Down Regulation of TOPK, PBX3, SRPK, DDX21 and CLC Genes in Newly Diagnosed, Early-Chronic Phase Chronic Myeloid Leukemia Patients Treated with Imatinib Mesylate.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4650-4650
Author(s):  
Angela Poerio ◽  
Marilina Amabile ◽  
Simona Soverini ◽  
Matteo Renzulli ◽  
Gianantonio Rosti ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Chromosome 9 ◽  
Pcr Analysis ◽  
Newly Diagnosed ◽  
Rt Pcr

Abstract Background: Due to the BCR/ABL chimeric protein expression in chronic myeloid leukemia (CML), several genes and signaling pathways have been reported to be activated. These alterations in gene expression contribute to the pathophysiology of p210BCR/ABL-mediated transformation. Some of the overexpressed genes are implicated in cellular processes known to be disturbed in CML, including the mitogen-activated protein kinase or the ubiquitin phatway, whereas overexpression of other genes may implicate new cellular pathways involved in CML. The characterization of new genes which could be implicated in the pathophysiology of CML, may lead to the identification of potentially novel therapeutic targets for CML. In a previous study we adopted a microarray analysis to study the gene expression profiling of CD34+ cells taken from 5 de novo CML patients and treated in vitro with Imatinib (data not shown). Five genes had shown a greater down-regulation and a possible involvement in the in vitro effects of Imatinib. Aims: Aim of the present study was to confirm these data in vivo through a quantitative real time PCR analysis in 10 CML patients enrolled in a multicenter clinical trial of the GIMEMA CML working party (newly diagnosed, early-CP CML patients treated with Imatinib 400mg/die and peghilated interferon). Five patients were known to have chromosome 9 deletion. Methods: this study was performed on 10 bone marrow samples from 10 consecutive patients enrolled in the protocol. Bone marrow bone was collected prior to treatment (baseline) and after 3 and 6 months of Imatinib therapy. Five genes (TOPK, PBX3, SRPK, DDX21 and CLC) were analyzed at the RNA levels by means of a quantitative RT-PCR analysis (TaqMan). β2 microglobuline was used as control gene. Results were expressed as a median ΔCt value. Expression of these 5 genes before treatment with Imatinib and after 6 months of therapy was compared. Statistical significance was tested by the student’s T-Test. Results: Quantitative RT-PCR analysis confirmed that the 5 tested genes were downregulated after 6 months of treatment with Imatinib. About 1 log reduction was observed. The differences between the baseline median values and median values after 6 months of treatment were statistically significant (p>0.005). We were not able identify any differences between patients who showed chromosome 9 deletion and patients who did not. Conclusions: treatment of CML patients with the ABL-specific tyrosine kinase inhibitor Imatinib decreased expression at the mRNA level of all the genes tested. This suggests that increased gene expression could be in some case tyrosine-kinase dependent and it could be implicated in the pathophisiology of CML. Study supported by: COFIN 2003 (M. Baccarani), AIRC, AIL, Fondazione del Monte di Bologna e Ravenna, FIRB 2001.

SPTBN1-FLT3 in Atypical Chronic Myeloid Leukemia Transforms Ba/F3 Cells to IL-3 Independence and Is Sensitive to Both Tyrosine Kinase Inhibitors and Immunotherapy.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2009-2009
Author(s):  
Francis H. Grand ◽  
Sameena Iqbal ◽  
Lingyan Zhang ◽  
Nigel H. Russell ◽  
Andrew Chase ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Fusion Gene ◽  
Kinase Domain ◽  
Tyrosine Kinase Domain ◽  
Unrelated Donor ◽  
Rt Pcr

