Stages Of Chronic Myeloid Leukemia Research Articles

Bioequivalence/bioavailability study of a new formulation of imatinib mesylate 100 mg coated tablets versus Glivec® in Brazilian healthy male volunteers in fed state

Introduction: Imatinib mesylate is currently the first-line oral treatment for all stages of chronic myeloid leukemia (CML) and is also used in some cases of gastrointestinal stromal tumor (GIST) and acute lymphoblastic leukemia (ALL). Objective: Investigate the bioavailability of two products containing imatinib mesylate, 100 mg coated tablet, to determine if they are bioequivalent. Method:The study was conducted using an open-label, randomized, balanced design and the formulations were administered orally in a single dose to 48 healthy adult males, in fed state, followed by sequential blood withdraws for the next 72 hours. Forty-eight male healthy volunteers were selected to participate in the study. Test formulation from Eurofarma Laboratórios S.A. Brazil was compared to that from Novartis Biociências S.A. The comparative bioavailability of the formulations was assessed based on statistical comparisons of relevant pharmacokinetic parameters obtained from drug concentration data from collected blood samples measured using an analytical method based on high-performance liquid chromatography coupled to mass spectrometry. Results: The ratio of the geometric means between the test and the reference, with a 90% confidence interval, of pharmacokinetic parameters for Cmax was 102.26% (94.17-111.04%) and for AUC0-t was 101.24% (95.19-107.68%). Conclusion: Imatinib mesylate 100 mg (test product) from Eurofarma Laboratórios S.A. was considered bioequivalent to the reference Glivec® 100 mg manufactured by Novartis Biociências S.A, and the test product can be interchangeable with the reference, based on their pharmacokinetic performance.

A Nucleus-Targeting WT1 Antagonistic Peptide Encapsulated in Polymeric Nanomicelles Combats Refractory Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is recognized as a classic clonal myeloproliferative disorder. Given the limited treatment options for CML patients in the accelerated phase (AP) and blast phase (BP), there is an evident need to develop new therapeutic strategies. This has the potential to improve outcomes for individuals in the advanced stages of CML. A promising therapeutic target is Wilms' tumor 1 (WT1), which is highly expressed in BP-CML cells and plays a crucial role in CML progression. In this study, a chemically synthesized nucleus-targeting WT1 antagonistic peptide termed WIP2W was identified. The therapeutic implications of both the peptide and its micellar formulation, M-WIP2W, were evaluated in WT1+ BP-CML cell lines and in mice. The findings indicate that WIP2W can bind specifically to the WT1 protein, inducing cell cycle arrest and notable cytotoxicity in WT1+ BP-CML cells. Moreover, subcutaneous injections of M-WIP2W were observed to significantly enhance intra-tumoral accumulation and to effectively inhibit tumor growth. Thus, WIP2W stands out as a potent and selective WT1 inhibitor, and the M-WIP2W nanoformulation appears promising for the therapeutic treatment of refractory CML as well as other WT1-overexpressing malignant cancers.

Assessment of regulatory T cells (Tregs) and Foxp3 methylation level in chronic myeloid leukemia patients on tyrosine kinase inhibitor therapy.

The key cell population permits cancer cells to avoid immune-surveillance is regulatory T cells (Tregs). This study evaluates the level of Tregs in chronic myeloid leukemia (CML) patients and the effect of Tyrosine kinase inhibitor (TKI) on Treg levels, as a pathway to understand the immune response and behavior among advance stage and optimal response CML patients using imatinib therapy. Blood samples were collected from 30 CML patients (optimal response to TKI), 30 CML patients (failure response to TKI), and 30 age- and gender-matched controls. Analysis involved measuring percentages of Tregs (CD4 + CD25 + FOXP3 +) by flow cytometer and demethylation levels of FOXP3 Treg-specific demethylated region (TSDR) by PCR. The data revealed that Tregs and the FOXP3-TSDR demethylation percentages significantly increased in failure response group in comparison to the optimal response and control groups, while no significant difference between optimal response and control groups. Tregs and FOXP3 TSDR demethylation percentages showed high sensitivity and specificity, suggesting powerful discriminatory biomarkers between failure and optimal groups. An assessment of the Tregs and demethylation percentage among different BCR-ABL levels of CML patients on TKI revealed no significant differences in parameter percentage in the optimal response to TKI patients with different molecular responses (log 3 reduction or other deeper log 4.5 and 5 reduction levels). Our findings demonstrate an effective role of functional Tregs among different CML stages. Also, the study suggests that the major molecular response to therapy at level 0.1% of BCR-ABL transcript could be enough to induce immune system restoration in patients.

