Deep Molecular Remission Research Articles

Management of Chronic Myeloid Leukemia that is Intolerant or Resistant to Front-Line Treatment

With advances in treatment for chronic myeloid leukemia (CML), the natural history of chronic phase (CP) CML has changed, with most individuals expected to live a normal life expectancy. The goal of therapy for most is to achieve a long-term deep molecular response (DMR) with the potential for medication discontinuation and treatment-free remission (TFR).1 Currently, six oral therapies have been approved for CP-CML in Canada: (1) imatinib, a first-generation tyrosine kinase inhibitor (TKI); (2) dasatinib, (3) nilotinib, and (4) bosutinib, the second-generation TKIs (2G-TKIs); (5) ponantinib, a third-generation TKI; and (6) asciminib, specifically targeting the ABL Myristoyl pocket (STAMP) inhibitor. Classically, treatment for CP-CML has consisted of front-line imatinib and switching to a 2G-TKI upon treatment resistance or intolerance. Increasingly, patients are being prescribed an upfront 2G-TKI with the goal of achieving quicker and deeper molecular remissions and a TFR.2 Challenges arise in CML when treatment with either two TKIs (imatinib + 2G-TKI) or one 2G-TKI fails, given the lack of evidence to inform clinical decision-making at this juncture. This paper aims to define TKI failure and help guide the selection of second-line treatment after failure of front-line therapy.

Prognostic Role of Human Leukocyte Antigen Alleles and Cytokine Single-Nucleotide Polymorphisms in Patients with Chronic Myeloid Leukemia Treated with Tyrosine Kinase Inhibitor Drugs.

Tyrosine kinase inhibitor (TKI) drugs have significantly improved chronic myeloid leukemia (CML) outcomes. Neopeptides from CML cells may induce specific immune responses, which are crucial for deep molecular (DMR) and treatment-free remission (TFR). In this study of Ethiopian patients with CML (n = 162), the HLA alleles and single-nucleotide polymorphisms of five cytokines revealed significant associations with clinical outcomes. Clinically unfavorable outcomes correlated with HLA alleles A*03:01/02, A*23:17:01, B*57:01/02/03, and HLA-DRB4*01:01 (p-value = 0.0347, p-value = 0.0285, p-value = 0.037, and p-value = 0.0127, respectively), while HLA-DRB4*01:03:01 was associated with favorable outcomes (p-value = 0.0058). After assigning values for the 'low', 'intermediate', and 'high' gene expression of the SNPs' respective cytokine genes, Kaplan-Meier estimates for relapse-free survival, adjusted for age, treatment duration, and relapse risk among patients after the administration of TKIs, indicated that a gene expression ratio above the overall median of TNF-α, IL-6, and the combination of TGF-β1/IL-10, IFNγ, and IL-6/IL-10 TGF-β1 was correlated with a higher likelihood of treatment failure ((RR: 3.01; 95% CI: 1.1-8.3; p-value = 0.0261) and (RR: 2.4; 95% CI: 1.1-5.2; p-value = 0.022), respectively). Multi-SNPs, surpassing single-SNPs, and HLA allele polymorphisms showed promise in predicting outcomes of patients with CML during TKI treatment, prompting further exploration into their potential utility.

Maternal and Foetal Outcomes of Newly Diagnosed Chronic Myeloid Leukemia During Pregnancy and its Long-Term Impact.

Diagnosing Chronic Myeloid Leukemia (CML) during pregnancy presents challenges for both haematologists and obstetricians. Limited data exists regarding the management of pregnancy-associated CML in low- and middle-income countries (LMICs). This study aimed to assess pregnancy, foetal, and long-term disease outcomes in female patients newly diagnosed with CML in the chronic phase (CML-CP) during pregnancy. A retrospective analysis was conducted on female CML-CP patients presenting between January 2002 and December 2022 at our institution. Inclusion criteria encompassed patients newly diagnosed with CML-CP during pregnancy. Data pertaining to pregnancy outcomes, foetal development, and disease progression were analysed through a comprehensive review of medical records. Among the female CML patients, thirteen were diagnosed with CML-CP during pregnancy. The median patient age was 24years (range: 20-35). Diagnoses occurred in the first trimester for six patients, the second trimester for three, and the third trimester for four. Outcomes included five elective terminations (38.5%), five pre-term deliveries (38.5%), and three full-term deliveries (23.1%). Management included observation in 6 (46.3%), hydroxyurea in 3 (23.1%), imatinib in 3 (23.1%) and Interferon-α (IFN-α) in 1 (7.7%) patients. Noteworthy obstetric complications encompassed threatened abortion with intrauterine foetal death (IUFD) (1 patient), intrauterine growth retardation (IUGR) (3 patients), oligohydramnios (2 patients), antepartum haemorrhage (1 patient), placental abruption (1 patient), and postpartum haemorrhage (3 patients). At a median follow-up duration of 10.7years, 11 patients were at a major molecular remission (including 2 patients with deep molecular remission) and two patients progressed to the accelerated phase. The diagnosis and management of CML during pregnancy is a complex and challenging task that requires collaboration between haematologists and obstetricians. Effective management of newly diagnosed CML-CP during pregnancy is contingent on the trimester of presentation. Further research and collaboration are warranted to develop standardised guidelines for managing pregnancy-associated CML, particularly in LMICs.

European Stop Tyrosine Kinase Inhibitor Trial (EURO-SKI) in Chronic Myeloid Leukemia: Final Analysis and Novel Prognostic Factors for Treatment-Free Remission.

Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned co-primary or secondary analyses are not yet available. Clinical Trial Updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported.The European Stop Kinase Inhibitors (EURO-SKI) study is the largest clinical trial for investigating the cessation of tyrosine kinase inhibitors (TKIs) in patients with chronic myeloid leukemia in stable deep molecular remission (DMR). Among 728 patients, 434 patients (61%; 95% CI, 57 to 64) remained in major molecular response (MMR) at 6 months and 309 patients of 678 (46%; 95% CI, 42 to 49) at 36 months. Duration of TKI treatment and DMR before TKI stop were confirmed as significant factors for the prediction of MMR loss at 6 months. In addition, the type of BCR::ABL1 transcript was identified as a prognostic factor. For late MMR losses after 6 months, TKI treatment duration, percentage of blasts in peripheral blood, and platelet count at diagnosis were significant factors in multivariate analysis. For the entire study period of 36 months, multiple logistic regression models confirmed duration of treatment, blasts, and transcript type as independent factors for MMR maintenance. In addition to the duration of treatment, transcript type as well as blasts in peripheral blood at diagnosis should be considered as important factors to predict treatment-free remission.

