Tyrosine Kinase Inhibitors Discontinuation Research Articles

Analytical Performance Evaluation of a Digital Real-Time PCR for Quantifying Major BCR::ABL1 Transcripts.

Accurate quantification of the BCR::ABL1 transcripts is essential for measurable residual disease (MRD) monitoring in chronic myeloid leukemia (CML) after tyrosine kinase inhibitor (TKI) treatment. This study evaluated the newly developed digital real-time PCR method, Dr. PCR, as an alternative reverse transcription-PCR (qRT-PCR) for MRD detection. The performance of Dr. PCR was assessed using reference and clinical materials. Precision, linearity, and correlation with qRT-PCR were evaluated. MRD levels detected by Dr. PCR were compared with qRT-PCR, and practical advantages were investigated. Dr. PCR detected MRD up to 0.0032%IS (MR4.5) with excellent precision and linearity and showed a strong correlation with qRT-PCR results. Notably, Dr. PCR identified higher levels of MRD in 12.7% (29/229) of patients than qRT-PCR, including six cases of MR4, which is a critical level for TKI discontinuation. Dr. PCR also allowed for sufficient ABL1 copies in all cases, while qRT-PCR necessitated multiple repeat tests in 3.5% (8/229) of cases. Our study provides a body of evidence supporting the clinical application of Dr. PCR as a rapid and efficient method for assessing MRD in patients with CML under the current treatment regimen.

Why are not all eligible chronic myeloid leukemia patients willing to attempt tyrosine kinase inhibitor discontinuation? A Czech nationwide analysis related to the TKI stopping trial HALF

Why are not all eligible chronic myeloid leukemia patients willing to attempt tyrosine kinase inhibitor discontinuation? A Czech nationwide analysis related to the TKI stopping trial HALF

Tyrosine Kinase Inhibitor Discontinuation in Chronic Myeloid Leukemia: Strategies to Optimize Success and New Directions.

The discovery that patients suffering from chronic myeloid leukemia who obtain deep and long-lasting molecular responses upon treatment with tyrosine kinase inhibitors may maintain their disease silent for many years after therapy discontinuation launched the era of treatment-free remission as a key management goal in clinical practice. The purpose of this review on treatment-free remission is to discuss clinical advances, highlight knowledge gaps, and describe areas of research. Patients in treatment-free remission are a minority, and it is believed that some may still retain a reservoir of leukemic stem cells; thus, whether they can be considered as truly cured is uncertain. Strengthening BCR::ABL1 inhibition increases deep molecular responses but is not sufficient to improve treatment-free remission, and we lack biomarkers to identify and specifically target residual cells with aggressive potential. Another level of complexity resides in the intra- and inter-patient clonal heterogeneity of minimal residual disease and characteristics of the bone marrow environment. Finding determinants of deep molecular responses achievement and elucidating varying biological mechanisms enabling either post-tyrosine kinase inhibitor chronic myeloid leukemia control or relapse may help develop innovative and safe therapies. In the light of the increasing prevalence of CML, targeting the residual leukemic stem cell pool is thought to be the key.

KIR-генетические факторы и ответ на терапию ингибиторами тирозинкиназ при хроническом миелоидном лейкозе

The development of tyrosine kinase inhibitors (TKIs) and their introduction into clinical practice considerably improved the prognosis for chronic myeloid leukemia (CML) patients. About 50 % of patients with achieved deep molecular response are eligible for safe TKI discontinuation. Despite these advances, no reliable biomarkers are known to predict a response and sustaining treatment-free remission after TKI withdrawal. As TKIs do not destroy leukemic stem cells, which can be responsible for relapse, critical importance in CML is attached to natural killers (NK-cells) having antitumor activity. Functional activity of NK-cells is evaluated by expression level and repertoire of killer cell immunoglobulin-like receptors (KIR). Current studies demonstrate that a patient’s KIR genotype affects the probability of achieving early and deep molecular responses to first- and second-generation TKIs, progression-free and overall survivals, and sustaining treatment-free remission. On that ground, KIR-genetic factors can be regarded as promising predictors of response to TKI therapy in CML. Early clinical studies, which dealt with monoclonal antibodies blocking the inhibitory KIR in order to increase NK-cell activity, revealed an acceptable safety profile and efficacy in some hematological diseases (such as acute myeloid leukemia, multiple myeloma, Т-cell lymphoma) if used in combination with cytostatic drugs or antitumor monoclonal antibodies. KIR genotype determination can contribute to the development of effective therapies of this malignant hematological tumor.

The Contemporary Role of Hematopoietic Stem Cell Transplantation in the Management of Chronic Myeloid Leukemia: Is It the Same in All Settings?

Hematopoietic stem cell transplantation (HSCT) for chronic myeloid leukemia (CML) patients has transitioned from the standard of care to a treatment option limited to those with unsatisfactory tyrosine kinase inhibitor (TKI) responses and advanced disease stages. In recent years, the threshold for undergoing HSCT has increased. Most CML patients now have life expectancies comparable to the general population, and therefore, the goal of therapy is shifting toward achieving treatment-free remission (TFR). While TKI discontinuation trials in CML show potential for achieving TFR, relapse risk is high, affirming allogeneic HSCT as the sole curative treatment. HSCT should be incorporated into treatment algorithms from the time of diagnosis and, in some patients, evaluated as soon as possible. In this review, we will look at some of the recent advances in HSCT, as well as its indication in the era of aiming for TFR in the presence of TKIs in CML.

Planned Discontinuation of Tyrosine Kinase Inhibitor Therapy in Metastatic Renal Cell Carcinoma: Lessons for the Era of Immunotherapy.

