Relapse Of Chronic Myeloid Leukemia Research Articles

Peripheral blood quantitation of CD26 positive leukemic stem cells as a predictor of tyrosine kinase inhibitor response in chronic myeloid leukemia.

Leukemic stem cells (LSCs) are the transcriptionally low/silent cells which are resistant to the tyrosine kinase inhibitor. These have been found to play a pivotal role in disease relapse in chronic myeloid leukemia (CML) cases. The present study evaluated the correlation of absolute CML-LSC count in the peripheral blood (PB) at diagnosis and achievement of major molecular response (MMR) at 12 months in patients of CML-CP. This was a prospective, observational, non-interventional single center study including newly diagnosed adult (>18 yrs) CML-CP patients. Absolute CD26 + CML-LSC quantification was done by multiparametric flow cytometry. Patients were treated with Imatinib treatment and subsequently monitored at 3-month intervals for BCR::ABL transcript levels. MMR was defined as a BCR::ABL1 transcript level of less than 0.1% on international scale. A total of 89 patients were enrolled in the study out of which 40.5% achieved MMR at 12 months. There was a significant difference in the median absolute CML-LSC count of the patients who achieved MMR at 12 months as compared to those who did not (58.5 vs 368.1 cells/μL; p value <0.001). Using a ROC analysis, a count of <165.69 CML LSC/μL was identified to have a sensitivity of 83.8% and specificity of 72.4%, in predicting the MMR at 12 months. Absolute CML-LSC count at diagnosis in the PB predicts the MMR achievement at 12 months. An absolute count of less than 165 cells/μL is highly predictive of achieving MMR at 12 months.

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.

Late relapse of chronic myeloid leukemia after allogeneic bone marrow transplantation points to KANSARL (KANSL1::ARL17A) alteration: a case report with insights on the molecular landscape

Chronic myeloid leukemia is a myeloproliferative neoplasm characterized by the presence of the Philadelphia chromosome and the consequent BCR::ABL1 oncoprotein. In the era before the introduction of tyrosine kinase inhibitors (TKIs), the only potentially curative treatment was allogeneic hematopoietic stem cell transplantation (HSCT). Here, we present the case of a patient affected by CML who experienced a relapse 20 years after allogeneic HSCT. Following relapse, the patient was treated with imatinib and bosutinib, resulting in a deep molecular response and successfully discontinued treatment. Additional analysis including whole-exome sequencing and RNA sequencing provided some insights on the molecular mechanisms of the relapse: the identification of the fusion transcript KANSL1::ARL17A (KANSARL), a cancer predisposition fusion gene, could justify a condition of genomic instability which may be associated with the onset and/or probably the late relapse of his 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

Role of Lama4 Expression in Bone Marrow Niche for the Progression and Treatment Response of Chronic Myeloid Leukemia

Although tyrosine kinase inhibitors (TKIs) have dramatically improved the treatment outcomes of chronic myeloid leukemia (CML), TKIs cannot eliminate CML-initiating stem cells (LSCs), leading to CML persistence and relapse post TKI-discontinuation. Thus, it is imperative to develop more effective therapeutic options to overcome the treatment challenges. Targeting the bone marrow (BM) niche has become one of the research focuses in drug discovery since increasing evidence suggests that BM niches protect CML LSCs. Laminin alpha4 chain (Lama4), a functional chain for several laminin isoforms, is widely expressed in bone marrow (BM). We have recently shown the critical impact of LAMA4 expression in the microenvironment for hematopoiesis regeneration including megakaryocyte maturation and acute myeloid leukemia progression in mice (Cai et al., Blood, 2022). Furthermore, we have by RNA sequencing shown downregulation of LAMA4 mRNA in freshly isolated BM mesenchymal stem cells (MSCs) from newly diagnosed patients with CML (Dolinska, Cai, Mansson, et al., Blood, 2023). However, the functional impact of the LAMA4 expression on CML LSCs remains to be investigated. We have here explored the role of LAMA4 in CML development and drug response by using both mouse models and primary patient BM samples. To first assess the in vivo impact of LAMA4 expression in BM niches on CML development, we transplanted BM cells (CD45.2) from Scl- tTA× TRE-BCR-ABL1 (BCR-ABL1) mice prior or following tetracycline withdrawal into irradiated Lama4 +/+ and Lama4 -/- CD45.1mice and analyzed CML progression and BCR-ABL1 LSC activity by flow cytometry and droplet digital PCR (ddPCR). Following transplantation of unfractionated BM cells from the BCR-ABL1 mice with CML induced by tetracycline withdrawal, Lama4 -/- recipients showed significantly increased platelet counts in blood compared to Lama4 +/+ recipients at 2-8 weeks post-transplantation (P=0.005 at week 2, P= 0.008, n=7-8, at week 8), which is in contrast to the impaired platelet recovery in non-transplanted Lama4 -/- mice following sublethal irradiation. This suggests that Lama4 deficient microenvironment is favorable for platelet production from BCR-ABL1 CML cells. Consistent with this, megakaryocyte-erythrocyte progenitors (MEPs) derived from BCR-ABL1 + cells were significantly increased in Lama4 -/- BM at the endpoint (8 weeks, p=0.02, n=3). Megakaryocyte progenitors (MkPs) seemed to be increased in Lama4 -/- recipient BM (P=0.05, n=3). Intriguingly, when LIN -SCA1 +KIT + CD150 + hematopoietic stem cells from BCR-ABL1 mice without prior tetracycline withdrawal were transplanted, the donor-derived MEPs were significantly reduced in Lama4 -/- BM (P=0.02, n=4-6), which is accompanied with a trend of reduction in MkP but no changes in platelets. ddPCR analysis indicated that the ratio of BCR-ABL1 + cell fraction to total CD45.2 + cells (detected by FACS) seemed to be lower in Lama4 -/- recipients (33.6%) than that in Lama4 +/+ recipients (41.5%), raising a possibility that Lama4 -/- niches is less suitable for maintenance of BCR-ABL1 + cells. Together, these data point to a possible differential regulation of CML stem and progenitor cells by the Lama4 expression. More work will be done to dissect the regulatory mechanisms through in vitro co-cultures, transplantation and confocal imaging. In parallel, the functional impact of LAMA4 on human CML LSC survival was evaluated by a revised long-term culture-initiating cell assay using CML patient derived MSCs. The preliminary result indicated that recombinant LAMA4 peptides appeared to inhibit CML patient BM-derived CD34 +CD38 - cell growth. In conclusion, LAMA4 is downregulated in BM MSCs and endothelial cells of CML patients at diagnosis. Lama4 -/- microenvironment might act differentially in regulating BCR-ABL1 + hematopoietic stem and progenitor cell activity post-transplantation. However, more work is required to validate the finding and the effect of LAMA4 peptides on the megakaryocyte differentiation and maturation.