VEXAS syndrome: complete molecular remission after hypomethylating therapy

The VEXAS syndrome, a genetically defined autoimmune disease, associated with various hematological neoplasms has been attracting growing attention since its initial description in 2020. While various therapeutic strategies have been explored in case studies, the optimal treatment strategy is still under investigation and allogeneic cell transplantation is considered the only curative treatment. Here, we describe 2 patients who achieved complete molecular remission of the underlying UBA1 mutant clone outside the context of allogeneic HCT. Both patients received treatment with the hypomethylating agent azacitidine, and deep molecular remission triggered treatment de-escalation and even cessation with sustained molecular remission in one of them. Prospective studies are necessary to clarify which VEXAS patients will benefit most from hypomethylating therapy and to understand the variability in the response to different treatment strategies.

ELN Treatment Milestones in Chronic Myeloid Leukemia Are Prognostic for Achieving Deep Molecular Response and Treatment-Free Remission in Routine Care: Results of the German CML Registry

Introduction: Tyrosine kinase inhibitor (TKI) induced molecular response (MR) within the first months after diagnosis of chronic myeloid leukemia (CML) is critical for long-term outcome prediction. Aim: To analyze the prognostic relevance of MR according to European Leukemia Net (ELN) treatment milestones in CML patients in routine care. Methods: Within the German CML registry, adult patients (pts) with CML newly diagnosed between 2013 and 2019 were registered and followed annually. Endpoints included time to MR, progression and death. MR was analyzed according to the ELN recommendations 2013 with the focus on the BCR::ABL1 (International Scale, IS) milestones 10% (EMR, early molecular response) at 3 months (M3), 1% (MR 2) at 6 months (M6) and 0.1% (MMR, major molecular response) at 12 months (M12) using the cumulative incidence function. MR at milestones were correlated to achievement of DMR (deep molecular remission, defined as BCR::ABL1 IS< 0.01%). Results: A total of 540 pts have been registered from 91 centers. Thirty pts had to be excluded (inclusion failure), 510 pts were evaluable for baseline analyses and 490 pts for MR analyses. Median age at diagnosis was 59 years (range 16-100), 264 pts of 510 pts (52%) were male. Median follow-up was 5.4 years. First-line treatment included imatinib ([n=209 (42%)], nilotinib [n=141 (29%)], dasatinib [n=146 (29%)], HU only (n=2 (0,4%)], and was unknown for 12 pts. In total, 473 (97%) of 490 pts achieved an EMR after a median of 3 months (mth) [range (r) 0-55 mth], 461 (94%) pts MR 2 [median 5 mth (1-67 mth)], and 446 (91%) pts MMR [median of 9 mth (1-82 mth)]. DMR was achieved in a total of 384 (78%) pts after a median of 15 mth (1-96 mth) of which at least 282 (58%) had a MR level according to MR 4.5 (BCR::ABL1 <0.0032%). Milestones M3, M6, and M12 were achieved in 245 (50%), 292 (60%), and 297 (61%) pts, respectively (see Table). DMR was achieved in 208 (85%), 259 (89%) and 277 (93%) pts who reached M3, M6, and M12, respectively. In all other pts only 176 (72%), 125 (63%), and 107 (55%) pts achieved DMR. 61 (12%) pts stopped TKI-treatment with the aim of treatment-free remission (TFR). Of these pts, 40 (66%), 47 (77%), and 52 (85%) pts achieved M3, M6, and M12 before TKI stop, respectively. 26 (43%) pts lost MMR in TFR. Progression to either accelerated phase (AP) or blast phase (BP) was documented for 9 patients (6 BP, 3 AP). Progression was more frequent in pts without achievement of milestones. At M3 2.4% vs. 1.2%, M6 3.5% vs. 0.7% and M12 4.7% vs. 0% of pts progressed. 48 deaths were reported. Only one death was clearly CML-related. This pt reached M3 but not M6 and M12, and died in BP. 7 pts developed BCR::ABL1 mutations. According to milestones, mutations were found in 1.2%, 1%, and 1% with achievement of M3, M6 and M12 vs. 1.6%, 1.5%, and 1.6% of pts not achieving the respective milestones. In addition, 126 pts (29%) lost MMR without being in TFR regardless of whether the milestones had been reached or not. Reasons for MMR-loss were variable (dose-reduction because of intolerance, incompliance, age) so that no correlation to treatment milestones can be made. Conclusion: Achieving ELN milestones in CML in routine care demonstrated a better outcome for pts with higher DMR rates, lower rates for progression rates and kinase domain mutations. DMR is of importance for stopping treatment in CML. Consequently, the rate of pts who could start TFR after achieving the ELN-milestones was 2-3 times higher than of pts who failed milestones or with unknown MR results.

Phase 2 Study of Dasatinib with and without Venetoclax in Patients with Early Chronic Phase Chronic Myeloid Leukemia (ECP-CML)

