Chronic Myeloid Leukemia Patients Research Articles

Epigenetic Modulation of DDIT3 and MGMT Expression Acts Synergistically with Resistance to Imatinib towards CML Disease Progression: A Hospital based Study.

Imatinib Mesylate is an authenticated drug that aids in the treatment of Chronic Myeloid Leukaemia and Philadelphia patients which is recognized as a BCR-ABL tyrosine kinase inhibitor. Indeed, DNA Methylation occupies a key role in the stability of chromosomes. Changes in the methylation status of genes may impart to the advancement of Chronic Myeloid Leukaemia. The present investigation aims to assess the role of expression analysis and methylation status of DDIT3 and MGMT genes in imatinib-resistant and nonresistant cases. The Imatinib resistance was screened through RFLP. In this case maximum number of patients were recorded in the chronic phase belonging to the age group 40-59 and the accelerated and blast phase is more common in elderly patients showing the progressive nature of the disease with age. Hemoglobin and platelet count are found to be higher in cases where WBC count was minimal. A history of long-term alcohol consumption is found to be associated with the progression of the disease. The maximum level of expression of the DDIT3 gene was recorded in the chronic phase regardless of upstream (67.8%) and downstream (57.9%) regulation. The highest MGMT expression regulation was also observed in the case of chronic phase in both upstream (78.9%) and downstream (44%) regulation. Further, the MGMT gene showed the highest methylation of 6.6% and DDIT3 showed 3.3% in CML cases. In the present study notable depletion of survivality was established in the Imatinib resistance patients manifesting genetic malfunction of BCR-ABL transcripts among the North East Indian inhabitants and advocating for the expansion of the disease.

Expression of genes potentially involved in loss of response in patients with chronic myeloid leukemia

Chronic Myeloid Leukemia (CML) is a hematological malignancy characterized by the presence of the BCR::ABL1 fusion gene, which leads to uncontrolled cell growth and survival. Tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of CML, but a significant proportion of patients develop resistance or lose response to these drugs. Understanding the molecular mechanisms underlying treatment response and resistance is crucial for improving patient outcomes. This study aimed to analyze the expression patterns of genes involved in treatment response and resistance in CML patients receiving TKI therapy. The expression levels of MET, FOXO3, p15, p16, HCK, and FYN genes were examined in CML patients and compared to healthy donors. Gene expression levels were compared between optimal responders (OR) and resistant patients (R) vs. healthy donors (HD). The MET and FOXO3 OR group showed significant differences compared with the HD, (p < 0.0001) and (p = 0.0003), respectively. p15 expression showed significant differences between OR and HD groups (p = 0.0078), while no significant differences were found in p16 expression between the HD groups. FYN showed a statistically significant difference between R vs. HD (p = 0.0157). The results of HCK expression analysis revealed significant differences between OR and HD (p = 0.0041) and between R and HD (p = 0.0026). When we analyzed OR patients with undetectable BCR::ABL1 transcripts, a greater expression of HCK was observed in the R group. These findings suggest that monitoring the expression levels of MET and FOXO3 genes could be valuable in predicting treatment response and relapse in CML patients. Our study provides important insights into the potential use of gene expression analysis as a tool for predicting treatment response and guiding treatment decisions in CML patients. This knowledge may ultimately contribute to the development of personalized treatment strategies to improve patient outcomes in CML.

Evaluation of serum taurine level as a biomarker for early diagnosis and follow up of chronic myeloid leukemia

Evaluation of serum taurine level as a biomarker for early diagnosis and follow up of chronic myeloid leukemia

Overcoming BCR::ABL1 dependent and independent survival mechanisms in chronic myeloid leukaemia using a multi-kinase targeting approach

BackgroundDespite improved patient outcome using tyrosine kinase inhibitors (TKIs), chronic myeloid leukaemia (CML) patients require life-long treatment due to leukaemic stem cell (LSC) persistence. LSCs reside in the bone marrow (BM) niche, which they modify to their advantage. The BM provides oncogene-independent signals to aid LSC cell survival and quiescence. The bone-morphogenetic pathway (BMP) is one pathway identified to be highly deregulated in CML, with high levels of BMP ligands detected in the BM, accompanied by CML stem and progenitor cells overexpressing BMP type 1 receptors- activin-like kinases (ALKs), especially in TKI resistant patients. Saracatinib (SC), a SRC/ABL1 dual inhibitor, inhibits the growth of CML cells resistant to the TKI imatinib (IM). Recent studies indicate that SC is also a potent ALK inhibitor and BMP antagonist. Here we investigate the efficacy of SC in overcoming CML BCR::ABL1 dependent and independent signals mediated by the BM niche both in 2D and 3D culture.MethodsCML cells (K562 cell line and CML CD34+ primary cells) were treated with single or combination treatments of: IM, SC and the BMP receptors inhibitor dorsomorphin (DOR), with or without BMP4 stimulation in 2D (suspension) and 3D co-culture on HS5 stroma cell line and mesenchymal stem cells in AggreWell and microfluidic devices. Flow cytometry was performed to investigate apoptosis, cell cycle progression and proliferation, alongside colony assays following treatment. Proteins changes were validated by immunoblotting and transcriptional changes by Fluidigm multiplex qPCR.ResultsBy targeting the BMP pathway, using specific inhibitors against ALKs in combination with SRC and ABL TKIs, we show an increase in apoptosis, altered cell cycle regulation, fewer cell divisions, and reduced numbers of CD34+ cells. Impairment of long-term proliferation and differentiation potential after combinatorial treatment also occurred.ConclusionBMP signalling pathway is important for CML cell survival. Targeting SRC, ABL and ALK kinases is more effective than ABL inhibition alone, the combination efficacy importantly being demonstrated in both 2D and 3D cell cultures highlighting the need for combinatorial therapies in contrast to standard of care single agents. Our study provides justification to target multiple kinases in CML to combat LSC persistence.