Abstract We have identified a patient who presented with BCR-ABL negative chronic myeloid leukemia (CML) and an acquired 46XX, t(2;13;2;21) (p13;q12;q33;q11.2) in all bone marrow metaphases examined. Fluorescence in situ hybridization (FISH) using probes flanking the FLT3 gene at 13q12 suggested that this gene was disrupted. 5′-RACE PCR using primers to the region of FLT3 encoding the tyrosine kinase domain identified a novel in-frame mRNA fusion between exon 3 of SPTBN1 (spectrin, beta, non-erythrocytic 1 isoform 2, NM 178313) on chromosome 2p16 and exon 13 of FLT3 (NM 004119). Juxtaposition of SPTBN1 and FLT3 was confirmed by two color FISH and amplification of the genomic DNA breakpoint confirmed a fusion between intron 3 of SPTBN1 and intron 12 of FLT3. The SPTBN1-FLT3 fusion gene is predicted to be translated into a 570 amino acid chimeric protein that retains two coiled-coil domains from SPTBN1 and 424 amino acids from FLT3, including the entire tyrosine kinase domain. Since the t(2;13) is readily visible by cytogenetic analysis but has not been reported previously it seems likely that SPTBN1-FLT3 is uncommon. However to test if FLT3 might be involved more widely in BCR-ABL negative CML we analysed 40 cases by RT-PCR. Two cases were positive for the FLT3 internal tandem duplication (ITD) but mutation of residue D835 was not observed. Expression of the SPTBN1-FLT3 fusion transformed the interleukin 3 (IL-3)-dependent cell line Ba/F3 to growth factor independence and was accompanied by constitutive phosphorylation of the fusion protein and the downstream substrate ERK1/2. The growth of transformed cells was inhibited in a dose-dependent fashion by SU11567 and PKC142, but not by imatinib mesylate. The patient was initially treated with hydroxyurea and subsequently underwent an unrelated donor bone marrow transplant. She relapsed cytogenetically at 4 years but responded to donor lymphocyte infusion (DLI), achieving sustained cytogenetic and molecular (nested RT-PCR) remission. We conclude that SPTBN1-FLT3 is a rare abnormality in BCR-ABL negative CML that is responsive to both targeted signal transduction therapy and immunotherapy by DLI.

scholarly journals Concurrent Diagnosis of Chronic Myeloid Leukemia and Follicular Lymphoma: An Unreported Presentation

2018 ◽  
Vol 2018 ◽  
pp. 1-4
Author(s):  
Amy G. Starr ◽  
Sushma R. Jonna ◽  
Joeffrey J. Chahine ◽  
Bhaskar V. Kallakury ◽  
Chaitra S. Ujjani
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Follicular Lymphoma ◽  
Myeloid Leukemia ◽  
Chronic Phase ◽  
Mediastinal Lymphadenopathy ◽  
Pcr Analysis ◽  
Molecular Testing ◽  
Fish Analysis ◽  
Rt Pcr ◽  
Non Hodgkin Lymphoma

Lymphadenopathy in chronic myeloid leukemia (CML) is usually due to extramedullary involvement with accelerated or blast phases of the disease. The occurrence of non-Hodgkin lymphoma (NHL) as a synchronous malignancy with CML is rare. We report a case of a 73-year-old male who presented with dyspnea and right-sided lower extremity edema in the setting of leukocytosis. Bone marrow evaluation indicated a chronic phase chronic myeloid leukemia (CML), confirmed by molecular testing. Imaging of the chest for persistent dyspnea revealed supraclavicular and mediastinal lymphadenopathy. Biopsy of the cervical node showed expanded lymphoid follicles with atypical germinal centers that were positive for CD10, BCL-2, and BCL-6, consistent with follicular lymphoma (FL). Nodal PCR demonstrated clonal IGH and IGK gene rearrangements, and FISH analysis was positive for IGH-BCL-2 fusion. Together, these tests supported the diagnosis of FL. Additionally, the lymph node showed paracortical expansion by maturing pan-hematopoietic elements, no blastic groups, and positive RT-PCR analysis for BCR-ABL1, indicating concomitant involvement by chronic phase-CML. To our knowledge, this is the first reported case of a patient with a concurrent diagnosis of CML and FL.

Kinetics of the Graft-Versus-Leukemia Response After Donor Leukocyte Infusions for Relapsed Chronic Myeloid Leukemia After Allogeneic Bone Marrow Transplantation

Blood ◽  
1998 ◽  
Vol 92 (10) ◽  
pp. 3582-3590 ◽  
Author(s):  
Herrad Baurmann ◽  
Stefan Nagel ◽  
Thomas Binder ◽  
Andreas Neubauer ◽  
Wolfgang Siegert ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Transplantation ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Allogeneic Bone Marrow Transplantation ◽  
Allogeneic Bone ◽  
Rt Pcr ◽  
Molecular Remission ◽  
Graft Versus Leukemia ◽  
Kinetics Of

