BCR-ABL Fusion Gene Research Articles

Comparation of Digital PCR and Quantitative Pcr for Molecular Monitorization of Chronic Myeloid Leukemia Patients

Introduction: Chronic myeloid leukemia (CML) is a neoplasm produced by the formation of the BCRABL fusion gene. Tyrosine kinase inhibitors (TKI) have changed dramatically the clinical course of the disease. Quantitative PCR (qPCR) is the reference technique for measuring response in CML patients. Droplet digital PCR (ddPCR) is, in brief, a technology based on partitioning of the sample in a high number of small fractions which, in a further step, are read as positive or negative by its fluorescence. ddPCR performs an absolute quantification of the number of transcripts and their results do not depend on PCR efficiency. In spite of these theoretical advantages over qPCR, ddPCR is not routinely applied for BCRABL monitoring. In this study, we sought to compare qPCR and ddPCR techniques in similar amplification conditions.Methods: We included samples from 43 CML patients with different levels of response determined by qPCR: 23 patients with BCRABL/ABL1 levels <15%, 7 patients with MR3.0, 10 patients with MR4.0 and 3 patients who showed MR4.5 (according to European LeukemiaNet guidelines). Additionally, 3 non-CML patients were included. High-Capacity cDNA Reverse Transcription Kit was employed, following manufacturer instructions, in a unique reaction for both subsequent technologies utilized. qPCR was carried out with primers and probes for BCRABL and ABL1 from Europe Against Cancer report with 5 microL of non-measured cDNA in duplicates. The standards were BCR-ABL P210 ELITe Standard (ELITech Group) and the conversion factor was obtained with the Philadelphia P210 RNA Reference (Elitech Group). qPCR assys were run in an ABI 7500 platform in standard quantitation mode. EUTOS recommendations for BCRABL quantification were followed.ddPCR reaction was performed with the same primers and probes. Median cDNA concentration was 4,485ng/µL (1,771-7,787ng/µL). In order to avoid saturation of the technology, 2microL of cDNA at a concentration of 600ng/microL (i.e. 1,200ng) were added and run in duplicates in a C1000 touch Thermal Cycler (Bio-RAD) with the same amplification conditions used in qPCR. The same RNA reference material was used to obtain the calibration factor for the ddPCR technique. Bio-Rad QX200 Droplet Digital PCR System was employed for analysis. We used the mode of analysis for rare event detection and thresholds were set individually in each reaction. Qualitative comparations were carried out using Kappa coefficient with quadratic weight. For quantitative comparations, we applied Pearson correlation, intraclass correlation coefficient for absolute agreement and Passing-Bablok tests. A subgroup analysis, including samples with MR3.0 or deeper, was also performed.Results: All the samples showed ≥10000 ABL copies per well by qPCR. More than 10000 events were analyzed in every ddPCR reaction well. Good replicates were observed in qPCR and ddPCR. Adequate separation of positive and negative events could be performed and no saturation was observed in the ddPCR reations. No signal from BCRABL was detected in non-CML patients in ddPCR. The Kappa coefficient between qPCR and ddPCR measurements was 0.76 (P< 0.001, 95% confidence interval (CI): 0.63-0.88). In the 20 samples with MR 3.0 or deeper, kappa=0.13 (P >0.05 ) .For quantitative comparison, 32 samples with detectable BCRABL were included. Pearson test yielded an R2=0.91 (P< 0.001). The intraclass correlation coefficient for absolute agreement showed a value of 0.96 (P <0.001). Passing-Bablok test provided a non significant constant difference (0.041, CI 95%: -0.04-0.05) and a significant proportional difference (0.8, CI 95%: 0.7-0.96) (i.e. ddPCR results= 0.041 + (0.8*qPCR results). There were only 9 samples with a BCRABL/ABL1 ratio and quantitative test were not performed.Conclusion: Although a global excelent qualitative correlation is observed, in patients with MR3.0 or deeper which are the more important to classify correctly, qualitative correlation is absent. The R2 and intraclass correlation coefficient for absolute agreement showed a high concordance but Passing-Bablok test yielded a significant proportional difference among qPCR and ddPCR. We sought to test both technologies in similar conditions but different ddPCR strategies could be implemented in order to improve reproducibility of both techniques: higher amount of replicates or, given the lack of saturation, a higher cDNA concentration in the reactions. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Presence of Philadelphia Chromosome Does Not Confer Poor Prognosis in Adult Pre-B Acute Lymphoblastic Leukemia in the Tyrosine Kinase Inhibitor Era - a Surveillance, Epidemiology, and End Results Database Analysis