Several regimens combining immunotherapy and tyrosine kinase inhibitors (TKIs) have recently been validated for the first-line treatment of patients with metastatic renal cell carcinoma (mRCC). While immunotherapy is typically discontinued after 2 years in patients who neither progress nor experience limiting toxicity, according to the protocols of most recent phase III clinical trials, TKIs are to be continued until disease progression or the emergence of limiting toxicity. However, the prolonged use of TKIs is associated with significant toxicity and financial costs. This has sparked considerable debate about whether TKIs can be safely discontinued, particularly in mRCC patients who have achieved a verified complete response. This concise review examines the available evidence on TKI discontinuation in the context of mRCC management.

Treatment-free remission after a second TKI discontinuation attempt in patients with Chronic Myeloid Leukemia re-treated with dasatinib – interim results from the DAstop2 trial

Tyrosine kinase inhibitor (TKI) discontinuation in chronic myeloid leukemia (CML) has become part of routine care for patients with a sustained deep molecular response (DMR). Approximately 50% experience a molecular relapse upon TKI cessation. Most of them quickly regain DMR upon TKI resumption. Whether these patients can achieve a second treatment-free remission (TFR) remains unclear. DAstop2 (ClinicalTrials.gov ID: NCT03573596) is a prospective study including patients with a failed first TFR attempt re-treated with any TKI for ≥ one year. Upon entering the study, patients received the TKI dasatinib for additional two years. Patients with sustained DMR for ≥1 year qualified for a second TKI stop. Ninety-four patients were included between Oct 2017-Dec 2021. At the time of data analysis, 62 patients had attempted a 2nd stop. After a median follow-up of 27 months from 2nd stop, TFR rates were 61, 56 and 46% at 6, 12 and 24 months respectively. No progression to advanced stage disease was seen and 87% had re-achieved MR4 within a median of 3 months from TKI re-initiation. In summary, we show that a 2nd TFR attempt after dasatinib treatment is safe, feasible and TFR rates seem in the range of those reported in trials of a first TKI stop.

Successful Pregnancy and Delivery After Frozen-thawed Embryo Transfer Following the Third Discontinuation of Tyrosine Kinase Inhibitor in a Woman with Chronic Myeloid Leukemia

Successful Pregnancy and Delivery After Frozen-thawed Embryo Transfer Following the Third Discontinuation of Tyrosine Kinase Inhibitor in a Woman with Chronic Myeloid Leukemia

[Discontinuation of tyrosine kinase inhibitors before epiphyseal closure leading to improved short stature in pediatric chronic myelogenous leukemia].

A 3-year-old boy was referred to our hospital with splenomegaly. Blood tests revealed hyperleukocytosis and bone marrow examination showed major BCR::ABL1 fusion, leading to the diagnosis of chronic myelogenous leukemia (CML). Due to intolerance, the tyrosine kinase inhibitor (TKI) was changed from imatinib to dasatinib to nilotinib. The patient achieved molecular remission but became markedly short in stature, measuring 129.3 cm (height standard deviation score [SDS] -3.3) at the age of 12. TKI therapy was discontinued at age 12 years and 10 months, which was 9 years and 8 months after the start of TKI and 1 year and 6 months after achievement of MR4.0, as discontinuation before epiphyseal closure would not improve short stature. At 2 years and 6 months after discontinuation, the patient's height improved to 156.1 cm (SDS-2.0) without relapse. Growth suppression by TKIs is a problem in the management of pediatric CML. This case illustrates how improvement in severe short stature can be achieved by discontinuing TKI therapy before epiphyseal closure.

Treatment patterns in patients with chronic lymphocytic leukemia/small lymphocytic lymphoma post covalent Bruton tyrosine kinase inhibitor treatment: a Japanese claims database study.

Standard treatment has not been established for patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) after discontinuation of covalent Bruton tyrosine kinase inhibitor (cBTKi) therapy. This retrospective, administrative database (Medical Data Vision) study described the patient characteristics, treatment patterns, and factors associated with receiving post-first-cBTKi treatment in Japanese patients with CLL/SLL. Patients aged ≥18 years with confirmed CLL/SLL diagnosis and treated with anti-neoplastic drugs indicated for CLL/SLL between March 2013 and February 2022 were included. Patient characteristics at baseline (first line), first cBTKi exposure (first-cBTKi), post-first-cBTKi treatment received, and the treatment sequence of CLL drugs received first line through third line, were described. Time-to-event analyses used the Kaplan-Meier method. Multivariable logistic regression analysis was used to explore factors associated with receiving post-first-cBTKi treatment among patients who discontinued first-cBTKi treatment. Among 2,424 eligible patients (median age: 72.0 years, 61.9% male), 450 (18.6%) received cBTKi in any treatment line. Among patients treated with cBTKi, 273 (60.7%) discontinued treatment; 56.0% of them (n = 153/273) received subsequent treatment. Median duration of post-first-cBTKi treatment was 2.2 months (95% confidence interval [CI]: 1.8, 3.5). The most common regimens post-first-cBTKi were cBTKi therapy (47.7%), bendamustine-based therapy (17.0%), and venetoclax-based therapy (13.1%). Patients aged <75 years (odds ratio [OR] [95% CI]: 2.0 [1.2, 3.4]) and those who did not receive blood transfusion during cBTKi treatment (OR [95% CI]: 2.3 [1.3, 4.1]) were more likely to receive post-first-cBTKi treatment. In conclusion, Japanese patients with CLL/SLL received various treatments for short duration after first-cBTKi discontinuation.