Real World Population- Based Data from Nationwide Registry Support the Existence of Accelerated Phase in Newly Diagnosed Chronic Myeloid Leukemia (CML) Patients Even in Tyrosine Kinase Inhibitors (TKIs) Era

Background For decades, CML has been considered to be a triphasic disease recognizing 3 distinct stages: chronic phase (CP), accelerated phase (AP), and blast crisis (BC). Since the discovery of TKIs, the prognosis of CML patients has rapidly improved and less than 10% of patients diagnosed in CP experience disease progression on the therapy. Based on low incidence of CML progression and the fact that CML biological behavior seems biphasic, the new WHO 2022 classification of CML deemed AP less relevant and suggested the omission of AP. Many experts, however, still advocate for its inclusion in CML classification (e.g. ELN 2020, ICC 2022, NCCN 2023), even though the universal definition of AP is not established and differs in between publications. Methods This retrospective, real-world study is based on the Czech nationwide CML registry INFINITY with the data of newly diagnosed CML patients who agreed and provided their written consent. Recruited subjects were adult patients diagnosed in years 2005 - 2022 with sufficient data to determine the phase of the disease at diagnosis and follow-ups to assess their overall survival (OS), disease specific survival (DSS) and progression-free survival (PFS). The phase of the disease at the time of diagnosis was determined using the classification according to ELN 2020, ICC 2022, WHO 2016, and WHO 2022. Whenever CP was established, EUTOS Long-Term Survival (ELTS) score was calculated, assigning the patient to low risk (LR), intermediate risk (IR) and high risk (HR) group. OS and PFS are defined according to published guidelines. DSS is defined as the time from diagnosis to death due to CML disease or CML treatment. Results During the studied time period, 1660 new cases of CML were reported in INIFINITY registry. Of them, 1500 patients had data sufficient to classify the phase of the disease at the time of the diagnosis according to ELN 2020 guidelines and WHO 2022 classification and 1395 patients had data to assess the phase according to WHO 2016 and ICC 2022 classifications. When using ELN 2020 guidelines, groups were assigned as follows: LR CP- 784 patients (52.3%), IR CP- 421 patients (28.1%), HR CP- 227 patients (15.1%), AP- 42 patients (2.8%), BC- 26 patients (1.7%). There were significant differences in representation by gender, age, comorbidities and performance score amongst patient groups. When comparing just HR CP and AP CML patients, there were no significant differences. Calculated estimates of 5- and 10- year OS, respectively, were 92.5% and 87.2% for LR CP, 83.5% and 65.9% for IR CP, 76.9% and 65.5% for HR CP, 59.2% and 45.9% for AP, and 32.9% for BC, p= 0.031 for HR CP versus AP (Figure 1). In the case of WHO 2016 and ICC 2022 classifications, 714 (51.2%) patients were diagnosed in LR CP, 374 (26.8%) in IR CP, 154 (11.0%) in HR CP, 125 (9.0%) in AP, and 28 (2.0%) in BC. Again, there were significant differences in representation by gender, age, comorbidities and performance score amongst patient groups. When comparing just HR CP and AP CML patients, only age at the time of diagnosis was significantly different. Calculated estimates of 5- and 10- year OS, respectively, were 93.2% and 87.5% for LR CP, 84.9% and 67.7% for IR CP, 80.0% and 69.5% for HR CP, 65.7% and 55.6% for AP, and 39.7% for BC. We also performed propensity score matching according to age for patients in HR CP and AP and calculated estimates of 5- and 10- year OS, respectively, were 85.4% and 74.4% for HR CP and 60.2%, and 52.0% for AP, p= 0.003 (Figure 2). Similar results were obtained when testing for PFS and DSS. Summary Real-world data obtained from 1500 patients diagnosed in Czechia over 17 years, in our opinion, support the need to recognize the existence of AP at the time of diagnosis, even though a singular definition of AP is not agreed upon. Patients in AP have worse survival scores (OS, DSS, PFS) compared to patients in CP and/or HR CP. Moreover, because ELTS risk score was calculated on patients in CP, it may not be suitable for patients in AP, which would be changed to CP according to the new WHO 2022 classification. In conclusion, based on the real-world data with long-term follow-up, we believe it would be reasonable to keep the AP as part of CML classification of newly diagnosed patients. This publication was supported by the grant number MUNI/A/1224/2022, Programme EXCELES, ID Project No. LX22NPO5102, and by the Ministry of Health of the Czech Republic grant number 00023736.

Sars-Cov-2 and Chronic Myeloid Leukemia: A Systematic Review

Introduction Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus causing the coronavirus disease of 2019. Numerous mutations had developed in SARS-CoV-2 virus that resulted in strains (Alfa Beta Delta Omicron) with varying degrees of virulence disease severity. In coronavirus disease of 2019 (COVID-19) patients with chronic myeloid leukemia (CML), there is a controversy regarding the effect of tyrosine kinase inhibitors (TKI) on SARS-CoV-2 infection and patients' outcomes. Some studies showed that CML patients with COVID-19 had better outcomes; other reports showed no significant difference. Method Following the PRISMA (preferred reporting items for systematic reviews and meta-analyses) guidelines, PubMed, Scopus, and Google Scholar databases were searched for published articles through 20 April 2023 for qualified studies. The search included retrospective, prospective studies, reviews, case series, and case reports. The inclusion criteria were English literature with CML patients above 18 years who had SARS-CoV-2 infection. Articles in languages other than English and articles with non-sufficient information were excluded, and patients with bone marrow transplants were excluded. Search terms were (chronic myeloid leukemia) OR (chronic myelogenous leukemia) AND (SARS-CoV-2) OR (COVID-19). Two eligibility checks were done on the included studies. Title and abstract screening was completed first, and the full text of articles that met the requirements for inclusion was subsequently obtained. The search included all articles published up to 20 April 2023. The review is registered in PROSPERO (registration number is: CRD42022326674). Results We reviewed 36 articles of published literature up to April 2023 and collected data for a total of 686 CML patients with COVID-19. The vast majority of patients were in the chronic phase; seven patients were in the accelerated phase, and eight patients were in the blast phase. Disease severity was classified according to WHO criteria. Twenty-seven patients were managed at home, and 21 patients were in ICU (intensive care unit) during the hospital course. Twenty-six patients required oxygen therapy; among them, seven patients required intubation and mechanical ventilation, and six patients required non-invasive ventilation. Mortality was seen in 47 patients, and there were no reports of thrombotic events, but some patients developed AKI, autoimmune hemolytic anemia, rhabdomyolysis, and DKA during COVID-19. Limitations The major limitation is the lack of details about the use or hold of TKIs during SARS-CoV-2 infection in the majority of the reports. Also, the specific or predominant strain of SARS-CoV-2 was not mentioned. Few studies mentioned the variant of the virus, which make it difficult to compare the outcome of the different variant of the SARS-CoV-2 virus in patients with CML. Discussion/Conclusions Hematological cancers are associated with an increased risk of thrombosis. Thrombosis risk is expected to increase in patients with COVID-19; interestingly, patients with CML have not been reported to have a significant increase in the risk of thrombosis during COVID-19. The crucial point is, does TKI continuation during COVID-19 beneficial or harmful? Few patients continued TKIs during Covid-19 without serious adverse events. For patients who had the TKI continued, there were no mortality or serious adverse events. In vitro, TKIs were found to have a significant effect on inhibiting viral replication, which might be the reason behind the better outcome in CML patients infected with SARS-CoV-2 virus. On the other hand, there is a theoretical risk of increased severe infection in CML patients taking TKI due to targeted inhibition of kinases involved in immune cell function. This might result in suppressed cellular immune response that facilitates viral replication. Despite that, CML patients with COVID-19 have no significant increase in mortality or ICU admission compared to other hematological malignancies. in fact, our review showed that the mortality of CML patients with COVID-19 has significantly improved from 0.12% in the pre-vaccination period to 0.04% in the post-vaccination era. The available data indicates that, COVID-19's effect on patients with chronic myeloid leukemia (CML) still needs to be better understood due to the limited data.