Successful delivery using interferon α for molecular relapse of chronic myeloid leukemia after interruption of tyrosine kinase inhibitor

A tyrosine kinase inhibitor (TKI) was used to treat the patient, a 35-year-old woman who was diagnosed with chronic myeloid leukemia at the age of 22 years. Since a four-year deep molecular response (DMR) was obtained, spontaneous pregnancy was planned under TKI withdrawal. Even though her disease had advanced to MR2.0 at the time of pregnancy confirmation, 2 months from TKI cessation, interferon α therapy was initiated in light of the patient's history. Later, the patient reached MR3.0, gave birth to a healthy baby, and maintained MR3.0-4.0. TKI was resumed after about 6 months of breastfeeding. Treatment-free remission (TFR) is required for natural conception despite the teratogenicity and miscarriage risks associated with BCR::ABL1 TKIs. When planning a pregnancy, it is also necessary to take the patients' backgrounds, disease states, and medical history into account.

Novel therapeutic strategy for targeting chronic myeloid leukemia stem cells via IRAK1/4 inhibition

Chronic myeloid leukemia (CML) stem cells have been identified to promote CML relapse due to their quiescent cell cycle and tyrosine kinase inhibitor resistance. Therefore, their eradication is important for the cure of CML. We herein identified the quiescent CML stem cell fraction using a G0 marker that can visualize quiescent cells. Whole-transcriptome analysis of imatinib-resistant, quiescent CML stem cells revealed that NF-κB is activated via inflammatory signals in the same cells. The combination of imatinib and an inhibitor of this inflammatory signal (IRAK1/4 inhibitor) effectively eliminated CML stem cells and attenuated PD-L1 expression in CML stem cells. Furthermore, the combination of anti-PD-L1 antibody and imatinib effectively eliminated CML stem cells in the presence of T-cell immunity, indicating the importance of creating an environment in which T cells can attack CML stem cells. Thus, IRAK1/4 inhibitors exert two effects: blocking CML stem cell survival and proliferation signals by inhibiting NF-κB and blocking T cell immune evasion by reducing PD-L1 expression in CML stem cells. Collectively, their combination may be one of the attractive strategies for achieving a radical cure for CML. Discussions regarding the possibility of future medications seem warranted.

