Cases Of Chronic Myeloid Leukemia Research Articles

BCR-ABL Negative CML-Like Disorder

Chronic myeloid leukemia (CML) has been defined for many years as BCR-ABL positive disease only, but older publications refer to a poor prognosis, clinically heterogeneous entity termed ‘BCR-ABL negative CML’ constituting about 5% of CML cases. Apart from very rare CML cases with cytogenetically cryptic, atypical variant BCR-ABL fusions that had been inadvertently missed during the diagnostic work up, most of these cases would now be classified as a subtype of myelodysplastic/myeloproliferative neoplasm (MDS/MPN) such as atypical CML (aCML), chronic myelomonocytic leukemia (CMML) or chronic neutrophilic leukemia (CNL). A minority would be classified as systemic mastocytosis with an associated hematological neoplasm (SM-AHN) or myeloid/lymphoid neoplasms associated with eosinophilia and rearrangement of PDGFRA, PDGFRB, or FGFR1 or with PCM1-JAK2 (MLN-eo).1

Prognostic significance of micro RNA 150 marker in BCR-ABL positive chronic myeloid leukaemia patients on imatinib mesylate

Background: Micro-RNAs control gene expression by destabilizing targeted transcripts and inhibiting their translation. In chronic myeloid leukaemia (CML), abnormal expressions of miRNAs have been described. The current treatment for newly diagnosed cases of CML is imatinib mesylate which produces rapid haematological responses. It is currently impossible to predict whether a patient will develop resistance to imatinib mesylate. This makes identification of predictors of resistance to imatinib an important goal in management of patients with CML. MicroRNA expression patterns can be used to predict outcome which can be remission or relapse. This study therefore, was set to assess the possible use of microRNA 150 for prognostication.Methods: Fifty peripheral blood samples previously collected from CML patients who were being treated with imatinib mesylate and stored in the refrigerator at +4°C were analyzed for the expression of microRNAs 150. Total RNA was extracted from guanidium isothiocynate (GITC) lysate of the blood samples using RNeasy mini spin column. The total RNA was converted to complimentary DNA by random hexamer priming using Murine Moloney Leukaemia Virus Reverse Transcriptase. Real time Multiplex PCR was used for detecting Breakpoint Cluster Region-Abelson Murine Leukaemia (BCR-ABL) transcript type.Results: The patients’ samples showed an expression of miRNA-150. Correlation of BCR-ABL ratio with miRNA-150 was done and the Spearman correlation coefficient (Rho) between BCR-ABL1 and miRNA-150 was 0.442 (p = 0.001; CI = 0.18-0.65) showing that there was a positive correlation between BCR-ABL1 and miRNA-150. The coefficient of determination was 20% (CI = 3-42%), which implies that about 20% of BCR-ABL1 ratio could be accounted for by the miRNA-150 values.Conclusions: Therefore, once patients who are on imatinib achieve molecular remission of the CML, the miRNA-150 can be useful in predicting outcome which could be relapse or complete molecular remission but is weak at diagnosis in predicting such outcome.

A rare case of three-way complex variant translocation in chronic myeloid leukemia t(6;9;22)(p21;q34;q11): A case report.

Philadelphia (Ph)-positive chromosome or Ph translocation has been recognized in 90-95 chronic myeloid leukemia (CML) cases worldwide. However, only 5-8% CML patients show complex variant translocations. In the present study, hematological tests for a 47-year-old female CML patient were performed to determine the hemoglobin, platelets and total leukocyte values. A FISH test was carried out to recognize the BCR/ABL gene fusion, and a cytogenetic analysis was performed. The hematological results showed an increase in WBC (414000/mm3) and a decrease in hemoglobin (8.9 mg/dl), indicating the anemic condition in the CML patient. Furthermore, cytogenetic karyotyping results showed 46,XX,t(6;9;22)(p21;q34;q11) and positive for Ph chromosome. In conclusion, in the present study, we report a rare three-way complex variant translocation in a CML patient.

Variation of LNK Gene in Chronic Myeloid Leukemia

To compare the mutation and single nucleotide polymorphism (SNP) of LNK gene between chronic myeloid leukemia(CML) and control groups, and to explore the relationship between LNK gene variation and the occurrence of CML. A total of 36 patients with CML were selected, 46 healthy persons were used as normal controls. DNA was extracted from bone marrow and peripheral blood, BCR/ABL1 fusion gene was detected by Q-PCR. The whole exon of LNK gene was amplified by PCR. The amplified sequences included the Rs3184504 (C/T) and Rs78894077 (A/C/G/T) affecting the expression of amino acids in LNK gene, and the Rs7973120 (A/T) unaffecting the expression of the amino acids. The mutations and SNP of LNK gene were analyzed by DNA sequencing. Thirty-six cases of CML had BCR/ABL1 mutation, while no mutation was found in the control group. One case of CML had LNK heterozygous mutation (A300V), and the mutation rate was 2.8%, no mutation was seen in normal control group. Rs3184504: C/T allele frequency was 50%/50% in the control group, 94.4%/5.6% in the CML group, and the C allele in CML group was significantly higher than that in the control group; CC genotype accounted for 94.4% (P<0.01). Rs78894077: C/T allele in the control group was 9.8%/90.2%, in CML group was 16.7%/83.3%, the difference was not statistically significant(P>0.05); but CC genotype in CML group was very statistically significant higher than that in control group(P<0.01). Rs7973120: A/T allele frequency was 10.9%/89.1% in the control group, 25%/75% in the CML group, the LNK A allele in CML group was very significantly higher than that in control group (P<0.01). CML patients have been confirmed to have LNK mutation; the SNPs of LNK are related with the development of CML, and the most CML patients carry the LNK Rs3184504 C allele and the Rs7973120 A allele.