Background Treatment-free remission (TFR) is a therapeutic goal for patients with ECP-CML once patients maintain a deep molecular remission (DMR) on tyrosine kinase inhibitors (TKIs). Only a fraction of patients (pts) achieved sustained (3+ years) molecular remission with 4.0- to 4.5-log reduction (MR4.5). Preclinical studies showed that BCL-2 is a key survival factor for CML stem/progenitor cells. Therefore, the combination of TKIs and venetoclax, targeting BCR::ABL1 and BCL-2, has the potential to significantly improve DMR and subsequently the rates of TFR. The aim of this study is to evaluate the safety and efficacy of the combination. Methods Patients with ECP-CML or accelerated phase (AP; defined by clonal evolution only) were eligible; pts with < 1 month of prior TKI therapy were eligible. Pts received single-agent dasatinib 50 mg/d for the first 3 months. From Month 4 and onwards, venetoclax was administered from Days 1 to 14 each month concomitantly with dasatinib for a total of 3 years. The first 16 patients received venetoclax 200 mg/d, with a rapid ramp-up starting 50 mg on Day 1, then 100 mg on Day 2, then 200 mg daily on Day 3 and onwards. Due to neutropenia observed with continuous dose of venetoclax 200 mg/d, the protocol was ameded: from patient #17 and onwards, venetoclax was administered at 200 mg/d for 14 days per month and upon achievement of MR4.5, venetoclax was reduced to 100 mg/d for 7 days per month. After 3 years of combination, patients received single-agent dasatinib. TFR was offered after ≥ 3 years of DMR. The primary endpoint was the rate of major molecular response (MMR) by 12 months of the combination. The secondary endpoints were the 12-month rate of MR4.5 and the cumulative overall rate of MR4.5; the rates of MR4.5 at 6-, 12-, 18-, 24, and 36-months of the combination; the proportion of patients with sustained MR4.5 of 3 years and more; the TFR rate; overall survival; and the safety of the combination. Cumulative response rates were calculated with treatment failure as a competing event. Failure-free survival (FFS) was calculated from the initiation of therapy to loss of major cytogenetic response (MCyR), transformation to AP or blast phase, discontinuation of therapy for any reason except of TFR, or death. Results Sixty-five pts were enrolled and treated between April 2018 and August 2021. The median age at treatment initation was 46 years (range, 23-73) and 57% of the pts were females. Twenty-one (32%) pts had splenomegaly, 46 (71%) had low Sokal risk disease and 3 (5%) had high Sokal risk disease (Table 1). By 12 months of the combination, 85% of the pts had achieved MMR or deeper response. The median time to MMR, MR4, and MR4.5 were 6.2 months, 13 months, and 23.9 months from the time of TKI start, respectively. After a median follow-up of 25.7 months (range, 8.7-48.1), two pts discontinued therapy due to adverse events (GI intolerance, and pleural effusion); the 2 -year FFS was 96%. One pt switched TKI therapy due to the lack of 18-month complete cytogenetic response (CCyR); no loss of CCyR, transformation, or death was observed. We then compared the outcomes with the combination to the outcomes of 85 pts treated with single-agent dasatinib. From the start of TKI therapy, the 12-month cumulative MMR rates were 78% and 79% in the dasatinib single-agent and dasatinib + venetoclax groups, respectively. The cumulative 24-month rates of CCyR, MMR, MR4, and MR4.5 are summarized in Table 2. In the dasatinib single-agent group, 3 pts switched TKI therapy due to the lack of 12-month MCyR, 18-month CCyR, and the acquisition of ABL1 mutation (1 each); 3 pts switched TKI therapy due to loss of CCyR; 2 pts died during therapy (deaths unrelated to CML). Within one year of therapy, the incidence of grade 1/2 neutropenia (absolute neutrophil count, below lower range of normal to 1,000 x10 9/L) was 21% and 52% in the dasatinib single agent and dasatinib + venetoclax groups, respectively ( P<0.001); the incidence of grade 3/4 neutropenia (absolute neutrophil count, below 1,000 x10 9/L) was 8% and 17%, respectively ( P<0.001). There was no incidence of neutropenic fever in the dasatinib + venetoclax. Conclusion The combination of dasatinib 50 mg/d and venetoclax is safe and effective in pts with ECP-CML. With a median follow-up of 2 years, the cumulative resposne of dasatinib + venetoclax is similar to that of dasatinib single-agent without adding significant toxicities. Further follow-up is needed for the evaluation of TFR.

Orelabrutinib, Fludarabine, Cyclophosphamide, and Obinutuzumab (OFCG) for First-Line Treatment of Chronic Lymphocytic Leukemia: A Multicenter, Investigator-Initiated Study (cwCLL-001 Study)

Background: A phase II trial has shown, first-line treatment with iFCG (ibrutinib, fludarabine, cyclophosphamide, and obinutuzumab) led to a bone marrow (BM) undetectable minimal residual disease (uMRD) rate of 98% (44/45) as best response in treatment-naïve patients with chronic lymphocytic leukemia/small lymphocytic leukemia (CLL/SLL). But this study[1] does not include patients with unmutated immunoglobulin heavy⁃chain variable region gene ( IGHV) and/or del(17p)/ TP53 mutation, and ibrutinib was the first generation BTK (BTKi) which had relatively high off-target adverse effects (AEs). Our study has demonstrated the efficacy and safety of the second generation BTKi orelabrutinib in patients with relapsed/refractory CLL [2]. Therefore, we used orelabrutinib (O), and initiated the phase II OFCG trial in younger, fit patients with previously untreated CLL/SLL unrestricted by IGHV mutation status and del(17p)/ TP53 aberrations (cwCLL-001 study, NCT05322733). Methods：This is a multicenter, open-label, non-randomized phase II study for previously untreated patients with CLL/SLL without restriction by del(17p)/ TP53 aberrations and/or IGHV mutation status. A 7-day lead-in period with single-agent oral orelabrutinib（150 mg daily）was given in all patients, followed by OFCG regimen which consisted of orelabrutinib, intravenous Obinutuzumab, fludarabine and cyclophosphamide for up to three 28-day cycles. MRD was detected by 8 multi-color flow cytometry (FCM) at 10 -4 sensitivity and cloneSEQ using NGS (next generation sequencing) assay with a sensitivity of up to 10 −6. If the patients could achieve undetectable MRD in BM, they will discontinue FC and receive 3 cycles of OG (orelabrutinib and obinutuzumab). Otherwise, the patients will receive another 3 cycles of OFCG. Patients will discontinue obinutuzumab if they achieve BM uMRD after 6 cycles and continue six additional cycles of orelabrutinib; all other patients will receive six cycles of OG. Patients who achieved BM uMRD after 12 cycles discontinue all treatments, otherwise will continue orelabrutinib for up to additional 1 year (Fig. 1). The primary endpoint is the percentage of uMRD in BM after 6 cycles by FCM (at 10 -4 sensitivity). The secondary endpoint is the percentage of uMRD in BM and peripheral blood (PB) at other key time points by FCM, best overall response rate (ORR), complete response rate (CRR), progression-free survival (PFS), 2- and 3-year PFS rate, duration of response (DOR), overall survival (OS), 3-year OS rate as well as safety. The percentage of uMRD by NGS is an exploratory endpoint. Results：From 30 May 2022 to 30 Jun 2023, 25 patients were enrolled. Median age was 49 years old (range, 23-62). Unmutated IGHV, TP53 mutation/del(17p) and del(11q) were detected in 56.5% (13/23), 16.0% (4/25), and 24.0% (6/25) of patients, respectively. At data cut off (30 Jun 2023), 19 patients completed 3 cycles of OFCG and were evaluable. 75% (15/20, 1 patient who off study was assessed at the end of C2 cycle) of patients achieved PB uMRD and 57.9% (11/19) achieved BM uMRD by FCM, ORR and CR/CRi rate was 100% (18/18) and 38.9% (7/18) (Fig. 2). One patient died after cycle 3 due to COVID-19 infection. 16 patients completed 6 cycles, 14 of them were evaluable and achieved deeper response irrespective of different strategies during cycle 4 to 6, PB uMRD and BM uMRD was 100% (14/14) and 85.7% (12/14) by FCM, and the ORR and CRR was 100% (14/14) and 57.1% (8/14), respectively. In IGHV unmutated subgroup, ORR was 100% (10/10) with 50% (5/10) CRR, 87.5% (7/8) patients achieved BM uMRD by FCM after cycle 6. The results of MRD response by NGS assay will be presented in the formal report. 91.7% patients experienced at least one treatment emergent AE at the data cutoff. Most AEs were mild and tolerable. The most frequent AEs (≥10% of any grade) were neutropenia, leukopenia and thrombocytopenia. No bleeding or cardiovascular AEs greater than grade 3 occurred in these patients. Overall, OFCG demonstrated a favorable safety profile. Conclusion：This is the first clinical trial exploring the efficacy and safety of the second generation BTKi plus chemoimmunotherapy in patients with CLL. The OFCG regimen shows a rapid and deep molecular remission with a pleasant safety profile in the TN CLL patients including the ones with unfavorable factors.

