Factor In Chronic Myeloid Leukemia Research Articles

Prognosis in Chronic Myeloid Leukemia: Baseline Factors, Dynamic Risk Assessment and Novel Insights.

The introduction of tyrosine kinase inhibitors (TKIs) has changed the treatment paradigm of chronic myeloid leukemia (CML), leading to a dramatic improvement of the outcome of CML patients, who now have a nearly normal life expectancy and, in some selected cases, the possibility of aiming for the more ambitious goal of treatment-free remission (TFR). However, the minority of patients who fail treatment and progress from chronic phase (CP) to accelerated phase (AP) and blast phase (BP) still have a relatively poor prognosis. The identification of predictive elements enabling a prompt recognition of patients at higher risk of progression still remains among the priorities in the field of CML management. Currently, the baseline risk is assessed using simple clinical and hematologic parameters, other than evaluating the presence of additional chromosomal abnormalities (ACAs), especially those at "high-risk". Beyond the onset, a re-evaluation of the risk status is mandatory, monitoring the response to TKI treatment. Moreover, novel critical insights are emerging into the role of genomic factors, present at diagnosis or evolving on therapy. This review presents the current knowledge regarding prognostic factors in CML and their potential role for an improved risk classification and a subsequent enhancement of therapeutic decisions and disease management.

Fibrinogen Level among Sudanese Patients with Chronic Myeloid Leukemia

Background: Chronic myeloid leukemia (CML) is a malignancy of white blood cells, distinguished by the Philadelphia chromosome's presence. The biological profile of sufferers with CML at diagnosis is nevertheless missing in Africa, especially in Sudan. Therefore, the study pursued to measure the fibrinogen level among Sudanese patients diagnosed with CML attending the Radiation and Isotopes Centre of Khartoum. Methods: This descriptive perspective and the analytical casecontrol study were performed at the Radiation and Isotopes Centre of Khartoum from August to September 2021. In this study, fifty patients with CML were utilized as a case, and 50 healthy people were used as a control group. The blood samples from groups were collected in sodium citrate anticoagulant containers. Fibrinogen levels in the blood were determined by the Clauss technique using a coagulometer device. Results: There was a significant increase in fibrinogen levels between case and control groups. Further, age, gender, family history, and presence of chronic disease were non-significantly variations in CML patients. Moreover, smoking was considered a risk factor in CML. Conclusions: Fibrinogen level was significantly elevated in CML patients; additionally, there were significant differences associated with smoking, and there were non-significant differences related to age, gender, family history, presence of chronic disorder, and treatment.

Killer immunoglobulin-like receptor genotypes and chronic myeloid leukemia outcomes after imatinib cessation for treatment-free remission.

Background Natural Killer (NK) cells are innate lymphoid cells that can be cytotoxic toward a large panel of solid tumors and hematological malignancies including chronic myeloid leukemia (CML). Such a cytotoxicity depends on various receptors. Killer immunoglobulin‐like receptors (KIR) belong to these receptors and are involved in maturation process, then in the activation abilities of NK cells. Methods: We investigated the prognostic impact of the KIR2DL5B genotype in 240 CML patients included in two clinical trials investigating tyrosine kinase inhibitors (TKI) discontinuation: STIM and STIM2. Results: After adjustment for standard risk factors in CML, we found that the inhibitory receptor KIR2DL5B‐positive genotype was independently related to a delayed second deep molecular remission (HR 0.54, 95% CI [0.32‐0.91], P = 0.02) after TKI rechallenge but not to time to first deep molecular remission or treatment‐free remission rates. Conclusion: These results suggest that KIR2DL5B could carry a role in lymphocyte‐mediated control of leukemic residual disease control in patient with CML relapse.

Prognostic factors for overall survival in patients with chronic myeloid leukemia treated with imatinib at the National Cancer Institute - Mexico, from 2000 to 2016.