Outcomes of blast phase chronic myeloid leukemia (BP-CML) in the tyrosine kinase inhibitor (TKI) era.

e18546 Background: Primary goal in management of chronic myeloid leukemia (CML) is to prevent progression to BP-CML. Even with the use of induction chemotherapy with a TKI, outcome remains dismal in BP-CML. We describe our experience with the management of BP-CML. Methods: Retrospective chart review was performed on CML patients treated between 2001 and 2016. Out of 585 CML cases, 58 (10%) had a diagnosis of BP-CML. Kaplan-Meier method with log-rank test was utilized for survival calculations. All p-values calculated were two sided. Results: The overall survival (OS) of BP-CML cohort was 31.9 months (mo) from diagnosis. In the patients diagnosed in chronic or accelerated phase, median time to progression to BP-CML was 19.1 mo. The median OS from the BP-CML diagnosis was not different when progressed from an earlier phase compared toBP-CML (10.8 vs 11.0, p 0.62). Trend toward worse OS was noted in myeloid BP-CML compared to lymphoid BP-CML (9.2 vs 17.5 mo, p 0.32). The median OS in the single agent TKI cohort (n=21) was 12.8 mo vs 10.9 mo (p 0.72) in the combination cohort (n = 36). Treatment with a single agent TKI vs combination therapy did not significantly impact OS in either myeloid (9.2 vs 9.1 mo, p 0.32) or lymphoid (14.5 vs 18.3 mo, p 0.24) BP-CML. Out of 26 patients (44.8%) who received allogeneic stem cell transplant, 26% (n=6) received a single agent TKI prior to transplant and 76.9% (n=20) received combination therapy. A trend toward improved OS was noted in the single agent TKI cohort (128.5 vs 24.0 mo, p 0.23). When stratified by the presence of standard Philadelphia chromosome or with additional cytogenetic aberrations, no difference in OS was noted (10.9 vs 12.1 mo, p 0.51). Monosomy 7 was present in greater frequency in lymphoid BP-CML than myeloid BP-CML (35.7% vs 6.3%, p 0.02). Conclusions: Our data suggest no survival difference when BP-CML is treated with a single agent TKI compared to a combination therapy, regardless of histology type. Therefore, single agent TKIs should be considered as an effective frontline therapy option for BP-CML, which may prevent the potential toxicity associated with chemotherapy. These findings need further validation in a larger prospective cohort.

A novel BCR-ABL1 fusion gene with genetic heterogeneity indicates a good prognosis in a chronic myeloid leukemia case

BackgroundChronic myelogenous leukemia (CML) is a pluripotent hematopoietic stem cell disorder caused by the fusion of the BCR and ABL1 genes. Quantitative RT-PCR (qRT-PCR) is a routinely performed screening technique to identify BCR-ABL1 fusion genes, but a limitation of this method is its inability to recognize novel fusions that have not been previously characterized. Next-generation sequencing (NGS) is an effective and sensitive detection method for the determination of novel BCR-ABL1 fusion genes as well as previously characterized ones. The oncoprotein tyrosine kinase BCR-ABL1 is a constitutively active kinase involved in the activation of a number of signaling pathways, and it has been the therapeutic target for tyrosine kinase inhibitors (TKIs) such as imatinib. Reports have presented opposing viewpoints about the effect of the disrupted Src homology 3 (SH3) domain on TKI efficacy.FindingsWe here report that using NGS we identified a novel BCR-ABL1 fusion gene with breakpoints in the BCR intron 14 and the ABL1 intron 2, leading to partial deletion of its SH3 domain. In the present case, the patient received targeted therapy with the TKI imatinib at 400 mg/day and no adverse reaction was reported. The patient eventually entered remission with decreased proliferation of karyocytes and granulocytes. We also identified mutations in genes, including TP53, FLT3, ASXL1, SETBP1, CEBPA and CBL, that seemed to have an influence on the outcome of TKI therapy targeting the BCR-ABL1 protein.ConclusionsTogether with previously reported results, it is clear that the genetic heterogeneity of CML patients significantly affects the presentation of the disease and its progression and therefore should inform the design of the therapeutic strategy.