Real-World Evidence of Treatment-Free Remission in Chronic Myeloid Leukemia at an Academic Center in the Deep South

Background: Treatment-free remission (TFR) is increasingly becoming a treatment goal for patients with chronic myeloid leukemia (CML) in chronic phase (CP). Although strict criteria guide tyrosine kinase inhibitor (TKI) discontinuation, there exists a knowledge gap in understanding patient and provider experience regarding TFR in real-world clinical practice. Here, we report our TFR experience in patients with CML-CP, treated at an academic center in the Deep South. Methods: A retrospective chart review was performed to evaluate outcomes of patients with CML-CP at the University of Alabama at Birmingham. The NCCN Version 2.2023 criteria for TKI discontinuation was utilized. Institutional review board approval was obtained. Results: We identified 204 patients with CML-CP through electronic medical records (EMR) . The median follow-up for the entire population was 95m (range 2-246m). The median age was 54y (range 21-75y) and majority of the patients were females (62%) and non-Hispanic whites (NHWs) (70%). A diagnostic bone marrow biopsy was performed on 59% of the patients. Most of the patients had p210 breakpoint (93%). Frontline therapy mainly consisted of imatinib (42%), dasatinib (32%) and nilotinib (16%). Out of the 204 patients, 44 (22%) met all the criteria for TKI discontinuation (Table 1). The median age for these patients was 53y (range 20-75y) and majority of the patients were females (68%) and NHWs (68%). Frontline therapy consisted of imatinib (59%), dasatinib (23%) and nilotinib (18%). Out of the 44 patients eligible for TFR attempt, 35 (80%) were offered TKI discontinuation by their provider whereas 9 (20%) were not. Reasons for not offering TKI discontinuation were not documented in the EMR. Twenty-four out of 35 patients attempted TKI discontinuation. The median time to TKI discontinuation for these patients was 114m (range 45-246m). Frontline therapy consisted of imatinib (n=13), dasatinib (n=7) and nilotinib (n=4) for those that attempted TFR. Ten patients had to resume TKI (imatinib=5, dasatinib=1, nilotinib=4) due to an increasing transcript after a median of 8m (range 2-29m). The remaining 14 patients (58%) remain in TFR to date with a median follow-up of 29m (6-51m). Eleven patients, eligible for TKI discontinuation, were offered a trial of TFR but did not attempt it. Three patients were satisfied with ongoing therapy and did not want to attempt TKI discontinuation or dose-reduction. Eight patients were hesitant to discontinue therapy and agreed to reduce the dose of the current therapy after discussing with their provider. Seven of those eight patients remain on a reduced TKI dose and 1 had to increase the dose due to an increase in transcript. Conclusion: In our single-center experience, we report that although most of the patients eligible for TKI discontinuation were offered a TFR attempt by their provider, there remains room for improvement. More than half the patients that attempted TKI discontinuation remain off therapy. Patient preference was the main reason for not attempting TKI discontinuation but there was willingness to attempt dose-reduction. Further studies are needed to understand both patient and provider perspectives regarding TFR to identify opportunities for improvement.

Single-Cell Analysis of Immune Recognition in Chronic Myeloid Leukemia Patients Following Tyrosine Kinase Inhibitor Discontinuation

Background: Modern treatment goal for patients with chronic myeloid leukemia (CML) is to maintain treatment-free remission (TFR) following tyrosine kinase inhibitor (TKI) discontinuation. The control of residual leukemia cells may occur with immunological mechanisms as several studies have shown that a higher proportion of CD56 dim NK cells before TKI discontinuation is associated with a better probability of TFR. In addition, CD8+ T cell abundances and phenotypes have been associated with responses to TKIs. However, the more detailed phenotypes and mechanisms and how to translate these findings to increased TFR rates have remained unknown. Methods: We analyzed 55 single-cell RNA and T cell receptor (TCR) sequenced (scRNA+TCRαβ-seq) samples from patients with CML ( n=13, N=25), other cancers (AML n=11, CLL n=13, solid tumors n=4), and healthy ( n=7). Additionally, we profiled 90 CML and 786 healthy TCRβ-seq samples, and sorted T cells specific to leukemia-associated antigen PR1 and matched these back to the scRNA+TCRαβ-seq and TCRβ-seq data. To validate and refine our findings, we performed co-culture of primary and expanded NK cells with CML cell lines (K562 and LAMA84) with a multiplexed scRNA-seq readout. Results: In the pan-cancer scRNA+TCRαβ-seq comparison of &amp;gt;250,000 cells, CML was separated from other cancers and healthy by the high number and active phenotype NK cells. The other phenotype more pronounced in CML was CD8+ T EM/T EMRA phenotype. Most NK cells in CML had an active CD56 dim phenotype with high expression of GZMA/B, PRF1, CCL3/4 and IFNG. In the off-TKI samples from patients with TFR, these NK cells upregulated NK cell activation genes such as TNFRSF4/OX40, TNFRSF9/4-1BB, and TNFRSF18/GITR. The most important predicted interactions between NK cells and CD34+ leukemic cells were inhibitory LGALS9- TIM3 and PVR- TIGIT interactions. Following TKI discontinuation, the inhibitory markers HAVCR2/TIM3 and TIGIT decreased in patients with TFR, which was not seen in patients with relapse following TFR. Accordingly, in our co-culture data of NK cells with CML cell lines, we noted activation of NK cells to a similar phenotype as noted in ex vivo off-TKI samples. In co-culture with CML target cells, NK cells gained an activation by upregulation of genes like TNFRSF4/OX40, TNFRSF9/4-1BB, TNFRSF18/GITR and the inhibitory markers HAVCR2/TIM3 and TIGIT. In co-culture with NK cells, CML target cells upregulated the IFN-γ response pathway including elevated HLA-expression and accordingly, upregulation of LGALS9 and PVR. The CD8+ T EM/EMRA cells that were expanded in CML diagnosis were suppressed following TKI treatment and decreased following TKI discontinuation. To track anti-CML T cells, we created a classifier to identify TCRs targeting leukemia-associated antigen PR1 from the TCRb-sequenced samples. As a validation of our classifier, we noticed that anti-PR1 T cells were more prevalent in CML than in healthy ( P adj&amp;lt;0.0001) or melanoma ( P adj&amp;lt;0.0001), more expanded than similar anti-viral T cells ( P adj&amp;lt;0.0001) and enriched in bone marrow samples compared to peripheral blood ( P adj&amp;lt;0.0001). From different TKIs, dasatinib expanded anti-PR1 T cells the most ( P adj&amp;lt;0.01). In the scRNA+TCRαβ-seq data, anti-PR1 T cells were enriched in the mature, cytotoxic CD8+ T EMRA phenotype ( P adj&amp;lt;0.0001). In a patient maintaining TFR, anti-PR1 T cells had a highly cytotoxic profile ( GZMB, PRF1) throughout the therapy with upregulated NK receptors like FCGR3A/CD16 and KIR2DL2, even more so than T cells targeting other targets e.g., different viruses. In comparison, the anti-PR1 T cells in a patient with early relapse had an exhausted profile with upregulation of memory marker GZMK and exhaustion markers like LAG3 and HAVCR2/TIM3. Conclusions: With our in-depth analysis of cellular and molecular immune responses in CML, we identified the active NK cells and anti-PR1 T cells that could help maintain TFR in patients discontinuing TKI treatment. Different immune-checkpoint blockade therapies such as anti-TIM3 could be considered as future strategies to improve the TFR rates.