New Pattern of Emerging Somatic Mutations in Optimal Responders Following Tyrosine Kinase Inhibitor Therapy in Chronic Myeloid Leukemia Patients Evidenced from Mutational Kinetic Analysis Based on Pairwise Comparison

Introduction Recent advances in genomics in chronic myeloid leukemia (CML) patients (pts) provide new insights in understanding somatic mutation (SM) and their prognostic implications following tyrosine kinase inhibitor (TKI) therapy. DNMT3A, TET2, or ASXL1 (DTA) mutations are most frequently detected following TKI therapy and have adverse prognosis. Five longitudinal patterns of changes in SM after TKI therapy were suggested with its clinical significance (Kim, Blood 2017): Pattern 1, persistent mutation burden despite optimal TKI response; Pattern 2, emerging mutation which correlates with TKI resistance/CML progression; Pattern 3, mutational clearance with mixed clinical outcomes; and Pattern 4 and 5 having some mutation in germline control as well as CML cells. Clonal evolution (CE) in Philadelphia negative (Ph -) clone is found in up to 10% of CML pts who respond to TKI therapy optimally but develop +8 or -7 changes in Ph - clone. This is diagnosed only by metaphase cytogenetic test on bone marrow samples. Alternatively, fluorescence in situ hybridization (FISH) test can be used. With recent advances in next-generation sequencing (NGS), we hypothesize that SM profiling can capture a clone carrying a SM in the Ph - population. We sequenced and analyzed mutation profiles in paired samples taken prior to TKI therapy and at longitudinal follow-up. Patients and methods The present study adopted single molecule-tagging and molecular inversion probe (smMIP)-based sequencing methods to analyze the DNA extracted from the mononuclear cell fraction of peripheral blood samples collected from CML pts at initial diagnosis and during follow-up. While conventional NGS has a relatively high intrinsic sequencing error rate and is limited in detecting mutations with a variant allele frequency (VAF) of less than 2%, smMIP-based sequencing overcomes this issue by tracking all sequence reads from a single strand and can detect mutations with a limit of detection up to 0.1%. The in-house CML-specific smMIP panel used in this study included 40 genes with 332 amplicon probes covering genes involved in epigenetic modifiers, activation signaling, myeloid transcription factor, spliceosome, tumor suppressor, cohesion, and miscellaneous functions. To evaluate kinetics of mutations, we calculated doubling time of mutations. Using the mutational VAFs from 2 time points, t1 and t2, the doubling time was calculated according to the following equation: = (-2 -1) log(2) /log(-2) - log(-1), where VAF1 and VAF2 is the VAF of the mutant at time points t1 and t2, respectively. Results We performed targeted deep sequencing on a total of 119 serial samples from 51 CML pts (median of 405.5 days following TKI therapy). 41 pts had an optimal response, 7 were non-responders, and 3 were non-evaluable for response. Pattern 2 was observed in 1 of 7 non-responders. In 12 pts pattern 3 (mutation clearance with mixed clinical outcomes) was observed. 22 pts carried 36 mutations. At baseline 9 pts had somatic mutations. Emergence of somatic mutations in optimal responders was observed in 13 out of total 51 (25%) pts, with DNMT3A (n=5), TET2 (n=4), ASXL1 (n=4), and EZH2 (n=2). While JAK2, SF3B1, U2AF1, PHF6, TP53, BRAF, and CBL were observed in one patient each (Figure 1). Particularly, those with emerging DTA mutation were all optimal responders to TKI therapy. This pattern of mutational change is novel and has never been described before. The median doubling time was 77 days (range 21-1909 days) in the group with emergence of somatic mutation in follow-up samples. The doubling time was 56.1, 93.7, 74.8 days in DNMT3A, ASXL1, and TET2 mutations, respectively. Based on kinetics of mutations (doubling time), to capture 10-fold increase (i.e. 1 log increase) of certain mutation, NGS profile can be monitored every 6-7 months. This pattern of emergence of mutations (mostly DTA mutation) in pts responding optimally to TKI therapy is new and does not fit into the five patterns mentioned in our previous paper (Kim, Blood 2017). Thus, we propose this sixth novel pattern called “Emerging mutational clones in CML pts responding optimally to TKI therapy”. Conclusion We suggest a new pattern of emerging mutations in optimal responders to TKI therapy, which could derive from expansion of Ph- clone carrying some clones with emerging mutations. Longitudinal NGS monitoring in CML patients is feasible with frequency every 6-7 months.

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.

Comparison of Characteristics of Chronic Myeloid Leukemia Combined with Different Degrees of Myelofibrosis : A Real-World Multicenter Retrospective Study from China