Chronic Myeloid Leukemia: A retrospective study of clinical and pathological features

Chronic myeloid leukemia (CML), is one of the myeloproliferative disorders with a characteristic cytogenetic abnormality resulting in the BCR-ABL fusion gene. Imatinib Mesylate is an effective agent for treating patients in all stages of CML. According to the annual Iraqi cancer registry 2019, the total number of chronic myeloproliferative disorders was 338. The percentage and incidence rates were 0.94% and 0.86%, respectively, with a higher incidence rate in males than females (1.12 in males and 0.60 in females). In this registry, no details about CML, so this study aimed to estimate the number of CML patients who attended the national center of hematology from 2005 until 2020 and investigate their epidemiological and clinic-pathological data. All data were obtained from the National Center of Hematology /Baghdad. Data regarding annual admission, number of deaths, and information about age, regions, and sex, were collected from patient cards. Interview and history taking from the patient and reviewing their clinical features at diagnosis were performed, and then statistical analysis was achieved. Two hundred sixteen patients were registered as CML; among them, 163 are still alive, while 53 died. The mean age at diagnosis was 40.39 ±14.30, with a male-to-female ratio of 1:1.16. The pediatrics age group comprises 3.7% of total cases and 1.8 % and 9.4% in the alive and dead groups, respectively. The percentages of death were higher in the pediatric age group, 62,5%( 5 /8), followed by the patient group who were ≥60 years, 42.1%(8/19), while it was the least in adults < 60 years, 21.1% (40/189). In conclusion, the studied patients with CML were younger than the globally reported ages at diagnosis, with predominant female patients. Females had better survival, while children and the elderly were the worse. Most patients were symptomatic at diagnosis; however, a good outcome is achieved when treated with IM. Keywords: CML, Imatinib, leukemia,

Itrifal-e-Aftimoon potentiates imatinib-induced anti-leukemic effect by influencing FAK/STAT/Akt/ERK signalling pathways against chronic myeloid leukaemia in vitro.

Limited treatment options are available for advanced stages of chronic myeloid leukaemia (CML). Moreover, patients' relapse after a short remission period, which prompts them to identify a potent drug with the least toxicity. An Unani herbal formulation, Itrifal-e-Aftimoon (IEA) is used for certain neurological disorders, however, its antitumor potential has not been reported yet in any malignancy, including CML. The aqueous extract of IEA was characterized by HPLC/LC-MS and used alone or in combination with standard drug, imatinib in CML cell lines (K562, KU812) in vitro to assess its effect on cancer-associated parameters such as cytotoxicity, cell cycle, apoptosis, oxidative stress, inflammation, angiogenesis, and certain signalling pathways. LC-MS characterization of IEA showed the presence of antitumor compounds including catechin and caffeic acid. Treatment with IEA caused cytotoxicity and arrested cells in the sub-G0/G1 phase. Subsequent assays confirmed apoptosis-mediated cell death with mitochondrial membrane depolarization and alleviation of oxidative stress. IEA abrogates IL-6, VEGF, angiopoietin-2, and alters Th1/Th2 cytokines. IEA potentiated the effect of imatinib even at lower doses by affecting FAK/STAT/Akt/ERK pathways. IEA possesses antitumor potential against CML and increases the efficacy of imatinib when used in combination, suggesting utilization of IEA as an adjuvant therapy for better management of CML in the future.

Identification of a Highly Deregulated eIF4F Translation Initiation Complex in Drug-Resistant BCR-ABL + Cells By a Phospho-Proteomic Antibody Microarray

Protein-tyrosine kinase inhibitors (TKIs) have been effective for treatment of early stages of chronic myeloid leukemia (CML). However, BCR-ABL-dependent resistance mechanisms and TKI-unresponsive quiescent leukemic stem cells (LSCs) can result in drug resistance and disease relapse. We have demonstrated that Abelson helper integration site-1 (AHI-1) is a highly deregulated protein in CML LSCs. It interacts with BCR-ABL through the AHI-1 WD40 domain and with other proteins, like dynamin-2 (DNM2), through its SH3 domain, to enhance leukemic-initiating activities and TKI resistance. To uncover downstream effects of the AHI-1-BCR-ABL-DNM2 complex and its biological role in mediating TKI resistance in CML stem/progenitor cells, an advanced antibody microarray analysis was then used to investigate differences in the proteome and phosphorylation landscape of BCR-ABL + cells co-transduced with wild-type Ahi-1 or Ahi-1 SH3 Δ mutant in the presence or absence of imatinib (IM). The microarray simultaneously quantified the differences in expression and phosphorylation sites of proteins in multiple signaling pathways using 1325 antibodies in duplicate measurements, and each microarray analysis was performed in duplicate. Significant changes in antibody signals for protein expression or phosphorylation were determined using limma. This analysis revealed that the overexpression of wild-type Ahi-1 (WT Ahi-1) has a profound differential effect, compared to the SH3 domain deleted Ahi-1 (Ahi-1 SH3 Δ), on the expression and phosphorylation status of proteins in BCR-ABL + cells with and without IM treatment. BCR-ABL + cells co-expressing WT Ahi-1 had a greater number of significantly differential antibody signals (7 increases, 42 decreases) when compared to BCR-ABL + cells, while Ahi-1 SH3 Δ expressing cells resulted in fewer significantly differential antibody signals (12 increases, 2 decreases) compared to BCR-ABL + cells. IM treatment resulted in WT Ahi-1 expressing cells having the greatest number of significantly differential antibody signals (7 increases, 56 decreases) compared to BCR-ABL + cells (5 decreases) and those co-expressing Ahi-1 SH3 Δ (2 increases, 9 decreases). Pathway enrichment analysis, using gProfiler, identified that the targets with significantly increased differential antibody signal after IM treatment in WT Ahi-1 cells were related to the regulation of translation initiation complex (p>0.0001). Interestingly, our RNA-seq dataset analysis further identified several members of the eukaryotic initiation factor 4F (eIF4F) complex to be significantly upregulated in CD34 + CML patient cells compared to normal bone marrow, particularly eIF4G1, the scaffold protein of the eIF4F complex involved in translation initiation (2-fold, p=0.001), as well as mTOR, a key regulator that controls the assembly of the eIF4F complex. This finding prompted us to further explore the regulation of translation initiation and the members of the eIF4F complex in BCR-ABL + cells. Immunoblotting demonstrated that BCR-ABL + cells co-transduced with WT Ahi-1 showed increased expression of eIF4G1 (3-fold) and eIF4B (2-fold), a cofactor that regulates the helicase activity of the eIF4F complex, compared to BCR-ABL + cells. Additionally, Cyclin D3, a gene reported to be sensitive to eIF4F translational activity, was found to have slightly increased expression (1.4-fold) in WT Ahi-1 cells compared to BCR-ABL + cells. Most interestingly, these results were also demonstrated in IM-resistant CML cells as compared to IM-sensitive cells, with an increase in eIF4G1 expression (2-fold), phosphorylation of eIF4B (5-fold, p=0.003), and Cyclin D3 expression (4-fold, p<0.05). Mechanistically, eIF4G1 knockdown by shRNA impaired survival (5-fold, p<0.0001) and increases TKI sensitivity in IM-resistant cells (3-fold, p<0.05). A new protein interaction between eIF4G1 and the mRNA cap-binding protein eIF4E was further identified in these cells using a proximity ligation assay. Thus, we have uncovered that the eIF4F complex, the key regulator of the mRNA-ribosome recruitment phase of translation initiation, has increased activity in IM-resistant cells, which may contribute to the regulation of TKI resistance in CML. DisclosuresNo relevant conflicts of interest to declare.