Predictive Value of BCR::ABL1 Transcripts Kinetics in the Molecular Relapse of Chronic Myeloid Leukemia Patients after Tyrosine Kinase Inhibitors Discontinuation: Results from the DES-CML Study

Duration of previous treatment, duration and depth of molecular response, previous resistance, BCR::ABL1 transcript, and lymphocyte subtypes are known predictive factors of molecular recurrence after tyrosine kinase inhibitors (TKI) discontinuation in chronic myeloid leukemia (CML) patients (pts). Few studies explored the kinetics of BCR::ABL1 during a dose de-escalation phase before discontinuation in predicting molecular relapse. This study aimed to evaluate the kinetics of BCR::ABL1 transcripts during the de-escalation phase of the DES-CML trial and to correlate with molecular relapse after TKI discontinuation. Methods: This prospective, multicenter, single-arm, phase 2 study included CML pts treated DES-CML Study. Inclusion criteria: Adult CML pts treated with TKI for a minimum of 3 years that achieved sustained deep molecular response (DMR), defined by a minimum duration of MR4.5 for two years, confirmed by four real-time quantitative reverse-transcription polymerase chain reaction (qPCR) tests with six months interval. Exclusion criteria: pts with atypical BCR::ABL1 transcripts, previous or current advanced stage CML, previous resistance to TKI or ABL mutations. The TKI dose was reduced by 50% for six months before discontinuation (de-escalation phase). Molecular monitoring was performed by qRT-PCR, using ABL as the control gene. The results were reported using the International Scale. BCR::ABL1 transcripts were evaluated monthly during the de-escalation phase. After discontinuation, assessments were performed monthly for six months, every two months at months 7-24, and then every three months. Therapy was restarted at the same dose before dose reduction if loss of MMR (qPCR&amp;lt;0.1%). Statistical analysis was performed by using SPSS software. Rates of MRFS were calculated by Kaplan-Meier analysis, and groups were compared using a log-rank test. P values of &amp;lt;.05 were considered statistically significant. This trial is registered at ReBEC (RBR-6f5xbq; UTN code: U1111-1252-7312) and was approved by the ethics committees of the participating centers. All pts signed informed consent. Results: This study is ongoing. Forty-one pts were enrolled from September 2020 to July 2023, with a median age of 61 years (23-85), 60% men, 33% Sokal low risk, 50% intermediate, 17% high; 78% with b3a2 BCR::ABL1 transcript type; 85% using imatinib, 7.5% dasatinib, 5% nilotinib, and 2.5% bosutinib. For 3 pts, it was the second attempt at stopping TKI. The median duration of TKI treatment until discontinuation was 11 years (3-20). The cutoff date was July 2023, with a median follow-up of six months (1-26) after TKI discontinuation. One patient had a screen failure; another was enrolled but has not started the de-escalation phase yet. Five patients are in the de-escalation phase, and 25 are in the discontinuation phase. Two patients did not complete the de-escalation phase due to a lack of medication and went to the discontinuation phase. One patient died from causes unrelated to CML in the discontinuation phase and was censored in the MRFS analysis. 15/33 (45%) patients lost MR4.5 at any time in the de-escalation phase, and 4/15 (31%) lost MMR after discontinuation. Three of the 18 patients who did not lose MR4.5 during de-escalation relapsed (16%, P=0.05). The two patients who did not undergo de-escalation were censored in this analysis. Nine pts lost MMR: one during de-escalation and 8/39 (20%) after discontinuation. The median time between discontinuation and loss of MMR was two months (0-10). The median time to MMR recovery was 1.5 months (1-5). The overall MRFS was 72% (95% CI: 56-88%)(Figure A) and 81% in the first-attempt discontinuation group. All pts in the second attempt relapsed (3 patients) (P&amp;lt;0.0001). There was no difference in MRFS by gender, BCR::ABL1 transcript type, Sokal, and ELTS. Molecular response status at discontinuation: 23 (74%) had MR4.5, 6 (19.5%) MR4.0, and 2 (6.5%) MMR. Patients with MR4.5 at discontinuation had a higher MRFS than those in MR4.0 and MMR (78% vs. 62% vs. 0%, respectively, P=0.012)(Figure B). No patient lost hematologic response or had disease progression. Conclusions: Loss of MR4.5 during the de-escalation phase or molecular status not in MR4.5 before discontinuation was predictive of subsequent loss of MMR. Treatment-free remission was not successful in the second attempt of discontinuation.

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 &amp;lt; 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 &amp;gt; 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.