Chronic myeloid leukemia (CML) is a hematological malignancy characterized by excessive proliferation of myeloid cells, which belongs to the group of myeloproliferative neoplasms (MPNs). Myelofibrosis (MF) is a significant prognostic indicator of CML, and its discovery at the time of diagnosis aids in decisions regarding the patient's course of treatment, according to a descriptive cross-sectional study. Nevertheless, the influence of MF is not clear. Our study is to investigate clinical characteristics and prognosis of CML pantients with different degrees of myelofibrosis, exploring the influence of MF. We retrospectively analyzed 1757 CML patients with bone marrow biopsy results and compared the characteristics of CML combined with different degrees of MF in 44 Chinese institutions from January 2010 to October 2022(ChiCTR2200061208). A total of 1757 CML patients with MF, including 1090 males (62.0%) and 667 females (38.0%) with a median age of 49 (6-92) years. 1757 CML patients were treated with TKIs, as shown in the picture ( Figure A).The median WBC count was 119.8×10 9/L (0.1-910.8×10 9/L) in peripheral blood at diagnosis, the median HB level was 111.5 g/L (12.6-181 g/L), the median PLT count was 512.1×10 9/L (5-4384×10 9/L), the median EOS% was 3.3% (0.0-31.1%), the median BAS% was 4.3% (0.0-19.0%), the median spleen size was 4.10 cm (0.0-26.0 cm). Patients with MF were older (p&amp;lt;0.001); had higher leukocyte counts (p&amp;lt;0.001) and platelet counts (p&amp;lt;0.001); were more prone to anemia (p&amp;lt;0.001), acidophilia (p&amp;lt;0.001), alkalophilia (p&amp;lt;0.001), and splenomegaly (p&amp;lt;0.001); and a higher proportion of peripheral blood blast cells (p&amp;lt;0.001). There was no significant difference between the two groups in the male to female ratio. Patients without MF were more likely to reach 3-month EMR (p&amp;lt;0.001), 6-month BCR::ABL IS ≤ 1% (p&amp;lt;0.001), and 12-month MMR (p&amp;lt;0.001). The proportion of medium / high-risk patients with MF according to the Sokal score (p&amp;lt;0.001), Hasford score (p&amp;lt;0.001), ELTS score (p&amp;lt;0.001) and EUTOS score (p=0.002) was significantly increased ( Figure B). CML patients with MF had lower OS and PFS than patients without MF (p=0.007, p=0.006, Figure D, E). CML patients with MF were more prone to treatment failure with imatinib as the first-line treatment (p&amp;lt;0.001, Figure F). There was no statistically significant difference in treatment failure between first-generation TKI (1G-TKI) and second-generation TKI (2G-TKI) (p=0.222, Figure G).With increasing degree of MF, CML patients had higher leukocyte counts were more susceptible to anemia and splenomegaly, and had more peripheral blood blast cells. However, there was no significant difference in sex ratio, age, platelets, eosinophils, and basophils. CML patients without MF were more likely to achieve 3-month EMR, 6-month BCR::ABL IS ≤ 1%, and 12-month MMR. As the degree of MF increased, the proportion of intermediate / high risk patients according to Sokal score, Hasford score, and ELTS score increased significantly ( Figure C), OS and PFS of CML patients decreased (p&amp;lt;0.001, p&amp;lt;0.001, Figure H, I) and the failure rate of first-line treatment increased (p&amp;lt;0.001, p&amp;lt;0.001, Figure J, K). In conclusion, we performed 1757 CML patients with MF and compared the characteristics of CML combined with different degrees of MF. As the degree of MF increased, the proportion of intermediate / high risk patients according to Sokal score, Hasford score, and ELTS score increased, the efficacy of TKIs was lower, the prognosis of patients was worse. So MF is an independent risk factor affecting the prognosis of patients with CML. Acknowledgement: This research was funded by the Key R&amp;D Program of Zhejiang, No. 2022C03137; Public Technology Application Research Program of Zhejiang, China, No. LGF21H080003; Zhejiang Medical Association Clinical Medical Research special fund project, No. 2022ZYC-D09. *Correspondence to: Dr Jian Huang, Department of Hematology, The First Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310003,Zhejiang, Peoples R China.E-mail: househuang@zju.edu.cn

Long-Term Follow-up of the AST Trial of Treatment-Free Remission in Chronic Myeloid Leukemia Patients

Background: Treatment-free remission (TFR) is a new goal for chronic myeloid leukemia (CML) patients who achieved a long deep molecular response (DMR) with better quality of life, reduced adverse events and high economic impact. The primary objective of the AST trial was to estimate molecular relapse rate at 24 month in CP-CML patients who discontinued TKI. Secondary objective to explore predictive markers for sustained TFR through clinical and immunological analysis ( innate inmune response). This update provides longer follow-up data and includes a new cohort of patients. Patients and Methods: AST-Argentina Stop Trial (NCT05926128) is a multicenter prospective study that included patients from 15 centers. Recruited patients ≥ 18 years, treated with TKI for at least 4 years, sustained MR 4.0 for ≥ 2 years and confirmed typical BCR-ABL1 b3a2 and/or b2a2 transcripts. Molecular studies were centralized in 2 internationally standardized laboratories and were performed monthly during the first 6 months, every 2 months until the first year, and every 3 months during the second year. TKI was restarted due to loss of major molecular response (MMR). Expression of multiple NK cell immunophenotypic markers(CD3-CD56+) was analyzed by flow cytometry at discontinuation time and 3 months later. The nonparametric Mann-Whitney test was used to compare the differences between relapsed and responding patients. Molecular relapse-free survival was estimated using the Kaplan-Meier method and optimal cut-off points were obtained using ROC curves. Comparisons between survival curves were made using the Log rank test method. The differences were considered statistically significant with p-values less than 0.05. Multivariate analysis was performed using the COX regression model. Results: A total of 81 patients divided into 2 cohorts were evaluated: 46 patients recruited between February 2018 and July 2020 (1st cohort) and 35 patients recruited between July 2022 and April 2023. Between these two periods, recruitment was stopped due to the COVID19 pandemic. This analysis focuses on the 1st cohort that has completed 24 months in the study. The median age was 57.5 years (24-86), median duration of treatment before discontinuation was 10.5 years (4.2-20). TKI treatment prior to discontinuation was as follows: Imatinib in 34 (73%) patients, Dasatinib 8 (17%), and Nilotinib 4 (9%). According to Sokal risk score: 22 (48%) were low risk, 14 (30%) intermediate, and 10 (22%) high risk. Seventeen patients (37%) lost MMR, leading to a 63% molecular relapse-free survival rate with a median follow-up of 47 months (41-52). All patients who lost MMR recovered it after restarting the same TKI, with a median time to response of 2.8 months (1-7). With a prolonged follow-up, 2 late relapses were observed (18 and 42 months) (Fig 1). For a univariate analysis, the continuous variables were dichotomized seeking to maximize the difference between the different cohorts. The differences were significant in the variables time in DMR (cut-off point: 51 months, p=0.0166)(HR=0.97 (0.94 - 0.99); p=0.013). Two of the NK cell markers showed significant differences between groups at month 3 after starting treatment, PD1 (Mann-Whitney p=0.035; Log-Rank test p=0.033) and NK-p44 (Mann-Whitney p=0.015; Log -Rank test p=0.020). Multivariate analysis using the Cox model that included variables as gender, age at diagnosis, time in DMR, time in TKI prior to discontinuation, Sokal score, and type of inhibitor, the clinical predictor of time in DMR was the only predictor, significantly associated with a higher rate of TFR, (HR = 0.97 (0.95 - 0.99); p=0.020). No patient experienced disease progression to advanced stages or death for any reason. Conclusion:This is the first multicenter study of TKI discontinuation in CML patients in Argentina showing that TKI can be safely discontinued in those who achieve and maintain a DMR before discontinuation. Molecular relapse-free survival rate was slightly higher than that reported in the international literature, and longer follow-up confirmed the sustained response. DMR before discontinuation showed to be a robust predictor of TFR. Although the sample size was increased, longer follow-up is needed to validate these findings in the second cohort. Potential advantages of TFR will not only impact in patients' quality of life but also show considerable reduction in the use of economic resources.