Mutational Profiles during the Progression of Chronic Myeloid Leukemia

Background:Despite significant improvements in the prognosis of chronic myeloid leukemia (CML) achieved by targeted therapy with tyrosine kinase inhibitors (TKIs), a small proportion of cases may not respond to TKIs or may relapse after an initial response, and then progress from chronic phase (CP) to blastic crisis (BC), characterized by a dismal prognosis. It remained uncertain whether the genetic lesions in addition to the BCR-ABL1 fusion could predict clinical outcomes of CML in the TKI era.Aim:To study the mutational profiles at each stage of CML and the prognostic significance of somatic mutations in addition to the BCR-ABL1 fusion in the TKI era.Patients and Methods:We performed targeted sequencing in 81 CML patients chosen retrospectively. 10 patients had optimal response to TKIs by European LeukemiaNet criteria and maintained durable major molecular response more than 5 years. 71 patients had progressed to accelerated phase (AP) or BC, of whom 43 had sequencing performed at paired CP and AP/BC samples, 28 at AP or BC samples. Totally, we analyzed 53 CP, 20 AP, and 61 BC samples. The targeted resequencing gene panel, covering 386 genes which were recurrently mutated in hematologic malignancies, were performed on a HiSeq 4000 NGS platform (Illumina).Results:Among the 53 CP samples, 20 (37.7%) had mutations involving 14 genes, and the number of mutated genes in each patient was 0-3 (median 0). ASXL1 was the most commonly mutated gene, 10/53 (18.9%) patients had this mutation, followed by KMT2D (4/53, 7.5%), PC (2/53, 3.8%), ERBB4 (2/53, 3.8%). ASXL1 mutation mainly existed in 43 patients with progressed disease , while only one case carried this mutation in 10 patients responsive to TKIs (20.9% vs 10%). 17/20 (85%) AP samples (including 10 patients progressed to AP and the other 10 patients who eventually progressed to BC from AP ) carried mutations involving 18 genes, the number of mutated genes in each patient was 0-6 (median 1.5). ABL1 was the most commonly mutated gene, and 8/20 (40%) patients had this mutation. The second was the ASXL1 mutation, 7 (7/20, 35%) patients carried this mutation. The other genes mutated in more than 2 patients included BCORL1 (3/20, 15%), RUNX1 (2/20, 10%), PHF6 (2/20, 10%), KMT2D (2/20, 10%), ATM (2/20, 10%). 54/61 (88.5%) BC samples (44 with myeloid crisis, 14 with lymphoid crisis, 3 with mixed phenotypic crisis) carried mutations, involving 41 genes, and the number of mutated genes in each patient was 0-9 (median 2). Similar to the mutation status in AP, the most commonly mutated gene was also ABL1, 24/61 (39.3%) patients carried this gene mutation, followed by ASXL1 mutation (13/61, 21.3%), and the other genes were in order, RUNX1 (11/61, 18.0%), WT1 (8/61, 13.1%), GATA2 (6/61, 9.8%), MED12 (5/61, 8.2%), IDH1 (5/61, 8.2%), TP53 (4/61 , 6.6%), KMT2D (4/61, 6.6%), etc. (Figure 1A)Among all the samples, 34 nonsynonymous variants in the ASXL1 gene were identified in 31 samples of 21 patients ( 3 samples with two variants). All the variants were frameshift and nonsense mutations, localized at the last exon of the ASXL1 gene. 13/21 patients with ASXL1 mutations had multi-stage samples. The median VAF of the ASXL1 mutations in the advanced stage was 31.4% (0-47.0%), which was significantly higher than that in CP at diagnosis (7.0%, 0-27.2%, P=0.033). Most of the ASXL1 mutations detected in CP expanded at the advanced disease, and were accompanied with other additional gene abnormalities, such as ABL1, RUNX1 and WT1 mutations, with the VAF similar to or lower than that of the ASXL1 mutations. In a few cases, the ASXL1 mutant clones in the CP disappeared, suggesting that some ASXL1 mutations may be clonal hematopoiesis unrelated to disease progression.(Figure 1B)In order to evaluate the effects of ASXL1 mutations on sensitivity to TKIs in vitro. We co-expressed P210-BCR-ABL1 fusion and ASXL1 mutation (G646Wfs*12) in Ba/F3 cells. Compared to Ba/F3 cells co-expressing BCR-ABL1 fusion and ASXL1 mutation (Ba/F3-BA/As), Ba/F3-BCR-ABL1 cells without ASXL1 mutation (Ba/F3-BA/Ve) showed higher sensitivity to TKIs, including imatinib, dasatinib and nilotinib.(Figure 1C)Conclusions:These results demonstrated the genetic lesions accumulated during the progression of CML from CP to BC. ASXL1 mutations were the most common genetic lesion in CP at diagnosis and may confer a poor prognosis, as it reduced the sensitivity to TKIs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Функціональний стан печінки у пацієнтів на стадіях прогресування хронічної мієлоїдної лейкемії