To determine potential predictors of long‐term survival in a large set of Hispanic (Mexican) patients with chronic myeloid leukemia (CML) treated with imatinib. We conducted an analysis with data from 411 patients with CML treated at the National Cancer Institute – Mexico, between January 2000 and December 2016. We found a median age at diagnosis of 40 years (range: 18‐84 years). The survival rate at 150 months was 82.02%, and we found that phase at diagnosis (β: 0.447, 95% Confidence Interval [95% CI]: 0.088, 0.806; P = 0.015), prognostic scales (Sokal [P = 0.021] and Hasford [β: 0.369, 95% CI: 0.049, 0.688; P = 0.024]) and hematological response at 3 months (β: 0.717, 95% CI: 0.443, 0.991; P < 0.001), but not molecular response (P = 0.834 for 6 months, P = 0.927 for 12 months, P = 0.250 for 18 months), were independently associated with overall survival. Survival analysis in subsets, according to the initial phase (chronic, accelerated and blastic phase) did not show any effect according to prognostic scales (P > 0.05). Mexican patients with CML have repeatedly been diagnosed at earlier ages. Prognostic factors in CML may differ according to the ethnic or geographical context. We found that phase at diagnosis, prognostic scale and hematological response at 3 months were independent predictors of survival.

Single nucleotide polymorphism in PTEN-Long gene: A risk factor in chronic myeloid leukemia

The BCR-ABL1 oncogene is associated with chronic myeloid leukemia (CML) pathogenesis, but the molecular mechanisms that initiate leukemogenesis are still unclear. Cancer pathogenesis has been associated with genetic alterations that may lead to inactivation of tumor suppressor genes. Phosphatase and tensin homolog (PTEN) is frequently deleted or inactivated in various tumors. A recently discovered variant of PTEN, PTEN-Long (PTEN-L), results from an alternative translation initiation site located upstream of the canonic AUG and generates a protein of 576 amino acids instead the expected protein of 403 amino acids. A 16 bp perfect palindromic motif centered on the PTEN-L CUG513 start codon is required for translation initiation. A single nucleotide polymorphism (SNP) of PTEN-L gene rs12573787 is located on the first exon respect to the CUG initiation site. In this case-control study we evaluated the association of genetic variants in PTEN-L with CML risk and therapy response in the Argentine population. The allele A of SNP rs12573787 was found to be associated with CML risk OR (95% CI) 1.71 (1.11–2.63) p = 0.016, which resulted consistent by multivariate analysis adjusted by gender and age. According to previous evidence that CML is more frequent in males, we found that the genetic risk of CML was confined to this gender. Unexpectedly, we also found this association confined to CML patients older than 45 years old. To our knowledge, this is the first time that PTEN-L rs1257378 was studied in CML suggesting that the variant A allele is a risk factor for CML development but, no association with the failure to TKIs treatment was found.

Abstract 3378: Variance in the distance between chromosome territories 9 and 22 in lymphocytes from peripheral blood healthy subjects: A proof of concept for a possible risk factor in chronic myeloid leukemia

Abstract The objective of our study was to assess the variations in the 3D organization of Chromosome Territories (CT) of chromosomes 9 and 22 in lymphocytes from healthy subjects. The differences in the distances of CTs 9 and 22 between subjects may provide a new perspective in the predisposition for specific chromosome translocations. We quantified the intra- and inter-subject variation in the distance of CT 9 and 22 as a proof of concept for new questions on the physiological and pathological effects of these differences. Methods: Blood samples from healthy subjects were processed for the isolation of lymphocytes with Ficoll as the gradient media. After the isolation, we fixed the cells directly onto coverslips for a 3D Fish protocol with 9 and 22 whole chromosome probes. Our protocol has been standardized to maintain the integrity of the nucleus with only minor changes in its volume. After the hybridization, we used the Elyra System of Zeiss to obtain super resolution images in Z stack. The images were processed with the Imaris software for its 3D reconstruction, measurement (distances, volumes and colocalization) and analysis. Results: The CTs 9 and 22 behave differently in peripheral blood lymphocytes. The CT 22 has a more internal position (closer to the center of the nucleus) in comparison with the CT 9, which can be seen most of the times in contact with the border of the nucleus. The CT 22 occupies a smaller volume than CT 9, which has almost a twice as larger volume. The CTs 9 have a polarized distribution inside the nucleus, therefore the distance between the CT 9-9 was larger (~7.3 μm ± 0.9) than the distance between CT 22-22. Although the position of the CTs was highly variable in cells from the same individual, we found a statistically significant difference in the inter-individual distance between CT 9-9 and the minimal distance between CT 9-22. Conclusion: Our study suggests that there are differences in the distribution of CT in lymphocytes isolated from peripheral blood between our subjects. These differences could prove to be a new individual characteristic with important implications in the predisposition for specific diseases, mainly to those related with chromosomal rearrangements. Citation Format: David G. Vallejo, Eunice Fabian, Luis A. Herrera. Variance in the distance between chromosome territories 9 and 22 in lymphocytes from peripheral blood healthy subjects: A proof of concept for a possible risk factor in chronic myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3378.