Wnt and Notch Pathways Regulate Stemness of Myeloid Leukemia Cells Via IL33/ST2 Signaling

Therapy-resistant leukemic stem cells (LSCs) are a main cause of relapse in chronic and acute myeloid leukemia (CML and AML) patients. Stemness, a cellular state of LSCs characterized by their quiescence, pluripotency, and long-term self-renewal capacity, is thought to have major implications on therapy resistance and outcome in myeloid leukemias. However, signaling pathways that induce stemness are often not specific for LSCs and shared with normal hematopoietic stem cells (HSCs). Therefore, LSCs are difficult to be targeted therapeutically. IL-33/ST2 signaling may be a potential target to eradicate CML and AML LSCs. To investigate the ST2 gene (IL1RL1) expression in different hematological malignancies, we used the MILE gene expression dataset containing 2096 samples. The IL1RL1 high expressing samples (n=62) were exclusively represented by myeloid leukemias (CML and AML), and 50% of the IL1RL1 high samples harbored either the BCR/ABL1 (CML) or the AML1/ETO (AML) oncogenes. We confirmed the result from the MILE database by analyzing the ST2 protein expression on FACS-sorted CD34+ LSCs from AML and CML patient samples. Indeed, AML1/ETO and BCR/ABL1 harboring LSCs expressed ST2, while healthy hematopoietic stem cells (HSCs) and LSCs harboring other oncogenes were ST2-negative. Transfecting the ST2-negative cell line Ramos with the AML1/ETO and BCR/ABL1 oncogenes induced ST2-expression. Next, we performed a gene set enrichment analysis (GSEA), comparing the IL1RL1 high vs. IL1RL1 low samples in the MILE dataset. Genes involved in stemness (p<0.01), Wnt- (p<0.001), and NF-ΚB (p=0.054) signaling were upregulated, while the Notch-pathway (p<0.05) was downregulated. Since the NF-ΚB is a known downstream pathway of ST2, we hypothesized that Wnt and/or Notch signaling pathways regulate ST2 expression. Therefore, we treated FACS-sorted CD34+ AML1/ETO expressing LSCs with the Wnt pathway inhibitor XAV939 or HES1 and NOTCH1 shRNAs, silencing the Notch pathway. Treatment with XAV939 led to a decrease in ST2 expression in these cells, while silencing HES1 and NOTCH1 enhanced ST2-expression. To analyze the molecular mechanisms induced by IL-33/ST2 signaling, we performed a transcriptomic analysis using FACS-sorted CD34+ AML1/ETO LSCs upon treatment with or without IL-33. In total, 377 genes were up- and 139 genes downregulated after IL-33 stimulation. IL-33-induced genes were involved in the positive regulation of NF-ΚB (p<0.05) and canonical Wnt (p<0.001) signaling pathways, and the regulation of stem cell division and maintenance (p<0.001). To functionally test whether IL-33/ST2 signaling triggers stemness in myeloid leukemia, we performed a colony forming assay using CD34+ AML1/ETO or BCR/ABL1 expressing LSCs from leukemia patient samples. The colony numbers increased 1.5-3fold upon IL-33 treatment in three consecutive platings, indicating an increase in stem cell numbers. IL-33 had no effect on colony numbers of HSCs and LSCs harboring other oncogenes. To test the in vivo relevance of IL-33/ST2 signaling in leukemia development, we used a congenic AML1/ETO9a (AE9a) or BCR/ABL1 (CML) mouse model. Briefly, we transduced ST2-negative HSCs originating from BL/6 or ST2-knockout (ST2-/-) mice with AE9a-GFP or BCR/ABL1-GFP and transplanted those cells into recipients. In both models, the LSCs strongly expressed ST2, but IL-33/ST2 signaling had no influence on the disease development. Stemness supports resistance against standard therapy in both, AML and CML. Since we were able to show that IL-33/ST2 signaling promotes stemness in AML1/ETO and BCR/ABL1 LSCs, we hypothesized that IL-33 addition protects LSCs from treatment with standard therapeutics. Indeed, IL-33 rescued the reduced colony forming capacity of LSCs that were either treated with the chemotherapeutic agent cytarabine (AML1/ETO LSCs) or the tyrosine kinase inhibitor Nilotinib (BCR/ABL1 LSCs). Furthermore, ST2-deficiency led to a reduced LSC count in CML mice compared to control mice when treated with Nilotinib (p=0.047). We showed that ST2 is expressed on AML1/ETO and BCR/ABL1 harboring LSCs and that activated IL-33/ST2 signaling induced stemness in these LSCs. Targeting IL-33/ST2 signaling in these specific subtypes of AML and CML may eliminate LSCs and overcome resistance towards chemotherapy or TKIs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Splenic red pulp macrophages provide a niche for CML stem cells and induce therapy resistance

Disease progression and relapse of chronic myeloid leukemia (CML) are caused by therapy resistant leukemia stem cells (LSCs), and cure relies on their eradication. The microenvironment in the bone marrow (BM) is known to contribute to LSC maintenance and resistance. Although leukemic infiltration of the spleen is a hallmark of CML, it is unknown whether spleen cells form a niche that maintains LSCs. Here, we demonstrate that LSCs preferentially accumulate in the spleen and contribute to disease progression. Spleen LSCs were located in the red pulp close to red pulp macrophages (RPM) in CML patients and in a murine CML model. Pharmacologic and genetic depletion of RPM reduced LSCs and decreased their cell cycling activity in the spleen. Gene expression analysis revealed enriched stemness and decreased myeloid lineage differentiation in spleen leukemic stem and progenitor cells (LSPCs). These results demonstrate that splenic RPM form a niche that maintains CML LSCs in a quiescent state, resulting in disease progression and resistance to therapy.