Frequency of &amp;lt;i&amp;gt;L1721W&amp;lt;/i&amp;gt; Polymorphism in &amp;lt;i&amp;gt;TET2&amp;lt;/i&amp;gt; Gene Among a Cohort of Sudanese Patients with Myeloproliferative Disorders: Possible Roles in Pathogenicity and Leukemic Transformation

Transformation of myeloproliferative disorders (MPDs) to acute leukemia is an evitable event that represents a stumbling block in the management of patients. The Janus Kinase-2 JAK2V617F mutation of MDP does not clarify the phenotypic variability observed in this disorder. But, a mutations in Ten-eleven-translocation-2 (TET2), a putative tumor suppressor gene, was recently implicated in MPDs and other hematologic malignancies. TET-2 is believed to play a role in leukemic transformation. This study aimed to determine the frequency of L1721W polymorphism in TET2 gene in a cohort of Sudanese patients with MPDs. Following informed consent, 25 (25/50, 50%) patients with polycythemia rubra vera (PRV), thirteen patients (13/50, 26%) with essential thrombocythemia (ET), eleven patients (11/50, 22%) with chronic myeloid leukemia (CML), and one patient (1/50, 2%) with primary myelofibrosis (PMF) were enrolled. None of the patients was in the transformation phase. Patients were diagnosed based on clinical picture, hematological parameters and JAK2V617F and BCR_ABL molecular aberrations. JAK2V617F was detected in Ph-negative-MPDs cases as (24/25, 96%) in PRV, (10/13, 76%) in ET, and (1/1, 100%) in PMF. BCR_ABL fusion was detected in all (11/11, 100%) cases of CML. DNA was extracted using the guanidine chloride method, followed by (PCR-RFLP) analysis. Only one patient showed the presence of L1721W polymorphism of the TET2. It was inferred that the low frequency of this transformation within the study cohort [all in chronic phase] probably indicates that it plays a minor role in MPD pathogenesis, while its role in blast transformation needs further studies in MPD patients.

Peripheral Artery Occlusive Disease Among Patients With Chronic Myeloid Leukemia Receiving Tyrosine Kinase Inhibitors: A Cross-Sectional Case-Control Study.

Background:There were some reports of peripheral artery occlusive disease (PAOD) associated with nilotinib usage in chronic myeloid leukemia (CML). These complications in other tyrosine kinase inhibitors are revealed as unknown.Materials and methods:We determined the prevalence of PAOD in patients with CML as compared with matched-control population by cross-sectional case-control study. Peripheral artery occlusive disease was screened by ankle-brachial index (ABI).Results:In total, 78 CML and 156 matched-control patients were included. The median age was 55 years. In all, 61 (78.2%) were on imatinib and 13 (16.7%) were on nilotinib, whereas 4 patients (5.2%) were on dasatinib. Prevalence of low ABI (<0.9) was 9.0%, and nilotinib users had the highest prevalence of low ABI of 30.7%. All cases with low ABI were not shown to be clinically overt of PAOD. There were well-balanced characteristics between cases of CML and matched control except in higher levels of hypercholesterolemia in the control. Interestingly, CML had more amounts of pathologic ABI than the control (odds ratio: 2.09, 95% confidence interval: 0.71-6.21), and diagnosis of diabetes found it to be independent of the risk of PAOD.Conclusions:Peripheral artery occlusive disease was higher among patients with CML than the control, especially in patients who had diabetes.

Thrombosis and Cancer: Case Series of 25 Patients

The objective of this paper is to describe the clinical and therapeutic characteristics of thrombosis associated with cancers in patients in charge of the internal medicine-oncology department of the Ibn Sina Military Hospital in Marrakech. This is a retrospective, descriptive and analytical study over a two-year period from January 2013 to December 2014. Among 158 patients with cancer, we identified 25 with thrombosis confirmed by imaging. Characteristics of the population of origin, incidence, risk factors for venous thromboembolic disease (VTE), diagnostic means, distribution of thrombosis, location and histological type of cancer and therapeutic strategy were analyzed. The mean age of our patients was 55.89 years, with female predominance (56% vs 44%). The incidence of venous thrombosis in our series is 15.8% (n = 25) of which 76% (n = 19) are isolated, 12% (n = 3) are on central venous catheter (CVC) and 12% (n = 3) are associated with pulmonary embolism. The lower limbs were the most affected (80.3%), with predominance on the left side. Bilateral involvement was found in 7% of cases. Thrombosis was indicative of cancer in 16% of patients (n = 4). While it complicated the development in 84% of the cases (n = 21). Risk factors for VTE were dominated by females (56%), metastases (64%), chemotherapy (80%), and the presence of a CVC (80%). Solid tumors were the most frequent 72% (n = 18) with predominantly digestive cancers (28%), pulmonary (16%), bladder (16%) and breast (12%). The most predominant histological type was adenocarcinoma in 44% (n = 11). The seven cases of malignant hemopathies are: four non-Hodgkin's lymphomas, two multiple myelomas and one case of chronic myeloid leukemia. The initial curative treatment as a relay used low molecular weight heparin in 92% of patients (n = 23). Unfractionated heparin was used in two patients with severe renal failure, the only ones receiving vitamin K antagonist maintenance therapy. Short-term outcomes were favorable in 92% (n = 23), two deaths Have been attributed to severe pulmonary embolism. Significant progress has been made in the context of cancer-related VTE. Nevertheless, there remains a considerable field of unresolved clinical issues. Numerous studies are underway and may lead to advances not only in the treatment of VTE associated with cancer but also in the treatment of the cancerous disease itself.