Chronic myeloid leukemia is found in approximately 20% of newly diagnosed cases of leukemia in adults. In Europe and North America, chronic myeloid leukemia ranks third among leukemias (after acute leukemias and chronic lymphoblastic leukemia). The problem of diagnosis, treatment, rehabilitation and adaptation of patients with chronic myeloid leukemia is relevant, as there is a gradual increase in the incidence of this disease both in the world and in our country. Some progress has been achieved in the treatment of patients with oncohematological diseases, in particular chronic myeloid leukemia. Chronic myeloid leukemia is a clonal myeloproliferative disease in which as a result of specific translocation of chromosomal sites between 9 and 22 chromosomes, a khimeric BCR-ABL gene with tyrosine kinase activity is formed. The purpose of the study was to investigate the main biochemical parameters that characterize protein and pigment metabolism, the activity of key enzymes in patients with chronic myeloblastic leukemia at different stages of the disease for further prospects of optimizing diagnosis, predicting the course of the disease in clinical practice. Materials and methods. The functional state of liver in 77 patients at different stages of chronic myeloid leukemia was studied. All patients admitted to the hospital were examined with the use of clinical, laboratory, instrumental and special research methods, and were consulted by specialists of related specialties, if necessary. The main biochemical parameters that characterize protein and pigment metabolism, the activity of key enzymes in patients with chronic myeloid leukemia of peripheral venous blood was determined by the method of V. Menshikov (1987). Results and discussion. The content of biochemical parameters that characterize protein and pigment metabolism, the activity of key enzymes in patients with chronic myeloid leukemia has been studied. It was found that biochemical parameters that characterize protein and pigment metabolism, the activity of key enzymes in patients with chronic myeloid leukemia is associated with an increase in the studied indicator (p&lt;0,05). The article discusses the possible causes and pathogenetic mechanisms of the identified changes. As the disease progressed the changes of protein, pigment and enzyme kinds of metabolism were revealed. The article discusses possible pathophysiological mechanisms of the identified changes. Conclusion. In patients with chronic myeloid leukemia, there is an imbalance in protein, pigment metabolism, and the activity of some liver enzymes. With the progression of chronic myeloid leukemia, the functional state of the liver deteriorates

Evaluation and Characterization of Indian Chronic Myeloid Leukemia Patients on Imatinib

Background: Chronic myeloid leukemia (CML) is a clonal malignant disorder of hematopoietic stem cell characterized by the classical chromosomal translocation, Philadelphia (Ph) chromosome in more than 90% of the patients. CML is associated with significantly high granulocyte numbers in the bone marrow and peripheral blood. Materials and Methods: This is a retrospective study conducted at the All India Institute of Medical Sciences, New Delhi, India aimed to evaluate the incidence and characteristics of CML patients. We have evaluated the demographic, clinical, and hematological data of 158 consecutive patients who visited the hospital from 2012 to 2014. Results: The diagnosis and stage of CML were determined based on the World Health Organization criteria, followed by the polymerase chain reaction. 158 patients evaluated in this study, consist of males (n = 98, 62%) which was higher than females (n = 60, 38%)( M:F::1.6:1). The mean + SD of male and female was 35.6 + 12.7, mean + SD of males was ( 35.7+ 13.6) and mean + SD of females was ( 35.4+11.3). While 17 (10.7%) patients were below 20 years of age, 94 (59.4%) were between 21–40 years, 40 (25.3%) were between 41–60 years, and 7 (4.4%) were more than 60 years of age. The predominance of younger patients (59.4.%) were belong to the age group of 21-40 years. While the most predominant symptom was fatigue and bone pain, the most common clinical sign was hepato-splenomegaly, followed by remarkable weight loss, and epistaxis. Conclusion: A patient with an increased WBC count, weakness, bone pain, low LAP score and hepatosplenomegaly should clearly be evaluated for CML.