Burden of tyrosine kinase inhibitor failure in Chinese chronic myeloid leukemia patients: a systematic literature review.

Aim: To conduct a systematic literature review of real-world evidence on the burden of tyrosine kinase inhibitor (TKI) failure in Chinese patients with chronic myeloid leukemia (CML). Methods: We identified 155 references in Chinese- and English-language journals from 2001 to 2021. Results: The age-adjusted mortality rate in Chinese CML patients was decreasing. Imatinib treatment had a higher annual treatment failure risk than nilotinib (0.199 vs 0.041). Patients with TKI treatment failure tended to be young (median: 38.6years), have progressive disease (44.3%) and harbor BCR-ABL1 mutations (51.6%). The disease burden of TKI treatment failure included reduced health outcomes and increased health resource utilization and costs. Conclusion: CML relapse cases could continuously rise in China due to increasing TKI treatment failure over extended survival.

Eliminating chronic myeloid leukemia stem cells by IRAK1/4 inhibitors

Leukemia stem cells (LSCs) in chronic myeloid leukemia (CML) are quiescent, insensitive to BCR-ABL1 tyrosine kinase inhibitors (TKIs) and responsible for CML relapse. Therefore, eradicating quiescent CML LSCs is a major goal in CML therapy. Here, using a G0 marker (G0M), we narrow down CML LSCs as G0M- and CD27- double positive cells among the conventional CML LSCs. Whole transcriptome analysis reveals NF-κB activation via inflammatory signals in imatinib-insensitive quiescent CML LSCs. Blocking NF-κB signals by inhibitors of interleukin-1 receptor-associated kinase 1/4 (IRAK1/4 inhibitors) together with imatinib eliminates mouse and human CML LSCs. Intriguingly, IRAK1/4 inhibitors attenuate PD-L1 expression on CML LSCs, and blocking PD-L1 together with imatinib also effectively eliminates CML LSCs in the presence of T cell immunity. Thus, IRAK1/4 inhibitors can eliminate CML LSCs through inhibiting NF-κB activity and reducing PD-L1 expression. Collectively, the combination of TKIs and IRAK1/4 inhibitors is an attractive strategy to achieve a radical cure of CML.

Leukemia Stem Cells as a Potential Target to Achieve Therapy-Free Remission in Chronic Myeloid Leukemia.

Simple SummaryLeukemic stem cells represent a rare subpopulation of leukemic cells, which not only drive leukemia initiation and progression, but also contribute to drug resistance and/or disease relapse. To achieve permanent cures and prevent relapse, eradication of leukemia stem cells is essential. Chronic myeloid leukemia is a myeloproliferative disorder, and tyrosine kinase inhibitors have dramatically improved long-term outcomes and quality of life for patients. Point mutations of the kinase domain of BCR-ABL1 lead to drug resistance, and as a result, several new generations of tyrosine kinase inhibitor have been introduced to the clinic. Some patients develop drug resistance without known mutations, however, and the presence of leukemia stem cells is believed to be at least partially associated with resistance development and disease relapse. The identification of specific markers distinguishing leukemia stem cells from healthy hematopoietic stem cells, and the potential contributions of the bone marrow microenvironment to leukemia pathogenesis, have also been explored. In this review, we revisit the current knowledge regarding the roles of leukemia stem cells in response to pharmacological treatment and explore how durable treatment-free remission may be achieved after discontinuing tyrosine kinase inhibitor treatment.Leukemia stem cells (LSCs, also known as leukemia-initiating cells) not only drive leukemia initiation and progression, but also contribute to drug resistance and/or disease relapse. Therefore, eradication of every last LSC is critical for a patient’s long-term cure. Chronic myeloid leukemia (CML) is a myeloproliferative disorder that arises from multipotent hematopoietic stem and progenitor cells. Tyrosine kinase inhibitors (TKIs) have dramatically improved long-term outcomes and quality of life for patients with CML in the chronic phase. Point mutations of the kinase domain of BCR-ABL1 lead to TKI resistance through a reduction in drug binding, and as a result, several new generations of TKIs have been introduced to the clinic. Some patients develop TKI resistance without known mutations, however, and the presence of LSCs is believed to be at least partially associated with resistance development and CML relapse. We previously proposed targeting quiescent LSCs as a therapeutic approach to CML, and a number of potential strategies for targeting insensitive LSCs have been presented over the last decade. The identification of specific markers distinguishing CML-LSCs from healthy HSCs, and the potential contributions of the bone marrow microenvironment to CML pathogenesis, have also been explored. Nonetheless, 25% of CML patients are still expected to switch TKIs at least once, and various TKI discontinuation studies have shown a wide range in the incidence of molecular relapse (from 30% to 60%). In this review, we revisit the current knowledge regarding the role(s) of LSCs in CML leukemogenesis and response to pharmacological treatment and explore how durable treatment-free remission may be achieved and maintained after discontinuing TKI treatment.