Tyrosine Kinase Inhibitor Withdrawal Syndrome in Chronic Myeloid Leukemia Patients Participants of Two Discontinuation Clinical Trials

Discontinuation of tyrosine kinase inhibitors (TKI) is currently one of the main goals in CML treatment. One of the known adverse events after TKI discontinuation is withdrawal syndrome (WS), which can occur in about 30% of patients (pts), characterized by musculoskeletal pain that begins or worsens weeks after interruption and may be more severe in some cases, but rarely leading to the need for TKI reintroduction. Some studies suggest TKI dose reduction or optimization before stopping treatment may reduce WS rates. Aims: To estimate the incidence of WS after TKI discontinuation in CML in two clinical trials and to correlate the incidence of these events with patients' characteristics and molecular relapse-free survival (MRFS). Methods: We analyzed two cohorts of CML patients of two prospective, single-arm, phase II clinical trials: EDI-PIO (Clinicaltrials.gov: NCT02852486) and DES-CML (ReBEC UTN code : U1111-1252-7312). In both trials, inclusion criteria were CML in the chronic phase, treated with TKI for at least three years, and with deep molecular response (MR4.5) for two years. In the first trial, TKI was discontinued after using three months of pioglitazone, and in the DES-CML trial, after reducing the dose of the TKI by 50% for six months before discontinuation. TKI was reintroduced if the patient lost MR4.0 (EDI-PIO trial) or major molecular response (MMR) (DES-CML trial). Adverse events related to WS were evaluated until the fourth month after discontinuation. WS was defined as the onset of new musculoskeletal pain and/or an increase in pre-existing pain and/or the need to start any pain medication. The adverse event classification followed the Common Terminology Criteria (CTCAE- Version 4.0), musculoskeletal and connective tissue disorder. The data were collected and stored using the REDcap Platform (by Vanderbilt). MRFS was defined by discontinuation date until the last seen date or loss of MR4.0/MMR. The Kaplan-Meier method was applied for OS, whereas Log-Rank tests were applied to compare its curves. All statistical analysis were done by IBM-SPSS v. 24 and considered significant p-value &amp;lt; 5%. Results: Between September 2016 and April 2023, we analyzed 63 patients, 32 from the DES-CML study and 31 from the EDI-PIO trial. The median age at discontinuation was 58 years (24-79), 59% male, 52% Sokal low-risk, 35% intermediate, and 13% high; at discontinuation, 94% of the patients were using Imatinib, 3% Dasatinib, 1.5% Bosutinib and 1.5% Nilotinib. The median duration of TKI treatment until discontinuation was 10 years (3-20). Withdrawal syndrome occurred in 41% of cases and was more frequent in females (58% vs. 42%, P=0.02). Fourteen pts from the EDI-PIO study (45%) and 12 (37.5%) from DES-CML developed WS (P=0.61). According to CTCAE, the events were classified as grade 1, 36%, and 33%, while grade 2 was 57% and 58%; and grade 3 was 7% and 8% (P=0.90), respectively, in EDI-PIO and DES-CML trials. Symptoms started at month 1 in 20% of the patients and 73% in months 2 and 3. Seventeen (30%) pts used anti-inflammatory drugs, analgesics, steroids, or muscle relaxants (64% EDI-PIO vs. 66% DES-CML, P=0.90). There was no need to reintroduce TKI for treating WS. Molecular relapse free-survival (MRFS) at 12 months was 72% (95% CI 60-84%), with no difference by protocol (75% DES-CML, 70% EDI-PIO). MRFS at 12 months was 73% and 74% in the group with or without WS, respectively (P=NS). Conclusions: The incidence and severity of WS were similar in the two trials, corroborating data from the literature. WS was more frequent in females. The dose reduction did not reduce WS rates. However, this condition was self-limited and managed with drugs, without reintroducing TKI. There was no difference in MRFS between the groups with or without WS.

Interleukin 10 (IL10) and IL15 Significantly Decreased in Chronic Myeloid Leukemia Patients Presenting with Tyrosine Kinase Inhibitor (TKI) Withdrawal Syndrome (TWS). Results from the Prospective, Explorative and Multicenter « Kiwis » Study Designed for TWS. Clinicaltrial.Gov ID: NCT03996096; Study Number: 18-206