Up-regulation of miR-155 potentiates CD34+ CML stem/progenitor cells to escape from the growth-inhibitory effects of TGF-ß1 and BMP signaling.

microRNAs (miRNAs or miRs) play key roles in different stages of chronic myeloid leukemia (CML) pathogenesis. The present study aimed to demonstrate whether miR-155 enables CD34+ CML cells to escape from the growth-inhibitory effects of TGF-β1 and bone morphogenetic protein (BMP) signaling. Among differentially expressed miRNAs in CD34+ CML cells, miR-155 was highly up-regulated. QRT-PCR revealed an inverse correlation between miR-155 and two key members of the TGF-β pathway-TGF-βR2 and SMAD5. Results showed that SMAD5 is not only up-regulated through BMPs treatment, but recombinant TGF-β1 can also induce SMAD5 in CML cells. We also demonstrated that TGF-β1-mediated phosphorylation of SMAD1/5 was abolished by pre-treatment with the blocking TGF-βR2 antibody, suggesting a possible involvement of TGF-βR2. Additionally, overexpression of miR-155 significantly promoted the proliferation rate of CD34+ CML cells. Results showed that siRNA-mediated knockdown of SMAD5 had a promoting effect on CD34+ CML cell proliferation, suggesting that SMAD5 knock-down recapitulates the proliferative effects of miR-155. Importantly, TGF-β1 and BMP2/4 treatment had inhibitory effects on cell proliferation; however, miR-155 overexpression enabled CD34+ CML cells to evade the anti-proliferative effects of TGF-β1 and BMPs. Consistently, down-regulation of miR-155 augmented the promoting effects of TGF-β1 and BMP signaling on inducing apoptosis in CD34+ CML stem cells. Our findings demonstrated that targeting of SMAD5 and TGF-βR2 links miR-155 to TGF-β signaling in CML. Overexpression of miR-155 enables CD34+ CML cells to evade growth-inhibitory effects of the TGF-β1 and BMP signaling, providing new perspectives for miR-155 as a therapeutic target for CML.

Analysis of cellular models of clonal evolution reveals co-evolution of imatinib and HSP90 inhibitor resistances

Treatment relapse due to clonal evolution was shown to be an independent factor for poor prognosis in advanced stages of chronic myeloid leukemia. Overcoming secondary resistance arising due to clonal evolution is still an unmet need and lack of adequate pre-clinical models hampers the identification of underlying mechanisms and testing of alternate treatment strategies. The current study thus aimed to create cellular models to study molecular mechanisms underlying clonal evolution and identify strategies to overcome the secondary drug resistance. Analysis of cell lines derived from three independent cell-based screens revealed the co-evolution specifically of imatinib and HSP90 inhibitor (HSP90i) resistances despite their exposure to a single inhibitor alone. Molecular and biochemical characterization of these cell lines revealed additional cytogenetic abnormalities, differential activation of pro-survival signaling molecules and over expression of ABL kinase and HSP90 genes. Importantly, all the imatinib-HSP90i dual resistant cell lines remained sensitive to sorafenib and vorinostat suggesting their utility in treating patients who relapse upon imatinib treatment due to clonal evolution. In addition, we cite similar examples of dual resistance towards various kinase inhibitors and HSP90i in some cell lines that represent solid cancers suggesting co-evolution leading to secondary drug resistance as a pan-cancer phenomenon. Taken together, our results suggest the efficacy of HSP90i in overcoming drug resistance caused by point mutations in the target kinase but not in cases of clonal evolution.

Chronic myeloid leukemia - update 2020

Chronic myeloid leukemia - update 2020 Abstract. The discovery of specific inhibitors of the BCR-ABL tyrosine kinase more than 20 years ago has revolutionized the treatment of patients with chronic myeloid leukemia (CML). Prognosis and outcome of patients considerably improved and progress in the use of the tyrosine kinase inhibitors is ongoing. In comparison to imatinib, second generation inhibitors used in first line lead to faster and deeper molecular remissions accompanied by different adverse event profiles. An essential part of the management of CML patients is to assess regularly the remaining tumor load by standardized molecular methods. Based on several clinical trials definitions of optimal response to treatment and of treatment failure have been put forward and help guide the treatment of the patients. The concept of treatment free remission was investigated extensively and is now part of the management of CML patients. Advanced stages of CML are diagnosed less frequently but are still challenging to treat. In these cases, allogeneic transplantation still plays an important role in the attempt to control the disease.

The Hidden Pathogenesis of CML: Is BCR-ABL1 the First Event?

Identification of the BCR-ABL1 fusion oncogene in patients diagnosed with chronic myeloid leukemia (CML) led to the development of targeted therapy responsible for the dramatic survival benefits observed in the past two decades. However, despite these revolutionary findings, there remains marked disparity in patient outcomes. Why do some patients present de novo while others evolve to the more aggressive stages of CML? Why can select patients successfully discontinue therapy as part of a treatment-free remission attempt whereas others fail to meet specific molecular milestones? BCR-ABL1 kinase mutations are only identified in approximately 50% of patients with poor responses and disease progression, suggesting the presence of alternative resistance mechanisms. Numerous institutions have identified the presence of additional genomic events in addition to BCR-ABL1 with the increasing availability of next-generation sequencing. We explore the potential pathways and events that may cooperate with BCR-ABL1 to answer these questions but also challenge the fundamental tenet that BCR-ABL1 is always the sole event initiating CML.