Deciphering the Mechanisms of Osteoblast-Induced Resistance of Leukemic Stem Cell (LSC) in Ph+ CML: Role of PI3-Kinase, BRD4 and MYC and Development of Strategies to Overcome Osteoblast-Induced Resistance

Chronic myeloid leukemia (CML) is a stem cell neoplasms characterized by the chromosome translocation t(9;22) and the related BCR-ABL1 fusion gene. Therapy with BCR-ABL1 kinase inhibitors is highly effective in the treatment of CML and deep molecular responses are achieved in most patients. However, not all patients respond to these drugs due to resistance of leukemic stem cells (LSC). Recent data suggest that the disease-related microenvironment, known as the stem cell niche contributes to drug resistance and relapse in CML. So far, little is known about the resistance mechanisms protecting niche cells in the bone marrow of patients with CML. We have recently shown that osteoblasts are a major site of LSC-mediated resistance against BCR-ABL1-targeting drugs in CML. In the current study, we screened for drugs that are able to suppress growth and viability of osteoblasts and/or other niche-related cells and can thereby overcome drug resistance of CML LSC. Proliferation was analyzed by determining 3H-thymidine uptake in niche-related cells and apoptosis was measured by Annexin-V/DAPI-staining and flow cytometry. We found that the dual PI3 kinase (PI3K) and mTOR inhibitor BEZ235 and the selective pan-PI3K inhibitor copanlisib suppress proliferation of primary osteoblasts (BEZ235 IC 50: 0.05 µM; copanlisib IC 50: 0.05 µM), the osteoblastic cell line CAL-72 (BEZ235 IC 50: 0.5 µM; copanlisib IC 50: 1 µM), primary human umbilical vein endothelial cells (BEZ235 IC 50: 0.5 µM; copanlisib IC 50: 0.5 µM) and the endothelial cell line HMEC-1 (BEZ235 IC 50: 1 µM; copanlisib IC 50: 1 µM), whereas no comparable effects were seen with the mTOR inhibitor rapamycin. As determined by flow cytometry, BEZ235 and copanlisib suppressed the expression of phosphorylated (p) pAKT and pS6 in endothelial cells and osteoblasts whereas rapamycin downregulated pS6 expression but did not decrease expression of pAKT. Moreover, we found that BEZ235 and copanlisib cooperate with nilotinib and ponatinib in suppressing growth and viability of osteoblasts and endothelial cells. Furthermore, BEZ235 and copanlisib were found to overcome osteoblast-induced resistance of K562, KU812 cells, and primary CD34 +/CD38 − CML LSC against nilotinib and ponatinib. This effect was also seen when CAL-72 cells were first exposed to BEZ235 or copanlisib and washed before co-cultures with CML cells and BCR-ABL1 inhibitors were prepared, suggesting that osteoblast inhibition was a crucial event capable of disrupting LSC resistance in these co-cultures. Of all other drugs tested, only the BRD4-targeting drug JQ1 was found to suppress CAL72-induced resistance in the CML cell lines KU812 and K562, suggesting that osteoblast-induced resistance of CML cells is also mediated by a BRD4-MYC pathway. In a next step, we examined the expression of resistance-mediating immune checkpoint molecules on CML cells (KU812, K562, LSC) and on osteoblasts by flow cytometry. We found that CML cells and CAL72 cells constitutively express PD-L1 and that interferon-gamma (IFN-G) promotes the expression of PD-L1 in all cell types tested. Moreover, we found that the BRD4 blocker JQ1 and the BRD4-degrader dBET6 suppress the IFN-G-induced upregulation of PD-L1 in CML LSC and osteoblasts. In conclusion, our data show that osteoblast-induced resistance of CML stem cells is mediated by a PI3K-dependent pathway and BRD4/MYC, and that BRD4-inhibition or BRD4-degradation counteracts osteoblast-induced resistance of CML (stem) cells against BCR-ABL1 inhibitors and PD-L1 expression on CML LSC and osteoblasts. We hypothesize that checkpoint inhibition may assist in drug-induced eradication of CML LSC and thus in the development of curative drug therapies in Ph + CML. DisclosuresHoermann: Novartis: Honoraria. Gleixner: Pfizer: Honoraria; Abbvie: Honoraria; BMS: Honoraria; Incyte: Honoraria; Novartis: Honoraria. Sperr: AbbVie, BMS-Celgene, Daiichi Sankyo, Deciphera, Incyte, Jazz, Novartis, Pfizer, StemLine, Thermo Fisher: Honoraria, Research Funding. Valent: Novartis: Honoraria; Pfizer: Honoraria, Research Funding; Celgene/BMS: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; OAP Orphan Pharmaceuticals: Honoraria.

