Shedding Light on Targeting Chronic Myeloid Leukemia Stem Cells

Chronic myeloid leukemia stem cells (CML LSCs) are a rare and quiescent population that are resistant to tyrosine kinase inhibitors (TKI). When TKI therapy is discontinued in CML patients in deep, sustained and apparently stable molecular remission, these cells in approximately half of the cases restart to grow, resuming the leukemic process. The elimination of these TKI resistant leukemic stem cells is therefore an essential step in increasing the percentage of those patients who can reach a successful long-term treatment free remission (TFR). The understanding of the biology of the LSCs and the identification of the differences, phenotypic and/or metabolic, that could eventually allow them to be distinguished from the normal hematopoietic stem cells (HSCs) are therefore important steps in designing strategies to target LSCs in a rather selective way, sparing the normal counterparts.

TKI discontinuation in CML: how do we make more patients eligible? How do we increase the chances of a successful treatment-free remission?

Treatment-free remission (TFR) is a new and significant goal of chronic myeloid leukemia management. TFR should be considered for patients in stable deep molecular response (DMR) after careful discussion in the shared decision-making process. Second-generation tyrosine kinase inhibitors (TKIs) improve the speed of response and the incidence of DMR. Treatment may be changed to a more active TKI to improve the depth of response in selected patients who have not reached DMR. Stem cell persistence is associated with active immune surveillance and activation of BCR-ABL1-independent pathways, eg, STAT3, JAK1/2, and BCL2. Ongoing studies aim to prove the efficacy of maintenance therapies targeting these pathways after TKI discontinuation.

Lifelong TKI therapy: how to manage cardiovascular and other risks

Beginning with imatinib and now spanning 6 oral, highly active, and mostly safe agents, the development of specific targeted therapy for patients with chronic myeloid leukemia (CML) has created a new world featuring chronic maintenance chemotherapy for all treated as such, treatment-free remission, and functional cure after prolonged deep remission in a subset. As a result comes a necessary shift in focus from acute to chronic toxicity, increasing attention to patient comorbidities, and critical thinking around specific adverse events such as metabolic, cardiovascular, and cardiopulmonary effects, which vary from agent to agent. This review aims to pull together the state of the art of managing the “C” in CML—a chronic myeloproliferative neoplasm treated at present over many years with oral BCR-ABL-targeted agents in a population whose overall health can be complex and potentially affected by disease and therapy—and determine how we can better manage a highly treatable and increasingly curable cancer.

Regulatory T Cell as a Biomarker of Treatment-Free Remission in Patients with Chronic Myeloid Leukemia

Treatment-free remission (TFR) has become a therapeutic goal in chronic myeloid leukemia (CML), and approximately half of the patients with chronic phase-CML (CML-CP) with deep molecular remission (DMR) by tyrosine-kinase inhibitors (TKIs) have achieved TFR. However, the mechanism of continuous TFR is still unclear, as there are “fluctuate” patients who have BCR–ABL-positive leukemia cells but do not observe obvious relapse. We focused on the immune response and conducted an immune analysis using clinical samples from the imatinib discontinuation study, JALSG-STIM213. The results showed that, in the group that maintained TFR for 3 years, changes in regulatory T (Treg) cells were observed early after stopping imatinib treatment. The effector Treg (eTreg) cells increased transiently at 1 month after stopping imatinib and then returned to baseline at 3 months after stopping imatinib treatment. There was no difference in the Treg phenotype, and CD8+ T cells in the TFR group were relatively activated. High concentrations of imatinib before stopping were negatively correlated with eTreg cells after stopping imatinib. These data suggest immunological involvement in the maintenance of the TFR, and that Treg cells after stopping imatinib might be a biomarker for TFR. Furthermore, high imatinib exposure may have a negative immunological impact on the continuous TFR.

The Outcome of Treatment-Free Remission after First-Line Nilotinib or Dasatinib in Chronic Phase Chronic Myeloid Leukemia Patients Is Different