Haematological and cytogenetic changes in CML patients treated with imatinib mesylate in Western Libya

Background: Chronic myeloid leukemia (CML) is a clonal myeloid proliferative disorder of primitive haemopoietic progenitor cells. The incidence of CML ranges between 10 and 15 cases/106 /year without any major geographic or ethnic differences. Imatinib mesylate provides good results in the treatment of CML. Early studies demonstrated that Imatinib mesylate can produce clear hematologic and cytogenetic response when used as a treatment of CML patients with positive BCR-ABL gene. Nevertheless, some patient with different stages of CML (chronic, accelerated, or acute phases) either relapse or stay unchanged for a long time after initial doses of treatment. This phenomenon led to the fact that we must explore the possible changes expected to appear if we make some changes in the treatment strategy. Objectives: The present study aimed to evaluate hematologic and molecular responses of CML patients to Imatinib mesylate treatment. Methods: Eighteen CML patients in chronic phase aged (24–65 years) males and females were treated with Imatinib mesylate (400, 500 or 600 mg/day) for sixteen months. Hematologic and cytogenetic changes were analyzed periodically. Results: Overall 18 cases, hematologic response of 14 cases was complete white blood cells (WBCs) decrease to normal range within 4 months) with P value of less than 0.0001 whereas in 4 cases WBCs were decreased slowly (after 8 months). A major cytogenetic response was noticed in 4 cases while in others the response was partially or in minor range. The major hematologic and cytogenetic response was noticed when using 600mg/day of Imatinib mesylate. The correlation appeared as a significant positive correlation between the treatment doses and Hb, hematocrit, MCV, MCH, esinophils%, and monocytes %. And a significant negative correlation between the treatment doses and RDW %, platelets count, WBCs count, and basophils %. On the other hand, no correlation between the treatment doses and RBCs Count, MCHC, Neutrophil % and BCR/APL ratio % Conclusion: It can be concluded that treatment of CML patients with Imatinib mesylate caused complete WBCs decrease to normal range. The major hematologic and cytogenetic response was noticed when using a higher dose of Imatinib mesylate.

Haematological and cytogenetic changes in CML patients treated with imatinib mesylate in Western Libya

Background: Chronic myeloid leukemia (CML) is a clonal myeloid proliferative disorder of primitive haemopoietic progenitor cells. The incidence of CML ranges between 10 and 15 cases/106 /year without any major geographic or ethnic differences. Imatinib mesylate provides good results in the treatment of CML. Early studies demonstrated that Imatinib mesylate can produce clear hematologic and cytogenetic response when used as a treatment of CML patients with positive BCR-ABL gene. Nevertheless, some patient with different stages of CML (chronic, accelerated, or acute phases) either relapse or stay unchanged for a long time after initial doses of treatment. This phenomenon led to the fact that we must explore the possible changes expected to appear if we make some changes in the treatment strategy. Objectives: The present study aimed to evaluate hematologic and molecular responses of CML patients to Imatinib mesylate treatment. Methods: Eighteen CML patients in chronic phase aged (24–65 years) males and females were treated with Imatinib mesylate (400, 500 or 600 mg/day) for sixteen months. Hematologic and cytogenetic changes were analyzed periodically. Results: Overall 18 cases, hematologic response of 14 cases was complete white blood cells (WBCs) decrease to normal range within 4 months) with P value of less than 0.0001 whereas in 4 cases WBCs were decreased slowly (after 8 months). A major cytogenetic response was noticed in 4 cases while in others the response was partially or in minor range. The major hematologic and cytogenetic response was noticed when using 600mg/day of Imatinib mesylate. The correlation appeared as a significant positive correlation between the treatment doses and Hb, hematocrit, MCV, MCH, esinophils%, and monocytes %. And a significant negative correlation between the treatment doses and RDW %, platelets count, WBCs count, and basophils %. On the other hand, no correlation between the treatment doses and RBCs Count, MCHC, Neutrophil % and BCR/APL ratio % Conclusion: It can be concluded that treatment of CML patients with Imatinib mesylate caused complete WBCs decrease to normal range. The major hematologic and cytogenetic response was noticed when using a higher dose of Imatinib mesylate.

Chronic myeloid leukemia-derived exosomes attenuate adipogenesis of adipose derived mesenchymal stem cells via transporting miR-92a-3p.

Cancer-associated cachexia (CAC) has tremendous effects on the patient's tolerance to chemotherapy and the quality of life, especially in the advanced stages, such as the acute and terminal stages of chronic myeloid leukemia (CML). However, the underlying mechanisms and mediators remain unclear. Here, we showed that mice injected with CML-derived exosomes had significant weight loss and great drop of body fat rate. In the meanwhile, we found that CML-derived exosomes could be taken up by adipose tissue, and, in turn, suppressed the adipogenic ability of adipose-derived mesenchymal stem cells (ADSCs). By RNA sequencing, miR-92a-3p was found highly expressed in both CML cells and the derivative exosomes. Mechanistically, miR-92a-3p inhibited adipogenesis of ADSCs via posttranscriptionally decreasing C/EBPα expression when transferred into the ADSCs with the exosomes, and encapsulating miR-92a-3p inhibitor into CML exosomes blocked the antiadipogenic effects of CML exosomes. In addition, we also found that miR-92a-3p was highly expressed in exosomes from some other types of cancers that cause cachexia. These results demonstrate that adipogenesis inhibition by tumor-derived exosomes, mainly exosomal microRNAs like miR-92a-3p, are the main mediators for CAC.