Translocation between chromosome 9 and 22 results in a BCR-ABL1 fusion gene identified in the form of an abnormal chromosome 22 (Philadelphia chromosome or Ph) in cytogenetics and/or other molecular studies. Ph+ acute lymphoblastic leukemia (ALL) constitutes about 25% of patients with pre-B ALL with increasing incidence with age. Prior to the introduction of tyrosine kinase inhibitors (TKI), presence of Ph chromosome conferred a poor prognosis in patients with ALL. Based on post-hoc comparisons between prospective clinical trials conducted before and after the introduction of this class of drugs, it appears that the use of TKIs inhibiting BCR-ABL have significantly improved the survival in Ph+ ALL. However, there has been no large-scale population based study comparing the survival of Ph+ ALL with Philadelphia negative (Ph-) ALL in the period when TKIs were widely used for treating Ph+ ALL. This information is highly relevant because survival of patients on clinical trials often exceeds results seen in real world. This could be due to study exclusion criteria, disparities in the number of elderly patients who receive TKI therapy, omission of patients with CNS involvement, etc. Thus the apparent improvement in outcomes has awaited confirmation at the population level. We compared the survival of Ph + and Ph-ALL during the period when TKIs were universally available in the US for Ph+ ALL, using a Surveillance, Epidemiology, and End Results (SEER) Database analysis. Patients with pre-B ALL ≥18 years in age, diagnosed between 2010 and 2014 were identified in SEER registries. 2694 patients with pre-B ALL were identified (206 Ph+ALL and 2488 Ph-ALL). The median OS was 32 months in Ph+ALL (95% CI 18 months-NE) and 27 months (95% CI 24-30 months) in Ph-ALL (Log-rank test p-value 0.34). To evaluate the progress in the survival of Ph+ ALL compared to Ph- ALL with time, the OS was estimated separately for patients diagnosed between 2010 - 2012 and 2013 - 2014 (Figure 3B and 3C). For 2010-2012, there was no significant difference (Log-rank p-value = 0.86) in the OS between Ph+ ALL (median OS = 24.5 months, 95% CI = 17.0-43.0) and Ph-ALL (median OS= 27, 95% CI = 23.0 - 30.0). For patients diagnosed between 2013 - 2014 period, there was a trend towards better survival for patients diagnosed with Ph+ ALL (1-year survival rate: 78.8%, 95% CI= 67.1%-86.7%) compared to Ph- (1-year survival rate: 69.2%, 95% CI= 65.5%-72.6%), although the difference was not statistically significant (Log-rank p-value = 0.094). Older age was associated with worse prognosis in both Ph+ALL and Ph-ALL. Age-adjusted OS was inferior in Hispanics and African-Americans compared to non-Hispanic whites. Survival of pre-B ALL shows continued improvement with time. Philadelphia chromosome status does not confer poor prognosis in pre-B ALL in the TKI era. Prognostic factors in pre-B ALL should be re-evaluated in the light of this finding. [Display omitted] DisclosuresKelly:Amgen: Honoraria; Abbvie: Honoraria; Jannsen: Honoraria; Pharmacyclics: Honoraria.

Updated Population Based Survival Analysis in Patients with Chronic Myeloid Leukemia: Increased Survival but Continued Disparities for Older Patients

Background: Survival for patients with chronic myeloid leukemia (CML) has increased dramatically since the advent of the first tyrosine kinase inhibitors (TKI) targeting the fusion gene BCR-ABL. However, survival has improved more for younger patients than for older patients. The reasons for this disparity are unclear. Here, we examine changes in survival for patients with CML by age between 2007-10 and 2011-14. In addition, we perform a subgroup analysis of patients known to have received chemotherapy.Methods: Data were extracted from the Surveillance, Epidemiology, and End Results (SEER) database, using the SEER18 dataset. Patients with ICD-10 code C92.1 were included in the analysis. Period analysis was used to calculate 5-year relative survival. Limited data on use of chemotherapy (yes or no/unknown) is available in the SEER dataset.Results: A total of 9209 cases of CML were found in the SEER18 database from 2007-14. Of these, 6868 were documented to have received chemotherapy. Overall 5-year relative survival increased from 69.0% in 2007-10 to 74.4% in 2011-14 (see table). Age specific analysis showed that there was little or no change for patients age 15-44, but point estimates of survival increased between 2007-10 and 2011-14 for all other age groups, although the differences were not statistically significant for individual age groups. Survival for patients age 45-54 was lower than for those aged 15-44 in 2007-10 but similar in 2011-14. However, survival continued to be lower for those aged 55 and older compared to younger patients, despite the observed increases in point estimate of survival for these patients.When analysis was limited to those patients documented to have received chemotherapy, the pattern of survival observed, i.e. increases in survival in 2011-14 compared to 2007-10 in all groups except for age 15-44 but decreased survival with increasing age, was unchanged.Conclusions: Survival increased for patients with CML age 45 and older between 2007-10 and 2011-14. However, there was a persistent disparity in survival for older patients. This disparity decreased only minimally when only patients known to have received chemotherapy were included, suggesting that chemotherapy usage is not the primary problem. Older patients may be treated less optimally despite receiving at least some chemotherapy, may suffer more fatal adverse events in treatment, or may have more resistant disease compared to younger patients. Further research into the optimal treatment of CML in older patients is needed to improve survival in this population. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