Roles of Interferon Regulatory Factors in Chronic Myeloid Leukemia.

The Interferon Regulatory Factor (IRF) family consists of multiple transcription factors involved in the regulation of a variety of biological processes. Originally identified as transcriptional regulators of the type I interferon system, IRFs play a pivotal role in adaptive immunity, cell growth, differentiation and tumorigenesis. Hence, understanding IRF biology has important implications in the host response to cancer development and progression. Many lines of evidence suggest that different IRFs are involved in the pathogenesis of Chronic Myeloid Leukemia (CML), a myeloproliferative disorder caused by the BCR-ABL oncoprotein. BCR-ABL displays constitutive tyrosine kinase activity that favors cell proliferation, inhibits apoptosis and allows cell survival even in the absence of proper adhesion to the extracellular matrix. Different BCR-ABL tyrosine kinase inhibitors are currently available for CML treatment. These drugs are able to generate eight year CML-specific overall survival rates >90%, only a minority of patients will achieve molecular responses compatible with drug discontinuation. Thus, there is an unmet need for additional therapeutic targets that may lead to the cure of most patients diagnosed with CML. A growing body of evidence has suggested a role for both IRF4 and IRF8 in the pathogenesis of CML. Furthermore, IRF1 is consistently deleted at one or both alleles in patients with leukemia and myelodysplasia. Finally, we have recently demonstrated that IRF5 is a target of BCR-ABL kinase activity and reduces CML cell proliferation. In this article, we provide an update on the current knowledge of the role of the IRFs in CML.

ChIP-seq Analysis of Human Chronic Myeloid Leukemia Cells.

Many transcription factors, chromatin-associated proteins and regulatory DNA elements are genetically and/or epigenetically altered in cancer, including Chronic Myeloid Leukemia (CML). This leads to deregulation of transcription that is often causally linked to the tumorigenic state. Chromatin-immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-seq) is the key technology to study transcription as it allows in vivo whole-genome mapping of epigenetic modifications and interactions of proteins with DNA or chromatin. However, numerous DNA/chromatin-binding proteins, including EZH2, remain difficult to "ChIP," thus yielding genome-wide binding maps of only suboptimal quality. Here, we describe a ChIP-seq protocol optimized for high-quality protein-genome binding maps that have proven especially useful for studying difficult to 'ChIP' transcription regulatory factors in Chronic Myeloid Leukemia (CML) and related malignancies.

Survivin and SIRT1: can be two prognostic factors in chronic myeloid leukemia?

Antiapoptotic molecules are important in the development, progression, and prognosis of malignant disorders. The aim of this study was to determine the expression of two distinct antiapoptotic molecules, SIRT1 and Survivin (SVV), in chronic myeloid leukemia (CML) and to compare them with hematological prognostic factors. In this study, we measured the expression of SVV and SIRT1 in 18 patients with typical Ph-positive chronic myeloid leukemia in chronic phases of the disease as well as in 18 matched healthy donors. SVV, SIRT1, and BCR/ABL expression were determined by quantitative-real-time polymerase chain reaction (Q-RT-PCR). HPRT was used as a reference gene to calculate SVV/HPRT and SIRT1/HPRT ratio, and ABL was used as an internal control for BCR/ABL expression. SVV was between 0.00 and 2.39-fold (median 0.204) and SIRT1 was between 0.00 and 33.32-fold (median 0.186). In our study, both SVV and SIRT1 were lower in patients than in the controls (p = 0.001 and 0.001, respectively). Although the expression of SVV and SIRT1 was decreased in patients with chronic phase (0.043 and 0.043-fold, respectively), a potent expression upregulation of both genes was found in K562, Jurkat, and U937 cell lines. Despite lower expression of both SVV and SIRT1 at chronic phase, a significant correlation was found between SVV expression levels and minor Hb concentration (<10) (p = 0.005). In addition, there was important correlation between BCR/ABL copy number and older age (≥60) and PLT count (p = 0.027 and p = 0.001, respectively). Of course, no specific clinicohematological or prognostic profile was associated with SIRT1 expression. In contrast with SIRT1 expression, high SVV expression and high BCR/ABL copy number could be introduced as prognostic factors in chronic phase of CML, but this finding must be evaluated in other phase of CML and with a higher number of subjects.