Impact of Additional Chromosomal Aberrations on the Disease Progression of Chronic Myelogenous Leukemia.

The emergence of additional chromosomal abnormalities (ACAs) in Philadelphia chromosome/BCR-ABL1 positive chronic myeloid leukemia (CML), is considered to be a feature of disease evolution. However, their frequency of incidence, impact on prognosis and treatment response effect in CML is not conclusive. In the present study, we performed a chromosome analysis of 489 patients in different clinical stages of CML, using conventional GTG-banding, Fluorescent in situ Hybridization and Spectral Karyotyping. Among the de novo CP cases, ACAs were observed in 30 patients (10.20%) with lowest incidence, followed by IM resistant CP (16.66%) whereas in AP and BC, the occurrence of ACAs were higher, and was about 40.63 and 50.98%, respectively. The frequency of occurrence of ACAs were compared between the study groups and it was found that the incidence of ACAs was higher in BC compared to de novo and IM resistant CP cases. Likewise, it was higher in AP patients when compared between de novo and IM resistant CP cases, mirroring the fact of cytogenetic evolution with disease progression in CML. In addition, we observed 10 novel and 10 rare chromosomal aberrations among the study subjects. This study pinpoints the fact that the genome of advanced phase patients was highly unstable, and this environment of genomic instability is responsible for the high occurrence of ACAs. Treatment response analysis revealed that compared to initial phases, ACAs were associated with an adverse prognostic effect during the progressive stages of CML. This study further portrayed the cytogenetic mechanism of disease evolution in CML.

Revisiting Early Stages of Chronic Myeloid Leukemia (CML): Evidence That Leukemic Progenitors Appear First in Peripheral Blood during Molecular Recurrence

The use of tyrosine kinase inhibitors (TKI) has dramatically changed the natural history and the prognosis of CML with a major improvement of survival in patients responding to therapy. However, tyrosine kinase inhibitor (TKI) discontinuation trials in patients with deep molecular responses (DMR), show that 50-60% of patients will have a molecular recurrence requiring to resume the TKI therapy. The great majority of these relapses occur during the first 6 months of TKI discontinuation. Although the sustained persistence of leukemic stem cells (LSC) is clearly the cause of the recurrent disease, cellular dynamics of these early relapses and the pattern of cells giving rise to them are not determined. We had the opportunity to study this pattern in 4 patients (pts) who presented molecular recurrence after Imatinib mesylate (IM) discontinuation (n= 3) or on TKI therapy with Dasatinib (n= 1) after obtaining a deep molecular response de grade MR 4.5. The total duration of TKI therapy before TKI discontinuation was 14 years (UPN1), 15 years (UPN2) and 30 months (UPN3). All three pts (Aged 47, 66, 87 respectively) had CML-CP treated with IM 400 mg as a first line therapy with induction of DMR in all three. The duration of DMR (MR4.5- was for 6, 5 and 2 years for UPN1, 2 and 3 respectively. The reasons for TKI discontinuation were the TKI-discontinuation protocol in French observatory (UPN1, UPN2) and suspicion of interstitial pneumonia (UPN3). The patient UPN4 was on Dasatinib as a second line therapy and in DMR for 3 years. At the time of detection of molecular recurrence, all 4 pts had normal clinical examination with normal complete blood counts (CBC) with no evidence of circulating immature blood cells. After IM-discontinuation, molecular relapses occurred at 2 months (BCR-ABL IS 27%) 3 months (BCR-ABL IS transcript level at 13.1%) and at 9 months (BCR-ABL IS transcript level at 2.7%.) in patients UPN1, 2 and 3 respectively. In UPN 4, the molecular recurrence (BCR-ABL IS transcript 10%) was documented at + 4 months. In all 4 patients, colony forming cell (CFC) assays were performed from the same blood samples in which the molecular recurrence was documented. At day + 14, clonogenic cells (CFU-GM, BFU-E, CFU-Mixed) were plucked and BCR-ABL expression in each individual or pooled (2-4) CFC was determined. For 5.104PBMC plated in methycellulose assays in culture, the mean numbers of CFC obtained varied between 12 and 29 CFC / dish. Despite the normal CBC, the presence of BCR-ABL-expressing CFC in blood was demonstrated in all 4 pts. In UPN1, all CFC (20/20) obtained from peripheral blood were positive for BCR-ABL with a heterogeneous expression amongst CFC (BCR-ABL /ABL ratio range 2- 25%, mean 33%). Interestingly in this patient, CFC and LTC-IC assays performed in a bone marrow aspirate prior to IM discontinuation showed no evidence of BCR-ABL-expressing progenitors, suggesting a rapid and massive expansion of a dormant LSC clone migrating to blood at the time of molecular relapse. In pt UPN2, 9 individual CFC were found to express BCR-ABL with a significant heterogeneity (BCR-ABL/ABL ratio 0.2% - 56% , mean = 15 %). In Pt UPN3, with the lowest levels of BCR-ABL during molecular relapse, 4/73 CFC expressed BCR-ABL. All three pts had a favorable response after resuming Imatinib (UPN1, 2, 3) or after switching therapy to Bosutinib (UPN4). On UPN4, 2 pools of 4 CFC/20 expressed BCR-ABL. In two pts with 100% clonogenic relapse (UPN 1 and UPN2) the estimated circulating leukemic CFC numbers were 607 / ml and 468 / ml of blood respectively, in the presence of normal appearing CBC. In UPN 3 and 4, with approximately 10% of CFC being BCR-ABL +, these numbers were estimated to be 507 and 45 per ml of blood, respectively. Thus, the study of these four patients demonstrates that circulating leukemic CFC are the first detectable abnormal cellular components during molecular recurrence and that the clonogenic relapse in peripheral blood precedes myeloid expansion. These results show also for the first time to our knowledge, that leukemic CFC egress from bone marrow to peripheral blood is an early event during early stages of CML and the CFC expansion takes place not only in bone marrow but also in peripheral blood. Besides shedding light into the pathophysiology of CML, these findings could be of major interest to predict relapse parameters and could lead to the design of novel biological follow-up protocols after TKI discontinuation. DisclosuresNo relevant conflicts of interest to declare.