Aims Treatment-free remission (TFR) has been included in practice guidelines as a new management goal for chronic myeloid leukemia (CML) patients, implying discontinuation of tyrosine kinase inhibitors (TKI). Paradoxically, TKI discontinuation gives rise to musculoskeletal pain and/or flushing, known as TKI withdrawal syndrome (TWS), in approximately 25% of patients with a reported median time of occurrence of 6 weeks. This syndrome is assumed to be caused by cytokine release, mast cell regrowth, or reactivation of an underlying inflammatory state. The KIWIS trial aimed at assessing these hypotheses by prospectively dosing biological markers before and after TKI discontinuation looking for any significant variation to be correlated to TWS. The secondary objectives were: description of the manifestations of TWS, assessment of TFR. Here, we will only focus on the primary objective. Methods The KIWIS study ( Clinicaltrial.govID: NCT03996096) was a multicenter prospective cohort of adult CML patients who had been treated with TKIs for at least 5 years, with a minimum of 2 years in deep molecular response (MR4.5). Patients with a history of inflammatory disease, those taking steroids, or those unable to complete the short form of the Brief Pain Inventory (BPI) were not included. BPI was used to assess pain severity and its impact on daily functioning. The arithmetic mean of the four severity items was used to score the severity of pain at different time points and identify patients with TWS. Blood samples were collected at baseline (M0), 1 month (M1), 2 months (M2), and 3 months (M3) post-discontinuation of TKI. We explored the following biomarkers: CRP, Rheumatoid Factor, tryptase, calcium, phosphate, Creatin Kinase, IL2, IL6, IL10, TGFβ, TNFα, IFNϒ, VEGF, PDGFβ, IL15, Crosslaps (CTX), Collagen pro-peptide type 1 (PINP1), and parathormone. Each patient served as their own control, and any variation of a biomarker by 20% from baseline was considered clinically significant. The BPI form was completed at the same time points as BCR-ABL follow-up: M0, M1, M2, M3, M4, M5, M6, M8, M10, and M12 following TKI withdrawal. The study was powered to include 50 patients. Results Out of the 50 patients, 16 (32%) developed TWS within the first 3 months, with a median onset time of 2 months. Patients' characteristics are displayed in table1. The baseline characteristics of patients in the TWS and non-TWS groups were similar. The duration of CML treatment was significantly longer in TWS patients compared to non-TWS patients (median: 9, Interquartile Range (IQR) [7-13] vs 7, IQR [6-8], p=0.039, respectively). No predictive factor for developing TWS were observed considering patients' characteristics and BPI scores at M0 in the univariate analysis. At M0, there were no significant differences between the two groups in terms of the concentration of each specific biomarker. In the TWS group, IL10, IL15, and IFNϒ levels decreased between inclusion and the M3 time point. For IL10, the median was 0.18 pg/ml (IQR: 0.12; 0.31) vs. 0.12 (IQR: 0.08-0.17), p=0.002; for IL15, 2.30 pg/ml (IQR: 1.77; 2.92) vs. 1.81 (IQR: 1.48; 2.25), p=0.026; and for IFNϒ, 5.92 pg/ml (IQR: 4.17; 12.49) vs. 4.24 pg/ml (IQR: 2.64; 7.99), p=0.036. In the non-TWS group, no significant differences were found. Changes in IL10 were positively correlated with changes in IL15 from M0 to M3 in all patients: r=0.42, p=0.009. No significant variations were observed in the two patient groups regarding the other biomarkers. Figure 1 exemplifies the evolution of IL10 (A: in TWS patients; B: in non-TWS patients) at M0 and M3. One patient missed the M3 time point sample. Conclusion This is the first study designed to prospectively investigate biomarkers associated with TWS. Our results suggest that TWS is not a cytokine-release-like syndrome but a distinct entity significantly associated with a decrease in IL10, IL15, and IFNϒ concentrations. It is worth noting that IL10 is an anti-inflammatory cytokine known to reduce pain; the role of IL15 and IFNϒ remains to be clarified. We hope that this study will pave the way for further investigations to fully understand the pathophysiology of TWS.

Successful Treatment-Free Remission in Low- and Middle-Income Countries

Introduction Discontinuation of tyrosine kinase inhibitors (TKIs) is feasible in a subset of chronic myeloid leukemia (CML) patients who have maintained a deep molecular response (MR) for ≥2 years. Numerous TKI discontinuation trials from high-income settings show that ~50% of patients relapse after discontinuing TKIs and need to restart treatment. However, most patients with CML worldwide are from low- and middle-income countries LMIC where TKI access and molecular testing make routine care and attempts of treatment-free remission (TFR) problematic. The successful implementation of TFR in LMICs would benefit both CML patients as well as the health economics of each country. This analysis describes the experience of attempting TKI discontinuation in the LMIC setting. Methods The Max Foundation's TFR Support Program provides financial and wrap-around support for patients attempting TFR in LMICs. The program operates in countries where The Max Foundation has existing partner institutions and physicians, imatinib and second line TKIs are available, and there is ≥1 reliable method of quantitative RT-PCR testing for BCR::ABL1. Eligible patients were &amp;gt;18 years old, diagnosed with chronic phase CML and treated with imatinib for ≥10 years with no history of resistance, and had a BCR-ABL level MR 4.5 or better (≤0.0032%) for ≥12 months (m) as demonstrated by 2 RT-PCR tests. Patients who consented were enrolled in the program and monitored by their treating physician. Upon starting TFR, patients' BCR::ABL1 levels were tested monthly for the first 6 m, then every 3 m for the following 6 m, then every 6 m. Patients who relapsed (BCR::ABL1 loss of MR 3 or &amp;gt;0.1%) after beginning TFR restarted imatinib and were tested approximately every 1-3 m until they achieved MR 3. We conducted a retrospective analysis on the outcomes of patients enrolled in The Max Foundation's TFR Support Program to better understand TFR outcomes in an LMIC setting. Results The molecular results pre- and post-TKI discontinuation are shown in Figure 1. In total, 39 patients from sites in 5 countries (Armenia, Honduras, India, Kenya, and Paraguay) were followed by 8 program physicians. The mean age of the patients was 51 years (range, 22-77), with 22 females and 17 males. Patients had been on imatinib for an average of 15 years (range, 10-20). At time of discontinuation, all but 1 patient was on 400 mg/day of imatinib (1 treated at 300 mg/day). Thirty-two patients had undetectable BCR::ABL1 for both pre-discontinuation tests, 6 had one test with a value of &amp;lt;0.01%, and 1 had both tests detectable at &amp;lt;0.01%. Of the 39 patients, 32 remain actively followed, with 14 patients completing &amp;gt;12 m of follow-up. Nine patients had a molecular relapse (23%) with a BCR::ABL1 &amp;gt;0.1% IS after a median of 2 m of discontinuation (range, 1-18 m). Of these, 2 patients have not had repeat testing to confirm molecular relapse, and 1 patient became undetectable on follow-up 1 m later. None of these 3 patients have restarted TKI therapy. Seven of 39 patients (18%) have restarted TKI treatment. Of these 7 patients, 3 were restarted on TKI, but have not had a follow-up BCR::ABL1 evaluation. Of the remaining 4 patients who restarted TKI, 2 had no detectable BCR::ABL1 at the 9 m and 12 m follow-up, and 2 remain with evidence of BCR::ABL1 at 12 m and 17 m follow-up. Conclusions The data suggest that under conditions that allow frequent follow-up examination and testing, patients attempting TFR in LMIC have similar outcomes to those patients described in controlled clinical trials from academic settings in high-income countries. Additional confirmation of these findings will likely impact both the health of CML patients and the health economics in these LMICs. Figure 1. BCR:ABL1 and outcomes following TKI discontinuation.