The BIM deletion polymorphism: A paradigm of a permissive interaction between germline and acquired TKI resistance factors in chronic myeloid leukemia.

Both germline polymorphisms and tumor-specific genetic alterations can determine the response of a cancer to a given therapy. We previously reported a germline deletion polymorphism in the BIM gene that was sufficient to mediate intrinsic resistance to tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML), as well as other cancers [1]. The deletion polymorphism favored the generation of BIM splice forms lacking the pro-apoptotic BH3 domain, conferring a relative resistance to the TKI imatinib (IM). However, CML patients with the BIM deletion polymorphism developed both partial and complete IM resistance. To understand the mechanisms underlying the latter, we grew CML cells either with or without the BIM deletion polymorphism in increasing IM concentrations. Under these conditions, the BIM deletion polymorphism enhanced the emergence of populations with complete IM resistance, mimicking the situation in patients. Importantly, the combined use of TKIs with the BH3 mimetic ABT-737 overcame the BCR-ABL1-dependent and -independent resistance mechanisms found in these cells. Our results illustrate the interplay between germline and acquired genetic factors in confering TKI resistance, and suggest a therapeutic strategy for patients with complete TKI resistance associated with the BIM deletion polymorphism.

Abstract 5103: RNA editing enzyme induces accelerated cell cycle in normal hematopoiesis

Abstract We have previously shown that inflammation-responsive RNA editase ADAR1 (ademosine deaminases acting on dsRNA) is a contribution factor in CML (chronic myeloid leukemia) disease progression. Whole transcriptome sequencing (RNA-Seq) data on primary CML chronic phase and CML blast crisis patient demonstrated over-representation of inflammatory IFN-pathways involved in hematological development. The resulting ADAR1 up-regulation plays important roles in both stem cell differentiation and self-renewal, as indicated by in vitro colony-formation assay and in vivo CML xenotransplantation mouse model. Though we have established ADAR1-mediated RNA editing as a novel theraputic target for treating CML, we do not yet understand the underlying mechanism of RNA editase's involvement in normal hematopoietic stem cells self-renewal and differentiation. In our new study, we describe ADAR1's role in cell cycle regulation of normal hematopoietic stem cell and its molecular editing targets. Normal cord bloods or aged bone marrow samples (CD34+) were lentivirally transduced with either backbone or ADAR1-overexpression vector. The cell cycle status of progenitors was analyzed by FACS and immunofluorescence. The expressions of genes in various cell cycle stages were analyzed by qRT-PCR. CML BC (CD34+) was transduced with lenti-shADAR1 and transplanted into immunodeficient mice. After 20 weeks, hematopoietic organs were harvested and analyzed for cell cycle status by FACS and serial transplantation. Our results demonstrated that ADAR1 accelerates G0 to G1 phase transition in normal hematopoietic stem cells, coupled with increased cell size and elevated expression of Ki67. The expanded population maintains stemness without any significant increase in differentiation. qRT-PCR microarray of cell cycle genes indicates that p21 expression level is reduced by &amp;gt;70% when ADAR1 is overexpressed. Moreover, shRNA knockdown of ADAR1 in CML BC sample shows a reduction of engraftment in bone marrow and spleen, and an enrichment of G0 population in the remaining cells. A decrease of self-renewal capacity as demonstrated by serial engraftment suggests the residual LSC failed to propagate. Our finding suggests carefully regulated A-to-I editing by ADAR1 is essential for the maintenance of proper cell grow and proliferation in HSC. It is plausible that the elevated expression level of ADAR1 observed in CML BC LSC contributes to false regulation of cell cycle that leads to the expansion of malignant leukemia stem cells. Citation Format: Qingfei Jiang, Marianna Zipeto, Angela Cournt, Heather Leu, Leslie Crews, Sheldon Morris, Catriona Jamieson. RNA editing enzyme induces accelerated cell cycle in normal hematopoiesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5103. doi:10.1158/1538-7445.AM2014-5103