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.

Outcomes of Adolescents and Young Adult Patients with Chronic Myeloid Leukemia in the Era of Second-Generation TKIs and Treatment-Free Remission

Background: While chronic myeloid leukemia (CML) is most often diagnosed in older adults, it is frequently diagnosed in adolescents and young adults (AYA). Defined by the NCI as individuals between the ages of 15-39 years old, AYA CML patients have been understudied by comparison to their older counterparts, with little known about their outcomes in the United States (US) in the era of second-generation tyrosine kinase inhibitors (TKIs) and treatment-free remission (TFR). We sought to determine the clinical outcomes of AYA patients with CML at our institution, a CML specialty center. Methods: We performed a single-center, retrospective cohort study of patients with CML who were diagnosed between the ages of 15-39 years old and seen in our adult hematology clinic between January 2012 and December 2022. Patients were identified by ICD-9 and ICD-10 codes from the electronic medical record and chart review. Descriptive statistics were used to summarize the data. Outcomes evaluating second-generation TKIs (2G-TKIs) started with patients diagnosed in June 2010. Outcomes evaluating molecular response included BCR-ABL1 PCR testing conducted after implementation of the International Scale (IS) in 2012. Molecular treatment milestones were defined per the 2023 NCCN guidelines for CML. Results: Of 396 CML patients evaluated at our institution, 136 (34.3%) were diagnosed as AYA at a median age of 31 years (range 15-39). The majority were male (62.5%; n=85/136) and English speaking (94.1%; n=128/136). The most frequently cited race/ethnicities were Caucasian (40.4%; n=55/136), Asian (20.6%; n=28/136), and Hispanic (19.1%; n=26/136). At time of diagnosis, 91.2% (n=124/136) were in chronic phase (CP), followed by 4.4% (n=6/136) in accelerated phase (AP) and 4.4% (n=6/136) in blast phase (BP). Of those diagnosed in advanced phase, 91.7% (n=11/12) were male and 66.7% (n=8/12) were Asian or Hispanic. Of CP patients evaluated, 75.8% (n=94/124) started treatment in the 2G-TKI era. Most CP patients were first prescribed dasatinib (61.7%; n=58/94), followed by imatinib (28.7%; n=27/94), and nilotinib (8.5%; n=8/94). Of 62 patients in CP with available molecular data after 2012, 64.5% (n=40/62) met a transcript level &amp;lt;10% at 3-months and 80.6% (n=50/62) met a transcript level &amp;lt;1% at 12-months. Major molecular response (MMR) was achieved and maintained in 59.7% (n=37/62) of these patients, with 25.8% (n=16/62) losing MMR and 14.5% (n=9/62) never achieving MMR. Reasons for loss of MMR included TFR attempts (31.3%; n=5/16), non-adherence from side effects (25%; n=4/16), lack of health insurance/inability to afford medication (18.8%; n=3/16), lack of response (18.8%; n=3/16), and pregnancy attempts (6.3%; n=1/16). Fertility was a notable issue for AYA patients, with 16 females (31.4%; n=16/51) and 18 patients overall (13.2%; n=18/136) interrupting or delaying start of TKI because of pregnancy/fertility. For patients who attempted TFR, 48.1% (n=13/27) remained off TKI in MMR at time of last follow-up. Of 130 AYA patients with CP or AP CML at diagnosis, 7.7% (n=10/130) experienced disease transformation, including 3.2% (n=3/94) of CP patients in the 2G-TKI era. Of 14 patients who presented or transformed into BP, 42.8% (n=6/14) underwent allogeneic stem cell transplantation and 64.3% (n=9/14) died. Of the entire cohort, 9.56% (n=13/136) patients died at a median age of 35 years old, including 5.4% who presented in CP (n=7/124) and 50% (n=6/12) of those who presented in AP/BP. Of the 13 who died, 11 (84.6%) died from CML-related complications. Conclusions: This represents the largest analysis of NCI-defined AYA CML patients in the US in the 2G-TKI era. Molecular response, progression and successful TFR rates in AYA CP CML patients appear to approximate those reported in prior large clinical trials that compared 2G-TKIs with imatinib. AYA patients can experience unique challenges that impact their management, including pregnancy/fertility and adherence, which can affect clinical outcomes. Patients may also present with aggressive advanced phase disease leading to mortality despite transplantation. Further study is warranted to better support the needs of this patient population. Evaluation of CML patients ≥ 40 years old at diagnosis at our institution is ongoing. A formal comparison between these groups will be presented.

Correlation between BCR::ABL1 Transcript LEVEL in Circulating Extracellular Vesicles and BOTH the Molecular Response and the Ongoing Therapy: A Study on Adult CML Patients