Baseline Features, Treatment Choice and Early Frontline TKI Permanent Discontinuation in Younger Patients with Chronic Myeloid Leukemia: A “Campus CML” Study

Introduction Median age at diagnosis in patients with Chronic Myeloid Leukemia (CML) is about 60 years: only few data are available in younger subjects at present. Aim To analyze initial features, choice of frontline Tyrosine-Kinase Inhibitor (TKI) and early adverse events leading to permanent TKI discontinuation in the first 12 months from TKI start in newly diagnosed subjects aged ≤ 40 years in a large and unselected real-life cohort of CML patients. Methods We retrospectively evaluated 1963 patients newly diagnosed from 1/2012 to 12/2019 at 38 Hematology Centers in Italy participating at the “Campus CML” project: according to age at diagnosis, 302 were ≤ 40 years [Younger Patients (YP)], 900 were &amp;gt; 45 &amp;lt; 65 years [Intermediate Aged Patients (IAP)] and 761 were ≥ 65 years [Elderly Patients (EP)]. Results Clinical features, frontline TKI and causes of early TKI discontinuation according to age at diagnosis are reported in the Table 1. As to baseline clinical features, YP had significantly higher WBC count, lower Hb levels and higher rate of spleen enlargement: moreover, as expected, low-risk Sokal score was more common and rate of concomitant diseases/number of concomitant drugs lower in YP. As to frontline TKI, 96 YP (31.1%) received imatinib (IM) and 206 (68.9%) a 2G-TKI [dasatinib (DAS) n=60, 19.9%; nilotinib (NIL) n=144, 47.6%]: choice of 2G-TKI as frontline treatment was significantly more common in YP compared to both IAP and EP (p &amp;lt; 0.001). Causes of permanent frontline TKI discontinuation during the first 12 months of treatment according to age at diagnosis are reported in the Table 1: among YP, 45 (14.9%) discontinued frontline TKI treatment due to hematologic toxicity (5 cases, 1.6%), extra-hematologic toxicity (11 cases, 3.7%), primary resistance (24 cases, 8.0%), secondary resistance (2 cases, 0.6%) or evolution in blastic phase (BP) (3 cases, 1.0%). Cumulative incidence of permanent discontinuation at 12 months was 14.6% (95%CI 10.7 - 18.5) in YP compared to 16.9% (95%CI 14.3 - 19.2) in IAP (p=0.388) and 24.0% (95%CI 20.9 - 27.1) in EP (p &amp;lt; 0.001) (Figure 1): considering only permanent TKI discontinuation due to treatment resistance (primary resistance + secondary resistance + evolution in BP), no difference was observed according to age at diagnosis [9.6% (95%CI 6.3 - 12.9) in YP versus 10.2% (95%CI 8.2 - 12.2) in IAP versus 11.8% (95%CI 9.6 - 14.0) in EP, p=0.589]. Conclusions Our data highlight the following peculiar clinical features at baseline and during the first year of treatment in YP with CML compared with IAP and EP: a delayed diagnosis, outlined by higher WBC count and rate of spleen enlargement; an expected choice of 2G-TKI as frontline treatment in the real-life, even if about one third of them was still treated with IM; a lower incidence of early frontline TKI permanent discontinuation compared to EP but not IAP, mainly due to toxic causes and not related to resistance. Data on early molecular response and further evaluations at 24 and 36 months are warranted to complete present analysis.

Biological Factors Associated with Treatment-Free Remission in Clinical Practice: Data from the Canarian Registry of CML

Introduction: The probability of maintaining treatment-free remission (TFR) after the discontinuation of tyrosine kinase inhibitors (TKI) is affected by clinical factors such as length of TKI treatment and the depth of molecular response. However, some individuals with similar treatment duration and response remain in TFR while others relapse. Thus, patient-specific factors must also play a role. Biological factors suggested to influence TFR include BCR::ABL1 transcript type, blasts at diagnosis, halving time (HT, first 3 months), BCR::ABL1 positivity at stop, and presence of somatic mutations. The aim of this study was to evaluate the biological factors associated with TFR in the clinical practice, using data from the Canarian CML Registry. Methods: The OMOP-standardized data of adult patients with chronic phase CML who discontinued TKI treatment included in the Canarian CML Registry (https://rclmc.es/rclmc/) were reviewed. Criteria to discontinue included TKI treatment ≥5 years and MR4.5 or better ≥3 years. TFR was defined as major molecular response (MMR), or deeper after stop. Molecular relapse was defined as loss of MMR in two consecutive tests. NGS was performed with the 30-gene panel Myeloid Solution (SOPHiA) on the MiSeq (Illumina) platform using 200 ng genomic DNA extracted from peripheral blood at diagnosis. Results: A total of 109 patients were included, with an average age of 50.5 years; 54 were male (49.5%). Median follow-up was 3.3 years. 86 patients discontinued from first-line, 21 from second- and 2 from third-line. 51% of patients (n=56) discontinued from imatinib, 31% from nilotinib (n=34), 16% from dasatinib (n=17), and 1% from bosutinib and ponatinib (1 patient each). The median TFR was 101 months ( Figure 1A; range 1.02-148.4 months). Thirty-two patients had molecular relapse and re-started TKI treatment: 36.7% with imatinib or dasatinib, 26.7% with nilotinib. Median time to regain MMR was 101 days (range 25-276). No difference was observed in the time to regain MMR for imatinib vs. 2G-TKI (106 vs. 144, p=0.419). No patients progressed to an accelerated phase. Differences were observed in duration of MR4 and MR4.5 prior to stop comparing the TFR vs. relapse groups (88.3 vs. 69.9 months, p=0.044; 74.2 vs. 58.6 months, p=0.079), but not total time on TKI (172.6 vs. 102.1, p=0.310), respectively. In terms of biological factors at diagnosis, 44 patients had blasts, 66.7% had a low Sokal risk. 23.9% expressed transcript e13a2 and 76.1% had e14a2. No differences in TFR were observed when blasts, Sokal or transcript were considered in chi-squared analyses. A long HT (&amp;gt;21.9 days, Shanmuganathan et al. Blood 2021;137) was of marginal significance for molecular relapse (p=0.076, OR 3.2) but no difference was found for short HT (&amp;lt;9.4 days). By Kaplan-Meier analysis, an MR3, 4, or 4.5 at stop was predictive of molecular relapse (n=42; Figure 1B) vs. an MR5 or undetectable (limit of detection 0.0001%). In multivariable analysis, only an MR5 or deeper at stop and withdrawal syndrome were predictive for TFR (p=0.003, HR 4.3; p=0.049, HR 0.20, respectively). NGS data was available for 47 patients. Myeloid mutations were detected in 21 patients (44.7%) at diagnosis, of these 11 had an epigenetic mutation (23.9%; in ASXL1, DNMT3A, TET2 or EZH2). 11 patients (23.9%) had a variant that was potentially germline (VAF 40-60%). By chi-squared and Kaplan-Meier analysis, no differences were observed between the median TFR for patients with or without a pathogenic somatic, epigenetic, ASXL1 or germline mutation. However, a greater separation of the curves was observed when considering TFR at 36 months for somatic (p=0.101) but not germline mutation. Conclusions: We confirmed previous observations that the biological factors BCR::ABL1 positivity at stop and longer HT were associated with molecular relapse although no difference was found for short HT. The impact of clinical factors on TFR, including duration of MR4 and MR4.5 prior to stop, were also confirmed in our real-life series with a long follow-up, while total TKI duration was not prognostic. Adnan Awad et al. associated the presence of epigenetic mutations at diagnosis with shorter TFR and higher relapse probability (Blood Cancer J. 2022;12). Here, a significant difference in TFR was not observed for epigenetic mutation at diagnosis; however, a tendency was observed between a pathogenic somatic but not a germline myeloid mutation and TFR at 36 months.