Regulation of the Interferon regulatory factor-8 (IRF-8) Tumor Suppressor Gene by the Signal Transducer and Activator of Transcription 5 (STAT5) Transcription Factor in Chronic Myeloid Leukemia

Tyrosine kinase inhibitors such as imatinib can effectively target the BCR-ABL oncoprotein in a majority of patients with chronic myeloid leukemia (CML). Unfortunately, some patients are resistant primarily to imatinib and others develop drug resistance, prompting interest in the discovery of new drug targets. Although much of this resistance can be explained by the presence of mutations within the tyrosine kinase domain of BCR-ABL, such mutations are not universally identified. Interferon regulatory factor-8 (IRF-8) is a transcription factor that is essential for myelopoiesis. Depressed IRF-8 levels are observed in a majority of CML patients and Irf-8(-/-) mice exhibit a CML-like disease. The underlying mechanisms of IRF-8 loss in CML are unknown. We hypothesized that BCR-ABL suppresses transcription of IRF-8 through STAT5, a proximal BCR-ABL target. Treatment of primary cells from newly diagnosed CML patients in chronic phase as well as BCR-ABL(+) cell lines with imatinib increased IRF-8 transcription. Furthermore, IRF-8 expression in cell line models was necessary for imatinib-induced antitumor responses. We have demonstrated that IRF-8 is a direct target of STAT5 and that silencing of STAT5 induced IRF-8 expression. Conversely, activating STAT5 suppressed IRF-8 transcription. Finally, we showed that STAT5 blockade using a recently discovered antagonist increased IRF-8 expression in patient samples. These data reveal a previously unrecognized BCR-ABL-STAT5-IRF-8 network, which widens the repertoire of potentially new anti-CML targets.

The BIM Deletion Polymorphism: A Paradigm Of a Permissive Interaction Between Germline and Acquired TKI Resistance Factors In Chronic Myeloid Leukemia

Germline polymorphisms and tumor-specific genetic mutations together contribute to the behavior of human cancers, including the response to therapy. However, few specific models allow for the detailed study of how inherited and acquired genetic factors interact to cause clinical drug resistance, nor how their interaction can be prevented or overcome. We recently reported a germline deletion polymorphism in the BIM gene that was sufficient to mediate intrinsic resistance to targeted therapies in cancer, including the example of imatinib in chronic myeloid leukemia (CML) (Ng et al. Nature Medicine, 2012). Mechanistically, the deletion polymorphism led to the generation of BIM splice forms that lacked the pro-apoptotic BH3 domain, and that were incapable of activating apoptosis in response to targeted therapy. Clinically, we found that CML patients with the deletion polymorphism experienced inferior responses to imatinib. Furthermore, within the cohort of resistant patients harboring the BIM deletion polymorphism, we found that the majority developed cross-resistance to second or third generation TKI (nilotinib, dasatinib, and bosutinib), and that TKI resistance-conferring BCR-ABL kinase domain mutations were less frequent in patients with the BIM deletion polymorphism than those without. These observations suggested that the BIM deletion polymorphism cooperates with both BCR-ABL-independent and -dependent TKI resistance mechanisms to produce broad resistance to TKI.We generated K562 cells with and without the BIM deletion polymorphism that were resistant to imatinib by using the method described by Mahon et al (Blood, 2000). We found that deletion-containing cells exhibited 3- to 4-fold increased viability as compared to wildtype cells, and this was due to reduced apoptosis in the deletion-containing cells. We then compared the panel of wildtype and deletion-containing cells for the development of known TKI resistance mechanisms. The table below summarizes our findings. Importantly, we also found that, in a number of cells, both ERK and LYN activation occurred independently of BCR-ABL.Known TKI resistance mechanismWildtypeHeterozygous for BIM deletion polymorphismHomozygous for BIM deletion polymorphismSomatic mutation in BCR-ABL kinase domain--G250EBCR-ABL gene amplification--+++LYN activation+++++ERK activation+++-++Next, we tested the ability of second-generation TKI, including dasatinib and nilotinib, to overcome resistance induced by long-term culture. We also determined if the addition of ABT-737, a BH3-mimetic, to the TKI could overcome resistance in our cell lines. First, we found that prolonged incubation with imatinib induced cross-resistance to both nilotinib and dasatinib, a result that reproduced our observation in patients with the BIM deletion polymorphism. Next, we found that a combination of second generation TKI, especially dasatinib, and ABT-737 induced maximal cell death in cells with the BIM deletion polymorphism. Based on the information generated from our cell lines, we evaluated the viability of TKI-resistant primary CML cells when treated with TKI with and without ABT-737. In TKI-resistant primary CML cells, regardless of their BIM deletion status, maximal reduction in cell viability was observed when both TKI and ABT-737 were used.We conclude that: 1. The BIM deletion polymorphism is permissive for the acquisition of somatic events that mediate both BCR-ABL-dependent and -independent mechanisms of TKI resistance. 2. The permissive action of the BIM deletion polymorphism is associated with reduced apoptosis, and likely accounts for the ability of BIM deletion polymorphism-containing CML cells to acquire secondary resistance-conferring events. 3. The combined use of TKI with ABT-737 represents a potential therapeutic strategy to overcome TKI resistance in CML patients with and without the BIM deletion polymorphism. 4. In our cell line system, dasatinib with ABT-737 induced maximum cell death. Finally, our results demonstrate that germline and acquired resistance factors can interact to produce resistance to targeted therapies in cancer, and that cell line systems allow us to both elucidate these mechanisms as well as devise therapeutic strategies to overcome such resistance. Disclosures:Chuah:Novartis: Honoraria; Bristol-Myers Squibb: Honoraria.