Small extracellular vesicles (sEVs) have been shown to play a function in tumor growth and have been studied in Chronic Myeloid Leukemia (CML). Analysis of sEVs and their contents offers a non-invasive strategy that might identify messages transmitted by persisting leukemic cells. The sEVs recovered from CML patients may contain the BCR::ABL1 transcript, but its relationship to clinical and biological factors is still unclear. This study aims to connect the disease status and several clinical features with the BCR::ABL1 transcript levels in sEVs (BCR::ABL1-EV). sEVs were isolated by a commercial kit from 104 plasma samples of adult CML patients in molecular response: 18/104 (17%) in MMR and 86/104 (83%) in DMR assessed by RT-qPCR following the international scale. At sampling, 21/104 (20%) patients were treated with imatinib, 14/104 (13.5%) with nilotinib, 6/104 (5.5%) with dasatinib, 1/104 with bosutinib (1%), 27/104 (26%) with intermittent TKI therapy, and 24/104 (23%) were in treatment free remission (TFR). Taking advantage of digital PCR's (dPCR) sensitivity, it was used to detect the presence of BCR::ABL1 transcript in sEVs. The undetectable samples by RT-qPCR on cells were 38/104 (36.5%), while by dPCR on sEVs were 13/104 (12.5%). This difference resulted statistically significant (p&amp;lt;0.0001) (Fig 1A). In terms of sensitivity, dPCR was able to detect more BCR::ABL1 transcript in the sEVs than in the cells (p= 0.0052), suggesting the release of sEVS shuttling BCR::ABL1 transcript by CML cells resident in the bone marrow (Fig 1B). Considering the quantity of BCR::ABL1 transcript molecules, the BCR::ABL1-EV showed a decrease at the deepening of the MR classes and there was a significant difference between MR3.0 and MR4.5 and MR5.0 (p= 0.0226 and p=0.0101, respectively) (Fig 1C). By gathering overall samples according to MMR and DMR definition, the BCR::ABL1-EV resulted statistically different (p= 0.0017), presenting an increased significance when compared with the analysis on the different MR classes (Fig 1D). A significant difference between BCR::ABL1-EV and the ongoing therapy was observed. For this analysis, nilotinib, dasatinib, and bosutinib were considered together as “2 nd generation TKI”. The BCR::ABL1-EV were significantly higher in samples from patients in intermittent therapy compared to those from patients treated with imatinib (p=0.0398). No statistical difference resulted by other comparisons, even if a trend can be appreciated (Fig 1E). This evidence may be due to the therapy duration, since the mean therapy duration is 111 months and 45 months for imatinib and 2 nd generation TKI, respectively. Despite that, no linear regression between therapy duration and BCR::ABL1-EV. Another fascinating hypothesis is that the difference could be related with mechanisms activated by the different drugs. Further analysis will clarify this point. Interestingly, when comparing the results obtained by dPCR on cells and sEVs, a significant difference was observed in samples of patients under intermittent treatment. A trend can be appreciated in the other conditions, with except for imatinib (Fig 1F). In conclusion, dPCR associated with sEVs analysis confirmed its higher sensitivity to detect even low levels of target and it is able to identify the presence of not-circulating leukemic cells releasing BCR::ABL1 positive sEVs. Moreover, the analysis of the BCR::ABL1 transcript on sEVs revealed a transcript reduction at the deepening of the MR classes, discriminated between MMR and DMR, and correlates with the ongoing therapeutic strategy. Further analysis will clarify the mechanisms at the bases of this correlation. This is the first report about the application of this approach on a cohort of adult CML patients, but strongly suggests how a liquid biopsy approach is able to identify active leukemic cells not otherwise detectable. It opens to the identification of different sub-sets of patients presenting different vesicular BCR::ABL1 transcript levels. The different sub-sets could be related to the therapy response as well as the eligibility to TKI suspension, two of the main hot points in the present CML patients' management.

Chronic Myeloid Leukemia Reduces Survival in Elderly Patients (age ≥ 75 years old): An International Multicenter Retrospective Study

Background Tyrosine kinase inhibitors (TKIs) have significantly improved the life expectancy of individuals with chronic myeloid leukemia (CML), bringing it closer to that of the age-matched general population 1. However, clinical trial data from the National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) Program indicates that only 3.8% of CML patients aged 75 or above participate in trials, despite this age group representing about 30% of CML patients 2. This study aims to assess the outcomes of elderly patients aged 75 years or older diagnosed with CML. Methods We conducted a retrospective study, using electronic medical records of consecutive CML patients aged ≥ 75 years, diagnosed between January 2002 and December 2021 at four hematological centers in Israel and Moffitt Cancer Center in Florida, United States (MCC). One-year and five-year overall survival (OS) rates were calculated for the whole cohort and for octogenarians (above 80 years). In addition, to assess if CML diagnosis affected life expectancy, we estimated the expected OS of the Israeli cohort based on life expectancy data from the central bureau of statistics (CBS). Kaplan-Meier curves were used to compare expected and observed median OS with the Log-rank analysis. Local Institutional Review Boards approved the study. Results A total of 123 patients (78 treated in MCC and 45 in Israel) aged ≥ 75 years were diagnosed with CML, with a median age of 79 (range: 75 - 100) years, and 55 patients (45%) were octogenarians. At the time of CML diagnosis 84% of had comorbidities, including cardiovascular risk factors in 90 patients (73%), while 50 patients (41%) had cardiovascular/cerebrovascular diseases. Most patients (93%) were diagnosed in chronic phase CML and high/intermediate EUTOS-LTS risk score (96%) treated with imatinib in the 1 st line treatment (69%). After a median duration of 15 (range:1-153) months on 1 st line treatment, 71 patients (58%) discontinued therapy primarily due to intolerance (n=51) while other causes included resistance (n=15), noncompliance/insurance issues (n=4) or progression to blast crisis (n=1). The 2 nd -line treatment included mainly 2 nd generation TKIs (n=53, 74%) (dasatinib-26, nilotinib -19 and bosutinib-8), while 9 patients received imatinib, 1 patient received nilotinib and imatinib, 5 patients received hydroxyurea and 3 patients were lost to follow-up. 35 patients (28%) reached 3 rd line of treatment (imatinib- 5, dasatinib- 3, bosutinib-12, nilotinib-5, ponatinib-2, asciminib-1, hydroxyurea -1, loss to follow up- 6) chiefly (28 patients) due to intolerance and others (7 patients) due to resistance. The best response assessed by RQ-PCR showed deep molecular response (DMR) in 50%, major molecular response (MMR) in 16%, and complete cytogenetic response (CCyR) in 11% of patients with a median time to maximal response of 19 (range: 0.5-111) months. Nevertheless, treatment-free remission (TFR) was rare (n=1). During a median follow-up of 45 (0.4-198) months, 55 (45%) patients died, with cardiovascular complications (n=9), disease progression (n=8), infection (n=7), and secondary malignancy (n=4) being the main known causes. The median OS for the whole cohort was 72.4 (53.1-91.7) months. Improved OS was documented in patients with an age adjusted charlson comorbidity index&amp;lt; 5 (vs. ≥5, p=0.007), those who achieved DMR (vs. no DMR, p=0.001) and median time to best response of 0-18 months (vs. ≥18, p=0.004). Moreover, OS was improved in patients who received 2 nd generation TKIs in 1 st line treatment, 91.1 (81.5-131) vs. 57.7 (35.4-79.9) months in those who received imatinib, (p=0.023). Older age did not affect OS; 1 and 5-year OS for the whole cohort was 86% and 29%, respectively, and 84.3% and 19.6% for the octogenarians, respectively (p=0.076). In the Israeli cohort, while the median expected OS was 103.7 (24-164.7) months, the median observed OS was only 54.77 (2.1-195) months (p=0.03). Conclusions Elderly CML patients were often diagnosed in chronic phase with high/intermediate risk scores. The majority received imatinib as 1 st line treatment and achieved CCyR and MMR, with half of patients achieving also DMR. Surprisingly, patients treated with imatinib had worse OS compared to those receiving 2nd generation TKIs as 1st line treatment. Furthermore, the Israeli cohort analysis supports reduced life expectancy in the very elderly patients with CML.