Synchronous Occurrence of Chronic Myeloid Leukemia and Mantle Cell Lymphoma

Chronic myeloid leukemia (CML) and mantle cell lymphoma (MCL) are hematologic malignancies that originate from different oligopotent progenitor stem cells, namely, common myeloid and lymphoid progenitor cells, respectively. Although blastic transformation of CML can occur in the lymphoid lineage and CML has been related to non-Hodgkin lymphoma on transformation, to our knowledge, de novo and synchronous occurrence of CML and MCL has not been reported. Herein, we report the first case of synchronous CML and MCL in an otherwise healthy 38-year-old man. Potential etiologies and pathological relationships between the two malignancies are explored, including the possibility that the downstream effects of BCR-ABL may link it to an overexpression of cyclin D1, which is inherent to the etiology of MCL.

A case of synchronous multiple myeloma and chronic myeloid leukemia.

A case of synchronous multiple myeloma and chronic myeloid leukemia.

Clinical and Hematological Characteristics of Patients with Chronic Myeloid Leukemia under Present-Day Conditions: Results of the Russian Part of International Multi-Center Prospective EUTOS Population-Based CML Study

Background. Much attention has been paid to molecule-genetic diagnostics of chronic myeloid leukemia (CML) and its treatment using new highly effective methods of therapy. The baseline characteristics of patients at primary CML diagnosis are hardly discussed in literature. Aim. To provide clinical, hematological, molecular genetic and demographic characteristics of patients obtained at primary diagnosis of CML. Patients &amp; Methods. Characteristics of CML patients are based on data gathered by the Russian Investigational Group for CML within the international project European Treatment and Outcome Study of CML in Europe (EUTOS, the European Treatment and Outcomes Study). The study included 197 patients with newly diagnosed CML in 6 regions of the Russian Federation (Mordovia, Kirov, Perm (2 sites), Bryansk, Irkutsk, and Chita) over the period from 2009 till 2012. Results. The study demonstrated that 94 % of CML cases were diagnosed in the chronic phase (CP) and 6 % of cases in the acceleration phase (AP) and the blast crisis phase (BC). In 40 % of patients there were no clinical symptoms, and CML was suspected only due to changes in the CBC test. Fatigue was the main subjective complaint presented by 77 % of patients in the CP and 100 % of patients with the AP and BC. Peripheral blood leukocytosis, left shift to immature myeloid cells and increased granulocytic lineage in bone marrow were typical for the patients. In all patients, the CML diagnosis was confirmed by cytogenetic or molecular tests. The social and demographic characteristics of CML patients and comorbidities at diagnosis were analyzed. Conclusion. Based on the results of the study, a modern «portrait of a CML patient» was obtained. The study demonstrated that cytogenetic and molecular methods allow to diagnose CML in most patients at early stages of the disease in the absence of clinical signs of progression. The data on comorbidities require a special attention while choosing a therapy considering its duration. Demographic and social characteristics of CML patients demonstrate that they are socially active, particularly interested in retaining the working capacity and quality of life.

Expression of transcription factors SOX4 and C/EBPα in chronic myeloid leukemia and their significances

Objective To investigate the expression of SOX4 and C/EBPα mRNA in chronic myeloid leukemia (CML) and their clinical significances. Methods Bone marrow samples from 68 cases of CML including 57 newly diagnosed patients and 11 patients treated with imatinib were collected, and peripheral blood mononuclear cells from 30 healthy people were collected as healthy control. The expression of SOX4 and C/EBPα mRNA and protein levels were detected by RT-PCR and Western blot, respectively. The relations between the expression of SOX4 and C/EBPα and the influences of imatinib on SOX4 and C/EBPα were analyzed. Results The expression level of SOX4 mRNA was increased in newly diagnosed CML patients compared with that of normal control group (6.545 5±1.495 2 vs. 0.059 6±0.018 8, t=3.139, P=0.002 3), but the expression level of C/EBPα mRNA was significantly decreased (0.238 8±0.033 8 vs. 0.810 5±0.056 2, t=9.240, P 0.05). The expression level of SOX4 mRNA in 5 patients treated with imatinib was decreased (0.120 6±0.044 9 vs. 0.557 9±0.144 8, t=2.885, P=0.020 4), and the expression level of C/EBPα mRNA was increased (0.330 3±0.042 4 vs. 0.150 5±0.046 5, t=2.855, P=0.021 3). The expression level of SOX4 mRNA in 6 patients who developed blast phase during the treatment of imatinib was increased (0.469 9 ± 0.123 0 vs. 0.050 2 ± 0.036 6, t=2.370, P=0.039 3), and the expression level of C/EBPα mRNA was decreased (0.197 9±0.064 7 vs. 0.378 7±0.042 9, t=2.327, P=0.042 3). The expression of SOX4 mRNA was negatively correlated with C/EBPα mRNA (t=-0.554 6, P=0.002 8). Conclusions In newly diagnosed CML, the expression level of SOX4 is increased, C/EBPα is decreased compared with that of healthy control, and both have negative correlation. In the patients in blast phase after imatinib treatment, SOX4 gene is up-regulated, and C/EBPα is down-regulated. C/EBPα-SOX4 axis may play a role in the occurrence and development of CML. SOX4 may be a new molecular target for the treatment of CML. Key words: Leukemia, myeloid, chronic; SOX4; C/EBPα; Prognosis; Targeted therapy