Cellular Barcoding of JAK2-V617F Hematopoietic Stem Cells Reveals No Substantial Preferences in the Contribution of Individual Stem Cell Clones to Erythroid Versus Megakaryocytic Lineages

JAK2-V617F is the most frequent driver gene mutation in patients with myeloproliferative neoplasms (MPN) but the phenotypic manifestation is heterogeneous with some patients presenting as polycythemia vera (PV), whereas others with essential thrombocythemia (ET) or myelofibrosis (MF). JAK2-V617F is acquired in a hematopoietic stem cell (HSC) and leads to clonal expansion of HSCs and progenitor cells that gain dominance over unmutated hematopoiesis. We tested the hypothesis that the preferential expansion of megakaryopoiesis in ET versus erythropoiesis in PV could be due to the acquisition of JAK2-V617F in different subsets of HSCs with inherent bias towards megakaryopoiesis or erythropoiesis. In addition, we addressed the question of whether sensitivity or resistance to interferon-α (IFNα), currently the only treatment that can induce a deep molecular remission in some MPN patients, could be also due to heterogeneity in HSC subpopulations. We used a mouse model of JAK2-V617F driven MPN in combination with molecular barcoding that allowed us to monitor expansion and lineage contribution of individual HSC subclones. First, we genetically barcoded HSCs from our tamoxifen-inducible SclCre;JAK2-V617F ( VF) mice with a lentiviral vector, and transplanted 2'000 HSCs into lethally irradiated recipient mice. After 12 weeks we sequenced the barcodes in bone marrow (BM) progenitors by next generation sequencing (NGS). We observed a strong selection of 1-2 HSCs clones per recipient mouse, which alone contributed to > 80% of all the myeloid cells. Since cytokine storm after irradiation and aplasia during the reconstitution with lentivirally transduced BM may favor such oligo-clonal dominance observed in this experiment, we characterized the clonal composition of JAK2-mutant HSCs in non-transplanted MPN mice. To this end, we crossed a CRISPR-based genetic barcoding mouse line ( CARLIN) (Bowling, S. et al, Cell 2020) with our VF mice to obtain VF;CARLIN mice. Cas9-mediated barcoding in VF;CARLIN or in CARLIN mice with wildtype Jak2(WT;CARLIN) was induced with doxycycline and immediately followed by the activation of the JAK2-V617F by tamoxifen (Figure 1A). After 12 weeks, when the mice developed full PV phenotype (hemoglobin 195 g/L; platelets 4.3x10 12 /L and neutrophils 10.5x10 9/L), we performed scRNAseq on lin- cKit+ BM cells using the 10X-platform to retrieve the cellular barcodes and transcriptomic profiles. MPN hematopoiesis in these non-transplanted VF;CARLIN mice was polyclonal (Figure 1B). A total of > 500 barcoded clones per mouse were detected and in both VF;CARLIN and WT;CARLIN mice, but ~5 individual barcodes accounted for 25% of the cells in all progenitor subsets analyzed (Figure 1B). Overall, clonal composition was similar between WT;CARLIN and VF;CARLIN mice, indicating that clonal selection was not more prominent in JAK2-mutant mice. Importantly, in the offspring of HSCs that accounted for 25% of the cells, we did not observe preferential presence of some barcodes in MkP versus EryP and other barcodes in EryP versus MkP, arguing against the model predicting that erythrocytosis and thrombocytosis originated in different subsets of biased HSCs. To investigate the question of whether sensitivity or resistance to IFNα could be also due to heterogeneity in HSC subpopulations, we transplanted BM cells from these barcoded VF;CARLIN mice together with an excess of WT competitor cells into irradiated WT recipient mice, and treated them with IFNα or vehicle for 16 weeks. IFNα normalized hemoglobin (122 g/L) compared to vehicle (177 g/L) and reduced VF;CARLIN chimerism in granulocytes from 85% to 44%. BM cells were harvested and the clonal composition is currently being analyzed by NGS in order to identify IFNα resistant and sensitive subclones. Our barcoding analyses of individual HSCs showed that, during recovery after BM transplantations, a few VF clones dominated, whereas induction of VF expression in situ without transplantation resulted in a polyclonal disease that showed a similar number of HSCs actively contributing to hematopoiesis as in a control experiment with WT mice. The lack of substantial preference in the clonal contribution of individual HSCs to erythropoiesis versus megakaryopoiesis in VF mice suggests that PV and ET are not caused by acquiring JAK2-V617F in HSCs with a pre-existing lineage bias.

Mechanism of Late Recurrences of Chronic Myeloid Leukemia after Discontinuation of TKI Therapy: BCR::ABL1 Ins35bp Loss-of-Function Splicing Mutation and Regulatory T Cells