Identification of Basophils as a Major Source of Hepatocyte Growth Factor in Chronic Myeloid Leukemia: A Novel Mechanism of BCR-ABL1-Independent Disease Progression

Chronic myeloid leukemia (CML) is a hematopoietic neoplasm characterized by the Philadelphia chromosome and the related BCR-ABL1 oncoprotein. Acceleration of CML is usually accompanied by basophilia. Several proangiogenic molecules have been implicated in disease acceleration, including the hepatocyte growth factor (HGF). However, little is known so far about the cellular distribution and function of HGF in CML. We here report that HGF is expressed abundantly in purified CML basophils and in the basophil-committed CML line KU812, whereas all other cell types examined expressed only trace amounts of HGF or no HGF. Interleukin 3, a major regulator of human basophils, was found to promote HGF expression in CML basophils. By contrast, BCR-ABL1 failed to induce HGF synthesis in CML cells, and imatinib failed to inhibit expression of HGF in these cells. Recombinant HGF as well as basophil-derived HGF induced endothelial cell migration in a scratch wound assay, and these effects of HGF were reverted by an anti-HGF antibody as well as by pharmacologic c-Met inhibitors. In addition, anti-HGF and c-Met inhibitors were found to suppress the spontaneous growth of KU812 cells, suggesting autocrine growth regulation. Together, HGF is a BCR-ABL1-independent angiogenic and autocrine growth regulator in CML. Basophils are a unique source of HGF in these patients and may play a more active role in disease-associated angiogenesis and disease progression than has so far been assumed. Our data also suggest that HGF and c-Met are potential therapeutic targets in CML.

Leukemic spleen cells are more potent than bone marrow-derived cells in a transgenic mouse model of CML

Spleen size ranks among the most important risk factors in chronic myeloid leukemia (CML), but the pathogenic mechanisms of splenic hematopoiesis in CML remain poorly defined. Here, we studied the biology of Bcr-Abl positive leukemia-initiating cells in the spleen, using an inducible transgenic mouse model of CML. Disease kinetics showed greater increases of immature leukemic cells in spleen vs bone marrow (BM). To assess how Bcr-Abl alters the behavior of spleen-derived CML cells, we transplanted these cells either before ('pre-uninduced') or 44 days after ('pre-induced') expression of the oncogene. Mice transplanted with pre-induced spleen cells showed significantly increased neutrophilia and splenomegaly compared with mice receiving pre-uninduced spleen cells, suggesting that Bcr-Abl expression in the donors had increased splenic tumor burden. However, pre-induction also altered the biology of these cells, as shown by a striking increase in erythropoietic potential. These results differ from those of BM-derived CML stem cells where pre-induction of Bcr-Abl had previously been shown to decrease disease transplantability. Moreover, splenic cells were less sensitive to imatinib than BM cells. In conclusion, Bcr-Abl alters the biology of splenic leukemic stem cells by a cell-autonomous mechanism, but the disease phenotype is also influenced by the microenvironment of these cells.