Significance of bcr-abl molecular detection in chronic myeloid leukemia patients

Important advances in the understanding of the molecular basis of chronic myeloid leukemia have resulted in the development of new therapies and changed the paradigm for managing this myeloproliferative disease. The reciprocal translocation of the abl (Abelson murine leukemia) proto-oncogene on chromosome 9 to the bcr (breakpoint cluster region) gene on chromosome 22 creates a transcriptionally active, chimeric bcr-abl gene and gives rise to the Philadelphia chromosome. 1&#x0D; The genetic hallmark of chronic myeloid leukemia (CML) is the Philadelphia chromosome (Ph), which occurs in at least 95% of CML cases as well as some cases of acute lymphocytic leukemia (ALL; approximately 5% for children, 20% for adults). It also has been reported in some other hematologic disorders, albeit rarely. 2-4 JMS 2016; 19(2):95-96

Enhanced and Accelerated Repopulation of Mutant C-Kit Immunodeficient Mice By Transplants of Primary Chronic and Acute Myeloid Leukemic Human Cells

Background: Assessment of the growth and differentiation of human hematopoietic cells transplanted into immunodeficient mice has become an important method for evaluating the numbers and properties of cells with engraftment potential and the progeny they can generate in an in vivo setting. Such xenograft models have undergone numerous modifications and have also been applied with increasing success to primary sources of many types of malignant as well as normal human hematopoietic cells. These advances include the incorporation of cDNAs encoding various human-specific growth factor genes into the genomes of the widely used and long-lived NOD-scid-IL2Rγcnull (NSG) mouse, the creation of regenerated human bone ossicles within the mice to more closely mimic the human marrow microenvironment, recognition of the superior supportive activity of female mice, and more recently of NSG mice additionally compromised by their genetic acquisition of a c-kit deficiency. Exploitation of these modifications to recipients of human hematopoietic cells has greatly increased our understanding of the complex composition of both normal and leukemic populations and their responses to various treatments. Nevertheless, many examples remain of both acute and chronic human myeloid leukemias that do not engraft or engraft very poorly, even in the most permissive mice thus far tested. These include samples of many cases of chronic phase chronic myeloid leukemia (CP-CML), myelodysplastic syndromes (MDS), and approximately one third of all cases of acute myeloid leukemia (AML). We have now created 2 new strains of immunodeficient mice that have the B, T, NK immunodeficiency determined by a SirpαNOD-Rag1-/--IL2Rγc-/- genotype (which is equivalent to that of NSG mice) and a c-kit deficiency obtained by replacement of the wild-type c-kit gene with a homozygous W41/W41genotype (SRG-W41 mice). The second strain has the same SRG-W41 genotype but is also transgenic for human IL3, GM-CSF, and SCF production (SRG-W41-3GS). NRG/SRG mice were chosen as the parental strain because they support similar levels of chimerism as NSG mice when given a radiobiologically equivalent dose of radiation, but can benefit from split or low dose rate irradiation protocols.Results: We first showed that leukemic cells from patients with AML and CP-CML produce progeny more rapidly in sub-lethally irradiated parental SRG-3GS mice than in SRG mice, and that SRG-W41 mice can support higher levels of human myeloid and B-lymphoid chimerism by normal human CD34+ cord blood (CB) and adult bone marrow (BM) cells than similarly suppressed or non-irradiated SRG controls. We then compared the chimerism obtained in different strains of mice transplanted each with 6x106 CD34+ cells from 1 CP-CML patient or ~106 CD34+ cells from 5 different AML patients pre-selected for their previously demonstrated poor or inability to engraft NSG-3GS mice. Within a few weeks, the level of the predominantly myeloid chimerism in 2 non-irradiated male SRG-W41-3GS mice and 2 sub-lethally irradiated female SRG-3GS recipients became equivalent and this equivalence was sustained for over a year. Only 3 of 5 AML samples tested engrafted either sex of sub-lethally irradiated SRG-W41-3GS or SRG-3GS mice, and in each case, the speed and level of engraftment was greatly enhanced in the SRG-W41-3GS mice as compared to sex-matched SRG-3GS hosts (with 20-30-fold and 2-3-fold higher levels at week 6 and 10 respectively in female as compared to male recipients of either strain). All the engrafted SRG-W41-3GS female hosts became moribund within 20 weeks post-transplant with a predominance of human leukemic cells in their BM and enlarged spleens filled with leukemic cells. In contrast, the SRG-3GS recipients of the same AML cells survived more than 20 weeks with a more delayed appearance of leukemic cells. Sublethally irradiated female SRG-W41-3GS recipients of 0.1-0.2x106 CD34+ cells from 3 of 3 chronic myelomonocytic leukemia (CMML) patients tested to date have also shown engraftment at either week 3 or 11 and, in one case, up to 17 weeks post-transplant.Conclusion: The introduction of a c-kit deficiency into highly immunodeficient SRG mice producing human IL3, GM-CSF and SCF transgenically offers new opportunities for examining the growth potential and sensitivity to treatment of a broader spectrum of primary human leukemias and neoplastic conditions than has been previously possible. DisclosuresDeininger:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; CTI BioPharma Corp.: Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; BMS: Consultancy, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Pharmacological IRS1/2 Inhibition Induces Apoptosis in BCR-ABL1T315I mutant Cells