Introduction: Treatment-free remission (TFR) as one of the goals of treatment of BCR::ABL-positive chronic phase chronic myeloid leukemia (CML) has been proposed. Actually, approximately 40-60% of CML patients maintain TFR without recurrence after discontinuation of tyrosine kinase inhibitors (TKIs). In some of such patients, there are fluctuated cases in which BCR::ABL positive clones continue to be detected for long periods at levels of international scale (IS) BCR::ABL < 0.1%. Such fluctuated cases show no apparent molecular recurrence and maintain TFR for a long time, but they have also been shown to be more likely to eventually develop late recurrence (Haematologica 2022, Blood adv. 2020). We have reported that a higher percentage of PD-1 positive CD8 + T cells before and after TKI discontinuation is associated with molecular recurrence, and together with the possibility of assessing host immune response capacity after TKI discontinuation by using regulatory T (Treg) cells (Fujioka, et al. Cancers 2021). On the other hands, mutations in BCR::ABL caused by the alternative splicing, such as BCR::ABL Ins35bp, are known to be one of the TKI resistance in CML. Loss of BCR::ABL kinase activity is thought to result in loss of TKI sensitivity and thus failure to achieve deep molecular remission (Yuda, et al. Cancer Science 2017). In this study, we analyzed the mutations that cause splicing variants such as BCR::ABL Ins35bp and host immune response in CML patients to elucidate molecular mechanisms in the long-term maintenance of TFR and late recurrence. Methods: Twenty-four CML patients who discontinued TKI treatment for any reason at Akita University Hospital were included in the study. The trends of IS level after TKI discontinuation were monitored, and immune profile analysis by multi-color flow cytometry (FCM) was performed in all patients. Mutation analysis of the BCR::ABL region including splicing variants was performed in selected patients. Mutation analysis was performed on samples with sufficient cell count and IS > 0.0032% (MR 4.5), and the BCR::ABL region of these samples was amplified by long nested PCR and then analyzed by the next-generation sequencer (NGS). Results: First, BCR::ABL Ins35bp was evaluated by mutation analysis. Among CML patients who underwent mutation analysis, BCR::ABL Ins35bp was detected in all cases in which the samples could perform analysis at newly diagnosis (ND). Even after TKI discontinuation, a certain percentage of BCR::ABL Ins35bp was detected in all patients, and the percentage of BCR::ABL Ins35bp increased at the time of recurrence regardless of whether early or late. These results suggest that function-dead BCR::ABL Ins35bp clones may remain in CML patients with detectable BCR::ABL under and after TKI treatment, and eventually leading to recurrence with transcriptional promotion of native BCR::ABL. On the other hand, there were cases in which the BCR::ABL Ins35bp was lost at the time of recurrence. There is a possibility of immunological involvement in such cases. CML patients with detectable BCR::ABL in the TFR for a long time had a decreased immunosuppressive Treg cells, while analysis using late recurrence increased Treg cells prior to recurrence (figure). These suggest the presence of cases in which early recurrence is suppressed by host immune responses. Conclusion: Although the loss of function mutations such as BCR::ABL Ins35bp was previously thought to occur after TKI administration as a cause of TKI resistance, this analysis reveals that the clone is included from the ND before TKI treatment. Examining the BCR::ABL Ins35bp clone at ND may be possible to predict the possibility of recurrence after TFR. Host immune capacity, as assessed by the kinetics of Treg after TKI discontinuation, may contribute to prolonged TFR. Thus Treg monitoring may be important in predicting molecular recurrence, especially in BCR::ABL Ins35bp cases.

A decade of CD4+ chimeric antigen receptor T-cell evolution in two chronic lymphocytic leukemia patients: were chronic lymphocytic leukemia cells present?

On Feb 2, 2022, Nature published the paper titled "Decade-long leukemia remissions with the persistence of CD4+ CAR T-cells" (Nature. 2022;602:503-9. doi: 10.1038/s41586-021-04390-6). According to the results presented, it could be argued that "chimeric antigen receptor (CAR) T-cells can actually cure patients with chronic lymphocytic leukemia (CLL)". CAR T-cells remained detectable more than ten years after infusion, and immunoglobulin heavy chain (IGH) rearrangement deep sequencing showed persistent deep molecular remission for both patients (no CLL clonotypes were detectable six months after CAR T-cell infusion and onwards). However, the existing actual disease status of both patients remained unclear, as it was unknown: (1) if CAR T-cells killed all leukemia cells during the initial anti-leukemic response phase, that is, soon after CAR T-cell infusion into both patients; (2) if few CLL cells survived, but persistent CAR T-cells had been able to destroy any leukemia cells before they reach detectable levels. In the first case, both patients could be considered definitely cured; in the second not and their decade-prolonged deep remission could be a consequence of the cytotoxic activity of the functionally active CD4+ CAR T-cells. The first version appears to be stronger and the supporting arguments have been included in a comprehensive commentary article. A new therapeutic intervention may emerge with the potential to fully improve the quality of life of both patients and in addition, ongoing research into CAR T-cells may turn in a new, more effective direction.

Anxiety exacerbation in a patient with chronic myeloid leukemia receiving dasatinib and venlafaxine.

Tyrosine kinase inhibitor (TKI) use leads to near-normal life expectancy in patients with chronic myeloid leukemia (CML); unfortunately for some patients, adverse drug effects (ADEs) and medication burden associated with TKI therapy can lead to decreased quality of life. Additionally, TKIs have drug interactions that may negatively impact patients' management of co-morbidities or lead to increased ADEs. A 65-year-old female with a history of anxiety treated and controlled with venlafaxine experienced increased and resistant anxiety and insomnia after starting dasatinib for CML. On dasatinib, the patient experienced worsening anxiety and insomnia. The stress of a new leukemia diagnosis, drug interactions, and ADEs from dasatinib were considered possible causes. Dose adjustments to dasatinib and venlafaxine were made to control the patient's symptoms. However, the patient's symptoms did not resolve. After being on dasatinib for 2.5 years, the patient discontinued TKI therapy due to being in a deep molecular remission and given ongoing challenges managing anxiety. Within 4 months of stopping dasatinib, the patient reported an improvement in anxiety and overall emotional wellbeing. She continues to feel better and remains in a complete molecular remission 20 months off treatment. This case demonstrates a possible previously unknown drug interaction with dasatinib as well as a possible rarely reported ADE of dasatinib. Additionally, it highlights the difficulties patients with psychiatric disorders may face on TKI therapy and challenges providers may have in identifying rare psychiatric ADEs, thus emphasizing the need for documentation of these types of cases.

A Case Report of Successful Kidney Transplantation in a Patient With Chronic Myelogenous Leukemia (CML) Who Has Been in Remission for 15 Years on Imatinib

For chronic myeloid leukemia (CML), a Philadelphia chromosome-positive myeloproliferative neoplasm, the introduction of tyrosine kinase inhibitors has transformed CML from a lethal disease into a manageable chronic disease with a close-to-normal life expectancy. Active malignancy is an absolute contraindication to kidney transplantation. However, it is controversial whether kidney transplantation can be safely performed in patients with a history of CML who are in remission. We describe the clinical course of a 64-year-old male patient with chronic kidney disease from diabetic nephropathy (DMN) who underwent living donor kidney transplantation. The patient was diagnosed with CML 15 years ago and promptly achieved cytogenetic and molecular biological remission after starting imatinib. After that, he continued imatinib treatment for 15 years and was in remission, but his chronic kidney disease from DMN gradually worsened. A preemptive living donor kidney transplant was performed in July 2020. Imatinib for CML was discontinued because the patient maintained deep molecular remission (DMR) of major molecular response for more than 15 years before kidney transplantation. After kidney transplantation, the transplanted kidney function remained good at approximate serum creatinine levels of 1.1 mg/dL without histopathologic rejection, and the 3 monthly BCR-ABL1 measurement results were negative and are in progress. Thus, he continues to maintain treatment-free remission status without imatinib for 26 months after renal transplantation. In conclusion, this result suggests that CML with long-lasting DMR on imatinib therapy can be considered an inactive malignancy and therefore a relative indication for kidney transplantation.