Role of the Hypoxic Microenvironment in the Antitumor Activity of Tyrosine Kinase Inhibitors

The tumor microenvironment is characterized, not only by marked gradients in drug concentration, but also by gradients in the rate of cell proliferation and by regions of hypoxia and acidity, all of which can influence tumor cell sensitivity to drug treatment. Hypoxia is also an important environmental factor in chronic myeloid leukemia (CML), because bone marrow is intrinsically hypoxic in nature. Systems-wide analyses of tumors have recently identified receptor tyrosine kinase coactivation as an important mechanism by which cancer cells achieve chemoresistance. Recent work suggests that Src activation might play a prominent role in the response to hypoxia to promote cell survival, progression, and metastasis of a variety of human cancer. Other studies also established a functional link between Bcr-Abl and the Src family tyrosine kinases. It is well known that mutations can also cause some tyrosine kinases to become constitutively active, a nonstop functional state that may contribute to initiation or progression of cancer as in CML. Leukemic cells carrying chromosomal alteration, are sensitive to imatinib that induces complete remission in most patients. This inhibitor is a highly selective Bcr-Abl tyrosine kinase inhibitor (TKI). There is a considerable interest in understanding how activated signaling pathways enhance tumor cell survival under hypoxia, because this might lead to the introduction of more effective treatments to target these resistant subpopulations. For all these reasons it is important to identify new TKIs which are also active in hypoxia, the real tumor microenvironment, as possible alternative therapy.

Deregulation of TWIST-1 in the CD34+ compartment represents a novel prognostic factor in chronic myeloid leukemia

AbstractThe mechanisms of resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) often remain obscure. Analysis of patient samples during disease progression revealed the up-regulation of the oncogene TWIST-1, also measured in primary samples from TKI-resistant patients. Moreover, we found that TWIST-1 was overexpressed in CML diagnostic samples of patients who later developed cytogenetic resistance to imatinib, even those without any detectable resistance mechanism. We confirmed the up-regulation of TWIST-1 at both RNA and protein levels in imatinib-resistant cell lines, irrespective of any other resistance mechanism. Analysis with specific small interfering RNA suggested TWIST-1 involvement in the resistance phenotype. Finally, the kinetics of TWIST-1 expression during the individual medical histories of CML patients indicated that TWIST-1 expression is down-regulated by TKIs and up-regulated with TKI resistance. We hypothesize that the overexpression of the TWIST-1 oncogene represents a novel key prognostic factor potentially useful for optimizing CML management in the TKI era.

Coexisting with Clonal Evolution and BCR-ABL Mutant in CML Patients Treated with Second-generation Tyrosine Kinase Inhibitors Predict the Discrepancy of in vitro Drug Sensitivity

Second-generation tyrosine kinase inhibitors (second TKIs) such as nilotinib and dasatinib control the activity of most ABL kinase domain mutations observed in patients with imatinib resistance. Although in vitro data show that both agents can inhibit all mutations except T315I, some discrepancies have been observed in a small subset of mutation clones. Cytogenetic clonal evolution is the important resistance mechanism of chronic myeloid leukemia (CML). Accordingly, we observed the clinical significance of coexisting with clonal evolution and BCR-ABL mutant in CML patients treated with second TKIs. We monitored BCR-ABL transcript kinetics, interrelationship of clones expressing non-mutated and mutant transcripts and clonal aberrations within Philadelphia (Ph) positive and negative clones, respectively, in eight patients with CML receiving dasatinib or nilotinib for 3~41 months. Clinical responses were correlated with in vitro sensitivity of the BCR-ABL mutants to the second TKIs in four patients. Four patients showed resistance to the second TKIs as compared to in vitro observations; three of them developed chromosomal abnormalities in the Ph chromosome positive or negative metaphases. Another patient lost the original mutation but acquired a more resistant new mutation and became resistant to the second TKI. Cytogenetic clonal evolution is an independent poor prognostic factor in CML, which could explain the onset of mechanisms for second TKIs resistance to ABL kinase domain mutations. The results indicate that an additional evaluation of chromosomal abnormalities is warranted when BCR-ABL mutants are more resistant than indicated by in vitro data.