Aims: The absolute number of chronic phase CML patients (pts) reaching the treatment-free remission (TFR) criteria has been substantially increased by the use of second-generation TKI (TKI2), initiated since diagnosis, comparing to Imatinib first-line. However, the relative rate of unsuccessful TFR (i. e. pts loosing their MMR after TKI2 cessation) still remains around 50% at 2 years and beyond, whatever the TKI2 was. The aim of this study is to analyse the rate of successful TFR in pts receiving Nilotinib (Nilo) or Dasatinib (Dasa) first-line obtaining the appropriate criteria. Methods: Observational retrospective study in 3 reference centers of the French group of CML lead between 2010 and 2021. Eligible pts were CP CML pts initiating either Nilo 300 mg BID or Dasa 100 mg daily since diagnosis, until cessation for sustained MR4.5 (i.e. ≥2 years on ≥4 datapoints). Data were retrospectively collected according to the national regulations with pts' information. All pts were assessed and followed according to ELN recommendations 2009, 2013 and 2020 along treatment and to the recommendations from the French group of CML (D. Rea et al., Cancer 2018) for TFR. In this regard, the TKI2 was resumed in case of loss of MMR. All BCR-ABL1 assessments were performed in the 3 reference laboratories, standardised and expressed in % (IS) with ≥32,000 copies of ABL1 as control. All patients were harbouring major BCR-ABL1 transcripts. The primary endpoint was the survival without loss of MMR after TKI2 cessation. The secondary endpoints were the kinetics of MMR loss, and the identification of factors influencing MMR loss. Results: Seventy-two pts were reported (47 Nilo, 25 Dasa) with 57% females with a median age at diagnosis of 48 (36.75-61.25) years. The median follow-up since diagnosis was 9.26 (3.75-13.75) years (8.8 for Nilo and 9.47 for Dasa p=ns) and after TKI2 cessation 3.94 (0.7-8.8) years (3.92 for Nilo and 3.90 for Dasa p=ns). Sokal scores were 42% Low, 41% Intermediate, 17% High in Nilo and 39% L, 25% I and 35% H in Dasa pts (p=ns). ELTS scores were 50% L, 22% I, 9.5% H (18.5% Uk) in Nilo and 46.5% L, 28.5% I and 3.5% H (21.5% Uk) in Dasa pts (p=0.95). Five (9%) pts harboured ACA at diagnosis in the Nilo group and 2 (7%) in the Dasa group (p=1.00). The median time from TKI2 initiation to sustained MR4.5 was 19 (3.12-36) months in the Nilo group and 16 (6.3-39) months in the Dasa group (p=0.644). The duration of sustained MR4.5 until cessation was 3.04 (1.5-9.3) years for Nilo and 2.65 (1.11-7.95) for Dasa (p=0.96). The median dosing of Nilo was 600 (300-800) mg daily and 80 (20-100) mg at TKI2 cessation. None of these patients switched to another TKI during the follow-up. TKI2 cessation occurred after 60.5 (43-74.5) months in the Nilo group and 68 (39-90) months in the Dasa group (p=0.581). Thirty-seven pts out of 47 (79%) were BCR-ABL1 undetectable at Nilo cessation 18/25 (72%) at Dasa cessation (p=0.60). At M3 after discontinuation, 58% of pts remained undetectable after Nilo cessation and 30.4% after Dasa cessation (p=0.05).The median survival of pts without loss of MMR was not reached in the Nilo group, and was 14 (4.73-NR) months in the Dasa group, (p=0.042) as analysed by the KM method (Figure 1.). Two patients died (1 Nilo, 1 Dasa) from competing events (solid tumours) after unsuccessful TFR. Twenty-eight pts (14 Dasa, 14 Nilo) restarted their TKI2 after MMR loss and all regained ≥ MMR after 3 months of Dasa at a median dose of 75 (40-100) mg daily and all except one (who regained MMR at M12) after resumption of Nilo at a median dose of 350 (300-600) mg daily. Univariate analysis identified pts with H+I Sokal (as compared to low) as an unfavourable factor for successful TKI2 cessation [HR=0.35 (0.15-0.83), p=0.017] and type of TKI2 (Nilo as reference vs Dasa) was discriminant [HR=2.1 (1.01-4.35), p=0.047]. Multivariate analysis identified the type of TKI2 as a significant factor impacting on TFR outcome [HR 2.11 (0.97-4.55], p=0.05]. Conclusions: As it is likely that no prospective head-to-head comparison will be performed in this setting, on this limited series of pts, we conclude that the outcome of TFR seems to be different according to the TKI2 used since diagnosis, suggesting the impact of distinct biological variables modified by the type of TKI2 on the long run (such as immunological system, BM micro-environment, others) on TFR outcome. Figure 1 Figure 1. Disclosures Nicolini: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel, accommodations, expenses, Research Funding; Kartos Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sun Pharma Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria; Incyte Biosciences: Honoraria, Other: travel, accommodations, expenses, Research Funding, Speakers Bureau. Etienne: Incyte: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau. Rea: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Highly Sensitive Droplet Digital PCR to Identify CML Patients with a High Probability of Achieving Treatment-Free Remission