Massive retinal infiltrates as the presenting sign of chronic myeloid leukemia: Clinical and imaging features of leukemic retinopathy.

To describe the clinical and optical coherence tomography (OCT) features of two cases with bilateral diffuse retinal infiltrates as the only presenting feature of chronic myeloid leukemia (CML) on initial diagnosis and upon relapse. We reported two patients with CML, one at initial diagnosis and one in remission who presented with bilateral subacute visual impairment. Fundal examination revealed bilateral symmetrical leukostatic appearance with increased vascular tortuosity, diffuse retinal infiltrates with size up to 6 disk diameters, retinal hemorrhages, and Roth's spots. OCT showed multiple intra-retinal hyper-reflective foci corresponding to intra-retinal hemorrhages, and outer retinal hyper-reflective foci in area corresponding to retinal infiltrate. The different retinal layers were relatively preserved and distinguishable. White cell count (WCC) were elevated in both patients ranging from 544 to 810 × 109/L. Bone marrow biopsy confirmed the diagnosis of CML in the patient without prior diagnosis and relapse of CML in another patient. Cytogenetic test detected Abelson murine leukemia (ABL) - breakpoint cluster region (BCR) fusion transcript in both cases. Both patients were started on oral imatinib, subsequently WCC returned to within normal values in both cases. Vision and OCT abnormalities improved and reduction in retinal hemorrhages and infiltrates were observed in follow up. This report highlights the important role of ophthalmologists and detailed fundus examination in making a prompt diagnosis of leukemia in patients with visual complaints. Appropriate systemic investigation and hematologist referrals for prompt treatment of CML may improve survival rate and preserve vision.

CML-011: Chronic Myeloid Leukemia: Clinico-Laboratory Patterns and Management Options in Relapsed and Refractory Cases

Context: The patients with the advanced and relapsed chronic myeloid leukemia (CML) experience marked disease burden in terms of symptoms, and unfavorable impact on their life quality and productivity. Objective: The study purpose was to evaluate the diagnosis and prognosis significance of lactate dehydrogenase (LDH) and T315I mutation, as well as the treatment options in the newly diagnosed and relapsed CML. Design: This cohort study included 27 CML patients, treated at the Institute of Oncology between 1995-2021. The venous blood samples were collected for lactate dehydrogenase (LDH) assay and detection of the ABL gene T315I mutation. Setting: The study was related to the outpatient and hospitalized care. Patients: The patients’ age ranged between 20-68 years (average age – 51.3±2, 14 years). The diagnosis of CML was asserted by the quantitative RT-PCR in chronic phase in 26 (96.3%) cases. Main Outcomes Measures: The survival and relapse rates may suggest the biological significance of LDH and T315I mutation. The ECOG-WHO score, p210 BCR-ABL transcript, and the survival should outline the response to tyrosine kinase inhibitors (TKIs). Interventions: Chemotherapy regimens included antimetabolites, alkylating antineoplastic agents, and TKIs. Results: The ECOG-WHO score was 1-3 at diagnosis. LDH ranged between 169–1107 U/L, and was increased in 14 (63.6%) patients, especially in those with leukocytosis over 100 x 109/l. The complete or major molecular responses followed under the TKIs chemotherapy in 16 (59.3%) cases, including 3 (11.1%) with T315I mutation. The relapses developed in 10 (71.4%) patients with the increased LDH and in 5 of 6 patients with T315I. One (3.7%) patient with T315I mutation evolved into the acute phase. The survival ranged between 23-236.8 months (median survival – 95.87±4.51 months) and was lower in cases with T315I mutation and higher LDH level. The ECOG-WHO score reached 0-1 under the treatment with TKIs. Conclusions: The elevated LDH may assess the activity at diagnosis and relapse of CML. T315I mutation and the increased LDH levels correlated with a higher rate of relapses and resistance to the first generation TKIs. Regardless of the chemotherapy line and in relapses, TKIs significantly improved the complete response rate, survival and ECOG-WHO score.

