Age-Related Clonal Hematopoiesis Mutations Detected at the Time of Stopping Tyrosine Kinase Inhibitor Therapy Predict the Achievement of Treatment-Free Remission for Patients with CML
Authors: Branford Susan , Wadham Carol , Shanmuganathan Naranie , Fernandes Adelina , Shahrin Nur Hezrin , Feng Jinghua , Toubia John , Wang Paul , Kok Chung Hoow , Maqsood Muneeza , Saunders Verity , King Rob , Lin Ming , Kenyon Rosalie , Pagani Ilaria Stefania , Yeung David T , Ross David M. , Yong Agnes S. M. , Hughes Timothy P 
Published: December 2023

Background
The gain of mutations with a competitive advantage in hematopoietic cells of healthy individuals is common with aging and termed clonal hematopoiesis of indeterminate potential (CHIP). Through sequencing studies we detected CHIP related mutants in chronic myeloid leukemia (CML) patients in deep molecular response. CHIP mutants may provide a selective growth advantage to non-leukemic cells, driving their clonal expansion in remission and potentially influencing treatment-free remission (TFR) if CHIP clones outcompete an emerging BCR:: ABL1 clone.
Aim
To determine if CHIP mutants detected at the time of stopping tyrosine kinase inhibitor (TKI) therapy could influence achievement of TFR.
Methods
An RNA-based sequencing method targeting 17 of the most frequently mutated CHIP genes was developed and applied to blood samples of 150 patients who attempted TFR. The median follow up of patients in TFR was 71 months. CHIP mutants met strict criteria for pathogenicity based on their potential to confer growth and survival advantages, i.e. the same criteria as cancer driver mutations. Variant allele frequency (VAF) of ≥0.4% was reproducibly detected.
CHIP at the time of cessation, termed CHIP TOC, was defined as 1) ≥1 CHIP mutant with VAF ≥2.0% (n=30 patients) or 2) a novel criterion for patients with multiple CHIP mutants where the combined VAF was ≥2.0% (n=7). Overall, 25/37 patients (68%) with CHIP TOC had multiple mutations, range 2-7.
Results
CHIP TOC was detected in 37/150 patients (25%). The average age of patients with CHIP TOC was 66.3 years vs 58.0 for those without, P=.0004, which is consistent with the age related nature of CHIP. No patient aged &lt;40 had CHIP TOC, whereas 44% of patients ≥70 had CHIP TOC. Twelve genes were mutated and the most frequent were TET2, DNMT3A, ASXL1 and PPM1D.
The overall probability of TFR was 60.4% at 12 months and 53.0% at 84 months. CHIP TOC was associated with TFR. At 12 months, 83.6% of patients with CHIP TOC maintained TFR versus 52.9% of patients without CHIP TOC, P=.002. At 84 months, 62.9% of patients with CHIP TOC maintained TFR versus 49.6% of patients without CHIP TOC, P=.014.
We previously demonstrated that e13a2 transcripts and a slow initial decline of BCR:: ABL1 after starting frontline TKI therapy, measured as the number of days over which BCR:: ABL1 halved, were our strongest predictors of relapse. Prognostic factors for the achievement of TFR at 12 and 84 months were examined in multivariable analysis. Candidate prognostic factors were CHIP TOC, BCR:: ABL1 halving time, BCR:: ABL1 transcript type, age at diagnosis, gender, duration of MR4.5 and time on TKI before TKI stop. The independent predictors of TFR at 12 and 84 months were the presence of CHIP TOC at TKI stop, a shorter BCR:: ABL1 halving time (indicating a more rapid initial BCR:: ABL1 decline) and the e14a2 (plus e14a2/e13a2) BCR:: ABL1 transcript type (Table 1).
The rate of late relapse, defined as loss of MMR &gt;12 months after cessation, was higher in patients with CHIP TOC. Overall, 85 patients had TFR at 12 months and late relapse occurred in 8. A landmark analysis demonstrated that the probability of maintaining TFR at 84 months for these 85 patients was 93.8% for those without CHIP TOC (n=57) and 75.2% for those with CHIP TOC (n=28), P=.007. The only variable associated with late relapse among the patients with CHIP TOC was fluctuation of BCR:: ABL1 within the first 12 months after cessation, defined as irregular rise and fall comprising 2 consecutive BCR:: ABL1 ratios with loss of MR4.5, or a single loss of MR4. Overall, 13 patients had fluctuation within the first 12 months and 5 of these had late relapse. All 5 had CHIP TOC. Patients with CHIP TOC and stable MR4.5 by 12 months had a 93.8% probability of TFR at 84 months (Table 2).
Conclusion
The presence of CHIP TOC at TKI cessation, a shorter BCR:: ABL1 halving time and e14a2 BCR:: ABL1 transcripts were independent predictors of TFR at 12 and 84 months. Delayed loss of MMR that occurred for a minority of patients with CHIP TOC may be associated with competition between CHIP clones and a residual leukemic clone. These patients had fluctuating BCR:: ABL1 over the first 12 months after cessation and prior to late relapse. Novel strategies may be warranted to reduce the risk of late relapse for these patients. Patients who sustained TFR by 12 months in stable MR4.5 had a reassuringly low risk of late relapse. CHIP status could be a key variable to guide TFR decision-making and monitoring.