prior treatment with imatinib or an approved second-generation TKI (Table). All pts will start treatment with ASC 80 mg QD. Pts achieving BCR::ABL1IS ≤1% at 6 months will continue at the same dose; pts not achieving BCR::ABL1IS ≤1% at 6 months will have their dose escalated to 200 mg QD. In pts not achieving MMR at 12 months, dose escalation from 80 to 200 mg QD or from 200 mg QD to 200 mg BID will occur or the pt will be taken off the study. If pts have grade ≥3 or persistent grade 2 toxicity refractory to optimal management, no dose escalation will occur, and pts will remain on ASC at their most current dose. This dose-escalation strategy applies to both the 2L and 1L cohorts. The primary endpoint is MMR rate at 12 months in pts using ASC as their 2L TKI. As all primary and secondary endpoints for the 2L cohort will be measured for the 1L cohort as secondary endpoints, the MMR rate at 12 months in pts receiving ASC as their 1L TKI is a secondary endpoint. Additional secondary endpoints are the same for both 2L and 1L cohorts and include MMR rates at 3, 6, 18, and 24 months; MMR rates by all scheduled time points; MR 2 ( BCR::ABL1IS ≤1%), MR 4 ( BCR::ABL1IS ≤0.01%), and MR 4.5 ( BCR::ABL1IS ≤0.0032%) at and by scheduled time points; time to and duration of MMR; time to treatment failure; progression-free survival; overall survival; and safety/tolerability. Exploratory endpoints include assessment of BCR::ABL1 mutations. ASC2ESCALATE is the first clinical trial designed to assess pts with CML-CP using ASC as their 2L TKI and will provide critical information on efficacy and safety of dose escalation in pts for whom certain response milestones are not met in 2L and 1L. Dose escalation of ASC in 2L and 1L is another treatment strategy and results from ASC2ESCALATE will add to the growing body of evidence for using ASC to treat pts with CML-CP. Recruitment for pts using ASC as their 2L TKI began November 2022, and as of July 10, 2023, 5 pts have been enrolled in this cohort; Up to 92 and 90 pts are estimated to enroll in the 2L and 1L cohorts, respectively, across 90 sites.

BCR::ABL1 kinase domain mutation testing and clinical outcome in a nationwide chronic myeloid leukemia patient population.

Acquired missense mutations in the BCR::ABL1 kinase domain (KD) may cause tyrosine kinase inhibitor (TKI) treatment failure. Based on mutation-specific in vitro derived IC50-values, alternative TKI may be selected. We assessed clinical practice of BCR::ABL1 KD mutation testing, clinical response in relation to IC50-values, and clinical outcome of tested patients. Patients from six Dutch CML reference centers and a national registry were included once a mutational analysis was performed. Reasons for testing were categorized as suboptimal TKI response, and primary or secondary TKI resistance. Four hundred twenty analyses were performed in 275 patients. Sixty-nine patients harbored at least one mutation. Most analyses were performed because of suboptimal TKI response but with low mutation incidence (4%), while most mutations were found in primary and secondary resistant patients (21% and 51%, respectively). Harboring a BCR::ABL1 mutation was associated with inferior overall survival (HR 3.2 [95% CI, 1.7-6.1; p < .001]). Clinically observed responses to TKI usually corresponded with the predicted TKI sensitivity based on the IC50-values, but a high IC50-value did not preclude a good clinical response per se. We recommend BCR::ABL1 KD mutation testing in particular in the context of primary or secondary resistance. IC50-values can direct the TKI choice for CML patients, but clinical efficacy can be seen despite adverse in vitro resistance.

The acquisition order of leukemic drug resistance mutations is directed by the selective fitness associated with each resistance mechanism

In Chronic Myeloid Leukemia, the transition from drug sensitive to drug resistant disease is poorly understood. Here, we used exploratory sequencing of gene transcripts to determine the mechanisms of drug resistance in a dasatinib resistant cell line model. Importantly, cell samples were collected sequentially during drug exposure and dose escalation, revealing several resistance mechanisms which fluctuated over time. BCR::ABL1 overexpression, BCR::ABL1 kinase domain mutation, and overexpression of the small molecule transporter ABCG2, were identified as dasatinib resistance mechanisms. The acquisition of mutations followed an order corresponding with the increase in selective fitness associated with each resistance mechanism. Additionally, it was demonstrated that ABCG2 overexpression confers partial ponatinib resistance. The results of this study have broad applicability and help direct effective therapeutic drug usage and dosing regimens and may be useful for clinicians to select the most efficacious therapy at the most beneficial time.

A phase 2 study of the combination of decitabine (DAC), venetoclax (VEN), and ponatinib in patients (Pts) with chronic myeloid leukemia (CML) in accelerated phase (AP)/myeloid blast phase (MBP) or Philadelphia-chromosome positive (Ph+) acute myeloid leukemia (AML).