Abstract Little is known about the mechanisms and the kinetics of the so-called graft-versus-leukemia (GVL) response induced by donor lymphocyte infusions (DLI) in patients with leukemic relapse after allogeneic bone marrow transplantation (BMT). We sought to elucidate this problem by sequentially studying three patients with relapsed chronic myeloid leukemia after sex-mismatched BMT from time before donor leukocyte infusion until achievement of complete molecular remission. Lineage-specific chimerism was assessed longitudinally by a combined fluorescent immunophenotyping and sex chromosome-specific in situ hybridization approach. Results were related to quantitative detection of bcr-abl transcripts by competitive differential reverse transcriptase-polymerase chain reaction (RT-PCR), qualitative bcr-abl RT-PCR, and multiplex PCR-based DNA donor/recipient chimerism. All patients had predominant donor lymphopoiesis at the time of DLI, suggesting a state of tolerance to recipient leukemic and/or normal cells. In contrast, granulopoiesis and erythropoiesis were mainly recipient derived in both patients with hematologic relapse and partly recipient derived in the patient with molecular relapse. Eighty percent, 90%, and 8% of CD34+cells, respectively, were found to be of recipient origin at relapse, and few donor stem cells predicted for cytopenia post-DLI. Responses were seen after a time lag of 5 to 13 weeks after DLI and resulted in reversal to full donor chimerism within a critical switch period of 4 to 5 weeks. A sudden decrease in recipient cells was paralleled by a sharp decrease in bcr-abl transcript numbers detectable several weeks before achievement of molecular remission and onset of clinical graft-versus-host disease (GVHD). This response pattern was confirmed by retrospective RT-PCR analysis in an additional five patients. Prospective monitoring of stem cell chimerism and response may enable us to individually tailor adoptive immunotherapy in the future.

Recurrent bone marrow aplasia secondary to nilotinib in a patient with chronic myeloid leukemia: A case report

2012 ◽  
Vol 18 (4) ◽  
pp. 440-444 ◽  
Author(s):  
Prathima Prodduturi ◽  
Anamarija M Perry ◽  
Patricia Aoun ◽  
Dennis D Weisenburger ◽  
Mojtaba Akhtari
Keyword(s):  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Chronic Phase ◽  
Bone Marrow Aplasia ◽  
Line Therapy ◽  
Grade 3

Nilotinib is a potent tyrosine kinase inhibitor of breakpoint cluster region-abelson (BCR-ABL), which has been approved as front-line therapy for newly diagnosed chronic myeloid leukemia in chronic phase and as second-line therapy after imatinib failure in chronic or accelerated phase chronic myeloid leukemia. Tyrosine kinase inhibitors have been associated with myelosuppression and grade 3 or grade 4 cytopenias are not uncommon in chronic myeloid leukemia patients treated with these drugs. There are a few reports of imatinib-associated bone marrow aplasia, but to our knowledge only one reported case of bone marrow aplasia associated with nilotinib. Herein, we report a 49-year-old male patient with chronic phase chronic myeloid leukemia, who developed severe bone marrow aplasia due to nilotinib. Possible mechanisms for this significant adverse drug reaction are discussed along with a review of literature.

In Vitro Sensitivity to Tyrosine Kinase Inhibitors Is One of Possible Predictive Parameters for Therapy Switch In Patients with Chronic Myeloid Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2747-2747
Author(s):  
Marketa Zackova ◽  
Tereza Lopotova ◽  
Zuzana Ondrackova ◽  
Hana Klamova ◽  
Jana Moravcova
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Transcript Level ◽  
Molecular Response ◽  
Newly Diagnosed ◽  
Therapy Switch