Heterogeneous BCR-ABL Signal Patterns Identified By Fluorescence in Situ Hybridization Can Predict Leukemic Clonal Evolution and Poor Prognosis in BCR-ABL-Positive Leukemia

BCR-ABL translocation occurs in approximately 90% of chronic myeloid leukemia (CML), 25% of acute lymphoblastic leukemia (ALL) and less than 5% of acute myeloid leukemia (AML) cases that produce BCR-ABL fusion proteins, which are responsible for proliferative signals and leukemogenesis. Although extensive use of tyrosine kinase inhibitors (TKIs) has resulted in high and durable response rates and prolonged survival times in BCR-ABL-positive CML and acute leukemia patients in recent years, relapse and drug resistance are still big challenges for clinicians. Monitoring the quantification of BCR-ABL fusion genes by quantitative PCR, minimal residual diseaseby flow cytometry and ABL kinase mutations by sequencing are effective means of predicting disease relapse and resistance; however, the prognostic impact of BCR-ABL signal modes by fluorescence in situ hybridization (FISH) in leukemia patients remains elusive. In the present study, we analyzed BCR-ABL signal modes using FISH in 243 CML-chronic phase (CML-CP), 17 CML-blast phase (CML-BP) and 52 ALL patients. A total of 52.9% (n=9) of the complex BCR-ABL signal modes (≥ two types of BCR-ABL signal modes) were observed in the CML-BP patients, followed by 30.8% (n=16) in the BCR-ABL-positive ALL patients, and only 2.1% (n=5) in the CML-CP patients. Conversely, typical single BCR-ABL signal modes (single 1R1G2F fusion signals) were observed in 29.4% (n=5) of the CML-BP, 53.8% (n=28) of the BCR-ABL-positive ALL and 73.7% (n=179) of the CML-CP patients. Patients with atypical single BCR-ABL signal modes (no 1R1G2F single fusion signal, such as 1R1G1F or 1R1G3F) were similar, and they accounted for 17.6% (n=3), 15.4% (n=8) and 24.3% (n=59) of the CML-BP, ALL and CML-CP patients, respectively. Additionally, multiple BCR-ABL signal modes presented very complexity and various. In total, twelve BCR-ABL signal modes were observed in this cohort: 1R1G2F, 1R1G1F, 2R1G1F, 1R2G1F, 2R2G1F, 1R2G2F, 1R1G3F, 1G3F, 2G3F, 1G4F, 1R1G4F and 1R4F. More importantly, the development of BCR-ABL signal patterns can indicate leukemic clonal evolution, disease progression and relapse by sequentially monitoring the BCR-ABL modes at different time points of disease progression using FISH. In this study, we observed five clonal evolution modes related to disease progression among our ALL patients. First, patients presented sensitive single clones at disease onset, which disappeared after treatment, whereas primary clones gradually progressed to drug-resistant clones until relapse during treatment. Second, patients presented sensitive single clones during disease onset that disappeared after treatment, whereas new single resistant clones were observed during relapse. Third, patients presented sensitive single clones during disease onset, whereas new and primary resistant subclones simultaneously occurred during relapse. Fourth, patients presented many different subclones during disease onset. Some preponderant subclones were sensitive and disappeared after treatment, whereas other minor subclones were resistant and gradually progressed to preponderant subclones until relapse. Fifth, patients simultaneously presented many different subclones during disease onset. Some minor subclones were sensitive and disappeared after treatment, whereas the preponderant subclones were resistant to TKIs or chemotherapy drugs. All of the BCR-ABL-positive ALL patients received a TKI combined with chemotherapy treatment. Furthermore, we analyzed the impact of complex BCR-ABL signal modes on survival time in the ALL patients. We observed that patients with complex BCR-ABL signal modes had very poor estimated overall survival (OS) times compared with patients with single BCR-ABL signal modes (typical and atypical single BCR-ABL signal modes) (5.0 vs. 15.0 month, P=0.006). Moreover, there were no OS differences between the BCR-ABL-positive ALL patients and the CML-BP patients (13.0 vs.10.0 month, P=0.984). Conclusion: Our data show that complex BCR-ABL signal modes usually occurred in patients with BCR-ABL-positive CML-BP, followed by ALL patients and rare in CML-CP patients. Complex BCR-ABL signal modes with various in forms predicted leukemic clonal evolution and poor prognoses. Monitoring BCR-ABL signal mode changes may be an effective means to provide prognostic guidance and treatment choices for patients with BCR-ABL-positive leukemia. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Treatment-free remission and immunity in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is caused by the reciprocal translocation t(9;22)(q34;q11), resulting in the BCR-ABL1 fusion gene. BCR-ABL1 tyrosine kinase inhibitors (TKIs) improve overall survival in patients with chronic phase CML (CML-CP). Approximately half of the patients who achieve a durable deep molecular response can achieve sustained treatment-free remission (TFR) after TKI discontinuation; thus TFR is now a therapeutic goal for most patients with CML-CP. Sensitive BCL-ABL1 transcript detection methods reveal that evidence of residual CML cells remains in patients who achieve sustained TFR, indicating that the host immune system protects against CML relapse. The human immune system is composed of innate and adaptive arms. Natural killer cells are major components of the innate immune system, while T cells are major components of the adaptive immune system. Myeloid-derived suppressor cells and regulatory T cells, both suppressors of the immune response, have important roles in the regulation of CML. Here, we review the current understanding of the immune response in CML, especially in TFR.