How Best to Express the Results of Serial RQ-PCR Studies to Measure Minimal Residual Disease in Treated Leukaemia Patients?.

The use of real time quantitative PCR (RQ-PCR) is now established for monitoring minimal residual disease (MRD) in patients with leukemia responding well to therapy. The level of the target gene is usually expressed as a percentage ratio normalised to the control gene. Measurement of a control gene is needed to exclude the possibility of false negative values due to sample (RNA) deterioration. The ideal control gene should be expressed in blood and/or bone marrow at intermediate level without significant variation between different patients; its RNA should have stability similar to that of the gene of interest; and it should not have a processed pseudogene. For monitoring the low levels of BCR-ABL transcripts characteristic of Ph-positive CML during treatment, different laboratories have quantified as controls (amongst others) ABL, BCR, G6PD, GAPDH,β-actin and β2-microglobulin. Despite the ABL gene being a component of the BCR-ABL fusion gene, it fulfils the criteria for a good control. Moreover as it is expressed at relatively low levels it is sensitive to sample deterioration. Analysis of 10,021 CML blood samples revealed that ABL transcript numbers were consistent when BCR-ABL transcript numbers were less than 20,000 (median 50,200), but above this level ABL quantification was influenced by BCR-ABL copy number (median 110,500). In MRD studies designed specifically to detect small numbers of residual transcripts, the contribution of amplifying BCR-ABL to the control value is not critical to the assessment of tumour load. A contemporaneous comparison of levels of BCR and ABL transcript levels in 831 blood samples taken from patients with CML at various stages of treatment showed a linear relationship (p=0.0001), but the median BCR expression was 0.7 log greater than the median ABL expression. In practice an assay using ABL as control gene for measuring the log reduction in BCR-ABL transcript levels following effective treatment compared with a BCR-ABL baseline value obtained before treatment yields values approximately equivalent to the log reduction obtained with an assay based on BCR as control gene. Nonetheless the variable expression of the commonly used control genes means that log reduction values may differ between centers. Thus we believe that the best method for reporting MRD values in CML patients undergoing treatment is as a percent ratio compared with previous values, i.e. the serial samples post treatment. Log reductions may be a useful ancillary for use within individual centres.

Targeting FEN1 Suppresses the Proliferation of Chronic Myeloid Leukemia Cells Through Regulating Alternative End-Joining Pathways.

Chronic myeloid leukemia (CML) is characterized by the formation of the BCR-ABL fusion gene. The BCR-ABL protein leads to an increased level of reactive oxygen species, which is a major cause of endogenous DNA double-strand breaks (DSBs). CML cells are prone to rely on a highly mutagenic alternative end-joining (Alt-EJ) pathway to cope with enhanced DSBs, which aggravates chromosomal instability. Hence, targeting dysregulated DNA repair proteins provides new insights into cancer treatment. In this study, we discovered the abnormal upregulation of Flap endonuclease 1 (FEN1) in CML, as well as FEN1's participation in the error-prone Alt-EJ repair pathway and its interplay with DNA Ligase1 and proliferating cell nuclear antigen in DSB repair. Knockdown of FEN1 by shRNA not only inhibited the proliferation and induced apoptosis but also enhanced the efficacy of imatinib (IM) in drug-resistant CML cell K562/G01. Moreover, excessive DSB accumulation was detected after FEN1 inhibition. In summary, our results demonstrated that FEN1 is a promising therapeutic target in CML treatment. This work extends the understanding of regulating abnormal DSB repair for cancer treatment.