SOHO State of the Art Updates and Next Questions | Update on Treatment-Free Remission in Chronic Myeloid Leukemia (CML).

Tyrosine kinase inhibitor (TKI) discontinuation, also known as treatment-free remission (TFR) is currently one of the main goals of chronic myeloid leukemia (CML) therapy. TKI discontinuation should be considered in eligible patients for several reasons. Specifically, TKI therapy is associated with reduced quality of life, long-term side effects, and a heavy financial burden on both the patients and society. TKI discontinuation is a particularly important goal for younger patients diagnosed with CML because of the treatment's effects on their growth and development in addition to potential long-term side-effects. Numerous studies with thousands of patients have demonstrated the safety and feasibility of attempting TKI discontinuation in a select group of patients who have achieved a sustained deep molecular remission. With current TKIs, approximately 50% of patients will be eligible for attempting TFR of which only 50% will achieve a successful TFR. Therefore, in reality, only 20% of patients with newly diagnosed CML will achieve a successful TFR, and the majority of patients will need to continue TKI therapy indefinitely. However, several ongoing clinical trials are investigating treatment options for patients to achieve deeper remission with the ultimate goal of a cure, which is defined as being off drug with no evidence of disease.

DNA Damage Response (DDR) Is Associated With Treatment-free Remission in Chronic Myeloid Leukemia Patients.

DNA Damage Response (DDR) Is Associated With Treatment-free Remission in Chronic Myeloid Leukemia Patients.

Feasibility of treatment-free remission with generic imatinib: Results of generic imatinib-free trial-in-chronic myeloid leukaemia-chronic phase study.

Both innovator and generic imatinib are approved for the treatment of Chronic Myeloid Leukaemia-Chronic phase (CML-CP). Currently, there are no studies on the feasibility of treatment-free remission (TFR) with generic imatinib. This study attempted to determine the feasibility and efficacy of TFR in patients on generic Imatinib. In this single-centre prospective Generic Imatinib-Free Trial-in-CML-CP study, twenty six patients on generic imatinib for ≥3 yr and in sustained deep molecular response (BCR ABLIS ≤0.01% for more than two years) were included. After treatment discontinuation, patients were monitored with complete blood count and BCR ABLIS by real-time quantitative PCR monthly for one year and three monthly thereafter. Generic imatinib was restarted at single documented loss of major molecular response (BCR ABLIS>0.1%). At a median follow up of 33 months (interquartile range 18.7-35), 42.3 per cent patients (n=11) continued to be in TFR. Estimated TFR at one year was 44 per cent. All patients restarted on generic imatinib regained major molecular response. On multivariate analysis, attainment of molecularly undetectable leukaemia (>MR5) prior to TFR was predictive of TFR [P=0.022, HR 0.284 (0.096-0.837)]. The study adds to the growing literature that generic imatinib is effective and can be safely discontinued in CML-CP patients who are in deep molecular remission.

Clonal Hematopoiesis Detected at the Time of Tyrosine Kinase Inhibitor Cessation Is Associated with Delayed Molecular Recurrence after Treatment-Free Remission in Patients with CML

Background Treatment-free remission (TFR) is a goal of therapy. However, ~50% of patients who attempt TFR will relapse, which mostly occurs by 6 months. The acquisition of somatic mutations in hematopoietic cells is common with age (clonal hematopoiesis, CH). Through next-generation sequencing studies we identified CH in patients in deep molecular remission. CH variants provide a selective growth advantage to cells, driving their clonal expansion. The clones also give rise to mutated immune effector cells with a proinflammatory profile that exacerbate diseases with a chronic inflammatory component, such as atherosclerosis. CH variants could potentially influence TFR, either by outcompeting an emerging residual CML clone or by altering immune function, which is known to play an important role in TFR. Aim To determine whether CH variants present at the time of therapy cessation could influence TFR. Methods An RNA-based hybridization capture sequencing method targeting 17 of the most frequently mutated CH genes was developed and applied to samples of 138 patients who attempted TFR. CH variants met strict criteria for pathogenicity based on their potential to confer growth and survival advantages. The CH status was confirmed by repeat testing of samples of 28 patients. The method reproducibly detected variants with a variant allele frequency (VAF) of ≥0.4%. Results The median follow-up for the 138 patients was 66 months from the TFR attempt. Molecular recurrence was defined as loss of major molecular response (MMR, BCR::ABL1 ≤0.1%). The probability of TFR was 64.5% at 6 months, 59.4% at 12 months and 51.0% at 84 months. CH variants were detected in 61/138 patients (44%): 107 variants in 12 genes. Multiple variants, range 2 - 7, were detected in 26 patients (19%). The median VAF was 0.9%, range 0.4 - 44%. Variants most frequently occurred in TET2 (44% of variants), DNMT3A (25%), PPM1D (7.4%) and ASXL1 (6.5%). The average age at cessation was 59.6 years, range 33 - 85. The average age of patients with CH was higher than those without CH: 64.7 versus 55.5 years, P < .0001. Only 3/13 patients (15%) aged ≤40 had CH compared with 6/7 patients (86%) ≥80. CH was significantly associated with TFR to 36 months after cessation. The optimal criteria for TFR prediction were at least one CH variant of VAF >2% or >2 variants (CH >2%/>2). Thirty-one of 138 patients (22%) had CH of VAF >2% (n = 24) or >2 variants (n = 7). The probability of TFR for these 31 patients was significantly higher than for the remaining patients when assessed at 6, 12, 24 and 36 months after cessation, Figure. The probability of TFR at 6 months was 90.3% for patients with CH >2%/>2 versus 57.0% for the remaining patients, P = .001. The probability of TFR at 36 months was 65.9% versus 51.2%, P = .049. A landmark analysis was performed at 12 months for the 74 patients who maintained TFR at 12 months. MMR was subsequently lost in 8/74 patients and the probability of TFR at 84 months was 86.9%. CH >2%/>2 occurred for 23/74 patients (31%). The probability of late molecular recurrence was higher among the patients with CH >2%/>2: 27.3% versus 5.5% for the remaining patients, P = .009. Overall, molecular recurrence occurred for 12/31 patients (39%) with CH >2%/>2 and 53/107 of remaining patients (50%). Time to molecular recurrence after cessation was longer for patients with CH >2%/>2 than the remaining patients: median 12.5 months versus 3.5 months, P < .0001. The longer time to molecular recurrence was linked to a different pattern of loss of MMR. The median time between first loss of MR4 (BCR::ABL1 ≤0.01%) and loss of MMR was 145 days for patients with CH >2%/>2 and 28 days for remaining patients, P = .0007. BCR::ABL1 values more frequently fluctuated before loss of MMR in patients with CH >2%/>2: 8/12 patients (67%, fluctuation over 120-1335 days) versus 6/53 (11%, fluctuation over 85-715 days) in the remaining patients, P =.016. Conclusion CH may improve the probability and duration of TFR, delaying the time to molecular recurrence, which has implications for patient selection and monitoring. Patients with CH who are eligible for therapy cessation have an excellent prospect of remaining drug-free for 1-2 years but their long term prospects of sustained TFR may be less certain compared to other patients, given the slower dynamics of relapse. Delayed relapse could be due to competition for clonal dominance between residual leukemic cells and larger CH clones, or modulation of the immune microenvironment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Anti-Leukemic Activity of STRO-002 a Novel Folate Receptor-α (FR-α)-Targeting ADC in Relapsed/Refractory CBF2AT3-GLIS2 AML