Mutation of Epigenetic Regulators at Diagnosis Is an Independent Predictor of Tyrosine Kinase Inhibitor Treatment Failure: A Report from the Residiag Study

Introduction Additional mutations at chronic myeloid leukemia (CML) diagnosis have been shown to variably affect tyrosine kinase inhibitor (TKI) response and were inconstantly detected at loss of response. Contradictory observations may have resulted from difficulties in reliably inferring CML clonal architecture from mutations quantified by NGS, BCR::ABL1 by qRT-PCR, ABL1-TK by RNA-Seq. The RESIDIAG study was designed to identify at diagnosis molecular markers predictive of TKI treatment failure and analyze the clonal dynamics using asymmetric capture sequencing (aCAP-Seq). Methods DNA samples from 60 TKI responders (median follow-up: 7.1 years) were sequenced at diagnosis by aCAP-Seq, allowing quantification and breakpoint sequencing of genomic BCR::ABL1 fusion together with 43 myeloid genes, including ABL1 with a limit of detection of ~0.1% for BCR::ABL1 and 0.5-1% for SNVs and Indels. In parallel, CML patients who experienced treatment failure (ELN 2020) were sequentially analyzed at diagnosis and at failure (n=53), at diagnosis only (n=1), and at failure only (n=3). Small deletions and BCR::ABL1 breakpoints were analyzed to search for microhomology-mediated end-joining (MMEJ) repair signatures. Mutation analysis followed strict medical standards. Results The proportion of patients receiving imatinib at diagnosis wasn't statistically different (p=0.08; Pearson's Chi-square test) between responders (70%) and the failure group (56%). At diagnosis, the number of mutations was higher (p&amp;lt;0.001, t-test with Welch correction) in the failure group (0.9±0.1) as compared with responders (0.18± 0.05). ASXL1, DNMT3A, and TET2 were more frequently mutated at diagnosis in the failure group (38.6%, p&amp;lt;0.001; 10.5%, p=0.01, and 4.3%, p=0.025 respectively; Pearson's Chi²test) as compared with responders (6.7%, 0% and 0% respectively) and the presence of a mutation in one of these genes was highly associated with a reduced failure-free survival (p&amp;lt;0.001, Log-ranked test, Figure 1). An MMEJ signature at BCR::ABL1 breakpoints was more frequent (p=0.014, Pearson's Chi² test) in the failure group (29.1%) than in the responder group (9.1%). and was significantly associated with the ELTS score (p=0.005, Pearson's Chi² test) but not the Sokal score (p=0.017, Pearson's Chi² test). At first failure, 69% of patients had additional mutations in ASXL1 (39%), ABL1-TK (38%), DNMT3A (18%), or TET2 (15%), while other genes were rarely (&amp;lt;5%) mutated. ASXL1 mutations found at diagnosis were still detectable in 18/22 patients at failure and VAF suggested that double mutant BCR::ABL1/ ASXL1 clones were driving TKI failure with or without BCR:: ABL1 tyrosine kinase domain mutation (TKD). ASXL1 mutations disappearing upon treatment were mostly at low frequency &amp;lt;5% at diagnosis and thus possibly not clonally related to CML. Furthermore, a stepwise selection of variables followed by multivariate analysis with the Fine-Gray model identified the age of patients (sdHR [95% C.I.] = 0.99 [0.97-1.00]; p=0.08) and the presence of a mutation in epigenetics regulators at diagnosis as factors affecting the onset of TKD mutations (sdHR = 3.01 [1.76-5.15]; p&amp;lt;0.001). Intriguingly, co-occurring mutations in DNMT3A and TET2 were found in five patients at TKI failure. Their allelic frequency kinetics suggested that these belonged to the same clone driving CML relapse in 4/5 CML patients, while one patient also had a TKD mutation likely involved in CML relapse. Finally, multivariate COX regression analysis identified two independent predictors of TKI failure at diagnosis: a high-risk ELTS score (p=0.001; hazard ratio=3.42 [1.62-7.22] with low-risk as baseline) and a mutation in ASXL1, DNMT3A or TET2 (p=0.002; hazard ratio=2.87 [1.49-5.51]). Conclusion Altogether these results strongly point to the contribution of epigenetic regulator mutations in the emergence of TKI failure in CML and warrant further biological studies to better understand the underlying mechanisms

KF1601, a Novel Orally Bioavailable Inhibitor of BCR-ABL1 T315I without Inducing Thrombotic Microangiopathy

Targeting Bcr-Abl with tyrosine kinase inhibitors (TKIs) has revolutionized the treatment of chronic myeloid leukemia (CML) by significantly improving patients' survival. However, the gatekeeper mutation T315I confers resistance to TKIs targeting Bcr-Abl, presenting a formidable task in CML's clinical management. Ponatinib significantly reduces the mortality of CML patients with Bcr-Abl T315I, but often accompanies severe, life-threatening side effects. Herein, we report KF1601, a novel orally bioavailable TKI: 1) KF1601 inhibited kinase functions of both Bcr-Abl WT and Bcr-Abl T315I with nanomolar IC 50 values; 2) KF1601 and ponatinib had similar potency in terms of inhibiting CRKL phosphorylation, which correlates to major molecular responses in CML patients. 3) In murine models using Ba/F3 Bcr-Abl T315I cell line, it showed promising in vivo antitumor efficacy, comparable to that of ponatinib; 4) In animal models, it did not cause thrombotic microangiopathy, a major mechanism of ponatinib's toxicity. In summary, KF1601 is a promising drug candidate for safely treating CML patients with drug-resistant Bcr-Abl T315I mutation.

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&amp;lt; 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 &amp;lt;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.

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.