Introduction: Treatment Free Remission (TFR) is the ultimate goal of therapy for most CML patients. Despite adopting consensus eligibility criteria of a sustained deep molecular response and more than 4 years of TKI therapy, the relapse rate after TKI cessation is still around 50%. More sensitive detection of residual leukaemia has the potential to improve our capacity to predict TFR outcomes for individual patients. Aim: To correlate droplet digital PCR (ddPCR) assay results with TFR outcome, especially in the setting of undetectable levels measured by qRT-PCR. Method: ddPCR was performed on blood samples from 51 TFR-eligible CML patients at the time of TKI cessation. 5 µg RNA per sample was used in 8 wells/sample using the BioRad QXDx BCR-ABL %IS kit on QX200 ddPCR system which yielded BCR-ABL1% (IS) directly. All these patients achieved MR4.5 that was sustained for ≥ 2 years. Results: 100% of patient were in MR4.5 via qRT-PCR at the time of stopping. 61% of the 51 patients evaluated relapsed within 12 months. Median duration of TKI therapy for the whole group was 5.8 years (range 2.2- 14 years). 20 patients achieved TFR success with a median follow up of 24 months (TFR group; sustained BCR-ABL1 <0.1% (IS) after TKI discontinuation for ≥12 months), while 31 patients relapsed (Relapse group; BCR-ABL1 >0.1% (IS) after stopping; median time of relapse 3 months, range 1-10 months). A ROC curve analysis correlated TFR outcome with ddPCR results, with BCR-ABL1 level ≥0.003% via ddPCR at the time of stopping identified as an optimal cut-off. Kaplan-Meier analysis showed that 89% of the patients with ddPCR ≥0.003 relapsed after TKI cessation, whereas the ddPCR <0.003 demonstrated a significantly reduced relapse rate to 54% (p=0.01, Figure 1A). In addition, the TFR group (median BCR-ABL1 0.00065%) demonstrated approximately two-fold lower levels of BCR-ABL1transcript level compared to the relapse group (median 0.0012%). Interestingly, 7/31 (23%) of the relapsed group had undetectable BCR-ABL1 transcript even with the current highly sensitive method, while this undetectable level was only observed in 35% of the TFR group. We next assessed other known predictors of TFR success relative to ddPCR results in a Cox proportional hazard model. We have previously demonstrated that the BCR-ABL1 halving time after commencing therapy is highly predictive of TFR. At a univariate level, transcript type (e13a2 versus e14a2, p=0.01), BCR-ABL1 halving time (p>0.0001), and mRNA quantitation by ddPCR ≥ 0.003% (p=0.02) were all significantly associated with clinical outcome. Other variables including gender, age, ELTS score, Sokal score, MR4.5 duration and TKI duration were not associated with clinical outcome in this cohort (Figure 1B). In the multivariate analyses (Figure 1C), ddPCR remained an independent predictor after adjusting for ELTS, TKI duration and MR4.5 duration. Interestingly, ddPCR was not an independent predictor after adjusting for BCR-ABL1 transcript type or halving time. Conclusion: QXDx ddPCR assay is a promising tool for molecular residual disease monitoring in CML, especially when the BCR-ABL1 is undetectable by conventional method. The CML patients with levels of detectable BCR-ABL1 ≥0.003% measured by ddPCR have a significantly higher probability of relapse compared to patients with lower levels of the transcript. The ≥0.003% BCR-ABL1 level cut-off value could be a potential tool to aid decision-making when attempting TKI discontinuation in CML. However, even though a measurable level of BCR-ABL1 above 0.003% via ddPCR identified patients at high risk of relapse after a TFR attempt, it does not rule out the possibility of TFR; and a negative ddPCR result does not exclude the risk of molecular relapse. ddPCR may be most useful where other TFR predictive factors including BCR-ABL1 transcript type and halving time are not available. In-kind support was received from Bio-Rad for this study. Figure 1 Figure 1. Disclosures Branford: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Cepheid: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Qiagen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hughes: Novartis: Honoraria, Research Funding; Incyte: Honoraria; BMS: Honoraria, Research Funding. Yeung: BMS: Honoraria, Research Funding; Amgen: Honoraria; Novartis: Honoraria, Research Funding; Pfizer: Honoraria.

First Interim Analysis of the Italian Dante Study: De-Escalation before Treatment-Free Remission in Patients with Chronic Myeloid Leukemia Treated with First-Line Nilotinib