Introduction: Chronic myeloid leukemia (CML) is a hematological malignancy associated with the BCR-ABL1 oncoprotein, which results in deregulated kinase activity-driven leukemic phenotype. Most CML cases are successfully treated with tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1, but a significant percentage of patients develop drug resistance and/or progress to blast crisis, a disease stage that is refractory to therapy. Furthermore, recent studies have provided evidence that the CML leukemia stem cell is resistant to imatinib treatment. In this sense, the identification of other crucial proteins that cooperate with BCR-ABL1 signaling pathways may represent secondary therapeutic targets optimize treatment strategies. Increased IRS1 mRNA expression has been previously identified as positively correlated with worse overall survival in BCR-ABL1-positive adult acute lymphoblastic leukemia. In K562 cells, IRS1 has been identified as a binding partner of BCR-ABL1 and is capable of activating the PI3K/Akt/mTOR and MAPK pathways. Recently, a pharmacological IRS1/2 inhibitor (NT157) has been developed and has shown promising results in preclinical studies on solid tumors. We have previously shown that NT157 treatment reduces colony formation of primary CML cells but not in normal cells, decreases cell viability and proliferation of K562 cells, and increases apoptosis of K562 cells in a time- and dose-dependent manner (ASH 2015 - Abstract #83876). To further characterize the mechanism of the sensitivity of CML cells to IRS1/2 inhibition, here we performed PCR-based gene expression analyses in CML cells in response to NT157 treatment and investigated the effects of IRS1/2 inhibition on cell proliferation and apoptosis in Ba/F3 cells expressing wild-type or T315I mutant BCR-ABL1.Materials and Methods: K562 cells treated with vehicle or with the IRS1/2 pharmacological inhibitor NT157 (1.6 µM) for 48 hours were submitted to PCR array analysis using the PI3K-AKT Signaling Pathway and CancerPathwayFinder RT2 Profiler PCR Array kit (#PAHS-033A and #PAHS-058A, respectively; SA Biosciences, Frederick, MD, USA). Each cDNA sample was processed in a 96-well plate containing 156 signaling pathway-related genes and 5 endogenous control genes. Ba/F3 parental, BCR-ABL1WT and BCR-ABL1T315I cells were subjected to IRS1/2 pharmacological inhibition using NT157 (0.2, 0.4, 0.8, 1.6 and 3.2 µM) for 24, 48 and 72 hours and were evaluated for cell viability (MTT assay), apoptosis (annexin V/PI), and protein expression/activation (Western blot). Statistical analyses were performed by Student’s t-test or Mann-Whitney test, as appropriate.Results: Sixteen genes were found to be differentially expressed (fold change ≥ 2); 5 upregulated genes (FOXO3A, CDKN1A, FOS, JUN, VEGF) and 1 downregulated gene (E2F1) were chosen for validation in a larger number of experiments. Notably, NT157 treatment also resulted in significant dose- and time-dependent decrease in viability in BCR-ABL1-expressing cells regardless of BCR-ABL1 mutation status, compared to parental Ba/F3 cells. NT157 treatment (1.6 µM) for 48 hours reduced cell viability of Ba/F3 BCR-ABL1WT and BCR-ABL1T315I cells to 64% and 76%, respectively (p<0.05). Identical treatment with NT157 also significantly induced apoptosis as noted by the percentage of cells positive for annexinV/PI staining compared to vehicle in Ba/F3 BCR-ABL1WT and BCR-ABL1T315I cells (84 vs. 13% and 92 vs. 22%, respectively; p<0.05). Similar results were observed after 24 and 72 hours of NT157 treatment for both cell lines. More importantly, NT157 treatment reduced phosphorylation of STAT3 at both serine (S)727 and tyrosine (Y)705 in Ba/F3 BCR-ABL1T315I in the absence of IL3.Conclusion: Pharmacological inhibition of IRS1/2 decreases cell viability, increases apoptosis, and reduces STAT3 activation in cells expressing wild-type or T315I-mutant BCR-ABL1 in a time- and dose-dependent manner. These findings suggest that targeting of IRS1/2 alone or in combination with ABL1 TKIs may offer further improved treatment strategies and outcomes for maximizing control of disease resistance and persistence in patients with CML. DisclosuresDruker:Agios: Honoraria; Ambit BioSciences: Consultancy; ARIAD: Patents & Royalties, Research Funding; Array: Patents & Royalties; AstraZeneca: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Other: travel, accommodations, expenses; BMS: Research Funding; CTI: Equity Ownership; Curis: Patents & Royalties; Cylene: Consultancy, Equity Ownership; D3 Oncology Solutions: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses ; Lorus: Consultancy, Equity Ownership; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Novartis: Research Funding; Oncotide Pharmaceuticals: Research Funding; Pfizer: Patents & Royalties; Roche: Consultancy.