BACKGROUND: CBF2AT3-GLIS2 (CBF/GLIS) oncogenic fusion is the underlying genomic cause of "RAM" phenotype AML, a megakaryoblastic subtype exclusively seen in infants and young children. CBF/GLIS AML is a highly refractory and uniformly fatal subtype of AML with limited response to conventional chemotherapy. We have demonstrated that FR-α, a known target in ovarian cancer, is expressed in CBF/GLIS AML cells and this expression is causally linked to the fusion oncogene. STRO-002 is currently under clinical investigation for the treatment of relapsed/refractory ovarian and endometrial cancer (NCT03748186). Preclinical studies have tested and confirmed the potent in vitro and in vivo anti-leukemia activity of STRO-002 in CBF/GLIS AML cell lines and xenograft models (Le et al. ASH 2021). Based on these compelling preclinical pharmacology observations, Sutro Biopharma evaluated patients on a case-by-case basis for consideration of a single patient IND (compassionate use) program for pediatric patients with CBF/GLIS AML. METHODS: Key criteria for consideration of STRO-002 were CBF2AT3-GLIS2 oncogenic fusion and FR-α expression by flow cytometry in AML sample. Key exclusion criteria were co-existent acute/uncontrolled infection and compromised organ function (pulmonary, hepatic, renal, etc). Between August 2021 and July 2022, 16 patients received STRO-002. Median age at treatment was 2 years (range 6 months-7 years). Median number of prior therapies was 3 (range 1-8). Eight patients had relapsed after stem cell transplant (SCT) and the remaining 8 had primary refractory (induction failure) or relapsed AML. RESULTS: In the 16 patients treated, 10 received STRO-002 as monotherapy, and 6 received combination with chemotherapy (fludarabine/cytarabine, decitabine, methotrexate (MTX), or dasatinib). All 16 pts were evaluable for response, best observed response included 7 CRs with 6 MRD negative remissions. Of these CRs, 3 occurred after monotherapy and 4 with combination therapy: MTX/fludarabine/cytarabine (n = 1), decitabine/DLI (n = 1), fludarabine/cytarabine/G-CSF (n = 1) and dasatinib/DLI (n = 1). The remaining 9 patients had stable disease or transient decrease in disease burden. Of the 7 patients who achieved a CR, 4 proceeded to SCT, 2 are receiving DLI and 1 will receive consolidation therapy. Ten (63%) of the 16 evaluable patients are alive and the other 6 died of progressive disease. Follow-up is limited for the surviving patients. Of note were 6 patients who achieved an MRD negative CR, two with primary refractory disease, and four who had relapsed after an allogeneic SCT. These six patients with significant disease had deep response to STRO-002 with MRD negative remission. One patient had a rapid decline in marrow blasts and had no evidence of disease by flow after 3 single agent every other week (QOW) doses of STRO-002. A second patient had a similarly rapid response to single agent STRO-002 with clearance of all detectable disease after a second QOW dose of STRO-002. ANC for both patients one week after last dose of STRO-002 was >1000. These patients were consolidated with allogeneic stem cell transplant or with DLI. STRO-002 was generally well tolerated. Collection and review of treatment-emergent AEs (TEAEs) is ongoing. Treatment-related events ≥ grade 3 included neutrophil count decreased (n = 3), platelet count decreased (n = 3), anemia (n = 2), WBC decreased (n = 2), hyperbilirubinemia (n = 1), bacteremia (n = 1), febrile neutropenia (n = 1), urticaria (n = 1), generalized edema (n = 1), aspartate aminotransferase elevation (n = 1), alanine aminotransferase elevation (n = 1). CONCLUSIONS: STRO-002 has promising activity in relapsed/refractory CBF2AT3-GLIS2 AML, a disease that tends to be highly refractory to all standard-of-care (SOC) therapies. Further, STRO-002 is well tolerated as a monotherapy agent and in combination with SOC therapies. Patients with low tumor burden were more likely to achieve CR and transition to SCT, DLI and CAR-T. Deep molecular and flow cytometric remission was achieved in a cohort of patients. Patients with primary refractory disease (no prior transplant), 1-3 prior lines of therapy, low disease burden, or without extramedullary disease appeared to have a more potent response to STRO-002. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Efficacy of Ropeginterferon Alpha 2b in Inducing Treatment Free Remission in Chronic Myeloid Leukemia - an International, Randomized Phase III Trial (ENDURE, CML-IX) of the German CML-Study Group