A Case Report, Suspected Megakaryoblastic Transformation In A Chronic Myeloid Leukemia Patient

Preliminary: Chronic myeloid leukaemia (CML) is a clonal hematopoiesis stem cell disorder, characterised by reciprocal translocation between chromosomes 9 and chromosomes 22, originally named the ‘Philadelphia chromosome’(Ph). The megakaryoblastic transformation of a CML is extremely rare, consisting <3% of transformed cases. Case description: A 68-year-old male presented with a CML history since 2000 and received Imatinib therapy. The patient currently showed anemia and splenomegaly. Laboratory results showed hemoglobin 8.1 g/dL, leukocyte count 31,740/?L, platelet count 7,000/?L, Immature Platelet Fraction (IPF) 29.2%, BCR-ABL gene fusion is positive. Bone marrow examination showed CML blastic crisis phase with 45% blasts which had megakaryoblast morphology so it was concluded as a CML that underwent a transformation into megakaryoblastic. The results of bone marrow aspirate using Sysmex-XN1000 showed a very high IPF value which indicated an increased megakaryopoietic proliferative activity. Immunophenotyping examination showed blast population with CD45 dim, low side scatter, with the expression of CD34+, CD33+, CD13+ corresponding to the megakaryoblast area. Examination of the CD41 and CD61 specific megakaryoblast markers was not performed due to reagent limitations. Discussion: Clinical features, morphology, immunophenotyping and molecular findings help the identification of CML with suspected megakaryoblastic transformation. Conclusion: A CML patient has been reported with suspected megakaryoblastic transformation based on the bone marrow morphology, immunophenotyping, molecular and elevated IPF examination.

Understanding and Monitoring Chronic Myeloid Leukemia Blast Crisis: How to Better Manage Patients.

Chronic myeloid leukemia (CML) is triggered primarily by the t(9;22) (q34.13; q11.23) translocation. This reciprocal chromosomal translocation leads to the formation of the BCR-ABL fusion gene. Patients in the chronic phase (CP) experience a good curative effect with tyrosine kinase inhibitors. However, cases are treatment refractory, with a dismal prognosis, when the disease has progressed to the accelerated phase (AP) or blast phase (BP). Until now, few reports have provided a comprehensive description of the mechanisms involved at different molecular levels. Indeed, the underlying pathogenesis of CML evolution comprises genetic aberrations, chromosomal translocations (except for the Philadelphia chromosome), telomere biology, and epigenetic anomalies. Herein, we provide knowledge of the biology responsible for blast transformation of CML at several levels, such as genetics, telomere biology, and epigenetic anomalies. Because of the limited treatment options available and poor outcomes, only the therapeutic response is monitored regularly, which involves BCR-ABL transcript level assessment and immunologic surveillance, with the optimal treatment strategy for patients in CP adapted to evaluate disease recurrence or progression. Overall, selecting optimal treatment endpoints to predict survival and successful TFR improves the quality of life of patients. Thus, identifying risk factors and developing risk-adapted therapeutic options may contribute to a better outcome for advanced-phase patients.

ND‑09 inhibits chronic myeloid leukemia K562 cell growth by regulating BCR‑ABL signaling.

Chronic myeloid leukemia (CML) accounts for approximately 15% of new adult leukemia cases. The fusion gene BCR-ABL is an important biological basis and target for CML. In the present study, a novel compound, ND-09, was developed and its inhibitory effect and mechanism of action on CML growth were evaluated using RT-PCR and western blot analysis. The results showed that ND-09 demonstrated a high level of inhibitory action toward CML cells overexpressing BCR-ABL and induced K562 cell apoptosis through the mitochondrial pathway. Notably, combined ND-09 and BCR-ABL siRNA treatment could better inhibit cell proliferation and induce apoptosis in K562 cells. Furthermore, this growth effect of BCR-ABL siRNA could be fully rescued by transfection with BCR-ABL. ND-09 exhibited a good fit within BCR-ABL and occupied its ATP-binding pocket, thus altering BCR-ABL kinase activity. Therefore, ND-09 downregulated the phosphorylation of BCR-ABL and ABL, ultimately inhibiting the downstream signaling pathways in K562 cells. These findings suggest that ND-09 induces growth arrest in CML cells by targeting BCR-ABL.

Leukemia Cell Lines: In Vitro Models for the Study of Chronic Neutrophilic Leukemia.

Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative neoplasm that is genetically characterized by the absence of both the Philadelphia chromosome and BCR-ABL1 fusion gene and the high prevalence of mutations in the colony-stimulating factor 3 receptor (CSF3R). Additional disease-modifying mutations have been recognized in CNL samples, portraying a distinct mutational landscape. Despite the growing knowledge base on genomic aberrations, further progress could be gained from the availability of representative models of CNL. To address this gap, we screened a large panel of available leukemia cell lines, followed by a detailed mutational investigation with focus on the CNL-associated candidate driver genes. The sister cell lines CNLBC-1 and MOLM-20 were derived from a patient with CNL and carry CNL-typical molecular hallmarks, namely mutations in several genes, such as CSF3R, ASXL1, EZH2, NRAS, and SETBP1. The use of these validated and comprehensively characterized models will benefit the understanding of the pathobiology of CNL and help inform therapeutic strategies.

Potential treatment for chronic myeloid leukemia using microRNA: in silico comparison between plants and human microRNAs in targeting BCR-ABL1 gene

BackgroundChronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by the expression of the BCR-ABL1 fusion gene. Tyrosine kinase inhibitors (TKI) are used to treat CML, but mutations in the tyrosine kinase domain contribute to CML chemo-resistance. Therefore, finding alternative molecular-targeted therapy is important for the comprehensive treatment of CML. MicroRNAs (miRNA) are small non-coding regulatory RNAs which suppress the expression of their target genes by binding to the 3′ untranslated region (3′UTR) of the target mRNA. Hypothetically, the miRNA-mRNA interaction would suppress BCR-ABL1 expression and consequently reduce and inhibit CML cell proliferation. Thus, our objective was to determine the target interaction of human and plant miRNAs targeting the 3′UTR region of BCR-ABL1 in terms of miRNA binding conformity, protein interaction network, and pathways using in silico analysis. The 3′UTR sequence of BCR-ABL1 is obtained from Ensembl Genome Browser while the binding conformity was determined using the PsRNATarget Analysis Server, RNA22, Target Rank Server, and DIANA TOOLS. Protein-protein interaction network and pathway analysis are determined using STRING, Cytoscape, and KEGG pathway analysis.ResultsFive plants and five human miRNAs show strong binding conformity with 3′UTR of BCR-ABL1. The strongest binding conformity was shown by Oryza sativa’s Osa-miR1858a and osa-miR1858b with −24.4 kcal/mol folding energy and a p value of 0.0077. Meanwhile, in human miRNA, the hsa-miR-891a-3p shows the highest miTG score of 0.99 with −12 kcal/mol folding energy and a p value of 0.037. Apart from ABL1, osa-miR1858a/osa-miR1858b and hsa-miR891a-3p also target other 720 and 645 genes, respectively. The interaction network of Osa-miR1858a/osa-miR1858b and hsa-miR891a-3p identifies nineteen and twelve ABL1’s immediate neighboring proteins, respectively. The pathways analysis focuses on the RAS, MAPK, CML, and hematopoietic cell lineage pathway.ConclusionBoth plant and human miRNAs tested in this study could be a potential therapeutic prospect in CML treatment, but thermodynamically, osa-miR1858a/osa-miR1858b binding to ABL1 is more favorable. However, it is important to carry out more research in vitro and in vivo and clinical studies to assess its efficacy as a targeted therapy for CML.Graphical abstract

Application of transcriptome sequencing and fusion genes analysis in the diagnosis of myeloid leukemia with normal karyotype

Objective: To analyze the possible fusion genes with high-throughput transcriptome sequencing in myeloid leukemia patients with normal karyotype. Methods: From May 2017 to January 2019, three cases of myeloid leukemia patients with normal karyotype and negative for common fusion genes from the First Affiliated Hospital of Nanchang University were selected as the research objects. The transcriptome sequencing of bone marrow mononuclear cells was performed by high-throughput gene sequencing technology. Defuse software was used to analyze the gene fusion sequence in the transcriptome data, reverse-transcription polymerase chain reaction (RT-PCR) and Sanger sequencing were used to verify the fusion gene with clear pathological significance. Results: All three patients were diagnosed with myeloid leukemia by clinical manifestations, bone marrow cell morphology, immunology, and histochemical staining. Cytogenetic tests showed normal chromosome karyotypes. Fluorescence in situ hybridization and RT-PCR were used to detect BCR-ABL1, PML-RARA, and other common fusion genes. The results were all negative. Transcriptome sequencing and fusion transcripts analysis revealed that these three patients carried rare fusion genes with clear pathological significance, which included BCR-FGFR1, CPSF6-RARG, and NUP98-RARG, respectively. Conclusion: Transcriptome sequencing can accurately analyze rare but pathologically significant fusion genes that may exist in myeloid leukemia patients with normal karyotypes.