No Significance of Derivative Chromosome 9 Deletion on the Clearance Kinetics of BCR/ABL Fusion Transcripts, Cytogenetic or Molecular Response, Loss of Response or Treatment Failure to Imatinib Mesylate Therapy for Chronic Myeloid Leukemia.

Background: Deletion of the derivative chromosome 9 (der 9; del-der9) is a poor prognostic factor in chronic myeloid leukemia (CML) patients treated with hydroxyurea or interferon. However, its prognostic implication in imatinib mesylate (IM) treated patients is unclear. One study reported that IM improved but did not fully reverse the poor prognosis of CML patients with del-der 9, while another suggested that IM might overcome the poor prognostic significance of del-der 9. The purpose of current study is to evaluate the prognostic implication of del-der 9 in CML patients in terms of hematologic, cytogenetic and molecular responses, loss of response to IM and IM treatment failure rate in addition to BCR/ABL mRNA quantitative PCR monitoring.Methods: The current study included 163 CML patients with available FISH results using the LSI-BCR/ABL-(ES) probe (Vysis, IL, USA) treated at the Princess Margaret Hospital (Toronto, Canada). End points of the study included hematologic response (HR), major or complete cytogenetic response (CyR; MCyR/CCyR), major or complete molecular response (MoR; MMoR/CMoR), loss of response (LOR), treatment failure (including primary hematologic or cytogenetic resistance and LOR), progression to accelerated phase or blast crisis (AP/BC), or time to IM dose escalation to achieve further response or to overcome LOR.Results: Of 163 patients, 22 (13.5%) had del-der 9 prior to initiation of IM. No differences were noted between those with and without del-der 9 in terms of disease phase or additional cytogenetic abnormalities. No differences were noted for the time to HR (p=0.598), MCyR (p=0.281), CCyR (p=0.883), MMoR (p=0.125), or CMoR (p=0.834). Times to LOR (p=0.974), treatment failure (p=0.455), progression to AP/BC (p=0.276), or dose escalation of IM (p=0.816) were not significantly different between those with and without del-der 9. The serial monitoring of BCR/ABL mRNA quantitative PCR showed similar patterns of BCR/ABL mRNA reduction between the 2 groups.Conclusion: The presence of del-der 9 in CML patients prior to IM therapy does not influence:1.the response to IM therapy in terms of HR, CyR and MoR;2.LOR, treatment failure or progression to AP/BC; and3.time to dose escalation of IM.Therefore, from the clinical point of view, the presence of del-der 9 does not impact on the management of CML patients treated with IM. [Display omitted]

BCR/ABL rearrangement in two cases of Philadelphia chromosome negative chronic myeloid leukemia: deletion on the derivative chromosome 9 may or not be present

The BCR/ABL gene rearrangement is the causing factor in chronic myeloid leukemia (CML). In most cases, it is cytogenetically visualized as a translocation between chromosomes 9 and 22, known as the Philadelphia (Ph) translocation. About 5–10% of CML patients lack cytogenetic evidence of the Ph translocation but show BCR/ABL fusion by fluorescence in situ hybridization (FISH) or reverse transcriptase-polymerase chain reaction. Deletions around the breakpoints on the derivative 9 including ABL and or BCR sequences occur in 10–15% of Ph+ CML patients and are thought to have prognostic significance. We describe two patients with CML and normal karyotype in whom cryptic rearrangements involving chromosomes 9 and 22 resulted in the causative BCR/ABL gene. FISH with a three-color probe combination revealed BCR/ABL fusion on chromosome 9 without deletion in one patient; the other patient had BCR/ABL on chromosome 22 with an associated derivative 9 deletion. We discuss the proposed mechanisms in the formation of BCR/ABL in the setting of a normal karyotype. Some authors reported that patients with the chimeric gene located on the derivative 9 have a poor clinical course. We suggest that deletion rather than location of the chimeric gene alone is more likely to be associated with prognosis.