Abstract Abstract 2747 Backround: Tyrosine kinase inhibitors (TKI) are very effective in chronic myeloid leukemia (CML) suppression, however, the problem with development of resistance in some patients exists. It is necessary to find optimal methods for therapy response prediction and for detection of resistance. Many studies on the resistance to imatinib therapy were performed on cell lines or model systems. However, these systems are not fully consistent with CML situation in vivo. Sensitivity to imatinib and its predictivity to molecular response in patients with de novo CML were tested in vitro on patients′ leukocytes by White et al. [Blood 2005; 106: 2520]. They found that IC50 values could be predictive mainly in patients with low Sokal score. Aims: To optimize in vitro method for evaluation of patients′ sensitivity to various TKIs and to test its predictivity for molecular response in therapy and/or after therapy change. Methods: The sensitivity to TKIs: imatinib, nilotinib and dasatinib were studied on leukocytes isolated from CML patients at diagnosis and various responses to treatment. Cell lines were used as controls. Isolated leukocytes/cell lines were cultivated with/without TKIs. Optimization of cultivation was performed on cell lines (ML-2, K562, CML-T2, JURL-MK1) and on leukocytes from CML newly diagnosed patients (15) and healthy donors (6). Various incubation times (4, 24, 48 and 72h) were tested. Concentrations of TKI were used in values near to physiological levels: 2 –3 concentrations for each inhibitor (1uM, 10uM imatinib, 0,5uM and 2uM nilotinib and 1nM, 10nM and 100nM dasatinib). In given time-points the cells were harvested and lysed for protein and mRNA analyses. Sensitivity to TKIs was tested by BCR-ABL kinase inhibition – via Crkl phosphorylation (western blots) and also by WT1 transcript level kinetics [Cilloni et al, Cancer 2004; 101: 979]. Quality of cultivation was tested by apoptosis level (RNA degradation, Annexin staining – Agilent Bioanalyzer 2100). Results: We found 48 h to be the optimal time for in vitro cultivation. This time was long enough to see TKIs dependent changes on protein as well as mRNA level. At this time the intensity of apoptosis was relatively low and did not influence results. The predictive ability of cultivation with TKIs was tested on patients at diagnosis (15), with optimal (5) and suboptimal response (5) and patient with therapy failure (13). The disease state of all patients was further monitored in range from 6 to 21 months (median 12 months) after cultivation. Mostly all of newly diagnosed patients were in vitro sensitive to all three TKIs, 10 of them achieved MMR (median 7 months, range 5 – 16) on imatinib. In patients with resistance to imanitib therapy the good sensitivity to one of 2nd generation TKI on in vitro tests represented the good response to this inhibitor, 4 patients from 10 on dasatinib achieved MMR (within 4 months), the other responded to therapy with continual decrease of BCR-ABL transcript level. Thus, the cultivation test can help with the therapy switch. However, the prognosis of patients with additive chromosomal aberration was poor even if they were sensitive to TKIs in vitro. Only one of 3 patients with 8 trisomy sensitive to dasatinib in vitro achieved MMR at 4th month after starting of dasatinib. Two patients with T315I were not sensitive to any of TKIs in vitro and in vivo, as it was expected. We continue to follow up of all patients. In conclusion, the results from in vitro cultivations of patients′ leukocytes with TKIs can help with the choice of efficient inhibitor for individual patient′s therapy, however, it is necessary to take into consideration the results of cytogenetic analyses of patients and other factors influencing CML. Supported by MZOUHKT2005. Disclosures: No relevant conflicts of interest to declare.

Quantitative Phosphoproteomics Identified a New Syk-Lyn-Axl Signalling Pathway Involved In Resistance to Nilotinib In Chronic Myeloid Leukemia Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3376-3376
Author(s):  
Romain Gioia ◽  
Cedric Leroy ◽  
Claire Drullion ◽  
Valérie Lagarde ◽  
Serge Roche ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Dependent Manner ◽  
Stable Isotope Labelling ◽  
Resistant Cells

Abstract Abstract 3376 Nilotinib has been developed to overcome resistance to imatinib, the first line treatment of chronic myeloid leukemia (CML). To anticipate resistance to nilotinib, we generate nilotinib resistant CML cell lines in vitro to characterize mechanisms and signaling pathways that may contribute to resistance. Among the different mechanisms of resistance identified, the overexpression of the Src-kinase Lyn was involved in resistance both in vitro, in a K562 cell line (K562-rn), and in vivo, in nilotinib-resistant CML patients. To characterize how Lyn mediates resistance, we performed a phosphoproteomic study using SILAC (Stable Isotope Labelling with Amino acid in Cell culture). Quantification and identification of phosphotyrosine proteins in the nilotinib resistant cells point out two tyrosine kinases, the spleen tyrosine kinase Syk and the UFO receptor Axl. The two tyrosine kinase Syk and Axl interact with Lyn as seen by coimmunopreciptation. Syk is phosphorylated on tyrosine 323 and 525/526 in Lyn dependent manner in nilotinib resistant cells. The inhibition of Syk tyrosine kinase by R406 or BAY31-6606 restores sensitivity to nilotinib in K562-rn cells. In parallel, the inhibition of Syk expression by ShRNA in K562-rn cells abolishes Lyn and Axl phosphorylation and then interaction between Lyn and Axl leading to a full restoration of nilotinib efficacy. In the opposite, the coexpression of Lyn and Syk in nilotinib sensitive K562 cells induced resistance to nilotinib whereas a Syk kinase dead mutant did not. These results highlight for the first time the critical role of Syk in resistance to tyrosine kinase inhibitors in CML disease emphasizing the therapeutic targeting of this tyrosine kinase. Moreover, Axl, which is already a target in solid tumor, will be also an interesting pathway to target in CML. Disclosures: No relevant conflicts of interest to declare.

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