Highlights
- Identification of gene expression signatures characterizing treatment-free remission (TFR) in chronic myeloid leukemia (CML) patients after tyrosine kinase inhibitor (TKI) discontinuation.
- Evidence of sustained intercellular communication between innate and adaptive immune cells in patients maintaining TFR, mirroring healthy controls.
- Disruption of immune cell interactions correlates with loss of treatment-free remission and molecular relapse.
- Definition of an FLT3 expression threshold distinguishing patients with significantly different risks for TFR loss, suggesting potential biomarker utility.
Background
Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder characterized by the presence of the BCR-ABL1 fusion gene resulting from the Philadelphia chromosome translocation. This oncogenic tyrosine kinase drives malignant proliferation. The introduction of targeted therapy with tyrosine kinase inhibitors (TKIs) revolutionized CML treatment, transforming a formerly fatal disease into a chronic condition with near-normal life expectancy. However, indefinite TKI treatment poses long-term toxicity and financial burdens.
Recently, treatment-free remission (TFR)—cessation of TKI therapy in patients who achieve deep molecular responses—has emerged as an important clinical goal. Despite successful TKI discontinuation in a subset of patients, approximately 50% experience molecular relapse necessitating therapy resumption. The molecular and immunological mechanisms maintaining durable TFR versus those underlying relapse remain incompletely understood. Elucidating these mechanisms is vital to optimize patient selection for TKI discontinuation and develop strategies to improve sustained remission.
Study Design
The European Stop Kinase Inhibitors (EURO-SKI) trial is a prospective, multicenter clinical study investigating TKI discontinuation in CML patients who attained stable deep molecular responses. This biomarker sub-study focused on molecular and immunological characterization of peripheral blood leukocytes at the time of TKI cessation.
The investigation included 240 CML patients divided into a training cohort (n=122) and a validation cohort (n=118), alongside 10 healthy controls. Whole transcriptome gene expression analysis was conducted using RNA sequencing and NanoString nCounter technology for absolute quantification of relevant transcripts. Additionally, an external dataset of 96 TKI-naïve CML patients was incorporated to delineate TKI-specific effects.
The primary objective was to identify molecular signatures and immune regulatory networks distinguishing patients who maintain TFR from those who relapse molecularly after therapy discontinuation. The study also sought predictive biomarkers associated with risk of TFR loss.
Key Findings
Molecular Signatures Associated with Treatment-Free Remission
TFR patients exhibited activation of transcriptional regulons controlled by GATA1, KLF1, and MYBL1, which are classical regulators of erythroid progenitor differentiation. This suggests that a reprogramming or persistence of erythroid lineage-associated expression patterns may represent a molecular hallmark of durable remission.
Immune Cell Interactions and Immune Surveillance
Analysis of cell-type specific gene signatures revealed that patients sustaining TFR maintained active communication between innate immune cells—including natural killer (NK) cells and dendritic cells—and adaptive immune cells such as CD8+ cytotoxic T lymphocytes, CD4+ helper T cells, and γδ T cells. This network resembles immune profiles observed in healthy donors, implying preserved immune surveillance mechanisms.
Conversely, patients experiencing molecular relapse demonstrated disruption of these immune cell interactions, indicating compromised immunological control potentially facilitating leukemic resurgence.
FLT3 Expression as a Predictive Biomarker
The study identified a specific threshold of FLT3 expression that segregates patients into distinct risk categories for TFR loss. Patients with FLT3 levels above this threshold showed significantly higher probability of molecular relapse, suggesting FLT3 as a promising biomarker for risk stratification upon TKI discontinuation.
Comparison to TKI-Naïve Patients
Incorporation of transcriptomic data from TKI-naïve CML patients demonstrated that gene expression patterns characteristic of TFR are not merely a baseline feature of the leukemic clone but evolve under TKI therapy. This insight highlights the influence of therapy on shaping molecular remission profiles.
Expert Commentary
This comprehensive molecular and immunological characterization advances our understanding of factors governing TFR in CML. The preservation of innate-adaptive immune crosstalk aligns with emerging concepts positing immune surveillance as essential for controlling minimal residual disease after therapy cessation.
The involvement of erythroid transcription factors in TFR is intriguing and may suggest previously unrecognized roles of hematopoietic lineage differentiation status in remission maintenance. Whether these signatures reflect direct effects on leukemic stem cell properties or an altered bone marrow microenvironment warrants further exploration.
Despite robust findings, limitations include reliance on peripheral blood samples, which may incompletely reflect bone marrow niche dynamics. Additionally, the FLT3 threshold requires validation in external cohorts and prospective settings before clinical implementation.
Current guidelines cautiously endorse TKI discontinuation in carefully selected patients but lack biomarkers for precise risk stratification. This study paves the way for biomarker-driven decisions and suggests therapeutic avenues targeting immune reactivation to promote durable TFR.
Conclusion
The EURO-SKI Biomarker Study provides pivotal insights into molecular and immune landscapes underpinning treatment-free remission and molecular relapse in CML patients discontinuing TKIs. Activation of erythroid progenitor-associated regulons and persistent functional interplay between innate and adaptive immunity emerge as hallmarks of sustainable remission, while disruption of these pathways predisposes to relapse.
FLT3 expression levels offer a candidate biomarker for predicting relapse risk, with potential utility for individualized patient management. Therapeutic strategies aimed at restoring or strengthening immune communication networks may enhance durable TFR rates, ultimately improving patient outcomes and quality of life.
Future research should focus on integrative multi-omics analyses, longitudinal immune monitoring, and interventional trials to translate these molecular insights into clinical practice.
Funding and Trial Registration
This study was supported by the EURO-SKI consortium and associated European research funding bodies. The clinical trial is registered under clinicaltrials.gov identifiers associated with the EURO-SKI program.
References
Rinaldetti S, Chepeleva M, Hedblom A, et al. Comprehensive molecular characterization of treatment-free remission and molecular relapse in chronic myeloid leukemia patients: the EURO-SKI Biomarker Study. Leukemia. 2026 Jun 18. doi:10.1038/s41375-026-xxxx-x. PMID: 42315627.
Mahon FX, et al. Discontinuation of Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia: Recommendations for Clinical Practice. Oncotarget. 2020;11(6):1345-1358.
Burchert A, et al. Immune mechanisms in treatment-free remission of chronic myeloid leukemia. Front Oncol. 2021;11:656586.