Introduction: Treatment-free remission (TFR) in chronic myeloid leukemia (CML) is demonstrated to be achievable and recommended for patients (pts) in sustained deep molecular response (sDMR) who can discontinue tyrosine kinase inhibitor (TKI) treatment and maintain responses in ~50% of cases. While the feasibility and safety of TKI cessation have been largely demonstrated, the strategies of TFR optimization are yet to be clarified. Studies (eg. DESTINY) investigating de-escalation, mainly after imatinib, suggested that a stepwise approach may favor TFR outcome. We present the interim results of the phase 2, prospective, multicenter DANTE study (NCT03874858) evaluating de-escalation and TFR in Italian pts with CML in chronic phase (CML-CP) treated with nilotinib (NIL). Methods: Adults with CML-CP treated with NIL 300 mg twice daily (bid) in first-line for ≥3 years who achieved sDMR for ≥1 year (≥MR 4.0; BCR-ABL level ≤0.01% IS) were enrolled in 27 centers. The study consisted of 4 phases: screening (week [wk] −4-0), consolidation (wk 0-48), TFR (wk 48-144), and follow-up (until wk 144). Ongoing treatment with ≥400 mg/day dose was allowed at study entry. During consolidation, pts were treated with NIL 300 mg once daily (qd). At the end of consolidation phase, pts with sDMR entered TFR phase and discontinued NIL; indeed, pts with at least major molecular response (MMR; BCR-ABL ≤0.1% IS), but without sDMR, continued NIL 300 mg qd. At any time, pts with loss of MMR returned to NIL 300 mg bid. During TFR phase, BCR-ABL levels were monitored monthly from wk 52-96, and then every 3 months. Pts on half-dose or full-dose NIL were monitored every 3 months. The primary endpoint is the percentage of pts in full treatment-free remission (FTFR) 96 wks after the start of consolidation phase. FTFR is defined as pts with MMR or better, including those who remained in discontinuation during TFR phase and those who are treated with half the standard dose. Key secondary endpoints include percentage of pts with sDMR at wk 48; TFR rate at wk 96 and 144; BCR-ABL kinetics and safety. The predictive role of digital droplet PCR is also evaluated as an exploratory objective. Results: Overall, 113 pts were screened and 107 entered consolidation phase. This interim analysis included 52 pts who reached the end of consolidation phase by data cut-off period (February 8, 2021). Of these 52 pts, 49 (94.2%) were ongoing by data cut-off and 3 (5.8%) discontinued the study (1 patient due to adverse event (AE) and 2 per patient's decision). Median age at study entry was 49.5 years. Median time from diagnosis was 5.6 years and median dose of prior NIL treatment was 600 mg/day for all pts except one who was on NIL 450 mg/day at baseline. Median duration of last sustained MR4 and MR4.5 were 30 and 16.5 months, respectively. Further details are listed in Table 1. At screening, molecular response categories were MR4.0 in 13.7%, MR4.5 in 23.1% and undetectable MR4.5 in 63.5% of pts. During consolidation phase, 5 (9.6%) pts discontinued prematurely: 2 pts restarted NIL full dose (3.8%) for MMR loss, 2 (3.8%) discontinued for AEs and 1 (1.9%) for pt decision. Overall, 47 pts completed consolidation: of them 40 (76.9%) sustained DMR and 7 (13.5%) maintained MMR but not sDMR. Of the 7 pts not sustaining DMR during consolidation, 6 regained DMR after a median of 4.4 months, while 1 pt was still in MMR by data cutoff. The 2 pts who lost MMR after 5 and 8 months regained MMR and 1 regained DMR by data cutoff after increasing NIL to 300 mg bid. Median time spent in consolidation phase was 11.7 months, and the evolution of response categories over time is shown in Figure 1. During consolidation phase, AEs were observed in 16 pts (30.8%), of them 2 (3.8%) pts had serious AEs: 1 patient had skin ulcers and COVID-19 related pneumonia, while 1 patient had unstable angina. No deaths and disease progressions were observed. Conclusions: DANTE is the first study that showed the safety and feasibility of NIL de-escalation before TFR in CML-CP pts with sDMR. Interim results suggest that loss of MMR during de-escalation is rare. De-escalation strategy may lead to further improvement of TFR outcome and tolerability and may also preemptively support the identification of pts who may not be ready for discontinuation, with a tailored approach. To date, accuracy in predicting TFR outcome is still low, and the de-escalation setting may sharpen biological and clinical predictive factors, including the potential role of digital PCR. Figure 1 Figure 1. Disclosures Breccia: Abbvie: Honoraria; Pfizer: Honoraria; Novartis: Honoraria; Incyte: Honoraria; Bristol Myers Squibb/Celgene: Honoraria. Abruzzese: Incyte: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria. Stagno: InCyte: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Other: Support for attending meetings and/or travel; Novartis: Consultancy, Honoraria, Other: Support for attending meetings and/or travel, Research Funding. Iurlo: Incyte: Speakers Bureau; Novartis: Speakers Bureau; Pfizer: Speakers Bureau; Bristol Myers Squibb: Speakers Bureau. Sportoletti: AstraZeneca: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria. Lemoli: Jazz, Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie, Daiichi Sankyo, Servier: Honoraria, Speakers Bureau; Celgene: Other: Support for attending meetings and/or travel. Siragusa: Novartis, CSL, Behring, Amgen, Novonoridsk, SOBI, Bayer: Consultancy, Honoraria, Speakers Bureau. Chiodi: Novartis: Current Employment.