Background: Only a minority of chronic myeloid leukemia (CML) patients can eventually successfully discontinue tyrosine kinase inhibitor (TKI) therapy after achieving a stable deep molecular remission. Mechanisms underlying a durable treatment free remission (TFR) are not well understood but supposedly involve restoration of anti-CML immunity. It was therefore hypothesized that interferon alpha (IFN) - a potent inducer of Th1-immunity and prior standard therapy in CML - might improve the TFR probability. Randomized trials to improve TFR rates in CML are currently lacking. ENDURE, CML-IX (NCT03117816) is a multicenter, international phase III trial evaluating the role of a novel form of pegylated proline interferon-alpha 2b (ropeg-interferon-alpha, ropeg-IFN) in inducing TFR. Patients and Methods: CML patients in stable deep molecular remission (MR4 or better for at least 12 months prior to screening) and with history of at least 3 years of TKI exposure were eligible to participate in this study. Patients in stable molecular remission could also be included if they had failed a former TKI discontinuation attempt (2nd stopper). Patients were randomized 1:1 to receive either ropeg-IFN 100μg s.c. every 2 weeks for 15 months (ropeg-IFN arm) or no further treatment after TKI stop treatment (no treatment arm). The primary efficacy endpoint of the trial was molecular relapse free survival (MRFS) with molecular relapse being defined as loss of major molecular remission (MMR), which is any increase of the BCR::ABL1 transcript level to >0.1% according to the international scale (IS). Time to relapse is defined as the time from randomization to relapse. Secondary endpoints were MRFS at month 6, 12 and 24 after TKI discontinuation. BCR::ABL1 measurable residual disease (MRD) was monitored from peripheral blood and centrally assessed for all centers in Germany and locally at the French centers. There was a post study follow up to collect clinical and molecular data. Results: Between May 2017 and June 2021, 203 evaluable patients (68 female, 135 male) with a median age of 55 years were randomized at 24 centers in Germany and 3 centers in France to receive ropeg-IFN (n=95) or no treatment (n=108) after TKI stop. For 77 patients in the ropeg-IFN arm (81%) and 86 patients in the no treatment arms (80%) it was their first TKI discontinuation attempt. At the time of data cut off in June 2022, the median observation time for all patients was 36 months. The hazard ratio (HR) of molecular relapse for the ropeg-IFN cohort versus the no treatment cohort was 1.03 (95% CI, 0.68 to 0.1.55; log-rank P=0.89). The Kaplan-Meier probabilities of molecular relapse free survival (MRFS) by 6, 12, and 24 months after TKI discontinuation were 73% (95%-CI: 62-81%), 64% (95-CI: 53-73%) and 56% (95%-CI: 45-66%) for the ropeg-IFN versus 67% (95-CI: 57-75%), 60% (95-CI: 50-69%) and 59% (95%-CI: 49-68%) for no treatment (Figure 1). The MRFS at months 6 and 12 after TKI stop (secondary endpoints) were 70% (95%-CI: 60-79%) and 64% (95%-CI: 53-73%) in 91 patients for ropeg-IFN group versus 65% (95-CI: 56-74%) and 59% (95-CI: 49-68%) in 107 and 104 patients for the no treatment group. Of 90 patients who were candidates for restarting TKI due to MMR loss, 82 patients were actually evaluable for TKI restart. Of those, 77 patients re-achieved at least MMR within 12 months (median time to reestablishing MMR was three months). Of the 5 patients who did not regain MMR, one patient withdrew consent and others were treated for only 1, 3, 4, and 11 months after TKI restart. Ropeg-IFN was well tolerated. Hematological and non-hematological WHO grade 3/4 toxicity occurred in 23 patients in the ropeg-IFN (31 adverse events) and in 20 patients in the no treatment arm (26 adverse events). Three deaths were reported on study which were unrelated to CML (cardiac arrest, fall from stairs, unknown). A detailed safety analysis will be presented at the meeting. Conclusion: Ropeg-IFN maintenance after discontinuing TKI-monotherapy does not increase the proportion of patients, who persistently maintain at least an MMR. The German CML-V (TIGER) trial currently explores the impact of IFN maintenance on TFR when patients receive a combination of TKI plus IFN before TKI stop. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Treatment Free Remission after Combination Therapy with Asciminib (ABL001) Plus Imatinib in Chronic Phase Chronic Myeloid Leukemia (CP-CML) Patients Who Relapsed after a Prior Attempt at TKI Discontinuation-H Jean Khoury Cure CML Consortium Study

Successful tyrosine kinase inhibitor (TKI) discontinuation is currently one of the main goals of CML therapy. Patients who are in sustained deep molecular remission can attempt TKI discontinuation; approximately 50% of such patients will achieve a successful treatment free remission (TFR) and the other 50% will have molecular recurrence , warranting reinitiation of TKI therapy. Molecular recurrence is defined as loss of major molecular response (BCR-ABL1 International Scale ratio >0.1%) . With a goal to increase the ultimate fraction of successful TFR, inquiry has begun into the rate and optimal approach to TFR after a second attempt at TKI discontinuation in patients with molecular recurrence after their first attempt. Molecular recurrence, is suspected to be driven by CML progenitor element(s) capable of, permitted to, and/or signaled to proliferate; different approaches to eradicate such a compartment have been proposed. One such potential means may be through alternative and synergistic BCR::ABL1 inhibition with the allosteric inhibitor asciminib in combination with approved TKIs for CML. Asciminib is a potent BCR::ABL1 inhibitor with a novel, allosteric mode of action. The activity of asciminib is a result of its ability to bind a vacant pocket on the kinase domain that is normally occupied by the myristoylated N-terminus of ABL, a negative regulatory element lost upon the fusion of ABL to BCR. Asciminib has an excellent selectivity profile, is several-fold more potent than current approved TKIs such as nilotinib, and in vitro was active in the low nanomolar range against a spectrum of clinically observed ATP site mutations. Asciminib demonstrated efficacy in phase I studies in resistant/intolerant chronic and accelerated phase CML, including T315I mutation positive and ponatinib pretreated patients. In a phase III trial, asciminib was more active than bosutinib in a phase III trial in patients who had received 2 prior TKIs Our primary objective in this study is to determine the 1-year TFR rate after 12 months of combination therapy with imatinib plus asciminib, followed by treatment cessation, in subjects who have previously had molecular recurrence first TFR attempt. This is a single arm phase II study that will enroll a maximum of 51 patients (NCT04838041). All subjects will have a confirmed diagnosis of CML-CP. Each subject must have previously attempted to discontinue imatinib, and were restarted on imatinib at the time of molecular recurrence in order to be eligible for this trial. This trial will use asciminib 40 mg PO daily plus imatinib at a maximum dose of 400 mg PO once daily in the combination treatment phase of this trial. All eligible subjects will be treated with asciminib and imatinib on cycle 1 day 1 of the combination therapy phase. Patients will remain on imatinib for 12 cycles. RQ-PCR to measure BCR::ABL1 transcripts in the peripheral blood at screening and at specific time points during the first 12 cycles on trial (combination therapy phase). At the end of 12 cycles, asciminib and imatinib will be discontinued in any subject who has continued in stable deep remission and thus meets criteria for TFR. This study will assess a wide range of PROs before and after stopping imatinib. These will be done in conjunction with RQ-PCR testing, though at fewer time points, utilizing online and/or phone questionnaires. The alternative hypothesis of relapse rate after second discontinuation of TKI combination treatment is 35% will be tested against a one-sided alternative of historical control of 55% relapse rate. Simon's two-stage admissible design will be used. The null hypothesis that the true TFR rate (no loss of MMR) is 45% will be tested against a one-sided alternative. In the first stage 29 patients will be accrued. If there are 15 patients or fewer who maintain TFR in these 29 patients, the study will be stopped. Otherwise, 18 additional patients will be accrued for a total of 47. The null hypothesis will be rejected if 27 or more patients maintain TFR in 47 patients. This design yields a type I error rate of 5% and power of 85% when the true TFR rate is 65%. To account for a potential dropout rate of 8% due to adverse events or loss of response, 51 patients will be enrolled to achieve the 47 patients needed.