Efficacy of Consolidation Therapy with Ponatinib 15mg on Treatment-Free Remission Rate in Patients with Chronic Myeloid Leukemia. Results of the Ponazero Trial

Introduction: Discontinuation of tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) has become part of routine clinical practice in recent years, thanks to growing evidence on their feasibility and safety. However, and despite adequate patient selection based on current recommendations, loss of response is observed in approximately 50% of patients. In this scenario, efforts are focused on finding strategies to increase the rate of successful discontinuations. Ponatinib has shown to induce deeper molecular responses compared with imatinib, but its risk of arterial occlusive events (AOE) limits its use in first line at its standard dose of 45mg. The risk of AOEs appears to be dose-related and following the results of the OPTIC study, dose reduction to 15mg in patients with good response is an increasingly popular strategy. Under these premises it has been postulated that a consolidation with ponatinib 15mg may increase the proportion of patients who could discontinue treatment successfully. Our aim is to evaluate the efficacy and safety of one year of consolidation therapy with ponatinib 15mg on treatment-free remission (TFR) rate in patients with CML who have achieved a deep molecular response with imatinib. Material and Methods: A multicenter open-label, single-arm, phase II, exploratory, prospective clinical trial (NCT 04043676) was conducted including patients with philadelphia-positive CML who met discontinuation criteria according to European LeukemiaNet recommendations, had received 4 years or more of imatinib therapy, with a documented grade 4 molecular response (MR4) at least 12 months prior to study entry. Uncontrolled arterial hypertension, active cardiovascular disease, or concomitant medication with potential for QTc prolongation were exclusion criteria. The study has two main phases: ponatinib consolidation (48 weeks), and ponatinib TFR phase (48 weeks); where patients remain without treatment. During the TFR phase, patients with a confirmed loss of MR4 or a single value above major molecular response (MMR) were scheduled to restart treatment with a TKI at investigator's discretion. The present abstract reports the results of the primary endpoint of the study; the proportion of patients with no loss of MR4 at the end of the TFR phase. Results: A total of 23 patients started treatment with ponatinib 15mg. One patient subsequently failed screening due to a major protocol deviation. Baseline patient characteristics were as follows: median age was 51.8 years (range 26-74), 65.2% were male. Ninety-five percent were Caucasian and 5% were Hispanic, ECOG was 0 in 83% and 1 in 17%, Sokal was low in 69.5%, intermediate in 17.5% and unknown in 13%. Eighteen patients were receiving imatinib 400mg daily (82%) at study entry, 2 patients 800mg and two 200mg. Median time on prior imatinib was 10 years (range 4-17), with a median time on MR4 of 3.6 years (range 1.9-13.3). Prior to initiation 13 patients (56.5%) had a grade 5 molecular response, 4 had a 4.5 molecular response (26%) and the remainder had a MR4. The adverse events (AEs) reported with ponatinib are summarized in Table 1, the most frequent being constipation (30%), asthenia (26%), myalgias and arthralgias (26% respectively) and skin rash (17%). A high rate of SARS-CoV2 infections was reported during the treatment period (26%), which should be interpreted considering that the study took place during the COVID-19 pandemic. Three patients out of 22 (13.6%) had serious AEs with ponatinib leading to treatment discontinuation; thrombophlebitis, erectile dysfunction, and intermittent claudication. Of the patients who entered the TFR phase, 14/19 (74%) continue with MMR without requiring treatment after a median follow-up of 12 months (68% with MR4) (table 1). Six patients (31%) lost MR4, five of them required reintroduction of an TKI (4 imatinib, 1 dasatinib; median time to reintroduction 5.4 months). All patients with MR4 loss were receiving the 400 mg dose of imatinib at study entry. All patients who restarted treatment recovered MMR. The sixth patient lost MR4 while maintaining an MMR without requiring treatment reinduction to date. Conclusions: Consolidation with 12 months of ponatinib 15mg in this study shows a high discontinuation success rate with an adequate safety profile, although not free of AEs. The results make this approach attractive for the design of further randomized clinical trials.