Long-Term Survival after Transplantation in Patients with Chronic Myeloid Leukemia from HLA-Compatible Unrelated Donors: May the Stem Cell Source Influence the Outcome?

IntroductionAllogeneic hematopoietic stem cell transplantation (alloSCT) is curative therapy for chronic myeloid leukemia (CML), but its long-term outcomes regarding graft sources are not well described.ResultsHere the outcomes of CML after alloSCT with bone marrow (BM, n=134) were compared to those of peripheral blood stem cell (PBSC, n=172) in the HLA-compatible unrelated donor setting. Patients were transplanted in 1st CP (Bone marrow transplantation (BMT), n=100, and Peripheral blood stem cell transplantation (PBSCT), n=103), in >1st CP (BMT, n=24, and PBSCT, n=52) and in blast crises (BMT, n=10, and PBSCT, n=17). Median follow-up were 54 months after BMT and 106 months after PBSCT. No significant differences were found in the incidence of acute and chronic graft-versus-host disease (GvHD) between both study-groups. The 5-year estimated probability of hematological relapse was 11% for patients in 1st CP CML and 49% for patients in advanced disease after BMT (p<0.001) and 18% for patients in 1st CP and 22% for patients in advanced disease after PBSCT (p= n.s.). The estimated probability for 10-year overall survival (OS) for patients in 1st CP and patients in advanced stages of CML were 58% and 20% after BMT and 59% and 52% after PBSCT, respectively (not significant for 1st CP and p<0.0001 for advanced stages). In the multivariate analysis patient age (age > 40 years), disease stage, acute GvHD, chronic GvHD and immunprophylaxis with use of ATG influenced OS significantly. For leukemia-free survival, the following risk factors were significantly in the multivariate analysis, graft source, patient age, gender constellation, disease stage, acute GvHD and chronic GvHD.ConclusionsThis large trial show significant difference between transplant recipients who received PBSC and those who received BM from unrelated donor. These finding may influence the selection of a graft source for alloSCT from unrelated donor. DisclosuresDührsen:Amgen: Research Funding; Gilead: Consultancy, Honoraria; Janssen: Honoraria; AbbVie: Consultancy, Honoraria; Roche: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Beelen:Medac: Consultancy, Other: Travel Support.

DNA methylation profiling reveals a pathological signature that contributes to transcriptional defects of CD34+ CD15- cells in early chronic-phase chronic myeloid leukemia.

Despite the high efficiency of tyrosine kinase inhibitors (TKI), some patients with chronic myeloid leukemia (CML) will display residual disease that can become resistant to treatment, indicating intraclonal heterogeneity in chronic‐phase CML (CP‐CML). To determine the basis of this heterogeneity, we conducted the first exhaustive characterization of the DNA methylation pattern of sorted CP‐CML CD34+CD15− (immature) and CD34−CD15+ (mature) cells at diagnosis (prior to any treatment) and compared it to that of CD34+CD15− and CD34−CD15+ cells isolated from healthy donors (HD). In both cell types, we identified several hundreds of differentially methylated regions (DMRs) showing DNA methylation changes between CP‐CML and HD samples, with only a subset of them in common between CD34+CD15− and CD34−CD15+ cells. This suggested DNA methylation variability within the same CML clone. We also identified 70 genes that could be aberrantly repressed upon hypermethylation and 171 genes that could be aberrantly expressed upon hypomethylation of some of these DMRs in CP‐CML cells, among which 18 and 81, respectively, were in CP‐CML CD34+CD15− cells only. We then validated the DNA methylation and expression defects of selected candidate genes. Specifically, we identified GAS2, a candidate oncogene, as a new example of gene the hypomethylation of which is associated with robust overexpression in CP‐CML cells. Altogether, we demonstrated that DNA methylation abnormalities exist at early stages of CML and can affect the transcriptional landscape of malignant cells. These observations could lead to the development of combination treatments with epigenetic drugs and TKI for CP‐CML.