PKC‐β/Alox5 axis activation promotes Bcr‐Abl‐independent TKI‐resistance in chronic myeloid leukemia

Bcr-Abl independent resistance to tyrosine kinase inhibitor (TKI) is a crucial factor lead to relapse or acute leukemia transformation in chronic myeloid leukemia (CML). However, its mechanism is still unclear. Herein, we found that of nine common protein kinases C (PKCs), PKC-β overexpression was significantly related with TKI resistance. Blockage of its expression in CD34+ cells and CML cell lines increased sensitivity to imatinib. Then, eighty-four leukemia related genes were compared between TKI-resistant CML cell lines with PKC-β silenced or not. Gene Ontology term and Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that Arachidonate 5-lipoxygenase (Alox5) and its relative pathway mainly participated in the resistance induced by PKC-β overexpression. It's also observed that Alox5 was increased not only in bone marrow biopsy but also in CD34+ cells derived from IM-resistant CML patients. The signaling pathway exploration indicated that ERK1/2 pathway mediates Alox5 upregulation by PKC-β. Meanwhile, we also proved that Alox5 induces TKI-insensitivity in CML through inactivation of PTEN. In vivo experiment, PKC-β elective inhibitor LY333531 prolonged survival time in CML-PDX mice model. In conclusion, targeted on PKC-β overexpression might be a novel therapy mechanism to overcome TKI-resistance in CML.

Interferon-α may help prevent molecular relapse of chronic myeloid leukemia after the discontinuation of tyrosine kinase inhibitors.

Background:Currently, the goal of chronic myeloid leukemia (CML) treatment is normal survival and good quality of life without life-long treatment, namely, “treatment-free remission” (TFR). At present, approximately only 50% of patients with CML with a deep molecular response are able to discontinue tyrosine kinase inhibitor (TKI) without experiencing molecular relapse [MR; loss of major molecular response (MMR)]. In addition, prior interferon (IFN) treatment is associated with a higher rate of TFR.Methods:We aimed to evaluate the feasibility of TKI discontinuation in Chinese patients with CML and determine whether IFN could prevent MR when used after TKI discontinuation in patients with 0.0032% <BCR-ABLIS ⩽0.1%. Therefore, we retrospectively analyzed the data of patients with CML who discontinued TKI treatment at our center.Results:Forty-nine patients who discontinued TKI therapy after achieving MR 4.5 were included in this study, and the median follow-up time from TKI discontinuation was 27 (7, 75) months. Nineteen patients eventually lost MMR, and the TFR rate of the 49 patients was 67% (95% confidence interval 53.6%, 80.3%) at 12 months. The duration of MR 4.5 ⩾54 months and duration of imatinib ⩾85 months were significantly associated with a higher TFR rate. Of the 22 patients with 0.0032% <BCR-ABLIS ⩽0.1%, 12 received IFN-α treatment. The median IFN-α therapy duration was nine (2, 18) months, and three patients eventually lost MMR. Three patients discontinued IFN-α after 2, 2.5, and 10 months, and maintained MMR for 9, 8, and 11 months after IFN discontinuation, respectively. Of the 10 patients not receiving IFN-α treatment, eight eventually lost MMR. The MR-free survival rate was significantly different between the patients treated with and those treated without IFN-α over 24 months (70.7% versus 15.0%, p = 0.002).Conclusion:These results indicate that after TKI discontinuation, IFN-α can be administered to patients with 0.0032% <BCR-ABLIS ⩽0.1%, which may help prevent MR.

Identification of Immunological Parameters as Predictive Biomarkers of Relapse in Patients with Chronic Myeloid Leukemia on Treatment-Free Remission.

BCR-ABL is an aberrant tyrosine kinase responsible for chronic myeloid leukemia (CML). Tyrosine kinase inhibitors (TKIs) induce a potent antileukemic response mostly based on the inhibition of BCR-ABL, but they also increase the activity of Natural Killer (NK) and CD8+ T cells. After several years, patients may interrupt treatment due to sustained, deep molecular response. By unknown reasons, half of the patients relapse during treatment interruption, whereas others maintain a potent control of the residual leukemic cells for several years. In this study, several immunological parameters related to sustained antileukemic control were analyzed. According to our results, the features more related to poor antileukemic control were as follows: low levels of cytotoxic cells such as NK, (Natural Killer T) NKT and CD8±TCRγβ+ T cells; low expression of activating receptors on the surface of NK and NKT cells; impaired synthesis of proinflammatory cytokines or proteases from NK cells; and HLA-E*0103 homozygosis and KIR haplotype BX. A Random Forest algorithm predicted 90% of the accuracy for the classification of CML patients in groups of relapse or non-relapse according to these parameters. Consequently, these features may be useful as biomarkers predictive of CML relapse in patients that are candidates to initiate treatment discontinuation.