e19044 Background: CML-AP/MBP and Ph+ AML are therapeutic challenges. Based on published pre-clinical data of synergism (Leonard et.al., Sci Transl Med 2016), we hypothesized that combining ponatinib with DAC and VEN might lead to high rates of response and facilitate allogeneic stem cell transplantation (SCT). Methods: The study (NCT04188405) enrolled pts ≥ 18 years of age with CML-AP, CML-MBP or Ph+ AML with ECOG PS ≤ 3 and adequate liver/renal function. Pts with significant uncontrolled cardiovascular comorbidities were excluded. Therapy was with DAC (20 mg/m2/day) x 5 days, VEN (400 mg equivalent dose/day) x 21 days, and ponatinib (45mg/day) x 21 days in cycle 1 and then continuously in cycles 2-24. Each cycle was for 28 days. Ponatinib was reduced to 30mg/day for pts in CR/CRi and to 15 mg/day for pts in CMR. Intrathecal prophylaxis with 4 doses of cytarabine was recommended. The primary objective was to determine the overall response rate (ORR) with the regimen. Results: From Jul 2020 to Jan 2023, 15 pts were treated, 4 with CML-AP, 10 with CML-MBP and 1 with Ph+ AML (Table). 11 pts (73%) had prior TKI exposure, including 4 pts with prior ponatinib. 5 of 13 tested pts (38%) had an ABL1 kinase domain mutation (T315I, Q252H, L384M in 1 pt each and E255K in 2 pts). The median number of cycles received was 3 (range, 1-7). 11 pts (73%) responded; 6 pts (40%) with CR/CRi and 5 (33%) with MLFS. Best response was achieved after cycle 1 in 9 of 11 (82%) responders. All 4 pts with CML-AP responded, and 9 of 11 pts (82%) with prior TKI exposure responded. Four pts underwent subsequent SCT. At a median follow-up of 9.8 months, the median overall survival (OS) was 11.0 months, and the estimated 10-month OS was 63%. The median relapse-free survival was 5.7 months. Among the 11 responders, 5 relapsed (one after SCT), and 6 are in ongoing response (3 after SCT). One pt had a related grade 3 mucositis. No grade 4-5 related non-hematological adverse events were observed. Of the 10 pts who received &gt; 1 cycle of therapy, 5 pts had delay (&gt;1 week) in initiation of subsequent cycles due to cytopenias. The 60-day mortality rate was 0%. Two pts died on study, one from leukemia and one from infection while in MLFS. Conclusions: A triplet combination of DAC, VEN and ponatinib was well-tolerated and resulted in an ORR of 73% in this poor-risk population of pts with advanced Ph+ leukemias. Clinical trial information: NCT04188405 . [Table: see text]

Droplet digital polymerase chain reaction improves the detection of BCR-ABL1 kinase domain mutation in Philadelphia chromosome-positive acute lymphoblastic leukemia.

Sanger sequencing (SS) is the most frequently used method for detecting ABL1 kinase domain (KD) mutations in patients with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). However, it cannot detect low levels of mutation. Recently, droplet digital polymerase chain reaction (ddPCR) has been developed as a sensitive technique for detecting mutations in hematological neoplasms. The aim of our study was to explore the value of ddPCR in detecting ABL1 KD mutations. We compared the results of SS and ddPCR in detecting ABL1 KD mutations in a consecutive cohort of 65 adolescent and adult patients with Ph+ ALL treated with intensive multiagent chemotherapy plus TKIs. At diagnosis, SS and ddPCR identified 1 (1.5%) and 26 (40%) out of 65 patients with positive ABL1 KD mutations, respectively. Patients with T315I mutations detected by ddPCR at diagnosis all developed SS-detectable T315I mutations during treatment with first- or second-generation TKIs, and non-T315I mutations detected by ddPCR at diagnosis displayed a limited prognostic impact. Our study demonstrates that ddPCR is a highly sensitive and accurate mutation detection method and the presence of T315I mutations before treatment shows prognostic significance in the context of first- or second-generation TKIs.

Impact of additional genetic abnormalities at diagnosis of chronic myeloid leukemia for first-line imatinib-treated patients receiving proactive treatment intervention.

The BCR::ABL1 gene fusion initiates chronic myeloid leukemia (CML); however, evidence has accumulated from studies of highly selected cohorts that variants in other cancer-related genes are associated with treatment failure. Nevertheless, the true incidence and impact of additional genetic abnormalities (AGA) at diagnosis of chronic phase (CP)-CML is unknown. We sought to determine whether AGA at diagnosis in a consecutive imatinib-treated cohort of 210 patients enrolled in the TIDEL-II trial influenced outcome despite a highly proactive treatment intervention strategy. Survival outcomes including overall survival, progression-free survival, failure-free survival, and BCR::ABL1 kinase domain mutation acquisition were evaluated. Molecular outcomes were measured at a central laboratory and included major molecular response (MMR, BCR::ABL1 ≤0.1%IS), MR4 (BCR::ABL1 ≤0.01%IS), and MR4.5 (BCR::ABL1 ≤0.0032%IS). AGA included variants in known cancer genes and novel rearrangements involving the formation of the Philadelphia chromosome. Clinical outcomes and molecular response were assessed based on the patient's genetic profile and other baseline factors. AGA were identified in 31% of patients. Potentially pathogenic variants in cancer-related genes were detected in 16% of patients at diagnosis (including gene fusions and deletions) and structural rearrangements involving the Philadelphia chromosome (Ph-associated rearrangements) were detected in 18%. Multivariable analysis demonstrated that the combined genetic abnormalities plus the EUTOS long-term survival clinical risk score were independent predictors of lower molecular response rates and higher treatment failure. Despite a highly proactive treatment intervention strategy, first-line imatinib-treated patients with AGA had poorer response rates. These data provide evidence for the incorporation of genomically-based risk assessment for CML.