A Novel Approach to Identify a Putative Leukemia Stem Cell Population in Acute Lymphocytic Leukemia (ALL) and Distinguish Philadelphia Chromosome-Positive ALL from Lymphoid Blast Crisis Chronic Myeloid Leukemia

Introduction: Recent evidence supports the clinical significance of leukemia stem cells (LSCs) in acute myeloid leukemia (AML). However, the identification of LSCs in acute lymphocytic leukemia (ALL) has proved challenging, as transplantation studies in immunocompromised mice have yielded conflicting results. The distinction between Philadelphia chromosome-positive (Ph+) ALL and lymphoid blast crisis (LBC) chronic myeloid leukemia (CML) is also controversial. We previously identified a clinically relevant CD34+CD38- population of LSCs with intermediate (int) levels of aldehyde dehydrogenase (ALDH) activity (CD34+CD38-ALDHint) in AML [Gerber, et al. Blood, 2012]. This population was not present in healthy controls and could be distinguished from normal hematopoietic stem cells (HSCs), which had higher levels of ALDH activity (CD34+CD38-ALDHhigh). We hypothesized that the same approach could be used to identify a putative LSC population in ALL. Furthermore, in contrast to most cases of AML, the chronic phase CML stem cell was found to reside in the same CD34+CD38-ALDHhigh population as normal HSCs [Gerber, et al. Am J Hematol, 2011]. We therefore also hypothesized that the presence of BCR/ABL mutations in the CD34+CD38-ALDHhigh population might help distinguish LBC CML from Ph+ ALL.Methods: Bone marrow and/or peripheral blood specimens were collected at diagnosis from patients with B cell ALL or LBC CML on an IRB-approved protocol. A total of 7 patients were evaluated: 2 Ph- ALL, 2 Ph+ ALL, and 3 LBC CML patients. CD34+ cells were isolated by magnetic bead and column selection, then analyzed by flow cytometry with respect to CD38 expression and ALDH activity. Sorted cell populations were analyzed by fluorescence in situ hybridization (FISH) for leukemia-specific abnormalities. Polymerase chain reaction was performed on clinical samples to determine the presence of a p190 vs. p210 transcript.Results: All patients harbored an aberrant CD34+CD38-ALDHint population, similar to that previously seen in AML. This population was ≥95% positive for BCR/ABL by FISH in all Ph+ ALL and LBC CML cases. It was similarly positive (≥75%) for other leukemia-specific FISH abnormalities (including trisomy 4, 8, 10, 12, and/or 21) in all four ALL cases, as well as one LBC CML case. Conversely, the CD34+CD38-ALDHhigh population (which typically contains the normal HSCs) lacked any of the other cytogenetic abnormalities in all of the cases, irrespective of Ph status or a diagnosis of ALL vs. CML. Notably, the CD34+CD38-ALDHhigh population was negative for BCR/ABL in the Ph+ ALL cases but was >95% positive for BCR/ABL by FISH in the LBC CML cases. The B cell differentiation marker, CD19, was expressed on the CD34+CD38-ALDHint but not the CD34+CD38-ALDHhigh population in all ALL cases, both Ph- and Ph+. In contrast, CD19 expression was variable in the LBC CML cases. Both Ph+ ALL cases possessed a p190 BCR/ABL transcript, whereas all of the LBC CML cases contained a p210 transcript. Also of note, the CD34+CD38-ALDHint population was persistently detectable in one of the LBC CML patients while in complete remission after induction therapy; that patient subsequently relapsed.Conclusions: An abnormal CD34+CD38-ALDHint population was identified in all cases of B cell ALL and LBC CML. This population is analogous to a previously identified, clinically relevant LSC population in AML and may represent a putative LSC population in ALL. The CD34+CD38-ALDHhigh population was normal by FISH in the ALL cases but contained the BCR/ABL mutation in the LBC CML cases, thus permitting distinction between Ph+ ALL and LBC CML (which also differed based on the presence of p190 vs. p210 transcripts, respectively). Additionally, clonal evolution from chronic phase to lymphoid blast crisis CML was apparent, based on the acquisition of additional cytogenetic abnormalities unique to the CD34+CD38-ALDHint population as compared to the CD34+CD38-ALDHhigh population. The presence of CD19 on the putative LSCs in the four cases of ALL suggest that CD19-directed therapies may target the LSCs and thus may have curative potential in those cases. This assay may serve as a means to evaluate other possible therapeutic targets. Lastly, the detection of the abnormal CD34+CD38-ALDHint population may have utility as a minimal residual disease assay for monitoring response to treatment. These findings warrant validation in a larger patient cohort. DisclosuresGerber:Janssen: Research Funding; Alexion: Membership on an entity's Board of Directors or advisory committees; Spectrum: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees. Grunwald:Alexion: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Incyte Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Medtronic: Equity Ownership; Janssen: Research Funding; Ariad: Membership on an entity's Board of Directors or advisory committees; Forma Therapeutics: Research Funding. Avalos:Seattle Genetics: Membership on an entity's Board of Directors or advisory committees.