Chromosomal Aberrations in Chronic Myeloid Leukemia: Response to Conventional TKIs and Risk of Blastic Transformation

Background and Purpose: Chronic Myeloid Leukemia (CML) is a common hematological malignancy. The characteristic molecular abnormality is the presence of Philadelphia chromosome or BCR-ABL fusion gene which is the result of 9:22 translocation. Tyrosine kinase inhibitors (TKIs) form the main stay of treatment in CML with excellent responses. The purpose of this study was to determine the impact of additional chromosomal abnormalities on outcomes in CML.Methods: This is a retrospective chart review of all patients who were diagnosed with CML in chronic phase (CP) with additional chromosomal abnormalities (ACAs) over a period of 5 years from 2010 to 2015 at Shaukat Khanum Memorial Cancer Hospital and Research Centre, Lahore, Pakistan. Results: A total of 283 patients were diagnosed with CML from January 2010 to January 2015. 31 patients out of these were found to have additional chromosomal abnormalities at the time of diagnosis in addition to BCR-ABL fusion gene or Philadelphia chromosome detection. Out of these 31 patients, 23 (74.2%) were males whereas 8 (25.8%) were females. 13 (41.9%) were in the age group of 31 to 50 years whereas the other two groups that is 18 to 30 years and 51 to 70 years had 9 patients each. After approval from the government which usually takes a standard 2-3 weeks’ time, these patients were started on tyrosine kinase inhibitors which was Imatinib in 30 (96.8%) and Nilotinib in 1 (3.2%) patient. Conventional cytogenetic analysis performed for each patient at the time of diagnosis revealed that 11 (35.5%) of patients had variant Philadelphia chromosome followed by 7 patients (22.6%) with trisomy 8. 5 patients (16.1%) had multiple chromosomal abnormalities including trisomy 8, deletion 1 and isochrome 17q. 2 patents each had isochrome 17q, inversion 3 and deletion 9 abnormalities. 1 patient had deletion 7 whereas 1 had variant Philadelphia chromosome with other chromosomal abnormalities. Conclusion: It was evident that frequently occurring ACAs In our CML population were Variant Philadelphia chromosome and trisomy 8.

Improved gene delivery to K-562 leukemia cells by lipoic acid modified block copolymer micelles

Although there has been substantial progress in the research field of gene delivery, there are some challenges remaining, e.g. there are still cell types such as primary cells and suspension cells (immune cells) known to be difficult to transfect. Cationic polymers have gained increasing attention due to their ability to bind, condense and mask genetic material, being amenable to scale up and highly variable in their composition. In addition, they can be combined with further monomers exhibiting desired biological and chemical properties, such as antioxidative, pH- and redox-responsive or biocompatible features. By introduction of hydrophobic monomers, in particular as block copolymers, cationic micelles can be formed possessing an improved chance of transfection in otherwise challenging cells. In this study, the antioxidant biomolecule lipoic acid, which can also be used as crosslinker, was incorporated into the hydrophobic block of a diblock copolymer, poly{[2-(dimethylamino)ethyl methacrylate]101-b-[n-(butyl methacrylate)124-co-(lipoic acid methacrylate)22]} (P(DMAEMA101-b-[nBMA124-co-LAMA22])), synthesized by RAFT polymerization and assembled into micelles (LAMA-mic). These micelles were investigated regarding their pDNA binding, cytotoxicity mechanisms and transfection efficiency in K-562 and HEK293T cells, the former representing a difficult to transfect, suspension leukemia cell line. The LAMA-mic exhibited low cytotoxicity at applied concentrations but demonstrated superior transfection efficiency in HEK293T and especially K-562 cells. In-depth studies on the transfection mechanism revealed that transfection efficiency in K-562 cells does not depend on the specific oncogenic fusion gene BCR-ABL alone. It is independent of the cellular uptake of polymer-pDNA complexes but correlates with the endosomal escape of the LAMA-mic. A comparison of the transfection efficiency of the LAMA-mic with structurally comparable micelles without lipoic acid showed that lipoic acid is not solely responsible for the superior transfection efficiency of the LAMA-mic. More likely, a synergistic effect of the antioxidative lipoic acid and the micellar architecture was identified. Therefore, the incorporation of lipoic acid into the core of hydrophobic-cationic micelles represents a promising tailor-made transfer strategy, which can potentially be beneficial for other difficult to transfect cell types.