Tgrx-678, a Novel Allosteric Inhibitor of BCR-ABL1, Demonstrates Preclinical Anti-Leukemia Activity, High Oral Bioavailability and Synergism with Ponatinib to Suppress the Highly Resistant Compound Mutations

Introduction Clinical resistance to the treatment of BCR-ABL1-positive chronic myeloid leukemia (CML) patients is often conferred by mutation(s) in the ATP-binding site of ABL1 kinase domain. ABL1 is subject to auto-inhibition mediated by myristoylation-triggered conformational change, which can be exploited to overcome resistance, as validated by allosteric inhibitors such as GNF2, GNF5 and asciminib. TGRX-678, a novel allosteric inhibitor designed to target the myristoyl pocket of ABL1, is currently being investigated in a phase I clinical trial (NCT05434312). Herein we report the selectivity, potency, anti-leukemia activity and in vivo pharmacokinetic (PK)/pharmacodynamic(PD) characteristics of TGRX-678. Methods For in vitro study, ABL1b 65-534 fragments were used following the protocol for Z'-LYTE kinase assay kit (Thermo Fisher). For cellular assays, CML cell lines or engineered Ba/F3 cells were incubated with titrating TGRX-678 for 72 hrs, then CellTiter Glo reagent (Promega) was added and the chemiluminescence was measured. For in vivo studies, SD rats received TGRX-678 or asciminib and plasma samples were collected for analysis. NOD-SCID mice were inoculated with CML or Ba/F3-BCR-ABL1 T315I cells and received oral TGRX-678. Tumor volumes and body weights were recorded for 3 to 5 weeks. Results TGRX-678 exhibited minimum off-target inhibition at 1 μM in a panel of 298 kinases, but inhibited the enzymatic activity of both ABL1 and the T315I mutant with IC50 values of 2.96 nM and 1.15 nM, respectively. The anti-proliferative activity of TGRX-678 was determined in CML cell lines including K562, KU812, KCL22-S, KCL22-R, LAMA-84 and MEG-01 with IC50 values ranging from 1.1 nM to 6.56 nM. TGRX-678 had no cytotoxicity in 29 cell lines representing other hematological malignancies and solid tumors. Using engineered Ba/F3 cell lines, TGRX-678 was active against the native BCR-ABL1 (IC50 = 4.11 nM) and the clinically prominent mutants including G250E (IC50 = 2.49 nM), Q252H (IC50 = 2.38 nM), Y253H (IC50 = 5.6 nM), E255K (IC50 = 1.01 nM), E255V (IC50 = 2.73 nM) and T315I (IC50 = 66.1 nM). The cellular activity of TGRX-678 was attenuated by mutations in the myristoyl pocket such as A337V, P465S, V468F and I502L or mutations in the SH3-kinase domain interface including P223S and K294E, underpinning its allosteric mode of action. In SD rats, oral administration of TGRX-678 at 20 mg/kg achieved a maximum plasma concentration (C max) of 2369 ng/mL, which was 1.3-fold higher than that of asciminib. The overall exposure (AUC last) of TGRX-678 (23,147 h·ng/mL) was also 1.7-fold higher than that of asciminib. The oral bioavailability of TGRX-678 was 62%. In xenograft-bearing mice, oral dosing of TGRX-678 at 1 mg/kg QD (for KU812) or 3 mg/kg QD (for K562) was sufficient to completely inhibit tumor growth. In the Ba/F3-BCR-ABL1 T315I xenograft mice, TGRX-678 induced dose-dependent tumor regression and reduced tumor volume by 67% at 45 mg/kg QD dose. We further examined the synergistic effect of TGRX-678 and ponatinib combination. While each agent was inactive to the Y253H/T315I mutant, the combination of TGRX-678 and ponatinib inhibited cell proliferation at suboptimal concentrations. The addition of TGRX-678 dramatically lowered the IC90 for ponatinib from 1489 nM to 60.9 nM, whereas asciminib only reduced the IC90 to 165 nM (Figure A). Similarly, while ponatinib alone had limited effect on cell viability of the E255V/T315I, Q252H/T315I and T315M mutants, the addition of TGRX-678 at a clinically achievable concentration of 720 nM significantly reduced cell viability to 0-10 % (Figures B-D). In comparison, the addition of asciminib at a much higher concentration of 6000 nM only reduced cell viability to 5-29% (Figures B-D). Conclusion Our findings support that TGRX-678, a novel allosteric inhibitor of BCR-ABL1, is potent against the CML cells and cell lines harboring clinical ATP-site mutations with minimum non-specific cytotoxicity. TGRX-678 exhibits a favorable PK profile and anti-tumor activity in vivo. Furthermore, TGRX-678 and ponatinib synergize to overcome the clinically challenging resistance conferred by T315M or T315I-inclusive compound mutations. This data warrant further clinical investigation of TGRX-678 for the treatment of resistant or refractory CML patients.

Multiomic Single-Cell Analysis Identifies Von Willebrand Factor and TIM3-Expressing BCR-ABL1+ Chronic Myeloid Leukemia Stem Cells

Characterization of the leukemic stem cell (LSC) population in chronic myeloid leukemia (CML) may identify therapeutic targets for sustained elimination of leukemic cells. High-throughput single-cell RNAseq (scRNAseq) has paved the way for detailed transcriptional assessment of CML LSC. Building on this technique, multiomic CITE-seq approaches carry additional benefit in that the paired protein expression information may enable identification of associated cell surface marker profiles, subsequent FACS-based isolation and functional characterization. However, detection of BCR-ABL1 transcripts, which is arguably the only unequivocal marker to distinguish CML LSC from healthy hematopoietic stem cells (HSC), remains challenging using current high-throughput 3' end capture-based scRNAseq methods. For the present study, we performed single-cell CITE-seq analysis of the expression of 597 genes and 51 proteins in &amp;gt;70,000 stem and progenitor cells from 16 chronic phase CML patients and five healthy donors. Furthermore, we developed and employed a strategy allowing parallel detection of BCR-ABL1 transcripts at the single-cell level. In bone marrow samples from CML patients, we observed pronounced expansion of erythroid and myeloid progenitors within the CD14 -CD34 + compartment, and substantial heterogeneity within the traditionally defined CD34 +CD38 -/low LSC compartment. In-depth analysis of the latter identified a group of immature BCR-ABL1 +cells displaying a CD45RA -cKIT -CD26 + TKI resistance phenotype sitting atop a hierarchy of myeloid progenitors. Within this group of potential LSC, expression of both previously reported ( e.g. CD25 and CD26) and unreported gene and protein markers was found to distinguish these cells from their healthy counterparts. Unlike HSC, the immature LSC showed high surface expression of TIM3 and high transcription of the von Willebrand factor gene ( VWF). While overexpression of VWF within the stem cell population may be linked to the aberrant myeloid-biased hematopoiesis characterizing the disease, the cell surface upregulation of TIM3 suggests that its targeting may have merit in CML. In conclusion, we here performed detailed multiomic characterization of CML stem and progenitor cells, paired with BCR-ABL1 detection at the single-cell level. Our findings revealed LSC-specific expression patterns that may have implications for the phenotypic definition of CML LSC. Additionally, the results identify TIM3 as a conceivable target for sustained elimination of immature LSC in CML.