Multi-Drug Resistant Chronic Myeloid Leukemia: Report of Two Cases and Review of Literature

Introduction: In the last one decade first generation tyrosine kinase inhibitor (TKI), imatinib mesylate was the choice of treatment in Chronic Myeloid Leukemia (CML) but recently, a considerable number of patients have developed resistance to first as well as second generation TKIs.&#x0D; Objective: To present two cases which were first and second generation TKIs resistant and to review multidrug resistant CML.&#x0D; Materials and Methods: Relevant articles and literatures were retrieved from different journals and web pages to find out the mechanism of development of drug resistance in chronic myeloid leukaemia.&#x0D; Discussion: Two cases of drug resistant CML were found in Combined Military Hospital (CMH), Dhaka cantonment. Extensive review of the literature showed that two mechanisms, namely Breakpoint Cluster Region-Abelson (BCR-ABL) dependent and independent mechanisms, are mainly responsible for multi drug resistant (MDR) CML. In Bangladesh, it is difficult to identify the exact mechanism of development of drug resistance in CML. In a few cases, sample from CML patients are sent overseas to detect any new mutation in TKI binding domain of BCR – ABL fusion gene but it is not possible to find out other mechanisms responsible for drug resistance in CML.&#x0D; Conclusion: To overcome the drug resistance in CML, it is important to use the conventional drugs used in the treatment of CML meticulously and regularly as irregular use and discontinuation of treatment will permit the development of drug resistance.&#x0D; Journal of Armed Forces Medical College Bangladesh Vol.12(2) 2016: 135-140

Downregulation of miR-451 in Tunisian chronic myeloid leukemia patients: potential implication in imatinib resistance

ABSTRACTObjectives: Resistance to imatinib has been recognized as a major challenge for the treatment of chronic myeloid leukemia (CML). Aberrant expression of miR-451 has been reported to participate in anticancer drug resistance. However, the role of miR-451 in imatinib resistance has not been investigated. The present study was undertaken to determine the expression of miR-451 in order to find a possible association between the expression of this miRNA and imatinib resistance in Tunisian CML patients.Methods: First, real-time RT-PCR was performed to identify the expression of miR-451 in peripheral leukocytes of 59 CML patients treated with imatinib. Then, bioinformatics analysis was carried out to understand the regulatory roles of miR-451 in imatinib-resistant process.Results: Downregulated miR-451 was observed in imatinib-resistant CML cases. In silico analysis identified MYC as a potential target of miR-451. We further revealed the existence of an MYC-binding site in MiR-451 promoter region. On the other hand, increased level of MYC was detected in imatinib-resistant CML cases which may explain the causative role of MYC in CML cases and the downregulation of miR-451.Conclusion and discussion: Taken together, our findings suggest that miR-451 and MYC form together a regulatory loop which may act as a potential therapeutic target, and disruption of suggested regulatory loop could help to improve CML therapy.

Chylothorax Related with Dasatinib in the Treatment of Chronic Myeloid Leukemia: Report of 3 Cases

To study the clinical features and prognosis of chylothorax related with dasatinib in treatment of chronic myeloid leukemia(CML). The clinical data from 3 cases of CML with chylothorax after the treatment with dasatinib were collected. The clinical characteristics were analyzed and compared in the light of published literatures. They were treated with imatinib 400 mg once daily after diagnosing the illness as CML. The patients achieved optimal response after switching to dasatinib 100 mg once daily when didn't resisted or endured to imatinib. However, the symptom of dyspnea occurred in the patient after dasatinib treatment for 8,19, and 24 months. The CT examination all showed pleural effusion. According to the results of the pleural effusion test, dasatinib-related chylothorax was diagnosed. Among them 1 case did not stop for dasatinib, only received the support treatment, up to now, the patient still in CMR. The effect of another case was not obvious after giving half dose of dasatinb. The symptoms was disappeared after stopping dasatinb, but there was still a small amount of pleural effusion. Currently, he was been given imatinib 400 mg once a day. The third case stopped taking dasatinib for 48 days, fasting for 35 days, supporting parenteral nutrition and basic therapy. Ligation of thoracic duct was also used to reduce pleural effusion. At present, the patient continued to dasatinib and the symptoms of chest distress disappeared, promising review to the hospital in 3 months. The second-generation TKI dasatinib can lead to serious chylothorax in treatment of CML, timely and appropriate intervention are necessary when finding early detection of lung symptoms.