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.

Development of BCR-ABL1 Transgenic Zebrafish Model Reproducing Chronic Myeloid Leukemia (CML) Like-Disease and Providing a New Insight into CML Mechanisms.

Zebrafish has proven to be a versatile and reliable experimental in vivo tool to study human hematopoiesis and model hematological malignancies. Transgenic technologies enable the generation of specific leukemia types by the expression of human oncogenes under specific promoters. Using this technology, a variety of myeloid and lymphoid malignancies zebrafish models have been described. Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasia characterized by the BCR-ABL1 fusion gene, derived from the t (9;22) translocation causing the Philadelphia Chromosome (Ph). The BCR-ABL1 protein is a constitutively activated tyrosine kinas inducing the leukemogenesis and resulting in an accumulation of immature leukemic cells into bone marrow and peripheral blood. To model Ph+ CML, a transgenic zebrafish line expressing the human BCR-ABL1 was generated by the Gal4/UAS system, and then crossed with the hsp70-Gal4 transgenic line. The new line named (BCR-ABL1pUAS:CFP/hsp70-Gal4), presented altered expression of hematopoietic markers during embryonic development compared to controls and transgenic larvae showed proliferating hematopoietic cells in the caudal hematopoietic tissue (CHT). The present transgenic zebrafish would be a robust CML model and a high-throughput drug screening tool.

Effective Downregulation of BCR-ABL Tumorigenicity by RNA Targeted CRISPR-Cas13a.

To induce BCR-ABL gene silencing using CRISPR Cas13a. CML is a clonal myeloproliferative disorder of pluripotent stem cells driven by a reciprocal translocation between chromosomes 9 and 22 forming a BCR-ABL fusion gene. Tyrosine- kinase inhibitor drugs like imatinib are the mainstay of treatment and cases resistant to these drugs have a poor prognosis in the absence of a compatible stem-cell donor. However with rapid advancements in gene-editing technologies most studies are now focusing on developing a translational model targeting single-gene disorders with a prospective permanent cure. To explore the potential application of the RNA targeting CRISPR-Cas13a system for effective knockdown of BCR-ABL fusion transcript in a CML cell line K562. CRISPR Cas13a crRNA was designed specific to the chimeric BCR-ABL gene and the system was transfected as a two-plasmid system into a CML cell line K562. The effects were enumerated by evaluating the expression levels of downstream genes dependent on the expression of the BCR-ABL gene. Also next-generation sequencing was used to ascertain the effects of CRISPR on the gene. The CRISPR system was successfully able to lower the expression of downstream genes [pCRKL and pCRK] dependent on the activated BCR-ABL kinase signal by up-to 4.3 folds. The viability of the CRISPR-treated cells was also significantly lowered by 373.83-fold [p-value= 0.000891196]. The time-dependent kinetics also highlighted the significant in-vitro suppressive activity to last up to 8 weeks [p-value: 0.025]. As per the cDNA sequencing data from the Oxford MinION next-generation sequencer the CRISPR treated cells show 62.37% suspected cleaved reads. These preliminary results highlight an excellent potential application of RNA targeting CRISPRs in Haematological neoplasms like CML and should pave the way for further research in this direction.

Outgrowth of a CSF3R-mutant clone drives a second myeloproliferative neoplasm in a chronic myeloid leukemia patient: a case report

BackgroundChronic myeloid leukemia (CML) and chronic neutrophilic leukemia (CNL) are two myeloproliferative neoplasms with mutually exclusive diagnostic criteria. A hallmark of CML is the Philadelphia chromosome (Ph), which results in a BCR-ABL1 fusion gene and constitutive tyrosine kinase activity. CNL is a Ph-negative neoplasm and is defined in part by the presence of CSF3R mutations, which drive constative JAK/STAT signaling.Case presentationHere, we report the exceedingly rare co-occurrence of two granulocytic myeloproliferative neoplasms in a 69-year old male patient. After an initial diagnosis of chronic myeloid leukemia, the patient’s clinical course was shaped by hematologic toxicity, the emergence of treatment-resistant BCR-ABL1 clones, and the expansion of a CSF3R-mutant clone without ABL1 mutations under selective pressure from tyrosine kinase inhibitors. The emergence of the CSF3R-mutant, neutrophilic clone led to the diagnosis of CNL as a second myeloproliferative neoplasm in the same patient.ConclusionsThis is the first reported case of CNL arising subsequent to CML, which occurred under selective pressure from targeted therapy in a patient with complex clonal architecture. Patients with such molecularly complex disease may ultimately benefit from combination therapy that targets multiple oncogenic pathways.

Targeting Abnormal Hematopoietic Stem Cells in Chronic Myeloid Leukemia and Philadelphia Chromosome-Negative Classical Myeloproliferative Neoplasms.

Myeloproliferative neoplasms (MPNs) are unique hematopoietic stem cell disorders sharing mutations that constitutively activate the signal-transduction pathways involved in haematopoiesis. They are characterized by stem cell-derived clonal myeloproliferation. The key MPNs comprise chronic myeloid leukemia (CML), polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). CML is defined by the presence of the Philadelphia (Ph) chromosome and BCR-ABL1 fusion gene. Despite effective cytoreductive agents and targeted therapy, complete CML/MPN stem cell eradication is rarely achieved. In this review article, we discuss the novel agents and combination therapy that can potentially abnormal hematopoietic stem cells in CML and MPNs and the CML/MPN stem cell-sustaining bone marrow microenvironment.

Mutations Encountered in Acute Lymphoblastic Leukemia: A Retrospective Study in a Teaching Hospital in Jeddah, Saudi Arabia.

BackgroundAcute lymphoblastic leukemia (ALL) is an invasive cancer that results from the malignant conversion and rapid replication of white blood cells and hematopoietic stem cells that supply multiple lymphocytes. Harmful gene mutations occur in more than two-thirds of patients with ALL; however, these mutations have not been extensively identified in Saudi Arabia.AimThe aim of this study was to identify the types of mutations in patients with ALL at King Abdulaziz University Hospital (KAUH) in Jeddah. In addition, we identified the most common mutations.MethodsA retrospective study was performed on patients who were diagnosed with ALL from January 2009 to January 2019 at the Department of Hematology at KAUH. Our target population comprised patients diagnosed with ALL, including all age groups and both sexes. Patients were excluded if they had Down syndrome or central nervous system involvement, Li-Fraumeni syndrome, or neurofibromatosis.ResultsOf the 130 patients with ALL, 101 (77.77%) were children. The number of men (n=81) was substantially more than that of women (n=49). The data showed that 13.1% of our patients had mutations, and they occurred more frequently in patients with B-cell lymphoblastic ALL (B-ALL) than in those with T-cell lymphoblastic ALL (T-ALL). Several mutations, including BCR-ABL and ETV6/RUNX1, were more common in B-ALL, whereas the MLL-F0X04mutation was more commonly observed in T-ALL. There was a significant difference between the types of ALL and the genes involved (p=0.039). One female patient had translocation t(X;11)(q26;q23) (MLL-F0X04), which is a rare mutation.ConclusionIn summary, 13.1% of our study population had mutations. The BCR-ABL fusion gene was the most frequent mutation in patients at KAUH, and it occurred at a higher rate in B-ALL. Moreover, we detected other mutations, such as ETV6/RUNX1 and MLL-F0X04. The gene mutations were significantly different between B-ALL and T-ALL.

A novel fusion circular RNA F-circBA1 derived from the BCR-ABL fusion gene displayed an oncogenic role in chronic myeloid leukemia cells

ABSTRACT The BCR-ABL fusion gene plays a crucial role in the leukemogenesis of chronic myeloid leukemia (CML). The BCR-ABL oncoprotein encoded by this fusion gene has been extensively studied. However, research on whether BCR-ABL also affects circular RNAs (circRNAs) is limited. This study aimed to explore the new fusion circRNAs produced by BCR-ABL and their role in CML cells. In this study, we identified a novel fusion circRNA, named F-circBA1, originating from BCR-ABL in K562 and K562/G01 cells using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Sanger sequencing. qRT-PCR of the nuclear RNA and cytoplasmic RNA were separated, indicating that F-circBA1 was mainly localized in the cytoplasm. Cell counting kit-8 assay and flow cytometry showed that F-circBA1 knockdown by shRNA prevented the proliferation of K562 and K562/G01 cells, and the cell cycle was arrested at G2/M. Mechanically, dual-luciferase reporter assay and western blotting assay showed that F-circBA1 sponged miR-148-3p and F-circBA1 silencing decreased CDC25B expression in vitro. Furthermore, the results of the murine leukemogenesis model showed that F-circBA1 knockdown suppressed leukemogenesis in vivo. Besides, we found the existence of F-circBA1 in some patients with BCR-ABL-positive CML. In conclusion, these results demonstrate the presence of F-circBA1 and its oncogenic role in CML cells.